Synthesis of dehydrodipeptide esters and their evaluation as inhibitors of cathepsin C

Article abstract of DOI:10.1007/s00044-015-1366-0

The procedures for the synthesis of esters of dehydropeptides containing C-terminal (Z)-dehydrophenylalanine and dehydroalanine have been elaborated. These esters appeared to be moderate or weak inhibitors of cathepsin C, with some of them exhibiting slow

Full text of DOI:10.1007/s00044-015-1366-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1366-0
ORIGINAL RESEARCH
Synthesis of dehydrodipeptide esters and their evaluation
as inhibitors of cathepsin C
Maciej Makowski1 Paweł Lenartowicz1 Bartosz Oszywa1
•
•
•
Michał Jewginski Małgorzata Pawełczak1 Paweł Kafarski1,2
2
•
•
´
Received: 7 October 2014 / Accepted: 27 February 2015
Ó The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract The procedures for the synthesis of esters of
dehydropeptides containing C-terminal (Z)-dehy-
2005; Battilani et al., 2011), hepatotoxins (microcystins
and nodularins) (Gulledge et al., 2002), phytotoxins (ten-
toxin and AM toxins) (Andre and Pinet, 1997; Jingfeng
et al., 2013) and antitumor agents (phenylahistin) (Kanoh
et al., 1999). This is because of both, the reactivity of their
side-chain double bonds (especially toward thiols) (Ferreira
et al., 2001; Seebeck et al., 2011) and of the ability to
undertake specific forms of three-dimensional structure
[they could be considered as foldamers (Goldman et al.,
2007)]. The latter properties cause the growing interest in
this class of compounds.
drophenylalanine and dehydroalanine have been elaborat-
ed. These esters appeared to be moderate or weak
inhibitors of cathepsin C, with some of them exhibiting
slow-binding behavior. As shown by molecular modeling,
they are rather bound at the surface of the enzyme and are
not submersed in its binding cavities.
Keywords Dehydropeptides Á Esterification Á
Enzyme inhibitors Á Molecular modeling
Although from some years we have been engaged in
studies on the dependence of three-dimensional structure of
dehydropeptides on their inhibitory activity toward cathep-
sin C, no clear structure–activity relationship could be drawn
(Makowski et al., 2001; Latajka et al., 2006, 2008). In this
paper, we present synthesis of esters of glycylZdehy-
drophenylalanine (Gly-ZDPhe), glycyldehydroalanine (Gly-
DAla) and L-phenylalanyldehydroalanine (Phe-DAla) and
evaluation of their action toward this enzyme.
Introduction
a,b-Dehydroaminoacids present in proteins contribute to
catalytic action in tyrosine aminomutase (Christenson
et al., 2003) and to properties of green fluorescent proteins
(Zimmer, 2002). They are also constituents of a variety of
peptidic allelochemicals of microbial origin, including
antimicrobial lantibiotics (nisin, subtilin, epidermin and
gallidermin) (Willey and van der Donk, 2007), neurotoxins
(roquefortine, oxaline and phomopsins) (Overy et al.,
Materials and methods
General
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1366-0) contains supplementary
material, which is available to authorized users.
All reagents and solvents were purchased from Sigma-
Aldrich, Avantor Performance Materials or Merck. Ethyl
acetate (EtOAc), dichloromethane (DCM), diethyl ether
(Et2O) and tetrahydrofuran (THF) were dried over P2O5
and then distilled. N,N-dimethyformamide (DMF) was
distilled under reduced pressure and stored over molecular
& Maciej Makowski
maciej.makowski@uni.opole.pl
1
Faculty of Chemistry, Opole University, Oleska 48,
45-052 Opole, Poland
˚
sieves 4 A. Other chemicals were used without purifica-
2
Department of Bioorganic Chemistry, Faculty of Chemistry,
Wroclaw University of Technology, Wybrzeze
Wyspianskiego 27, 50-370 Wroclaw, Poland
tion. Reaction progress was monitored by TLC on Merck
60 silica plates. The spots were visualized by placing
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Med Chem Res
chromatogram plate at chlorine vapor followed by spraying
with o-tolidine in water/acetic acid mixture. NMR spectra
were recorded on Bruker Ultrashield 400 MHz instrument,
operating at 400 MHz (1H) and 100 MHz (13C). Samples
were prepared in DMSO-d6 (99.8 % at. D). Chemical
shifts are reported in ppm relative to TMS used as internal
OMe in 94 %, Boc-(S)Phe-DAla-OEt in 94 % and Boc-
(S)Phe-DAla-OPri in 81 % yields. Deprotection of amine
group was performed in 20 % solution of TFA in DCM.
Deprotection of amine group of dehydrodipeptide esters
containing dehydroalanine required the use of anisole (3 %
v/v) for protection against oligomerization reactions.
Mixture was stirred at room temperature for 30 min, and
equivalent of p-toluenesulfonic acid was added. Mixing
was continued for 15 min, and solvent was removed under
reduced pressure. The residue was dissolved in dichlor-
omethane, and solvent was carefully evaporated to remove
the excess of trifluoroacetic acid. Products were crystal-
lized from mixtures of isopropanol/hexane.
standard or to the signal of solvent (1H NMR 2.5 ppm; 13
C
NMR 39.52 ppm for DMSO-d6), and coupling constant is
1
reported in Hertz. In the description of dipeptide H NMR
and 13C NMR spectra, the tosylate group is omitted for
better readability (Tos 1H NMR (DMSO, 400 MHz): d
7.48 (d, J = 8.0 Hz, 2H, ArH), 7.11 (d, J = 8.0 Hz, 2H,
ArH), 2.29 (s, 3H, CH3); 13C NMR (DMSO, 100 MHz): d
20.84 (CH3), 125.57, 128.14, 137.80, 145.71 (4 9 ArC).
The copies of all NMR spectra are available at electronic
supplementary material. Melting points were determined
on a Stuart SMP30 apparatus and are reported uncorrected.
Mass spectra were recorded on Bruker micrOTOF-Q II
high-resolution mass spectrometer with electrospray ion-
ization (ESI). IR spectra were recorded on Nicolet 6700
FT-IR spectrophotometer (Thermo Scientific) operating at
Gly-DAla-OMeÁTos 87 % yield (deprotection); mp =
151.5–155 °C with decomposition; 1H NMR d 9.92 (s, 1H,
NH), 8.02 (s, 3H, NH?3 ), 6.32 (s, 1H, CHAHB DAla), 5.84 (s,
1H, CHAHB DAla), 3.80 (s, 2H, CH2Gly), 3.78 (s, 3H,
OCH3). 13C NMR d 166.14 (C=Oamid.), 163.45 (C=Oest.),
132.02 (C=), 110.58 (CH2=), 52.88 (OCH3), 41.10
(CH2Gly). HRMS (ESI) m/z calcd for C6H11N2O3
(M ? H)? 159.0764; found 159.0767; IR (KBr, cm-1
)
resolution 2 cm-1 and scanning range 4000–400 cm-1
.
3700–2600 broad (H-bonding), 1733 (C=Oester), 1689 IAB
(C=Oamid), 1634 (C=C), 1551 IIAB (C–N and N–H),
1200–1171 (C–O–C and SO3), 919 (=CH2).
Samples were measured as KBr disks.
Synthesis of N-protected dehydrodipeptides
(S)Phe-DAla-OMeÁTos 98 % yield (deprotection); mp =
1
156–157 °C with decomposition; H NMR d 9.93 (s, 1H,
Boc-protected dehydrodipeptides containing C-terminal
dehydroalanine (DAla) or (Z)-dehydrophenylalanine (DZ-
Phe) were synthesized earlier by condensation of appro-
priate carboxamides with a-keto acids in benzene in the
presence of p-toluenesulfonic acid as catalyst (Makowski
et al., 1985).
NH), 8.25 (s, 3H, NH3?), 7.37–7.23 (m, 5H, ArHPhe), 6.27
(s, 1H, CHAHB DAla), 5.85 (s, 1H, CHAHB DAla), 4.42–4.34
(m, 1H, CHPhe), 3.76 (s, 3H, OCH3), 3.09 (ABX system,
J 13.9, 6.1 Hz, 1H, CHAHB Phe), 2.99 (ABX system, J 13.9,
7.8 Hz, 1H, CHAHB Phe). 13C NMR d 168.13 (C=Oamid.),
163.37 (C=Oest.), 134.60 (CArPhe), 131.92 (C=), 129.58,
128.60, 127.32 (3 9 CArPhe), 111.52 (CH2=), 53.72
(CHPhe), 52.86 (OCH3), 37.10 (CH2Phe). HRMS (ESI) m/z
calcd for C13H17N2O3 (M ? H)? 249.1234; found
249.1223; IR (KBr, cm-1) 3700–2700 broad (H-bonding),
1728 (C=Oester), 1694 IAB (C=Oamid), 1638 (C=C), 1538
IIAB (C–N and N–H), 1203–1166 (C–O–C and SO3), 919
(=CH2).
Synthesis of dehydrodipeptide methyl, ethyl
and isopropyl esters
Syntheses were based on procedure of Cossec et al. (2008).
Thus, Boc-Gly-DAla or Boc-(S)Phe-DAla was dissolved in
methanol (0.2 or 0.4 M, respectively), and 0.5 equivalent
of Cs2CO3 was added. The mixture was stirred for 1 h at
room temperature followed by evaporation of solvent. The
dipeptide cesium salt was dissolved in DMF (0.28 M), and
fivefold or fourfold excess (respectively) of methyl, ethyl
or isopropyl iodide was added in portions. After comple-
tion of the reaction (3–5 h, controlled by TLC), solvent
was evaporated under reduced pressure. The obtained
residue was dissolved in ethyl acetate and washed subse-
quently with: 1 M HCl, saturated KHCO3, 0.1 M Na2S2O3
and brine (each one in triplicate). Organic layer was dried
over anhydrous MgSO4. Product was crystallized from
mixtures of diethyl ether/hexane or ethyl acetate/hexane
providing Boc-Gly-DAla-OMe in 91 %, Boc-(S)Phe-DAla-
(S)Phe-DAla-OEtÁTos 85 % yield (deprotection); mp =
1
139–141 °C; H NMR d 9.91 (s, 1H, NH), 8.24 (s, 3H,
NH?3 ), 7.37–7.24 (m, 5H, ArHPhe), 6.27 (s, 1H, CHAHB
DAla), 5.84 (s, 1H, CHAHB DAla), 4.44–4.35 (m, 1H, CHPhe),
4.22 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.10 (ABX system,
J = 13.9, 6.2 Hz, 1H, CHAHB Phe), 2.99 (ABX system,
J = 13.9, 7.8 Hz, 1H, CHAHB Phe), 1.25 (t, J = 7.1 Hz,
3H, OCH2CH3). 13C NMR d 168.10 (C=Oamid.), 162.89
(C=Oest.), 134.60 (CArPhe), 132.10 (C=), 129.55, 128.60,
127.31 (3 9 CArPhe), 111.19 (CH2 =), 61.74 (CH2CH3),
53.70 (CHPhe), 37.10 (CH2Phe), 13.99 (CH2CH3). HRMS
(ESI) m/z calcd for C14H19N2O3 (M ? H)? 263.1390;
123

Med Chem Res
found 263.1395; IR (KBr, cm-1) 3700–2450 broad (H-
bonding), 1713 (C=Oester), 1691 IAB (C=Oamid), 1640
(C=C), 1535 IIAB (C–N and N–H), 1249–1167 (C–O–C
and SO3), 915 (=CH2).
(m, 1H, CH2=CH), 5.88 (s, 1H, CHAHB DAla), 5.40–5.33
(2 9 m, 1H, CH=CHAHB), 5.30–5.25 (2 9 m, 1H,
CH=CHAHB), 4.73 (m, 2H, OCH2), 3.81 (s, 2H, CH2Gly).
13C NMR d 166.11 (C=Oamid.), 162.63 (C=Oest.), 132.09
(CH=All), 132.01 (C=), 118.37 (CH2=All), 110.75
(CH2=DAla), 65.92 (OCH2), 41.08 (CH2Gly). HRMS (ESI)
m/z calcd for C8H13N2O3 (M ? H)? 185.0921; found
185.0919. IR (KBr, cm-1) 3600–2600 broad (H-bonding),
1718 (C=Oester), 1692 IAB (C=Oamid), 1649 (C=C), 1538
IIAB (C–N and N–H), 1198 broad (C–O–C and SO3), 922
(=CH2).
(S)Phe-DAla-OPriÁTos 80 % yield (deprotection); mp =
1
153–155 °C with decomposition; H NMR d 9.88 (s, 1H,
NH), 8.24 (s, 3H, NH3?), 7.39–7.21 (m, 5H, ArHPhe), 6.25
(s, 1H, CHAHB DAla), 5.81 (s, 1H, CHAHB DAla), 5.00 (hept,
J = 6.2 Hz, 1H, CH(CH3)2), 4.43–4.34 (m, 1H, CHPhe),
3.10 (dd, J = 13.9, 6.1 Hz, 1H, ABX system CHAHB Phe),
2.99 (dd, J = 13.9, 7.8 Hz, 1H, ABX system CHAHB Phe),
1.26 (d, J = 6.2 Hz, 6H, CH(CH3)2). 13C NMR d 168.07
(C = Oamid.), 162.44 (C = Oest.), 134.63 (CArPhe), 132.34
(C=), 129.55, 128.60, 127.31 (3 9 CArPhe), 110.91
(CH2=), 69.51 (CH(CH3)2), 53.70 (CHPhe), 37.11 (CH2Phe),
21.43 (CH(CH3)2). HRMS (ESI) m/z calcd for C15H21N2O3
Gly-DAla-OPrgÁTos 71 % global yield; mp = 141–
143.5 °C with decomposition; 1H NMR d 9.98 (s, 1H, NH),
8.05 (s, 3H, NH3?), 6.36 (s, 1H, CHAHB DAla), 5.87 (s, 1H,
CHAHB DAla), 4.89 (d, J = 2.3 Hz, 2H, OCH2), 3.81 (s,
2H, CH2Gly), 3.67 (t, J = 2.3 Hz, 1H, :CH). 13C NMR d
166.20 (C=Oamid.), 162.30 (C=Oest.), 131.69 (C=), 111.49
(CH2=), 78.48, 77.92 (2 9 C:CH), 53.34 (OCH2), 41.10
(CH2Gly). HRMS (ESI) m/z calcd for C8H11N2O3
(M ? H)? 277.1547; found 277.1545; IR (KBr, cm-1
)
3700–2450 broad (H-bonding), 1710 (C=Oester), 1690 IAB
(C=Oamid), 1640 (C=C), 1534 IIAB (C–N and N–H),
1226–1169 (C–O–C and SO3), 919 (=CH2).
(M ? H)? 183.0764; found 183.0771. IR (KBr, cm-1
)
3600–2800 broad (H-bonding), 2129 (C:C), 1732
(C=Oester), 1700 IAB (C=Oamid), 1638 (C=C), 1547 IIAB
(C–N and N–H), 1178 broad (C–O–C and SO3), 895
(=CH2).
Synthesis of allyl and propargyl esters of dipeptides
containing dehydroalanine
A Cs2CO3 0.163 g (0.5 mmol) was added to solution of
Boc-Gly-DAla 0.244 g (1 mmol) or Boc-(S)Phe-DAla
0.334 g (1 mmol) in 5 mL of methanol. The mixture was
stirred at room temperature for 2 h, and solvent was re-
moved under reduced pressure. Solid residue was dissolved
in 5 mL of THF for Boc-Gly-DAla or 5 mL of DMF for
Boc-(S)Phe-DAla, and allyl bromide 0.856 mL (10 mmol)
or propargyl bromide 1.114 mL (10 mmol) was added
dropwise over 15 min. When peptide substrate was con-
sumed (controlled by TLC), the solvent and excess of
bromide were removed under reduced pressure. The resi-
due was dissolved in 80 mL of ethyl acetate, filtrated and
washed with: 1 M HCl (4 9 5 mL), saturated KHCO3
(4 9 5 mL) and brine. Organic layer was dried over
MgSO4 and filtered, and 0.2 mL of anisole was added. The
solvent was removed under reduced pressure at 35 °C. The
residue was dissolved in 10 mL DCM, 1.5 mL of TFA was
added and the mixture was stirred for 1 h at room tem-
perature followed by addition of 0.190 g (1 mmol) of p-
toluenesulfonic acid. Stirring was continued for additional
20 min, and solvent was removed under reduced pressure.
The residue was evaporated two times with 20 mL of DCM
to remove TFA excess. Products were crystallized from
mixtures of isopropanol/hexane
(S)Phe-DAla-OAllÁTos 70 % global yield; mp = 123.5–
1
125 °C with decomposition; H NMR d 9.96 (s, 1H, NH),
8.24 (s, 3H, NH?3 ), 7.39–7.23 (m, 5H, ArHPhe), 6.30 (s, 1H,
CHAHB DAla), 6.03–5.91 (m, 1H, CH2=CH), 5.89 (s, 1H,
CHAHB DAla), 5.40–5.33 (2 9 m, 1H, CH=CHAHB),
5.30–5.25 (2 9 m, 1H, CH=CHAHB), 4.71 (m, 2H, OCH2),
4.40 (wide s, 1H, CHPhe), 3.10 (dd, J = 13.9, 6.2 Hz, 1H,
ABX system CHAHB Phe), 3.00 (dd, J = 13.9, 7.8 Hz, 1H,
ABX system CHAHB Phe). 13C NMR d 168.15 (C=Oamid.),
162.57 (C=Oest.), 134.59 (CArPhe), 132.08 (CH=All),
131.90 (C=), 129.55, 128.59, 127.31 (3 9 CArPhe), 118.39
(CH2=All), 111.71 (CH2=DAla), 65.92 (OCH2), 53.70
(CHPhe), 37.09 (CH2Phe). HRMS (ESI) m/z calcd for
C15H19N2O3 (M ? H)? 275.1390; found 275.1381. IR (KBr,
cm-1) 3600–2700 broad (H-bonding), 1722 (C=Oester), 1699
IAB (C=Oamid), 1637 (C=C), 1527 IIAB (C–N and N–H),
1231–1176 (C–O–C and SO3), 947 (=CH2).
(S)Phe-DAla-OPrgÁTos 65 % global yield; mp =
1
170–172 °C with decomposition; H NMR d 10.02 (s, 1H
NH), 8.24 (s, 3H, NH3?), 7.39–7.24 (m, 5H, ArHPhe), 6.30
(s, 1H, CHAHB DAla), 5.89 (s, 1H, CHAHB DAla), 4.87 (d,
J = 2.3 Hz, 2H, OCH2), 4.38 (wide s, 1H, CHPhe), 3.68 (t,
J = 2.3 Hz, 1H, : CH), 3.11 (dd, J = 13.9, 6.0 Hz, 1H,
ABX system CHAHB Phe), 3.00 (dd, J = 13.9, 7.8 Hz, 1H,
ABX system CHAHB Phe). 13C NMR d 168.20 (C=Oamid.),
162.22 (C=Oest.), 134.58 (CArPhe), 131.58 (C=), 129.56,
128.61, 127.34 (3 9 CArPhe), 112.52 (CH2=DAla), 78.48,
Gly-DAla-OAllÁTos 72 % global yield; mp = 159–
161.5 °C with decomposition; 1H NMR d 9.92 (s, 1H, NH),
8.04 (s, 3H, NH?3 ), 6.34 (s, 1H, CHAHB DAla), 6.05–5.92
123

Med Chem Res
77.89 (2 9 C : CH), 53.72 (CHPhe), 53.32 (OCH2), 37.07
(CH2Phe). HRMS (ESI) m/z calcd for C15H17N2O3
Efforts to synthesize dehydrodipeptide glycidyl
esters
(M ? H)? 273.1234; found 273.1224. IR (KBr, cm-1
)
3600–2850 broad (H-bonding), 2120 (C:C), 1745
(C=Oester), 1699 IAB (C=Oamid), 1632 (C=C), 1517 IIAB
(C–N and N–H), 1227–1168 broad (C–O–C and SO3).
Method I Boc-Gly-DZPhe 0.160 g (0.5 mmol), Et3N
0.196 mL (1.1 mmol) and (S)-glycidol 0.266 mL (2.0
mmol) were dissolved in 2.0 mL of acetonitrile, and TBTU
(Abdelmoty et al., 1994) 0.208 g (0.65 mmol) was then
added. Mixture was stirred at room temperature for 2.5 h,
and solvent removed under reduced pressure. The residue
was dissolved in 70 mL of ethyl acetate and washed sub-
sequently with: 1 M HCl (3 9 5 mL), saturated KHCO3
(3 9 5 mL) and brine. Organic phase was dried over
MgSO4 and filtered, and solvents were removed. We were
unable to purify a mixture of products obtained using
column chromatography with silica gel 60H (Merck) as
stationary phase and various eluents. Thus, crude mixture
was used in deprotection step. HRMS (ESI) indicated the
presence of the desired product as a major one: m/z calcd
for C19H24N2O6 (M ? Na)? 399.1526; found 399.1529.
Method II iso-butyl chloroformate 0.066 mL (0.5 mmol)
was added to solution of Boc-Gly-DZPhe 0.160 g (0.5
mmol) and Et3N 0.070 mL (0.5 mmol) in dichloromethane
when cooling in ice bath to -15 °C. After 1.5 min, gly-
cidol 0.133 mL (1.0 mmol) was added. The mixture was
left to warm to room temperature, and stirring was con-
tinued for next 24 h. Further steps of synthesis were per-
formed according to the methodology described for
Method I and afforded similar mixture of products.
Synthesis of allyl and propargyl esters of dipeptides
containing (Z)-dehydrophenylalanine
Boc-Gly-DZPhe 0.320 g (1.0 mmol) was dissolved in 5 mL
DMF, and Cs2CO3 0.163 g (0.5 mmol) was added. Mixture
was stirred for 3 h, and allyl bromide 0.856 mL (10 mmol)
or propargyl bromide 1.114 mL (10 mmol) was added
dropwise over 15 min. The reaction was continued for 12 h
stirring at room temperature. Further steps of synthesis
were done according to procedure described for allyl and
propargyl esters of Boc-Gly-DAla. The deprotection reac-
tion of amine group was performed without addition of
anisole and p-toluenesulfonic acid.
Gly-DZPhe-OAllÁTFA 88 % global yield; mp = 137–
1
138.5 °C with decomposition; H NMR d 10.19 (s, 1H,
NH), 8.19 (s, 3H, NH3?), 7.78–7.41 (m, 5H, ArHDPhe), 7.39
(s, 1H, CH DPhe), 6.08–5.90 (m, 1H, CH2=CH), 5.43–5.34
(2 9 m, 1H, CH=CHAHB), 5.30–5.23 (2 9 m, 1H,
H=CHAHB), 4.69 (m, 2H, OCH2), 3.81 (s, 2H, CH2Gly).
13C NMR d 166.22 (C=Oamid.), 164.08 (C=Oest.), 133.11,
132.87, 132.44, 130.18, 129.89, 128.79, 124.87, 117.99 (8
C atoms derived from (Z)-dehydrophenylalanine and allyl
group), 65.54 (OCH2), 40.38 (CH2Gly), (peaks derived from
TFA group are omitted for clarity). HRMS (ESI) m/z calcd
for C14H17N2O3 (M ? H)? 261.1234; found 261.1229. IR
Deprotection of amine group
Method I Trifluoroacetic acid 0.5 mL was added to solution
of Boc-Gly-DZPhe-OGdl 0.098 g (0.25 mmol) in 2 mL of
dichloromethane. Mixture was stirred for 20 min at room
temperature, and solvent was removed under reduced
pressure. The residue was evaporated three times with
20 mL of dichloromethane and 20 mL of diethyl ether to
remove the excess of trifluoroacetic acid. Mixture of
products was obtained as oily residue. HRMS (ESI) indi-
cated the presence of the two major products—desired
glycidol ester (Gly-DZPhe-OGdl(S)) and the product of
oxirane ring opening—Gly-DZPhe-OCH2CH(OH)CH2OH:
m/z calcd for C14H17N2O4 (M ? H)? 277.1183 and
C14H19N2O5 (M ? H)? 295.1288; found 277.1164 and
295.1266, respectively.
(KBr, cm-1
) 3600–2600 broad (H-bonding), 1723
(C=Oester), 1698 IAB (C=Oamid), 1625 (C=C), 1529 IIAB
(C–N and N–H), 1201–1180 (C–O–C), 922 (=CH2), 837
(=CHDPhe).
Gly-DZPhe-OPrgÁTFA 92 % global yield; mp = 145–
147 °C with decomposition; 1H NMR d 10.21 (s, 1H, NH),
8.20 (s, 3H, NH?3 ), 7.73–7.42 (m, 5H, ArHDPhe), 7.40 (s,
1H, CH DPhe), 4.84 (d, J = 2.4 Hz, 2H, OCH2), 3.81 (s,
2H, CH2Gly), 3.64 (t, J = 2.4 Hz, 1H, : CH). 13C NMR d
166.24 (C=Oamid.), 163.72 (C=Oest.), 133.88, 132.74,
130.27, 130.07, 128.83, 124.33 (6 C atoms derived from
(Z)-dehydrophenylalanine), 78.29, 78.16 (2 9 C:CH),
52.86 (OCH2), 40.38 (CH2Gly) (for clarity peaks derived
from TFA group are omitted); HRMS (ESI) m/z calcd for
C14H15N2O3 (M ? H)? 259.1077; found 259.1060. IR
(KBr, cm-1) 3600–2600 broad (H-bonding), 2132 (C:C),
1723 (C=Oester), 1698 IAB (C=Oamid), 1624 (C=C) 1531
IIAB (C–N and N–H), 1201–1179 (C–O–C), 837
(=CHDPhe).
Method II HCl in methanol (*3.8 M) solution was
prepared by bubbling dry HCl gas through methanol for 1 h
at 0 °C. Crude Boc-Gly-DZPhe-OGdl(S) 0.129 g (0.34
mmol) was dissolved in methanol (1.2 mL), and HCl–
methanol solution was added (1.3 mL). After 1 h at room
temperature, solvent was evaporated under reduced pres-
sure. The oil residue was evaporated three times with 5 mL
of dichloromethane. Product was crystallized from mixture
123

Med Chem Res
´
using the computer program provided kindly by dr Jozef
of isopropanol/diethyl ether/hexane (2:1), filtered and dried
in vacuo.
Hurek (University of Opole). The Ki values presented in
the Table 1 are the average ones calculated by using all
these methods. All measurements were taken in a three
repetitions.
In that manner, Gly-DZPhe-OCH2CH(OH)CH2ClÁHCl
was obtained as a white solid in 50 % yield (deprotection):
mp = 178–180 °C decomposition; 1H NMR d 10.28 (s,
1H, NH), 8.30 (s, 3H, NH?3 ), 7.74–7.40 (2 9 m, 2H and
4H, ArHD(Z)Phe overlapped with CHD(Z)Phe), 5.67 (d,
J = 5.1 Hz, 1H, OH), 4.18 (dd, J = 11.1, 5.2 Hz, 1H),
4.13 (dd, J = 11.1, 5.7 Hz, 1H), 4.05–3.97 (m, 1H,
CHOH), 3.79 (s, 2H, CH2Gly), 3.73 (dd, J = 11.3, 4.8 Hz,
1H), 3.66 (dd, J = 11.3, 5.5 Hz, 1H). Four dd at 4.18, 4.13,
3.73, 3.66 ppm derived from two CH2 groups which are
present at OCH2CH(OH)CH2Cl part of the molecule. 13C
NMR d 166.21 (C=Oamid.), 164.22 (C=Oest.), 133.44,
132.91, 130.24, 129.87, 128.78, 124.62 (6 C atoms derived
from (Z)-dehydrophenylalanine), 67.94, 66.14, 46.56
(OCH2CH(OH)CH2Cl), 40.37 (CH2Gly). HRMS (ESI) m/z
calcd for C14H18ClN2O4 (M ? H)? 313.0950; found
313.0950; intensity of ions: 313.0950 I = 100 %;
Molecular modeling
The structures of studied dehydropeptides were optimized
in Gaussian09 program at the B3LYP/6-311 g (d,p) level
(Frisch et al., 2004) in gas phase with using Merz-Singh-
Kollman scheme (Besler et al., 1990) to the determination
of the atomic charges. The calculations of the docking
process were performed using AutoDock program (Morris
et al., 2009). The starting geometry and charges of the
dehydropeptides were taken from the ab initio calculations.
The structure of cathepsin C was extracted from the
structure of human dipeptidyl peptidase I deposited EC
3.4.14 in Protein Data Bank (Turk et al., 2001). Structure
of the enzyme has been protonated on the H?? server
(Myers et al., 2006) at pH = 5.7, and also charges of all
enzymatic atoms have been assignment on this server.
During the docking process, main chain of the dehy-
dropeptide was fixed, whereas side chains and the terminal
groups were left as flexible. The coordinates of the SH
proton from the Cys234 were taken as a grid center in the
docking process. In the simulation, docking process was
performed 100 times. Analysis of the obtained results has
been performed by using AutoDock Tools (Morris et al.,
2009).
315.0926 I = 34.2 % (chlorine isotopes). IR (KBr, cm-1
)
3600–2550 broad (H-bonding), 1706 (C=Oester), 1680 IAB
(C=Oamid), 1636 (C=C), 1541 IIAB (C–N and N–H), 841
(=CHDPhe).
Enzymatic studies
Cathepsin C was isolated from bovine spleen by modified
method of McDonald et al. (1972). The KM value of
2.3 mM for the enzyme was measured using synthetic
substrate—glycine-L-phenylalanine-p-nitroanilide (Gly-L-
Phe-pNA). Purity of the enzyme was confirmed by
electrophoresis.
Results and discussion
Inhibitory studies
Cathepsin C (EC 3.4.14.1) is a lysosomal cysteine protease
expressed in majority of mammalian tissues and is pri-
marily responsible for activation of serine proteases in in-
flammatory and immune cells (Reiser et al., 2010). It
sequentially removes dipeptides from the N-termini of
protein and peptide substrates (Lindley, 1972; Pore˛ba et al.,
2014). Increasing evidence of the key role of DPPI in
various diseases, such as sepsis, asthma, Duchenne mus-
cular dystrophy, rheumatoid arthritis, basal cell carcino-
mas, chronic obstructive pulmonary disease and other
inflammatory disorders (Guay et al., 2010; Laine and
Busch-Petersen, 2010), stimulates interest in this enzyme
as the possible medicinal target.
Cathepsin C was activated for 0.5 h in a water bath at
37 °C in 1 % NaCl solution containing 1 mM EDTA-Na2
and 5 mM 2-mercaptoethanol. The enzymatic reaction was
carried out at 37 °C in 100 mM acetate buffer, pH 5.0,
containing 1 mM EDTA-Na2, 1 mM DTT and 30 mM
NaCl (all final concentrations). The progress of the reaction
was monitored spectrophotometrically (UV–Vis spec-
trophotometer Cintra 303) at a wavelength of 405 nm
against a control sample containing no enzyme. Attempting
mixture contained: solution of the synthetic substrate Gly-
L-Phe-pNA in acetate buffer at pH 5 containing 1 mM
EDTA-Na2, 1 mM DTT, 30 mM NaCl (substrate concen-
tration: 2.7–0.01 mM—final concentration), the solution of
inhibitor in reaction buffer (concentration of compound
depended on inhibitory potential), and enzyme.
Dehydropeptides appear to be weak inhibitors of the
enzyme (Latajka et al., 2006, 2008). In this paper, we
synthesized series of structurally variable esters of gly-
cylZdehydrophenylalanine and its analogs. We speculated
that the possible binding of the aromatic part of the in-
hibitor within S2 pocket of the enzyme might result in
Kinetic constants KM, Vmax and Ki and type of inhibi-
tion were determined by using Lineweaver–Burk, Dixon,
Hanes-Woolf and half-inhibitory concentration methods
123

Med Chem Res
Table 1 Inhibitory constants of the studied dehydrodipeptides toward cathepsin C
Compound
Ki (lM)
Compound
Ki (lM)
(S)Phe-AlaOMeÁTos
Gly-DAlaOMeÁTos
(S)Phe-DAlaOEtÁTos
Gly-DAlaOAllÁTos
(S)Phe-DAlaOAllÁTos
Gly-DAlaOPrgÁTos
(S)Phe-DAlaOPrgÁTos
416 10
NI
(S)Phe-DAlaOMeÁTos
(S)Phe-DAlaOPriÁTos
Gly-ZDPheOAllÁTFA
64
171
13
3
8
1
84
460 20
17
320 20
86
4
1
Gly-ZDPheOPrgÁTFA
33
2
4
Gly-DZPheO-CH2CH(OH)CH2ClÁHCl
5.5 0.5
NI—no inhibition up to 1245 mM
reaction between active ester (allyl, propargyl or glycidyl)
with thiol moiety of the active-site cysteine. Unfortunately,
obtained compounds exerted moderate inhibitory activity
acting as competitive inhibitors. More likely this results
from different than expected binding mode of these
compounds.
product with yield 91 %. Glycidyl esters seem to be more
interesting as inhibitors of cathepsin since they posses
oxirane ring, which is known to react preferably with the
enzyme active-site cysteine. In order to prepare these
esters, two standard methods, both basing on the activation
of carboxylic moiety, have been elaborated (Fig. 2). Un-
fortunately, the reaction afforded inseparable mixture of
glycidyl ester and some products of oxirane ring opening.
Efforts to remove Boc protection by trifluoroacetic acid
were unsuccessful and gave even more complex mixture of
products, whereas using hydrogen chloride in methanol we
were able to isolate 3-chloro-2-hydroxypropyl ester of Boc-
Gly-DZPhe.
Synthesis of inhibitors
Esters of dehydropeptides have been synthesized using
classical methods of peptide chemistry. The synthetic
schemes are outlined in Figs. 1 and 2. As seen from the
figures for each group of esters, specific method of their
preparation should be elaborated. Direct esterification of
Boc-Gly-DAla with DMTMM (Kunishima et al., 1999) as
coupling agent gave non-satisfactory results (30 % of
yield). Far better results for esterification of Boc-Gly-DAla
were obtained via nucleophilic substitution of alkyl halides
with dipeptide cesium salts (Fig. 1). This method gives
Inhibitory studies
Inhibitory activities of the synthesized esters are collected in
Table 1 and compared to action of methyl L-phenylalanyl-L-
Fig. 1 Synthesis of dehydrodipeptide methyl, ethyl, isopropyl, allyl and propargyl esters
123

Med Chem Res
Fig. 2 Synthesis of dehydrodipeptide glycidyl ester
Fig. 3 Dixon plot for the hydrolysis Gly-Phe-p-NA by bovine cathepsin C versus increasing concentration of Gly-ZDPheOAll
alaninate (Phe-AlaOMe). All the compounds appeared to be
competitive inhibitors, as shown in Fig. 3 for Gly-ZDPheOAll
trifluoroacetate as a representative example. The most active
appeared to be Gly-DZPhe-OCH2CH(OH)CH2ClÁHCl,
Gly-ZDPheOAllÁTFA and (S)Phe-DAlaOAllÁTos, which
inhibitory constants were in micromolar range. Quite
123

Med Chem Res
Fig. 4 a Most probable binding mode of Gly-ZDPheOAll by cathepsin C and found by molecular modeling. Catalytic triad is shown in green,
whereas inhibitor in white and gold. b Distance of allylic group of inhibitor from thiol moiety of active-site cysteine
interesting, six of the peptides—Gly-ZDPheOPrgÁTFA, Phe-
Conclusions
DAlaOMeÁTos, Phe-DAlaOEtÁTos, (S)Phe-DAlaOPriÁTos,
(S)Phe-DAlaOAllÁTos and (S)Phe-DAlaOPrgÁTos—inhibit
Synthesis of esters of dehydropeptides is not an easy task
cathepsin C according to slow-binding mechanism. This
mechanism is of B type and considers conformational re-
arrangement of inhibitor after binding to the enzyme
(Pawełczak and Hurek, 2014). From the data shown in
Table 1, it is also not possible to derive clear-cut structure–
activity relationship. Contrary to recent studies on the
structural requirements for the specific substrates for
cathepsin C (Pore˛ba et al., 2014), introduction of N-terminal
phenylalanine into peptide chain results in elevation of
affinity of Phe-DAlaOMeÁTos if compared with Gly-
DAlaOMeÁTos. This suggests that both dipeptide and de-
hydrodipeptide esters are bound differently than synthetic
substrate of this enzyme.
and requires the choice of specific method tailored to each
case. Esters of dehydrodipeptides containing C-terminal
dehydroalanine or (Z)-dehydrophenylalanine appeared to
be moderate or weak inhibitors of cathepsin C. As sug-
gested by molecular modeling, they are bound rather on the
surface of the enzyme than inside of the binding cavities of
the enzyme.
Acknowledgments These studies were supported by Wroclaw Re-
search Centre EIT? under the Project ‘‘Biotechnologies and ad-
vanced medical technologies’’—BioMed (POIG.01.01.02-02-003/
08)—financed from the European Regional Development Fund
(Operational Programme Innovative Economy, 1.1.2). We would like
_
_
to thank Dr inz. Bozena Fra˛ckowiak-Wojtasek for the HRMS ana-
lyses. Bartosz Oszywa and Paweł Lenartowicz are recipients of PhD
fellowships from a Project funded by the European Social Found.
Therefore, simple studies on their presumable binding
using AutoDock program had been undertaken.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
Molecular modeling
Simple molecular modeling using AutoDock has shown
that dehydrodipeptide esters are bound at the surface of the
enzyme in a non-typical manner. Their phenyl rings are
not, as expected, submerged in the cathepsin C cavity re-
sponsible for binding aromatic fragments of the substrates
and inhibitors but are rather placed at the surface of
the enzyme. The most probable binding mode of
Gly-ZDPheOAll is shown in Fig. 4. As seen from this fig-
ure, allylic double bond of the inhibitor, albeit directed
toward cathepsin C active-site cysteine 234, is too far away
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