The 1,4-naphthoquinone scaffold in the design of cysteine protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.04.068

A series of 1,4-naphthoquinone derivatives diversely substituted at C-2, C-3, C-5 and C-8, prepared by reaction of amines, amino acids and alcohols with commercial 1,4-naphthoquinones, has been evaluated against papain and bovine spleen cathepsin B. These

Full text of DOI:10.1016/j.bmc.2007.04.068

Bioorganic & Medicinal Chemistry 15 (2007) 5340–5350
The 1,4-naphthoquinone scaffold in the design
of cysteine protease inhibitors
a
a,
a
b,
*
*
Cl a´ udia Valente, Rui Moreira, Rita C. Guedes, Jim Iley,
c
c
Mohammed Jaffar and Kenneth T. Douglas
a
CECF, Faculty of Pharmacy, University of Lisbon, Avenida das For c¸ as Armadas, Lisboa 1600-083, Portugal
b
Chemistry Department, The Open University, Milton Keynes, MK7 6AA, UK
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
c
Received 9 March 2007; revised 18 April 2007; accepted 27 April 2007
Available online 6 May 2007
Abstract—A series of 1,4-naphthoquinone derivatives diversely substituted at C-2, C-3, C-5 and C-8, prepared by reaction of
amines, amino acids and alcohols with commercial 1,4-naphthoquinones, has been evaluated against papain and bovine spleen
cathepsin B. These 1,4-naphthoquinone derivatives were found to be irreversible inhibitors for both cysteine proteases, with sec-
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
ond-order rate constants, k
2
, ranging from 0.67 to 35.4 M
s
for papain, and from 0.54 to 8.03 M s for cathepsin B. Some
derivatives display a hyperbolic dependence of the first-order inactivation rate constant, kobs, with the inhibitor concentration, indic-
ative of a specific interaction process between enzyme and inhibitor. The chemical reactivity of the compounds towards cysteine as a
model thiol is dependent on the naphthoquinone LUMO energy, whereas papain inactivation is not. The 1,4-naphthoquinone deriv-
atives are inactive against the serine protease, porcine pancreatic elastase.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
associated with its substitution pattern. Although the
production of reactive oxygen species has been suggested
to play a prominent role in their actions, recent investiga-
tions have focused on the electrophilicity of quinones, in
order to explore the inhibitory potential of quinones
against enzymes containing a thiol residue at or near the
active site (topoisomerases and protein-tyrosine phos-
Quinones play vital roles in the biochemistry of living
cells, where they are involved in cellular respiration, pho-
tosynthesis and cellular defence against bacteria, fungi
and parasites. They became the subject of intense research
because of their interesting pharmacological, for exam-
ple, antitumour, trypanocidal, anti-inflammatory, antivi-
4
–6
phatases).
In spite of the potentially high reactivity
1
ral and antifungal, properties. Some have reached
2
associated with quinones, it should be possible to modu-
late this property by the appropriate choice of substitu-
ents, as shown by atovaquone, which has a relatively
clinical use as antineoplastic drugs and atovaquone, a
hydroxyl-1,4-naphthoquinone, is a broad-spectrum anti-
3
3
protozoal drug. Unfortunately, the potential usefulness
favourable side-effect profile and is tolerated by children.
of quinones is limited by their toxicity. Their actions
and toxicity are still difficult to understand, although at
least two competing mechanisms have been identified:
Among the enzymes dependent on a thiol nucleophile
(cysteine) at their active sites, the largest and best stud-
ied is the papain superfamily, which encompasses para-
7
(
i) their capacity to undergo redox cycling, generating
reactive oxygen species, and (ii) their capacity to act as
electrophiles via Michael-type addition, leading to cova-
lent modification of thiols or other vital physiological
components, proteins, DNA and RNA. Whether a qui-
none acts as an electrophile or a redox cycler is closely
site enzymes (falcipain, cruzain and rhodesain) and
mammalian enzymes (e.g., cathepsins and calpains).
The first group is crucial in the metabolism and repro-
ductive function of Plasmodium falciparum and Trypan-
osoma spp., while elevated levels of the second group of
enzymes are evident in pathological processes such as
muscular dystrophy, osteoporosis, chronic inflamma-
tory diseases and cancer. Therefore, papain-like cys-
teine proteases represent major therapeutic targets for
protease inhibitor development.
2
Keywords: Quinones; Papain; Cathepsin B; Cysteine proteases; Thiols;
Reactivity; LUMO energy.
8
*
Corresponding authors. Tel.: +44 1908653226; fax: +44 1908858327
J.I.); e-mail: j.n.iley@open.ac.uk
(
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.068

C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
5341
Bearing in mind the reactivity of thiols to quinones, we
have focused on the evaluation of the 1,4-naphthoqui-
none scaffold as a potential building block for the design
of irreversible inhibitors of cysteine proteases. To
achieve this goal, we have determined the inhibition
kinetics of papain, the best known cysteine protease
and as such a good model for this family of enzymes,
with a group of naphthoquinones, 1 (Table 1). For pa-
pain-like cysteine proteases, the most important element
of substrate specificity is the hydrophobic S2 subsite,
which is highly selective for bulky S-amino acids such
as Phe or the Leu side-chain of the epoxysuccinyl-Leu-
ethyl esters or EtOH. In the first approach to the synthe-
sis of amino acid derivatives 1a and 1b (Scheme 1),
2-bromo-1,4-naphthoquinone 1k was reacted with
S-phenylalanine ethyl ester hydrochloride (1 equiv) and
TEA (2 equiv) in DCM, but after stirring for 3 h at rt,
only starting material was recovered. Changing the sol-
1
1,12
vent to abs EtOH, as previously described,
the reaction of 1k with amine to proceed smoothly
allowed
1
3
through conjugate addition chemistry. This reaction
led to the formation, in a one-pot procedure, of the
mono-substituted 1,4-naphthoquinones (1a, 1d and 1g)
as the major products, along with their bromo ana-
logues (1b, 1e and 1h). This is a slightly different result
from that recently described for the reaction of 2-bro-
mo-1,4-naphthoquinones with S-a-amino acid methyl
esters, in which the non-bromo derivatives were the only
9
i-Am inactivator, E64c (i-Am = iso-amylamine). It is
also known that papain favours ligands based on the
1
0
N-Ac-S-Phe-Gly motif, and thus Phe, Leu-i-Am and
Gly moieties were attached to the naphthoquinone core
to probe the potential interaction between inhibitors and
papain. We have also investigated the inactivation of
bovine spleen cathepsin B and the reactivity of com-
pounds 1 towards cysteine to explore the relationship
between the intrinsic chemical reactivity and the enzyme
inhibition activity.
1
4
products isolated. However, it must be mentioned that
in those investigations reactions were performed under
reflux.
2.2. Reactivity studies
As 1,4-naphthoquinone derivatives may function as
covalent-binding inhibitors of cysteine proteases, infor-
mation on reactivity of these compounds towards thiols
is important. In an attempt to get insight into the factors
that could affect the chemical reactivity of each com-
pound, the LUMO energies for 1a–s were calculated
using density functional theory (DFT). This parameter
was chosen on the basis of two reported correlations;
first, between the reactivity of quinones and their
2
. Results and discussion
2
.1. Chemistry
,4-Naphthoquinone derivatives substituted at C-2 and
C-3 were prepared by condensation of commercially
1
available naphthoquinones with amines, S-a-amino acid
Table 1. LUMO energies, cysteine-catalyzed second-order rate constants, kcysꢀ , and papain inhibition data for naphthoquinones 1
R3
O
R1
R2
O
R4
1
R
2
R
3
R
4
R
kcysꢀ ðMꢀ1
ꢀ1Þ
Compound
ELUMO/Hartree
s
Papain
ꢀ1
ꢀ1
ꢀ3 ꢀ1
inact (10
k
2
(M
s
)
k
s
)
I
K (lM)
a
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
PheOEt
PheOEt
PheOEt
GlyOEt
GlyOEt
GlyOEt
NHMe
NHMe
Leu-i-Am
H
H
Br
Cl
H
Br
Cl
H
Br
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
ꢀ0.1146
ꢀ0.1214
ꢀ0.1216
ꢀ0.1097
ꢀ0.1172
ꢀ0.1174
ꢀ0.1070
ꢀ0.1149
—
25.9
—
NI
—
—
—
—
—
H
1.92
0.67
35.4
14.7
5.35
0.76
1.03
1.35
H
—
—
0.34 ± 0.05
H
0.49
—
9.57 ± 4.18
89.6 ± 7.08
266 ± 68.4
H
1.32 ± 0.03
1.42 ± 0.16
H
—
g
h
i
H
0.24
—
—
—
—
—
H
H
—
0.30 ± 0.04
—
222 ± 39.4
—
66.0 ± 20.0
b
j
H
ꢀ0.1245
ꢀ0.1320
ꢀ0.1370
ꢀ0.1161
ꢀ0.1193
ꢀ0.1133
ꢀ0.1170
ꢀ0.1114
—
2851.2
—
—
(49.5 ± 4.2)
33.3
6.39
k
l
Br
Cl
H
2.20 ± 0.31
—
H
—
—
m
n
o
p
q
r
Morpholinyl
Me
Me
H
93.1
114.3
—
9.29
4.90
—
—
H
—
—
a
NI
Me
H
—
—
c
OH
OEt
—
(82.0 ± 4.4)
11.6
—
H
10.9
—
1.34 ± 0.18
115 ± 43.7
b
H
OH
OAc
(60.4 ± 4.1)
14.6
—
—
—
—
s
H
ꢀ0.1335
—
a
b
c
No inhibition when tested up to their solubility limit.
Inhibition % at 65 lM after 30-min incubation.
Inhibition % at 1.3 mM after 20-min incubation.

5342
C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
O
O
O
O
changes and before any significant secondary reaction
was observed. The UV–visible spectra were also
recorded in the presence of buffer lacking cysteine; the lack
of change between scans obtained over 19 h excluded
any background reaction. For the reactions involving
cysteine, plotting the pseudo first-order rate constant,
(i)
R
1
j
1i R = Leu-
q R = OEt
i-Am
1
kobs, versus the total cysteine concentration, [cys] , gave
t
an intercept at [cys] = 0 indistinguishable from zero and
t
O
O
O
O
O
O
a slope, kcat, which, in principle, contains the contribu-
tions of both cysteine and its thiolate to the overall reac-
tion. For 1n, kcat values were determined at different pH
values (7.1, 7.9, 8.0 and 8.8) and then plotted against the
Br
Br
R
(ii) or (iii)
+
R
ꢀ
fraction of cysteine present as the free base, a = [cys ]/
[
cys] (Fig. 1). The linear correlation showed an intercept
1
k
1a R = PheOEt
1b R = PheOEt
1e R = GlyOEt
1h R = NHMe
t
1
1
d R = GlyOEt
g R = NHMe
at a = 0 indistinguishable from zero, indicating that
there is no detectable reaction for the undissociated
thiol. The second-order rate constant for the reaction
with the thiolate anion, kcysꢀ , was determined from the
intercept at a = 1. This result is consistent with previous
studies with quinones which claim thiolate anion as the
2
3
O
O
O
Cl
Cl
Cl
(ii)
R
O
1
8,21
reactive species.
values were determined by kcys
For the remaining compounds, kcysꢀ
¼ k cat=a (Table 1).
ꢀ
1
l
1c R = PheOEt
f R = GlyOEt
1
Naphthoquinone 1j, however, revealed a hyperbolic
dependence of kobs on the total cysteine concentration
(
reaction of dopaminoquinone with cysteine and has
been ascribed to an initial formation of a reversible
Scheme 1. Reagents: (i) TEA, abs EtOH for 1q and TEA, Leu-i-Am,
abs EtOH for 1i; (ii) PheOEt or GlyOEt hydrochlorides, TEA, abs
EtOH for 1a–f; (iii) R = MeNH , abs EtOH for 1g, h.
Fig. 2). Such a dependence has been reported for the
2
4
2
6
,15,16
LUMO energy,
gies and electronic descriptors thereby providing an
and second, between LUMO ener-
1
7
12
8
12
8
1
7–20
alternative approach to QSAR studies.
In the con-
r2 = 0.969
text of this latter observation, in the present study, we
observed a correlation between the LUMO energy of
compounds 1d, 1g, 1j, 1k, 1l, 1n and 1q (Table 1) and
the corresponding Hammett r constants for C-2 and
4
4
p
2
C-3 substituents (r = 0.910; data not shown).
0
0
0
The reaction between naphthoquinones and cysteine
was followed spectrophotometrically at 25 ꢁC. On mix-
ing the reactants, there was a progressive change in the
absorbance, followed by a plateau region. Subsequently,
the reaction was characterized by continuous spectral
changes. Some of the spectra did not present an isos-
bestic point. These observations are similar to previous
.0
0.2
0.4
0.6
0.8
1.0
Fraction of free base (α)
Figure 1. Plot of the catalytic rate constants, kcat, for the reaction of 1n
with cysteine, against the fraction of free base, a, at 25 ꢁC and
I = 0.5 M (KCl).
1
8
reports regarding the reactivity of quinones, which
related this pattern to the occurrence of complex second-
ary reactions involving the hydroquinone product that
results from the initial Michael addition. Since the
kinetic studies were performed without protection from
air, this complexity may arise from the aerobic oxidation
3
2
1
6
,21
of those products.
In order to simplify the kinetic
interpretation of the overall process, some of the previ-
ous investigations were performed with exclusion of
oxygen. To the best of our knowledge, spectrophotome-
try has been used only once to investigate naphthoqui-
r = 0.927
2
0
0
2
4
6
3
2
1,22
21
10 [cys] /M
t
nones,
and then under anaerobic conditions.
Nevertheless, it has been recognized that these condi-
tions may compromise any application of the results
to biological systems.
Figure 2. Plot of the pseudo first-order rate constant, kobs, for
the reaction of naphthoquinone 1j with cysteine, against the total
2
1
ꢀ2
cysteine concentration at pH 7.08 (a = 7.08 · 10
and ionic strength 0.5 M. Points are experimental and line is
) at 25.0 ꢁC
ꢀ6
ꢀ6
In the present work, the reaction was followed at a fixed
wavelength until completion of the initial spectral
theoretical for 1/Kcys 9.26 · 10 ± 3.16 · 10
2.64 · 10 ± 0.17 · 10
M
and kcys
ꢀ2
ꢀ2 ꢀ1
s .

C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
5343
5
3
1
depicted in Eq. 2, where EÆI represents the non-covalent
enzyme–inhibitor complex, E ꢂ I the covalently bound
1
j
r2 = 0.940
complex, K represents the dissociation constant of the
I
1
m
EÆI complex and kinact is the first-order rate-constant
for the chemical inactivation step. The compounds were
found to inactivate papain, at a rate dependent on the
concentration of the inhibitor as well as on the incuba-
tion time. This type of kinetic profile has been related re-
cently to the quinone behaving as an electrophile, in
contrast with a redox-dependent mechanism which was
much more affected by the concentration of the reducing
agent dithiothreitol (DTT) than by the inhibitor
1
q
1
n
1
a
1
g
1
d
-
1
3
-
-
0.125
-0.12
-0.115
LUMO (Hartree)
-0.11
-0.105
2
6
concentration.
Therefore, the present results are
Figure 3. Linear correlation between the LUMO orbital energies and
the second-order rate constants, kcysꢀ , for the reaction of 1 with
cysteine.
consistent with covalent modification of the enzyme.
The time dependency of inhibition also suggested an
irreversible process, which was confirmed by dialyses
carried out after reaction of the Gly derivative 1d with
papain, from which no enzyme activity could be
recovered.
tetrahedral intermediate, resulting from cysteine conju-
gate addition to C-6 (dopaminoquinone numbering),
which is then followed by the decomposition into the fi-
nal product. Assuming that reaction of 1j with cysteine
thiolate also involves initial reversible formation of an
kinact
E þ I ꢀ E ꢃ I ! E ꢂ I
ð2Þ
ð3Þ
K
I
adduct (e.g., 3), then kobs dependence on [cys] follows
t
kinact½Iꢁ
Eq. 1,
k
obs
¼
K þ ½Iꢁ
I
k
cys½cysꢁ
t
kobs
¼
ð1Þ
For compounds 1d, 1e, 1f, 1i, 1k and 1q, the pseudo
first-order inactivation rate constants, kobs, displayed a
hyperbolic dependence with [I] (Fig. 5a) indicative of
saturation inhibition kinetics, which suggests that the
inhibition process is preceded by a specific interaction
between enzyme and inhibitor in a Michaelis-type com-
plex (Eq. 3). In the case of derivatives 1b, 1c, 1h, 1i, 1l,
þ
f½ð1=KcysÞðK
where Kcys is the stability constant for the formation of
a
þ ½H ꢁÞ=K
a
ꢁ þ ½cysꢁtg
the adduct, kcys is the first-order rate constant for the
decomposition of the adduct and (K + [H ])/K is 1/a.
Kinetic data were computer-fitted to Eq. 1 allowing
+
a
a
5
ꢀ1
the determination of K
(1.08 · 10
M
), of kcys
) and thus of the second-order rate-con-
stant for the reaction with the thiolate anion
kcys
ð¼ kcysKcysÞ.
cys
ꢀ2
ꢀ1
s
1m, 1n and 1s, kobs varied linearly with inhibitor concen-
(
2.64 · 10
tration, suggesting that K ꢄ [I], the corresponding
I
slope being the apparent second-order rate constants
for the inactivation process k (=k
ꢀ
/K ) (Fig. 5b).
I
2
inact
Overall, 1,4-naphthoquinone derivatives inhibited pa-
pain with the second-order rate constants for the inacti-
O
H
S
ꢀ1 ꢀ1
vation process, k , ranging from 0.67 to 35.4 M
2
s ,
Cys
which represents ꢂ50-fold variation in their inhibitory
activity (Table 1). Unfortunately, the Phe and Leu-i-
Am fragments did not improve inhibitory potency,
although the observation of a hyperbolic dependence
for the Leu-i-Am derivative 1i supports a specific inhibi-
tion process. The phenylalanine derivative 1a did not
inactivate papain. The lack of activity of compounds
bearing a phenylalanine residue directly attached to
the reactive site has been previously observed for
vinylogous amino acid esters and ascribed to a binding
mode where the Michael acceptor is far from the
reach of the sulfur atom of Cys25 due to the interaction
O
3
The kcysꢀ values varied by up to four orders of magni-
tude, with naphthoquinone 1j as the most effective
acceptor for cysteine (Table 1). Plotting log kcys
ꢀ
against
the corresponding LUMO energies resulted in a linear
correlation (Fig. 3), which shows that the lower the
LUMO energy the more reactive the molecule. Calcula-
tion of the LUMO density for the glycine derivative 1d
(
Fig. 4), as well for other aminoacid derivatives, showed
between phenylalanine and the S2 hydrophobic
27
that for these compounds this orbital is largely centred
on the double bond of the quinone ring. Overall, these
observations are consistent with the occurrence of a Mi-
chael-type addition, which is dependent on the electro-
philicity of the naphthoquinone.
subsite of papain.
(
The glycine derivative 1d
) and 2-bromo-1,4-naphthoquinone
ꢀ
1
ꢀ1
ꢀ
k2 = 35.4 M
1
s
1
ꢀ1
k (k = 33.3 M
2
s
) were the most potent inhibitors.
Comparing the kinetic parameters of 1d and 1k, it is
clear that the first-order rate constant for the chemical
inactivation step, kinact, plays an important role in the
inhibitory activity of 1k. In fact, this compound showed
the highest kinact of all, consistent with the propensity of
halo-quinones to undergo nucleophilic substitution, as-
cribed to the increased electron affinity provided by
2
.3. Enzyme inhibition studies
Kinetic inhibition assays were performed by Kitz and
2
5
Wilson’s pre-incubation method. Accordingly, enzyme
inactivation may be represented by the minimal reaction
1
3
the substituent.

5344
C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
Figure 4. Frontier molecular orbitals of a low-energy conformation of the glycine derivative 1d.
1
0
0
0
.2
.9
.6
.3
0
Gln19 (Fig. 6). This orientation presents C-3 close
˚
2.8–2.9 A) to the sulfur atom of Cys25, as would be ex-
pected for Michael addition chemistry. The ethyl glycin-
ate moiety is pointing towards the S2 pocket, although it
does not reach this binding site.
a
(
2
r = 0.996
The suggestion that the ability of the inhibitor to fit in
the active site of papain may be much more important
than the intrinsic electrophilicity of the naphthoquinone
is further supported by the comparison of the kinetic
parameters for the glycine derivatives (1d–f), for which
the halo compounds 1e and 1f are weaker inhibitors
than the non-halo counterpart 1d (largely, though not
0
2
4
6
1
04[Ι]/M
3
2
1
0
b
exclusively, due to an increase in the K value). These
I
2
r = 0.994
observations also suggest that substitution both in the
C-2 and C-3 positions of the naphthoquinone core
may be detrimental to inhibitory activity. In fact, a
similar situation seems to occur in the case of the
methylamino bromo derivative 1h which, in contrast
to 2-bromo-1,4-naphthoquinone 1k, did not exhibit
saturation kinetics.
Naphthoquinone 1j, lawsone 1p and juglone 1r were
shown to be time-dependent inhibitors but, unfortu-
nately, graphical representation of v/v versus incuba-
0
tion time presented a scattering of points which were
poorly fitted to a pseudo-first-order kinetics, so their
inhibitory activity was evaluated at fixed time and con-
centration of the inhibitor. For compounds 1j and 1r,
this departure from the usual behaviour could result
0
1
2
3
04[Ι]/M
1
Figure 5. (a) Plot of the pseudo first-order inactivation rate constant,
obs, against inhibitor concentration for the inactivation of papain by
k
derivative 1e; (b) plot of the pseudo first-order inactivation rate
constant, kobs, for the inactivation of papain by derivative 1h against
inhibitor concentration.
The expected contribution of an electron-withdrawing
group to the increase of the activity is also clear in the
Phe and NHMe series, particularly in the case of the
Trp177
His159
ꢀ1
ꢀ1
brominated phenylalanine derivative 1b (1.92 M
s )
compared to its inactive non-halo counterpart 1a. In
contrast, the potency of the glycine derivative 1d is con-
nected with its K (9.57 lM), the lowest in the series.
I
2.5 2.4 2.9
This suggests that the Gly residue is able to promote sta-
bilization of the non-covalent inhibitor–enzyme com-
plex, directing the naphthoquinone moiety to the
active cysteine residue thereby enhancing the inhibitory
potency, even at low alkylation rate constants. Model-
ling the interaction of this compound with papain, using
the program GOLD, revealed that all 10 inspected con-
formations presented hydrogen bonding interactions be-
tween the carbonyl oxygen atom at C-4 and NH of
S2 pocket
Gln19
Cys25
Gly66
Figure 6. GOLD-generated active site interaction of papain and 1d
magenta). Figure generated using Pymol software: DeLano, W.L. The
(
PyMOL Molecular Graphics System (2002) DeLano Scientific, San
Carlos, CA, USA. http://www.pymol.org.

C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
5345
from instability of compounds in the reaction buffer
with DTT, which is consistent with the report that these
compounds have a high reactivity towards simple thi-
3. Conclusion
It has been shown that 1,4-naphthoquinone derivatives
are able to inhibit the cysteine proteases papain and
bovine spleen cathepsin B in an irreversible manner.
The results suggest that the inactivation process is
more affected by the specific recognition interactions
between inhibitor and enzyme than by the electrophilic-
ity of the naphthoquinone moiety. This observation is
quite important considering that intrinsic chemical
reactivity of naphthoquinones may be cited as a poten-
tial limitation to their applicability under biological
conditions, where other thiol species are expected to
occur at high concentrations, for example, glutathione,
promoting unspecific undesirable conjugations. Thus,
the 1,4-naphthoquinone scaffold appears to be a reli-
able approach to the development of papain-like cys-
teine protease inhibitors. By adjusting the inherent
chemical reactivity downwards and exploiting the spe-
cific binding requirements of the nucleophilic attack it
should be possible to tailor quinone and quinone-like
inhibitors to a variety of cysteine enzymes that are po-
tential therapeutic targets.
2
2
ols, and with observation that the incubation mixture
turned from pale yellow to brown, this colour change
only occurring in the presence of DTT. It is known that
2
6
quinones can transfer electrons from DTT to oxygen.
However, inhibition by 1r was not prevented by keeping
the incubation mixture under nitrogen, or by the addi-
tion of ascorbic acid to the reaction buffer. Therefore,
the possible existence of significant redox cycling can
be excluded.
In an attempt to assess how the electronic properties of
the substituents affect the rate of enzyme inhibition, we
analysed LUMO energy values along with the kinetic
parameters of papain inhibition (Table 1). One interest-
ing finding from compounds 1d–f, 1k and 1q is a trend in
which kinact values increase as the corresponding LUMO
energies decrease (Table 1), suggesting that the chemical
inactivation step of papain might be a function of the
LUMO energy of the inhibitor. Even so, as kinact values
vary only 7-fold (1k versus 1i), whereas K values vary
I
ꢂ30-fold (1d vs 1f), it would seem that the overall activ-
ity of the compounds towards papain is governed to a
large extent by the molecular recognition of the inhibitor
prior to the irreversible reaction step. Indeed, it was not
possible to establish any correlation between the appar-
4. Experimental
4.1. General information
ent second-order rate constants, k , and LUMO ener-
2
gies, indicating that inhibitory activity does not
parallel chemical reactivity.
All materials were used without further purification. Re-
agent grade chemicals were bought from Sigma–Aldrich
(naphthoquinones 1j, 1k, 1l, 1m, 1n and 1r, amino acid
The best inhibitors were tested against bovine spleen
cathepsin B, which shares about 80% homology with
ethyl esters, Boc-protected amino acid N-hydroxy-
succinimide ester, iso-amylamine) and Merck (methyl-
amine and solvents). Papain (Sigma P4762), bovine
spleen cathepsin B (Sigma C6286, 10 U), PPE (Sigma
E0258), benzoyl-S-arginine-4-nitroanilide (BAPA),
Z-S-Lys-ONp, Suc-Ala-Ala-Ala-4-nitroanilide and
0.1 M Hepes buffer (H9897) were obtained from Sig-
ma–Aldrich. DMSO and buffer materials for kinetics
measurements were of analytical grade and were pur-
chased from Merck (Germany). Derivatives 1o and 1s
2
8
the human liver variety. Cathepsin B, one of the best
characterized mammalian cysteine proteases, is impli-
2
9
cated in a variety of diseases. The presently reported
compounds were found to be time-dependent inhibitors,
with second-order rate constant k values 3–20 times
2
smaller than those for papain (Table 2). The most potent
inhibitor for papain, the glycine derivative 1d, was also
the most active against cathepsin B. Its dissociation
3
0
constant K is also the lowest in the series, with a similar
I
resulted from previous investigations. Dialysis experi-
ments were carried out with Spectra/Por 4 dialysis mem-
branes, 12000–14000 MW (Spectrum Laboratories Inc.
132697). Column chromatography was performed with
silica gel (Merck, 230–400 mesh ASTM). TLC was per-
formed on pre-coated silica gel 60 F254 (Merck) and
visualized under UV light. The IR spectra were deter-
mined using NaCl solution cells on a Nicolet Impact
400 FTIR spectrophotometer and only the most signifi-
cant absorption bands are reported. Low-resolution
mass spectra were recorded using VG Quattro LCMS
instruments. Elemental analyses were performed using
an EA 1110 CE Instruments automatic analyser. The
value to that with papain. In contrast, 2-bromo-1,4-
naphthoquinone 1k exhibited a linear dependence be-
tween kobs and [I], suggesting a drastic increase in the
K value. Against a serine protease, porcine pancreatic
I
elastase (PPE), this group of derivatives was shown to
have no activity when incubated for 2 h over the same
range of concentrations used against papain and cathep-
sin B; this suggests that 1,4-naphthoquinone derivatives
may be selective inhibitors for cysteine proteases.
Table 2. Second-order rate constants, k
B by 1
2
, for inactivation of cathepsin
1
13
H and C NMR were recorded on a Bruker Avance
C
1
00 NMR spectrometer ( H 400 MHz;
13
4
Compound
Bovine spleen cathepsin B
1
00.62 MHz) in CDCl solution; chemical shifts, d, are
ꢀ1
ꢀ1
ꢀ4 ꢀ1
inact (10
3
k
2
(M
s
)
k
s
)
I
K (lM)
expressed in ppm. Signal assignments were effected using
1
1
1
1
1
1
d
8.03
4.37
5.65
0.54
1.12 ± 0.06
2.44 ± 0.24
—
14.0 ± 3.07
55.9 ± 14.1
—
H– H COSY, HMQC and HMBC 2D NMR tech-
niques. Coupling constants (J) are reported in hertz
Hz). Kinetic measurements were carried out using
either Shimadzu UV UV-2100 or Shimadzu UV-1603
e
k
q
(
4.28 ± 0.63
797 ± 228

5346
C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
spectrophotometers, equipped with a temperature con-
troller (CPS).
(m, 2H, Ph), 7.27–7.34 (m, 3H, Ph), 7.66 (dt, 1H,
J = 1.2, 7.6, H-7), 7.74 (dt, 1H, J = 1.2, 7.6, H-6), 8.03
(
d
br d, 1H, J = 7.6, H-8), 8.15 (br d, 1H, J = 7.6, H-5).
C NMR 14.2 (CH CH ), 40.2 (CH Ph), 57.3
2 3 2
1
3
4
.2. Synthesis
(
CHCH Ph), 61.9 (CH CH ), 126.8 (C-8), 126.9 (C-5),
2 2 3
S-Phenylalanine ethyl ester hydrochloride (97 mg;
.4 mmol) and TEA (85 mg; 0.8 mmol) were added to
a solution of 2-bromo-1,4-naphthoquinone (102 mg;
.4 mmol) in abs EtOH (15 mL). After stirring at rt
for 1 h, the solvent was evaporated to yield crude prod-
uct (300 mg). Compounds 1a (61 mg) and 1b (16 mg)
were separated by column chromatography on silica
gel (30 g) using DCM as eluent (0.5 L). Compounds
127.6 (CH), 128.9 (CH), 129.4 (CH), 130.0 (C-8a),
132.3 (C-4a), 132.7 (C-7), 134.9 (C-6), 135.1 (C), 171.3
(CO), 176.8 (C-4), 180.1 (C-1); the following H and
0
1
1
3
0
C signal assignments are interchangeable: C-1 with
C-4, C-4a with C-8a, H-5/C-5 with H-8/C-8 and H-6/
+
C-6 with H-7/C-7. ESI-MS m/z (rel int.) 384 (MH )
+
(100), 386 (MH +2) (30). Anal. Calcd (C H ClNO ):
2
1
18
4
C, 65.71; H, 4.73; N, 3.65. Found: C, 65.73; H, 4.45;
N, 3.66.
1c, 1d, 1e and 1f were prepared using the same method
from the appropriate naphthoquinone and S-amino acid
ethyl ester.
4.2.4. N-(1,4-Naphthoquinon-2-yl)glycine ethyl ester (1d).
Compound 1d (19 mg) was prepared from 2-bromo-1,4-
naphthoquinone and glycine ethyl ester hydrochloride;
4.2.1. N-(1,4-Naphthoquinon-2-yl)-S-phenylalanine ethyl
ester (1a). Light orange gummy solid (42%); IR (film):
orange amorphous solid (9%); IR (film): m
1743, 1677, 1573 cm . d H NMR 1.34 (t, 3H,
= 3373,
max
ꢀ
1
1
ꢀ1
1
mmax = 3374, 1737, 1673, 1571 cm . d H NMR 1.24–
.27 (m, 3H, CH CH ), 3.19 (dd, 1H, J = 6.6, 14.0,
CHHPh), 3.28 (dd, 1H, J = 5.6, 14.0, CHHPh), 4.19–
1
J = 7.2, CH CH ), 3.94 (d, 2H, J = 5.2, NCH ), 4.31
(q, 2H, J = 7.2, CH CH ), 5.66 (s, 1H, H-3), 6.41 (br
2 3
2
3
2
3
2
4
7
7
1
8
.24 (m, 2H, CH CH ), 4.32 (br ddd, 1H, J = 5.6, 6.6,
3
s, 1H, NH), 7.64 (br t, 1H, J = 7.6, H-7), 7.73 (br t,
1H, J = 7.6, H-6), 8.05–8.10 (m, 2H, H-5 and H-8). d
2
.6, CH), 5.71 (s, 1H, H-3), 6.33 (d, 1H, J = 7.6, NH),
.16–7.18 (m, 2H, Ph), 7.25–7.34 (m, 3H, Ph), 7.64 (dt,
H, J = 1.2, 7.6, H-7), 7.74 (dt 1H, J = 1.2, 7.6, H-6),
.06 (brdd, 1H, J = 0.8; 7.6, H-8), 8.09 (brdd, 1H,
1
3
C
(CH CH ), 102.1 (C-3), 126.2 (C-8), 126.4 (C-5), 130.5
NMR 14.2 (CH CH ), 43.9 (NCH2), 62.1
2 3
2
3
(C-8a), 132.3 (C-7), 133.3 (C-4a), 134.8 (C-6), 147.2
(C-2), 168.3 (CO), 181.2 (C-1), 183.2 (C-4). ESI-MS
m/z (rel int.) 260 (MH ) (50). Anal. Calcd
1
3
J = 0.4, 7.6, H-5). d C NMR 14.1 (CH CH ), 37.5
2
3
+
(
CH Ph), 56.1 (CHCH Ph), 62.0 (CH CH ), 102.1 (C-
2
2
2
3
3
1
1
), 126.2 (C-8), 126.4 (C-5), 127.5 (CH), 128.8 (CH),
29.2 (CH), 130.5 (C-8a), 132.2 (C-7), 133.2 (C-4a),
34.8 (C-6), 135.1 (C), 146.6 (C-2), 170.3 (CO), 181.2
(C H NO ): C, 64.86; H, 5.05; N, 5.40. Found: C,
4
64.59; H, 5.01; N, 5.60.
1
4
13
+
(
(
C-1), 183.2 (C-4). ESI-MS m/z (rel int.) 350 (MH )
100). Anal. Calcd (C H NO ): C, 72.19; H, 5.48; N,
4.2.5. N-(3-Bromo-1,4-naphthoquinon-2-yl)glycine ethyl
ester (1e). Compound 1e (18 mg) was obtained together
with compound 1d; orange amorphous solid (6%); IR
2
1
19
4
4
.01. Found: C, 72.45; H, 5.61; N, 4.13.
ꢀ1
1
(
film): mmax = 3317, 1741, 1677, 1569 cm . d H NMR
4.2.2. N-(3-Bromo-1,4-naphthoquinon-2-yl)-S-phenylala-
nine ethyl ester (1b). Orange oil (9%); IR (film):
mmax = 3317, 1737, 1673, 1571 cm . d H NMR 1.26–
1.34 (t, 3H, J = 7.2, CH CH ), 4.31 (q, 2H, J = 7.2,
CH CH ), 4.62 (d, 2H, J = 5.6, NCH ), 6.54 (br s, 1H,
2 3 2
2
3
ꢀ
1
1
NH), 7.66 (dt, 1H, J = 1.0; 7.6, H-7), 7.73 (dt, 1H,
J = 1.2; 7.6, H-6), 8.05 (br d, 1H, J = 7.6, H-8), 8.15
1
4
.30 (m, 3H, CH CH ), 3.20–3.31 (m, 2H, CH Ph),
.21–4.27 (m, 2H, CH CH ), 5.58 (m, 1H, CH), 6.28
2 3 2
1
3
(br d, 1H, J = 7.6, H-5). d C NMR 14.2 (CH CH ),
2
3
2 3
(
3
br s, 1H, NH), 7.20–7.22 (m, 2H, Ph), 7.24–7.34 (m,
H, Ph), 7.65 (dt, 1H, J = 1.2, 7.6, H-7), 7.72 (dt, 1H,
J = 1.2, 7.6, H-6), 8.00 (br d, 1H, J = 7.6, H-8), 8.14
46.7 (NCH ), 62.1 (CH CH ), 127.0 (C-5 and C-8),
2 2 3
129.9 (C-8a), 132.0 (C-4a), 132.6 (C-7), 134.8 (C-6),
146.2 (C-2), 169.5 (CO), 176.5 (C-4), 179.8 (C-1); the fol-
1
3
1
13
(
4
br d, 1H, J = 6.8, H-5). d C NMR 14.1 (CH CH ),
lowing H and C signal assignments are interchange-
able: C-1 with C-4, C-4a with C-8a, H-5 with H-8 and
H-6/C-6 with H-7/C-7. ESI-MS m/z (rel int.) 338
2
3
0.0 (CH Ph), 57.6 (CHCH Ph), 61.9 (CH CH ), 126.9
2 2 2 3
(
1
C-5 and C-8), 127.5 (CH), 128.8 (CH), 129.4 (CH),
31.9 (C-4a), 132.6 (C-7), 134.7 (C-6), 135.0 (C), 171.2
CO), 176.4 (C-4), 179.8 (C-1); the following H and
+
+
(MH ) (78), 340 (MH +2) (100). Anal. Calcd
(C H BrNO ): C, 49.73; H, 3.58; N, 4.14. Found: C,
50.30; H, 3.60; N, 4.15.
1
(
1
4
12
4
1
3
C signal assignments are interchangeable: C-1 with
C-4, C-4a with C-8a, H-5 with H-8 and H-6/C-6 with
+
H-7/C-7. ESI-MS m/z (rel int.) 428 (MH ) (83), 430
4.2.6. N-(3-Chloro-1,4-naphthoquinon-2-yl)glycine ethyl
ester (1f). Compound 1f (11 mg) was prepared from 2,3-
dichloro-1,4-naphthoquinone and glycine ethyl ester
hydrochloride; orange amorphous solid (7%); IR (film):
+
MH +2) (100). Anal. Calcd (C H BrNO ): C, 58.89;
(
H, 4.24; N, 3.27. Found: C, 58.77; H, 4.44; N, 3.42.
2
1
18
4
ꢀ
1
1
4
.2.3. N-(3-Chloro-1,4-naphthoquinon-2-yl)-S-phenylala-
mmax = 3315, 1737, 1673, 1571 cm . d H NMR 1.34 (t,
3H, J = 7.2, CH CH ), 4.31 (q, 2H, J = 7.2, CH CH ),
4.61 (d, 2H, J = 6.0, NCH ), 6.53 (br s, 1H, NH), 7.66
nine ethyl ester (1c). Compound 1c (18 mg) was prepared
from 2,3-dichloro-1,4-naphthoquinone and S-phenylal-
anine ethyl ester hydrochloride; orange oil (11%); IR
2
3
2
3
2
(dt, 1H, J = 0.8; 7.6, H-7), 7.75 (dt, 1H, J = 0.8; 7.6,
H-6), 8.06 (br d, 1H, J = 7.6, H-8), 8.16 (br d, 1H,
ꢀ
1
1
(
film): mmax = 3315, 1737, 1673, 1571 cm . d H NMR
.27 (t, 3 H, J = 7.2, CH CH ), 3.24-3.27 (m, 2H,
CH Ph), 4.20–4.28 (m, 2H, CH CH ), 5.51 (ddd, 1H,
1
3
1
J = 7.6, H-5). d
C NMR 14.2 (CH CH ), 46.3
2 3
2
3
(NCH ), 62.1 (CH CH ), 126.8 (C-8), 126.9 (C-5),
2
2
2
3
2
3
J = 2.0, 6.0; 12.2, CH), 6.34 (br s, 1H, NH), 7.19–7.21
129.9 (C-8a), 132.3 (C-4a), 132.7 (C-7), 134.9 (C-6),

C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
5347
1
43.9 (C-2), 169.5 (CO), 176.9 (C-4), 180.1 (C-1); the fol-
NMR 0.90 [d, 6H, J = 6.8, 2 · CH (i-Am)], 0.95 [d,
3
1
13
lowing H and C signal assignments are interchange-
able: C-1 with C-4, C-4a with C-8a, H-5/C-5 with H-8/
C-8 and H-6/C-6 with H-7/C-7. The H NMR spectra
3H, J = 6.4, CH (Leu)], 1.02 [d, 3H, J = 6.0, CH
(Leu)], 1.37–1.42 [m, 2H, CH CH (i-Am)], 1.55–1.63
3
3
2
1
[m, 1H, CH (i-Am)], 1.70–1.79 [m, 2H, CHH and
CH(CH3)2 (Leu)], 1.88–1.92 [m, 1H, CHH (Leu)],
3.28–3.33 [m, 2H, NHCH (i-Am)], 3.89–3.92 [m, 1H,
of 1f are in agreement with those reported in the litera-
31
+
ture. ESI-MS m/z (rel int.) 294 (MH ) (100), 296
2
+
MH +2) (37). Anal. Calcd (C H ClNO ): C, 57.25;
(
H, 4.12; N, 4.77. Found: C, 57.30; H, 4.30; N, 4.60.
CHNH (Leu)], 5.82 (s, 1H, H-3), 6.13 [d, 1H, J = 6.8,
NH (Leu)], 6.28 [m, 1H, NH (i-Am)], 7.66 (dt, 1H,
J = 1.2, 7.6, H-7), 7.76 (dt, 1H, J = 1.2, 7.6, H-6),
1
4
12
4
1
3
4
(
.2.7. 2-(Methylamino)-1,4-naphthoquinone (1g) and 2-
methylamino)-3-bromo-1,4-naphthoquinone (1h). To a
solution of 2-bromo-1,4-naphthoquinone (283 mg;
.2 mmol) in abs EtOH (40 mL) was added an excess
of aqueous methylamine solution (40%; 182 mg;
.3 mmol). The mixture instantly turned orange. After
8.07-8.10 (m, 2H, H-5 and H-8). d C NMR 21.9
[CH (Leu)], 22.4 [2 · CH (i-Am)], 22.9 [CH (Leu)],
3
3
3
25.1 [CH(CH ) (Leu)], 25.9 [CH (i-Am)], 38.0 [NHCH
2
3
2
1
(i-Am)], 38.3 [CH CH (i-Am)], 41.8 [CH (Leu)], 55.9
2 2
[CHNH (Leu)], 102.4 (C-3), 126.3 (C-8), 126.5 (C-5),
130.4 (C-8a), 132.4 (C-7), 133.2 (C-4a), 135.0 (C-6),
147.4 (C-2), 170.3 [CO (Leu)], 181.2 (C-1), 183.2 (C-4).
2
vigorous stirring at rt for 40 min, the solvent was evap-
orated to yield crude product (330 mg). Compounds 1g
+
ESI-MS m/z (rel int.) 357 (MH ) (100). Anal. Calcd
(C H N O ): C, 70.76; H, 7.92; N, 7.86. Found: C,
70.95; H, 7.45; N, 7.53.
(
170 mg) and 1h (19 mg) were separated by column
2
1
28
2
3
chromatography on silica gel (40 g) using as eluents
DCM (0.2 L) and a mixture of DCM/EtOAc 3:1 (0.2 L).
4.2.9. 2-Ethoxy-1,4-naphthoquinone (1q). A solution of
Compound 1g; red amorphous solid (78%); IR (film):
mmax = 3335, 1678, 1566 cm . d H NMR 2.96 (d, 3H,
1,4-naphthoquinone (311 mg; 1.9 mmol) in abs EtOH
(40 mL) with TEA (192 mg; 1.9 mmol) was vigorously
stirred at rt for 1 h, during which time a red colour
was found to develop. After stirring for another 12 h,
the solvent was evaporated to yield crude product
(529 mg), which was fractionated by column chromatog-
raphy on silica gel (40 g) using DCM (0.45 L) as eluent
to afford 110 mg of 1q: yellow amorphous solid (29%);
ꢀ
1
1
J = 5.6, CH ), 5.74 (s, 1H, H-3), 5.97 (br s, 1H, NH),
3
7
7
1
3
.64 (dt, 1H, J = 1.2, 7.6, H-7), 7.75 (dt, 1H, J = 1.2,
.6, H-6), 8.06 (brdd, 1H, J = 0.8; 7.6, H-8), 8.12 (brdd,
1
3
H, J = 0.8, 7.6, H-5). d C NMR 29.2 (CH ), 100.7 (C-
3
), 126.3 (C-5 and C-8), 130.5 (C-8a), 132.0 (C-7), 133.7
(
C-4a), 134.8 (C-6), 149.0 (C-2), 181.9 (C-1), 182.9 (C-4).
ESI-MS m/z (rel int.) 188 (MH ) (100). Anal. Calcd
+
ꢀ1
1
IR (film): mmax = 1682, 1656, 1610 cm . d H NMR
1.55 (t, 3H, J = 6.8, CH CH ), 4.12 (q, 2H, J = 6.8,
(
C H NO ): C, 70.58; H, 4.85; N, 7.48. Found: C,
11 9 2
2
3
7
0.96; H, 4.88; N, 7.43.
CH CH ), 6.17 (s, 1H, H-3), 7.73 (dt, 1H, J = 1.6, 7.6,
2 3
H-7), 7.77 (dt, 1H, J = 1.6, 7.6, H-6), 8.10 (brdd, 1H,
J = 1.6; 7.4, H-8), 8.15 (brdd, 1H, J = 1.6, 6.8, H-5). d
Compound 1h; red amorphous solid (6%); IR (film):
mmax = 3315, 1678, 1571 cm . d H NMR 3.47 (d, 3H,
ꢀ
1
1
13
C NMR 13.9 (CH CH ), 65.4 (CH CH ), 110.2 (C-
3), 126.1 (C-8), 126.7 (C-5), 131.2 (C-8a), 132.0 (C-7),
133.3 (C-4a), 134.3 (C-6), 159.7 (C-2), 180.2 (C-1),
2
3
2
3
J = 5.6, CH ), 6.17 (br s, 1H, NH), 7.64 (dt, 1H,
J = 1.2, 7.6, H-7), 7.73 (dt, 1H, J = 1.2, 7.6, H-6), 8.04
3
þ
(
7
brdd, 1H, J = 0.8; 7.6, H-8), 8.17 (brdd, 1H, J = 0.8,
185.1 (C-4). ESI-MS m/z (rel int.) 422 ðM
2
NH4 Þ (82).
1
3
.6, H-5). d C NMR 33.1 (CH ), 126.9 (C-8), 127.1
Anal. Calcd (C H O ): C, 71.28; H, 4.98. Found: C,
12 10 3
3
(
(
C-5), 129.8 (C-8a), 132.4 (C-4a), 132.5 (C-7), 134.9
C-6), 147.0 (C-2), 176.5 (C-4), 180.2 (C-1); the follow-
71.30; H, 4.58.
1
13
ing H and C signal assignments are interchangeable:
C-1 with C-4, C-4a with C-8a, H-5/C-5 with H-8/C-8
and H-6/C-6 with H-7/C-7. ESI-MS m/z (rel int.) 266
4.3. Kinetics of reaction with cysteine
Thiol solutions were prepared immediately before use,
by dissolution of cysteine in 50 mM K HPO /KH PO
4
buffer, pH 7.1, containing KCl to maintain ionic
strength at 0.5 M. Solutions with a change in the final
pH value greater than 0.05 were rejected. The concentra-
tion of thiol in solution was measured just before use by
+
+
(
(
MH ) (86), 268 (MH +2) (100). Anal. Calcd
C H BrNO ): C, 49.65; H, 3.03; N, 5.26. Found: C,
2
4
2
1
1
8
2
4
9.87; H, 2.98; N, 5.21.
4
.2.8. 1-[(1,4-Naphthoquinon-2-yl)-S-leucylamido]-3-methyl-
3
4
butane (1i). The side-chain Leu-i-Am was prepared
under standard peptide coupling conditions from the
corresponding Boc-protected amino acid N-hydroxy-
succinimide ester and iso-amylamine, followed by the
removal of the N-Boc group with trifluoroacetic
acid.
added to a solution of 1,4-naphthoquinone (110 mg;
0
0
at rt for 1 h and then the solvent was evaporated. Com-
pound 1i (42 mg) was purified by column chromatogra-
phy on silica gel (40 g) using as eluents DCM (0.2 L) and
a mixture of DCM/EtOAc 19:1 (0.5 L): brown oil (18%);
Ellman’s method. In the case of menadione, 1n, the
rates were measured at three additional pH values using
3
5
sodium borate/HCl buffer (pH 7.9, 8.0 and 8.8). The
kinetic measurements were carried out at 25.0 ± 0.1 ꢁC
without protection from air. The naphthoquinone stock
solutions were prepared in DMSO. Reactions were initi-
ated by addition of an aliquot of naphthoquinone stock
(150 lL) to the buffer containing cysteine (2.85 mL), and
recorded against a blank solution of the buffer with cys-
teine and DMSO. Cysteine concentration ranged be-
tween 0.5 and 5 mM except in the kinetics of
naphthoquinone 1j, in which the range was 0.06–
5 mM. The initial concentration of the naphthoquinone
in the cuvette and the wavelengths used to monitor the
32,33
The resultant amine (131 mg; 0.7 mmol) was
.7 mmol) in abs EtOH (10 mL) with TEA (66 mg;
.7 mmol). The reaction mixture was vigorously stirred
ꢀ
1
1
IR (film): mmax = 3335, 1675, 1572, 1640 cm . d
H

5348
C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
kinetic reactions were as follows: 1j (50 lM; increase at
60 nm), 1q (50 lM; decrease at 282 nm),1 g (50 lM; in-
crease at 300 nm), 1a (32 lM; decrease at 265 nm), 1d
50 lM; increase at 277.3 nm), 1m (10 lM; decrease at
00 nm), 1n(50 lM; increase at 279.8 nm). The pseudo
first-order rate constants, kobs, were obtained from the
slopes of the plots of ln(dA) versus time, where dA rep-
resents the difference in absorbance between time t and
time infinity.
it to a cuvette containing 100 lL of the Z-S-Lys-ONp
stock solution (0.8 mM in DMSO) and 800 lL of
20 mM sodium acetate buffer, pH 5.0, containing
1.0 mM EDTA and 2.0 mM DTT. Enzyme activity
was monitored at 25.0 ± 0.1 ꢁC by following the
appearance of the p-nitrophenol product at 326 nm,
over a period of 3 min, against a blank of 20 mM so-
dium acetate buffer, pH 5.0, containing 1.0 mM EDTA
and 2.0 mM DTT. For PPE, the inhibitor (50 lL of
a stock solution in DMSO) was mixed with 750 lL
of 0.1 M Hepes buffer, adjusted to pH 7.2, and 200 lL
of a stock solution of enzyme (50 lM in 0.1 M Hepes
buffer, pH 7.2). Aliquots (100 lL) were withdrawn at
different time intervals and transferred to a cuvette
containing 5 lL of the Suc-Ala-Ala-Ala-4-nitroanilide
stock solution (12.5 mM in DMSO) and 895 lL of
0.1 M Hepes buffer. The absorbance change was
followed for 50 s at 390 nm against a blank of 0.1 M
Hepes buffer. Pseudo first-order inactivation rate con-
3
(
5
3
8
4
content was measured by the method of Ellman. In
.3.1. Determination of free thiol content. The free thiol
4
3
general, aliquots of the thiol solutions (650 lL) were
added to a cuvette containing a 0.1 mM solution of
0
5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) in 0.1 M
K HPO /KH PO buffer, pH 7.4, to give a total vol-
2
4
2
4
ume of 1 mL. Under these conditions, thiol concentra-
tions in the cuvette ranged between 20 and 120 lM
(
absorbance values between 0.3 and 1.7). The absor-
bance at 412 nm was measured after 5 min, against a
blank solution lacking thiol. A new blank was pre-
pared for every measurement. Thiol concentration
was calculated through the formation of the yellow
stants, kobs, were determined from plots of ln(v/v ) ver-
0
sus incubation time. Four to six inhibitor concentrations
were used to determine k , K and k values, with the
inact
2
I
exception of the k value for 1c, which was calculated
2
dianion
1
of
5-thio-2-nitrobenzoic
acid
(e =
using a single inhibitor concentration due to solubility
problems. In the case of hyperbolic dependences, KI
3
ꢀ1 ꢀ1 34
s ).
4.15 · 10 M
and k
values were determined by computer-fit (Sig-
inact
ꢂ
4
.4. Papain, cathepsin B and PPE inhibition studies
ma Plot ) using least squares nonlinear regression anal-
ysis to Eq. 3.
4
sured by Kitz and Wilson’s pre-incubation method,
.4.1. Kinetic studies. The inhibitory activity was mea-
2
5
4.4.2. Dialysis experiments. Papain was inactivated to
the extent of about 80% by the glycine derivative 1d in
an incubation mixture (2 mL) prepared as described
for the inhibition studies. This solution was then dia-
lysed at 4 ꢁC over a period of 24 h, against 0.8 L of
50 mM K HPO /KH PO buffer, pH 7.0, containing
3
6–38
using procedures based on the literature.
of control incubations were performed at the same time
Assays
3
6
as inhibitor incubations. Papain (1 mg/mL) was acti-
vated by incubation at 25 ꢁC, during 30 min, in 50 mM
K HPO /KH PO buffer, pH 7.0, containing 2.5 mM
2
4
2
4
2
4
2
4
EDTA and 2.0 mM DTT. Reactions were started by
the addition of the inhibitor stock solution in DMSO
2.5 mM EDTA and 2 mM DTT. This thiol was used
to maintain the reducing conditions essential for papain
activity. The enzyme activity was periodically assayed
during the 24 h of dialysis, along with the determination
(
(
50 lL) to the incubation solution containing papain
300 lL of the stock solution 1 mg/mL) and 650 lL of
3
9
5
0 mM K HPO /KH PO , pH 7.0, containing only
of papain concentration by the Bradford Reagent
2
4
2
4
2
.5 mM EDTA. At different times, aliquots (100 lL)
(Bio-Rad) using a microassay procedure. For compari-
son between the assays, the enzyme activity was ex-
pressed as (DA/Dt)/(lg papain), where DA represents
the increase in absorbance during the reading time
(Dt), due to the release of p-nitroaniline. No recovery
of enzyme activity was detected, while the control sam-
ple retained its activity. The protein recovery was always
superior to 80%.
from the incubation mixture were withdrawn and trans-
ferred to a cuvette containing 100 lL of BAPA stock
solution (10 mM in DMSO) and 800 lL of 50 mM
K HPO /KH PO buffer, pH 7.0, containing 2.5 mM
EDTA and 2.0 mM DTT. Enzyme activity was deter-
mined at 25.0 ± 0.1 ꢁC, by following the release of p-nit-
roaniline from BAPA at 410 nm during 4 min, against a
blank of 50 mM K HPO /KH PO buffer, pH 7.0, con-
2
4
2
4
2
4
2
4
taining 2.5 mM EDTA and 2.0 mM DTT. The assays in
the presence of ascorbic acid were carried out as de-
scribed above, except that the incubation buffer included
4.5. Molecular orbital calculations
4
0
Density functional theory (DFT) calculations were
41
carried out with the Gaussian 03 suite of programs.
3
7
ascorbic acid (2.0 and 30 mM). Cathepsin B (10 U/
2
2
1
mL) was incubated at 25 ꢁC, during 15 min in
0 mM sodium acetate buffer, pH 5.0, containing
.0 mM EDTA and 30 mM DTT. Reactions were
1,4-Naphthoquinone structures were optimised with-
out symmetry constraints using the hybrid B3LYP
functional, which is a combination of the Becke’s
4
2
started by addition of the inhibitor stock solution in
DMSO (50 lL) to the incubation solution containing
cathepsin B (20 lL of the stock solution 10 U/2 mL)
and 930 lL of 20 mM sodium acetate buffer, pH 5.0,
containing only 1.0 mM EDTA. At various time inter-
vals, cathepsin B activity was assayed by taking a
three-parameter (B3) exchange functional with the
4
Lee, Yang and Parr (LYP) correlation functional,
3
4
4
in conjunction with 6-311G(d,p) basis set. All geom-
etries were found to be minima on the respective po-
tential energy surfaces. Lowest unoccupied molecular
orbital (LUMO) energies were calculated at this level
of theory.
1
00 lL aliquot of the incubation solution and adding

C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
5349
4
.6. Molecular modelling
18. Hasinoff, B. B.; Wu, X.; Begleiter, A.; Guziec, L. J.;
Guziec, F., Jr.; Giorgianni, A.; Shaohua, Y.; Jiang, Y.;
Yalowich, J. C. Cancer Chemother. Pharmacol. 2006, 57,
Molecular docking enzyme-inhibitor studies were per-
formed with the flexible GOLD 3.0 (Genetic Optimisa-
2
19. Rietjens, I. M. C. M.; Soffers, A. E. M. F.; Hooiveld, G. J.
21.
4
5
tion for Ligand Docking) package. GOLD program
uses a genetic algorithm (which mimics the process of
evolution by applying genetic operators) to explore the
preferred orientations and conformations of the ligand
in relation to the receptor, and has been fully validated
against 305 diverse and extensively checked protein–li-
gand complexes from the PDB database. The docking
results were ranked based on the goldscore scoring func-
E. J.; Veeger, C.; Vervoort, J. Chem. Res. Toxicol. 1995, 8,
4
0. Aguilar-Mart ´ı nez, M.; Mac ´ı as-Ruvalcaba, N. A.; Bautis-
ta-Mart ´ı nez, J. A.; G o´ mez, M.; Gonz a´ lez, F. J.; Gonz a´ lez,
I. Curr. Org. Chem. 2004, 8, 1721.
21. Wilson, I.; Wardman, P.; Lin, T. S.; Sartorelli, A. C.
Chem. Biol. Interact. 1987, 61, 229.
¨
2. Ollinger, K.; Brunmark, A. J. Biol. Chem. 1991, 266,
21496.
23. Sykes, N. O.; Macdonald, S. J. F.; Page, M. I. J. Med.
Chem. 2002, 45, 2850.
4. Jameson, G. N. L.; Zhang, J.; Jameson, R. F.; Linert, W.
Org. Biomol. Chem. 2004, 2, 777.
5. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
6. Rodriguez, C. E.; Fukuto, J. M.; Taguchi, K.; Froines, J.;
Cho, A. K. Chem. Biol. Interact. 2005, 155, 97.
81.
2
2
4
6
tion (fitness function). The papain crystal structure
4
7
used in the calculations (PDB code 1PPN) was ob-
tained by deletion of one oxygen and the methanol
present in the crystal structure. The structure was en-
ergy-minimized. For each ligand (1,4-naphthoquinone
geometries were previously energy-minimized with
DFT) 50 docking runs were performed (100,000 genetic
algorithm operations, 5 islands).
2
2
2
27. Hanzlik, R. P.; Thompson, S. A. J. Med. Chem. 1984, 27,
11.
7
2
8. Meloun, B.; Baudy sˇ , M.; Pohl, J.; Pavlik, M.; Kostka, V.
J. Biol. Chem. 1988, 263, 9087.
Acknowledgment
2
9. Frlan, R.; Gobec, S. Curr. Med. Chem. 2006, 13, 2309.
0. Phillips, R. M.; Jaffar, M.; Maitland, D. J.; Loadman, P.
M.; Shnyder, S. D.; Steans, G.; Cooper, P. A.; Race, A.;
Patterson, A. V.; Stratford, I. J. Biochem. Pharmacol.
3
The authors thank Dr. Ana Paula Leandro (Faculty of
Pharmacy, University of Lisbon) for the support during
dialysis assays. We also thank FCT, POCTI, FEDER
2
004, 68, 2107.
(Portugal) as well as the British Council for financial
support and a Grant to CV (SFRH/BD/10815/2002).
3
1. Tandon, V. K.; Yadav, D. B.; Maurya, H. K.; Chatuverdi,
A. K.; Shukla, P. K. Bioorg. Med. Chem. 2006, 14, 6120.
2. Portela, M. J.; Moreira, R. F. A.; Valente, E.; Constan-
tino, L.; Iley, J.; Pinto, J.; Rosa, R.; Cravo, P.; do Ros a´ rio,
V. E. Pharm. Res. 1999, 16, 949.
3
References and notes
3
3
3
3. Gomes, P.; Ara u´ jo, M. J.; Rodrigues, M.; Vale, N.;
Azevedo, Z.; Iley, J.; Chambel, P.; Morais, J.; Moreira, R.
Tetrahedron 2004, 60, 5551.
4. Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.;
Simeon-Rudolf, V.; Reiner, E. Anal. Biochem. 2003, 312,
1
. dos Santos, E. V. M.; Carneiro, J. W. M.; Ferreira, V. F.
Bioorg. Med. Chem. 2004, 12, 87.
. O’Brien, P. J. Chem. Biol. Interact. 1991, 80, 1.
. Baggish, A. L.; Hill, D. R. Antimicrob. Agents Chemother.
2
3
2
24.
2
002, 46, 1163.
5. Perrin, D. D.; Dempsey, B. Buffers for pH and Metal ion
Control; Chapman and Hall Laboratory Manuals: Lon-
don, 1974.
4
. Nishikawa, Y.; Carr, B. I.; Wang, M.; Kar, S.; Finn, F.;
Dowd, P.; Zheng, Z. B.; Kerns, J.; Naganathan, S. J. Biol.
Chem. 1995, 270, 28304.
3
3
3
6. Zhao, G.; Zhou, Z. S. Bioorg. Med. Chem. Lett. 2001, 11,
5
6
. Ni, R.; Nishikawa, Y.; Carr, B. I. J. Biol. Chem. 1998, 273,
906.
2
331.
7. Bajkowski, A. S.; Frankfater, A. Anal. Biochem. 1975, 68,
19.
9
. Wissner, A.; Floyd, M. B.; Johnson, B. D.; Frase, H.;
Ingalls, C.; Nittoli, T.; Dushin, R. G.; Discafani, C.;
Nilakantan, R.; Marini, J.; Ravi, M.; Cheung, K.; Tan,
X.; Musto, S.; Annable, T.; Siegel, M. M.; Loganzo, F.
J. Med. Chem. 2005, 48, 7560.
1
8. Hinchliffe, P. S.; Wood, J. M.; Davis, A. M.; Austin, R. P.;
Beckett, R. P.; Page, M. I. Org. Biomol. Chem. 2003, 1, 67.
9. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
0. Parr, R. G.; Yang, W. Density Functional Theory of Atoms
and Molecules; Oxford University Press: Oxford, U.K,
3
4
7
8
9
. Veber, D. F.; Thompson, S. K. Curr. Opin. Drug Discov.
Dev. 2000, 3, 362.
. Lecaille, F.; Kaleta, J.; Br o¨ mme, D. Chem. Rev. 2002, 102,
459.
. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
1
989.
4
1. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Montgomery Jr., J.
A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.;
Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.;
Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda,
R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.;
Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.;
Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.;
Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz,
4
1
1
0. Liu, S.; Hanzlik, R. P. J. Med. Chem. 1992, 35, 1067.
1. Lin, T.-S.; Zhu, L.-Y.; Xu, S.-P.; Divo, A. A.; Sartorelli,
A. C. J. Med. Chem. 1991, 34, 1634.
1
1
1
2. Prescott, B. J. Med. Chem. 1969, 12, 181.
3. Kutyrev, A. A. Tetrahedron 1991, 47, 8043.
4. Tandon, V. K.; Yadav, D. B.; Singh, R. V.; Chaturvedi, A.
K.; Shukla, P. K. Bioorg. Med. Chem. Lett. 2005, 15, 5324.
5. Rozeboom, M. D.; Tegmo-Larsson, I.-M.; Houck, K. N.
J. Org. Chem. 1981, 46, 2338.
6. Ham, S. W.; Choe, J.-I.; Wang, M. F.; Peyregne, V.; Carr,
B. I. Bioorg. Med. Chem. 2004, 14, 4103.
7. Chan, A. W. E.; Golec, J. M. C. Bioorg. Med. Chem. 1996,
1
1
1
4
, 1673.

5350
C. Valente et al. / Bioorg. Med. Chem. 15 (2007) 5340–5350
J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
43. Lee, C. T.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785.
44. Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A.
J. Chem. Phys. 1980, 72, 650.
45. CCDC Software Ltd: Cambridge, UK.
46. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. J. Mol. Biol. 1997, 267, 727.
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-
Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challa-
combe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong,
M. W.; Gonzalez, C.; Pople, J.A.; Gaussian, Inc.:
Wallingford CT, 2004.
47. Pickersgill, R. W.; Harris, G. W.; Garman, E. Acta
Crystallogr., Sect. B 1992, 48, 59.
4
2. Becke, A. D. J. Chem. Phys. 1993, 98, 5648.