Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials

Article abstract of DOI:10.1016/j.ejmech.2012.05.022

A series of cinnamic acid/4-aminoquinoline conjugates conceived to link, through a proper retro-enantio dipeptide, a heterocyclic core known to prevent hemozoin formation, to a trans-cinnamic acid motif capable of inhibiting enzyme catalytic Cys residues, were synthesized as potential dual-action antimalarials. The effect of amino acid configuration and the absence of the dipeptide spacer were also assessed. The replacement of the D-amino acids by their natural L counterparts led to a decrease in both anti-plasmodial and falcipain-inhibitory activity, suggesting that the former are preferable. Molecules with such spacer were active against blood-stage Plasmodium falciparum, in vitro, and hemozoin formation, implying that the dipeptide has a key role in mediating these two activities. In turn, compounds without spacer were better falcipain-2 inhibitors, likely because these compounds are smaller and have their vinyl bonds in closer vicinity to the catalytic Cys, as suggested by molecular modeling calculations. These novel conjugates constitute promising leads for the development of new antiplasmodials targeted at blood-stage malaria parasites.

Full text of DOI:10.1016/j.ejmech.2012.05.022

European Journal of Medicinal Chemistry 54 (2012) 887e899
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Short communication
Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic
amino acids: Towards the development of potential dual action antimalarials
,
,b,
Bianca C. Pérez a 1, Cátia Teixeira a 1, Marta Figueiras a, Jiri Gut c, Philip J. Rosenthal c, José R.B. Gomes b,
,
Paula Gomes a
*
a Centro de Investigação em Química da Universidade do Porto, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, R. do Campo Alegre, 687,
P-4169-007 Porto, Portugal
b CICECO, Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
c Department of Medicine, San Francisco General Hospital, University of California, CA 94143-0811, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Cinnamic
acid/4-aminoquinoline
hybrids were synthesized as dual-
action drugs.
< Hybrids were linked via
a retro-
enantio dipeptide. Its absence was
also evaluated.
< Their power to inhibit hemozoin
growth, falcipain and blood-stage Pf
were assessed.
< Hybrids with dipeptide were active
against blood-stage Pf and hemo-
zoin growth.
< Molecules without spacer were
better falcipain-2 inhibitors.
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cinnamic acid/4-aminoquinoline conjugates conceived to link, through a proper retro-enantio
dipeptide, a heterocyclic core known to prevent hemozoin formation, to a trans-cinnamic acid motif
capable of inhibiting enzyme catalytic Cys residues, were synthesized as potential dual-action antimalarials.
The effect of amino acid configuration and the absence of the dipeptide spacer were also assessed. The
replacement of the D-aminoacids by their natural L counterparts led to a decrease in bothanti-plasmodial and
falcipaineinhibitory activity, suggesting that the former are preferable. Molecules with such spacer were
active against blood-stage Plasmodium falciparum, in vitro, and hemozoin formation, implying that the
dipeptide has a key role in mediating these two activities. In turn, compounds without spacer were better
falcipain-2 inhibitors, likely because these compoundsare smallerand have their vinyl bonds in closer vicinity
to the catalytic Cys, as suggested by molecular modeling calculations. These novel conjugates constitute
promising leads for the development of new antiplasmodials targeted at blood-stage malaria parasites.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 12 March 2012
Received in revised form
11 May 2012
Accepted 15 May 2012
Available online 28 May 2012
Keywords:
Antimalarials
Blood-schizontocidal
Chloroquine
Cinnamic acid derivatives
Molecular modeling studies
Abbreviations: AA, amino acid; ABC, adenosine triphosphate binding cassette; CQ, chloroquine; DCM, dichloromethane; DFT, density functional theory; DIEA, N,N-dii-
sopropylethylamine; DMF, N,N-dimethylformamide; HECIN, heterocyclic-cinnamic acid conjugate; HEDICIN, heterocyclic-dipeptide-cinnamic acid conjugate; mp, melting
point; PyAOP, 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate;
PyBrop, bromotripyrrolidinophosphonium hexafluorophosphate; PyClocK, 6-chloro-beenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; PyOxim,
[ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate; RBC, red blood cell; RF, rate of flow (thin-layer chromatography); rt, retention
time; SAR, structureeactivity relationships; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate; THF, tetrahydrofurane.
* Corresponding author. Tel./fax: þ351 220402563.
E-mail address: pgomes@fc.up.pt (P. Gomes).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.022

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B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
potential inhibitors of both falcipain protease activity and hemo-
zoin formation, according to the rationale below.
1. Introduction
Malaria remains one of the most important infectious diseases
in the world. Plasmodium falciparum, the most virulent species of
human malaria parasite, is responsible for about 1 million deaths
every year [1e3]. Once having invaded the human host through the
bite of an infected female Anopheles mosquito, the parasites repli-
cate silently in the liver, after which they invade red blood cells
(RBCs), where a second asexual reproduction cycle gives rise to the
clinical symptoms of the disease. P. falciparum causes the most
severe clinical manifestations of malaria, and is particularly deadly
to children under five years old, so impairment of development of
blood-stage P. falciparum has been the main target in antimalarial
chemotherapy [4e6].
Within infected RBCs, parasites digest host hemoglobin to obtain
free amino acids required for parasite nourishment and develop-
ment [7e9]. In this process, free heme is produced as a side product
that is toxic to the parasite. Hence, the parasite converts free heme
into a polymer, hemozoin, which is a non-toxic insoluble biocrystal
[8]. The classical antimalarial chloroquine (CQ) blocks hemozoin
formation and consequently kills malaria parasites [8,10].
Inhibition of blood-stage P. falciparum may be also achieved
through starvation, i.e., by inhibiting parasitic enzymes involved in
globin degradation. Amongst them, cysteine proteases, especially
falcipain-2 (FP2) and falcipain-3 (FP3), are key therapeutic targets
[7]. Therefore, compounds able to simultaneously impair hemozoin
formation and falcipain proteolytic activity may be potent agents
against blood-stage plasmodia with reduced propensity to elicit
parasite resistance [11]. In this regard, this paper provides the
synthesis, in silico study and in vitro evaluation of novel cinnamic
acid/4-aminoquinoline conjugates (8 and 9, Scheme 1) as potential
dual action antimalarials. These conjugates, named HEDICINs
(8, Scheme 1) and HECINs (9, Scheme 1) were conceived as
1.1. Rationale
HEterocyclic-DIpeptide-CINnamic acid conjugates (HEDICINs)
were designed to link, through a suitable spacer, (i) the CQ hetero-
cyclic core, known as relevant to inhibit hemozoin formation [12,13],
to a (ii) trans-cinnamic acid motif, as cinnamic acids have been
described to exhibit both antimalarial activity [14] and inhibiting
enzyme catalytic Cys residues [15]. Cinnamic acid derivatives, due to
their
a,b-unsaturated carbonyl moiety, can act as Michael acceptors
and inhibit cysteine proteases through S-alkylation [9,16,17]. Irre-
versible S-alkylation of the falcipain catalytic Cys has been consid-
ered the major mechanism behind the inhibitory and in vitro
anti-plasmodial activity of peptidyl inhibitors including leupeptin
and vinyl sulfones developed by Rosenthal and co-workers (Fig. 1)
[9,18]. The most active vinyl sulfones contained a dipeptide Leu-
hPhe spacer between a bulky moiety and the S-alkylating motif
[9,18e20]. Unfortunately, peptide-based inhibitors are prone to
proteolytic degradation [21], a problem that can be overcome by use
of suitable peptide delivery systems [22,23]. Still, such systems are
likely to impair efficient inhibition of the target enzyme.
One way to circumvent the limitations of peptidyl inhibitors is
the use of retro-enantio peptides, i.e., analogs where all amino acids
have a D configuration and are assembled in reversed order [24,25].
As these molecules have native side chain topology but reversed
amide bonds, they theoretically allow enzyme-ligand contacts
identical to those displayed by native peptides, while eluding
recognition by other proteases (Fig. 1).
The retro-enantio analog of Leu-hPhe seems the best mimic of
falcipain P2eP1 sites, linking the putative P3 motif (heterocyclic
core of CQ) to the Michael acceptor in P1
0 (cinnamic acid). However,
Cl
R
H
N
N
O
9a-k
(vii)
NH2
O
O
O
NH2
Cl
N
NH
HN
N
HN
N
(ii)
(iii)
(i)
O
Cl
Cl
N
Cl
Cl
3
4
5
2
(iv)
O
O
O
O
O
NH
NH
O
HN
NH2
(v)
HN
N
N
H
NH
(vi)
Cl
HN
N
N
O
O
H
O
N
Cl
Cl
R
7
6
8a-l
Scheme 1. Reagent conditions (reactions run at room temperature except otherwise indicated): (i) NH3 (g), phenol, 150 ꢀC, 2 h; (ii) TBTU, Boc-D-homoPhenylalanine, DIEA, DMF, 1
day; (iii) TFA, 2 h; (iv) PyBOP, DIEA, DCM, 1 day; (v) TFA, 2 h; (vi) PyBOP, corresponding cinnamic acid, DIEA, DCM, 3 days; (vii) PyBOP, corresponding cinnamic acid, DIEA, DMF, 1
day. Substituents R, in both HEDICINs 8ael and HECINs 9aek, are given on Table 1.

B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
889
Fig. 1. Structures of (A) leupeptin, a cysteine protease inhibitor, (B) a vinylsulfone falcipain inhibitor, and (C) HEDICIN structure originally designed, based on a cinnamic acid
building block linked to the chloroquine’s hetereocyclic core through the retro-enantio analog of Leu-hPhe (see text).
preliminary computational studies by our group on HEDICINs 1 and
8f (structures given below, Fig. 2A and B, respectively), differing
data, as described in the Experimental section. Global synthesis
yields were modest, due to the low nucleophilicity of the aniline
group in 4-amino-7-chloroquinoline (3), a problem that persisted
even when using highly effective coupling agents such as PyBroP,
PyClocK, PyAOP or PyOxim [26] in step (ii) of Scheme 1. Interestingly,
even though the use of 7-aza-1-hydroxybenzotriazole derivatives as
PyAOP has been reported to improve yields in couplings involving
poor nucleophiles [27], the best yields in step (ii) were achieved
when using the most common 1-hydroxybenzotriazole derivative,
TBTU, as a coupling agent. The low nucleophilicity of the aniline
4-amino-7-chloroquinoline (3) was also likely responsible for the
low yields obtained in the direct coupling of this heterocycle to
cinnamic acid derivatives, step (vii) in Scheme 1. In this case, the best
only in the order of the two
D
-amino acids, suggested that the
inverse sequence, i.e., -hPhe-
D
D-Leu, would allow a closer approxi-
mation of the electrophilic moiety to the catalytic center (Fig. S1).
These preliminary observations in silico were later supported
in vitro by anti-plasmodial activity tests against the CQ-resistant
Plasmodium falciparum strain W2, which revealed that 1 did not
display any antimalarial activity up to 10
parasite development with an IC50 of 5.43
m
M, while 8f inhibited
mM (Table 1). Therefore,
we engaged in the synthesis of HEDICINs with general structure
(Scheme 1) as potential dual-action drugs against blood-
8
stage parasites. Analogs lacking the dipeptide spacer, HECINs 9
(Scheme 1), were also prepared to assess the relevance of that
spacer. Compounds 8 and 9 were evaluated in vitro concerning their
ability to inhibit (i) heme polymerization to hemozoin; (ii) falcipain
activity; and (iii) development of blood-stage P. falciparum. In order
to support observed SAR and to rationalize the activity profile of the
novel compounds, we also performed molecular modeling calcu-
lations on computational models derived from X-ray structures of
FP2 (PDB code: 3BPF) and FP3 (PDB code: 3BWK) co-crystalized
with E64 and K11017, respectively [7,16].
results were achieved using PyBOP as a coupling agent. The
L-amino
acid analog of 8a, compound 10a, was also prepared by similar
methods.
2.2. In vitro studies
2.2.1. Inhibition of heme polymerization
The capacity of test compounds 8 and 9 to inhibit heme poly-
merization in vitro was assessed by previously reported methods
[28,29], given in detail under Experimental. The assays were run in
96-well microplates, where negative controls (water, DMSO) and
positive controls (1 mM CQ) were included. Test compounds were
assayed at 1 mM and data are given in Table 1. Interestingly, the
dipeptide spacer was required to block heme polymerization, i.e.,
while HECINs 9 were not active, HEDICINs 8 displayed variable
inhibitory efficiencies, with four out of the twelve compounds
2. Results and discussion
2.1. Chemical synthesis
Twenty-three novel compounds, 8ael and 9aek, were success-
fully obtained in high purity and with correct spectral and analytical
Fig. 2. Structures of compounds 1 (A, left) and 8f (B, right).

890
B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
Table 1
2.2.2. Anti-plasmodial activity
In vitro data on test compounds, HEDICINs (8) and HECINs (9). Compound 10a, the
amino acid analog of 8a, is also included.
L-
Activity against blood-stage CQ-resistant P. falciparum strain W2
was assessed as previously reported [30] and given in detail in
Experimental. Results demonstrated a complete lack of activity
displayed by HECINs 9, correlating with their inability to inhibit
heme polymerization (Table 1). In turn, eleven out of the twelve
HEDICINs 8 had IC50 values under 10 mM. Interestingly, three of
the four most active HEDICIN blockers of heme polymerization
(8b, 8j and 8l) were also among the four most active anti-
plasmodials, with IC50 below 2
mM. These results suggest that
inhibition of heme polymerization is, at least in part, responsible for
the anti-plasmodial activity of HEDICINs.
An obvious exception to correlation between inhibition of heme
polymerization and anti-plasmodial activity in HEDICINs is
compound 8c; this bears a bulky electron-donating p-isopropyl
group and did not inhibit heme polymerization in vitro, but dis-
played the highest anti-plasmodial activity. The inability of 8c to
inhibit heme polymerization could be related to the bulkiness of
the isopropyl group, but due to the higher hydrophobicity of this
substituent, 8c was the most lipophilic HEDICIN assayed. Though
we could not establish a full correlation between HEDICIN anti-
plasmodial activity and estimated clogP values (not shown), the
markedly higher lipophilicity of 8c, as compared to the other
Compound
R
Hemozoin IC50 Pf W2b IC50 FP2c IC50 FP3 (
m
M) LogPd
Inhibitiona
(
m
M)
(mM)
8a
10ae
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
9e
9f
H
H
e
4.89
8.00
1.96
0.830
10.8
3.28
5.43
4.67
2.89
2.55
1.66
2.10
1.23
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
ND
19.6
50.0
>50
>50
>50
20.3
23.1
50.0
>50
48.3
>50
28.1
>50
>50
28.5
>50
>50
>50
>50
41.3
33.1
23.6
14.2
>50
0.0246
ND
>50
ND
ND
ND
ND
>50
>50
ND
ND
>50
ND
>50
ND
ND
>50
ND
ND
6.6
6.6
7.1
7.9
6.5
5.8
6.8
6.8
7.2
7.4
6.6
6.6
6.6
4.3
4.8
5.6
4.2
4.5
4.5
4.9
5.1
4.3
4.3
4.3
NDf
þþ
e
p-Me
p-iPr
p-OMe
þ
p-NH2 þþ
m-F
e
e
e
e
þ
e
p-F
p-Cl
p-Br
o-NO2
m-NO2
analogs, could promote
a higher permeabilization of this
compound into the infected RBC. Kirk and co-workers have
demonstrated that P. falciparum parasites create new permeability
pathways in host RBC, leading to increased permeability to small
organic cations [31,32].
In summary, though clean correlations could not be drawn
between the anti-plasmodial activities displayed by the different
HEDICINs (8) in vitro and molecular descriptors such as stereo-
electronic factors (aryl substituents) or lipophilicity, it is demon-
strated that these compounds displayed anti-plasmodial activity,
whereas their HECIN counterparts (9), lacking the dipeptide spacer,
did not. HEDICINs (8) inhibited heme polymerization in vitro,
suggesting that this inhibitory activity is at least in part responsible
for their anti-plasmodial activity.
p-NO2 þþ
H
e
p-Me
p-iPr
p-OMe
m-F
p-F
p-Cl
p-Br
o-NO2
m-NO2
p-NO2
e
e
e
e
e
ND
ND
e
9g
9h
9i
e
>50
>50
>50
>50
ND
ND
ND
ND
e
e
9j
e
9k
E64
ART
CQ
e
ND
ND
þþ
e
0.00876
0.0760
e
ND
a
Ability of the test compounds to inhibit hemozoin formation in vitro was
calculated as a % of the inhibitory effect displayed by reference drug CQ in the same
experiment; test compounds were ranked as follows: <50%, not active (ꢁ); between
50 and 75%, moderately active (þ); ꢂ75%, highly active (þþ).
2.2.3. Falcipain inhibition in vitro
The very different activities of compounds 8 and 9 suggest that
the retro-enantio dipeptide spacer has a relevant role in deter-
mining anti-plasmodial activity. In view of this, we hypothesized
that the anti-plasmodial activity of HEDICINs could also be partly
due to falcipain inhibition [9,18]. Therefore, both HEDICINs 8 and
HECINs 9 were evaluated in vitro for inhibition of falcipains, using
previously described methods [9], given in detail under Experi-
b
Blood-stage anti-plasmodial activity was determined against the CQ-resistant
P. falciparum strain W2, using artemisinin (ART) and CQ as reference drugs.
c
Falcipain inhibition capacity was evaluated against falcipain-2 using E64 as
reference inhibitor and only positives (IC50 < 50 mM) were further evaluated against
falcipain-3.
d
The logP values were estimated with the MarvinSketch software [33].
Compound 10a is the L-amino acid analog of 8a.
ND, not determined.
e
f
mental. Only those compounds with IC50 < 50
mM against FP2 were
assayed against FP3, as it has been established that FP2 has a larger
catalytic cavity that accommodates a wider range of inhibitors than
FP3. Consistent with this assumption, none of the compounds that
(8b, 8e, 8j and 8l) highly active (i.e., comparable to the reference
drug, CQ). Though no clear trend could be established regarding
stereoelectronic properties of the aryl substituent in compounds 8,
it was clear that: (i) hydrogen (i.e., absence of a substituent) or
halogens in the para position were detrimental for activity, whereas
(ii) nitrogenated groups at either the ortho (8j, o-NO2) or the para
(8e, p-NH2; 8l, p-NO2) position of the aryl ring was beneficial, but
(iii) detrimental if placed in the meta position (8k, m-NO2).
Furthermore, while small alkyl groups in para (8b, p-Me) were
advantageous, bulkier groups as in 8c (p-iPr) led to complete loss of
activity.
Thus, replacement of CQ’s aliphatic chain by an adequate
dipeptidyl-cinnamoyl moiety as in 8b, 8e, 8j and 8l appears to
preserve the parent drug’s ability to inhibit hemozoin formation,
suggesting that these novel compounds could be promising leads
for new CQ surrogates.
inhibited FP2 in vitro displayed IC50 < 50
Falcipain inhibition results contrasted with those for inhibition
of heme polymerization or parasite development. HECINs
mM against FP3 (Table 1).
9
generally displayed more potent inhibition of falcipain than did
HEDICINs 8 (Table 1). The ability of the test compounds to inhibit
FP2 did not correlate with their anti-plasmodial activity. Although
none of the HECINs 9 displayed anti-plasmodial activity, many
inhibited FP2 and one of them, 9j, was actually the best FP2
inhibitor amongst the test compounds. Concerning HEDICINs, 8a,
8f and 8k, derived from cinnamic acid, m-fluorocinnamic acid, and
m-nitrocinnamic acid, respectively, were the most active HEDICINs
against FP2, with IC50 values of 19.7, 23.1 and 28.1 mM. Thus, ster-
eoelectronic effects from aryl substituents did not correlate with
inhibitory activities, as in HEDICINs 8 the most active compound
was unsubstituted (8a), followed by the meta-fluorinated

B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
891
compound (8f); in turn, in HECINs 9, the meta-fluorinated deriva-
as HEDICINs 8 are more lipophilic than HECINs 9 (due to the
D
-Leu
tive was inactive, whereas the most active of the set was 9j, which
bears a meta-nitro substituent.
and the D-hPhe hydrophobic side chains), and the most lipophilic
compound, 8c, had the greatest anti-plasmodial activity.
The effect of amino acid configuration on HEDICIN activity was
also assessed through synthesis and evaluation of 10a, the
L-amino
2.3. In silico studies
acid analog of 8a; interestingly, replacement of the -amino acids
D
by their natural L counterparts led to a clear decrease in both anti-
plasmodial and falcipaineinhibitory activity. Therefore, amino acid
configuration does influence compound behavior as either anti-
plasmodial agent or falcipain inhibitor and, in the particular case of
A thorough computational study was undertaken in order to
explain the unexpected superior ability of HECINs 9 to inhibit fal-
cipains, as compared to HEDICINs 8, and the inability to establish
any clear correlation between structure and activity of the test
compounds.
HEDICINs, data suggests that D-amino acids are preferable.
Of note, the compound with highest anti-plasmodial activity, 8c,
was completely devoid of inhibitory activity against FP2. This
compound likely exerts its anti-plasmodial action by mechanisms
other than inhibition of hemozoin formation or falcipain activity.
Data on compound 8e reinforce the idea that falcipain or hemozoin
inhibition are not the main mechanisms of action responsible for
HEDICINs anti-plasmodial activity: 8e, bearing a p-amino substit-
uent in the aryl ring, was the test compound which best reached
our original goal of a dual-action inhibitor, by joining high hemo-
2.3.1. Molecular docking and dynamic simulations
Docking calculations were performed to predict the structures
of complexes between falcipains and the two families of
compounds. The stabilities of the complexes were further evalu-
ated by molecular dynamics simulations. Additionally, DFT calcu-
lations of Fukui indices were used to quantify electrophilicity at
particular atomic sites. Molecular modeling calculations, given in
detail under Experimental, were carried out on a computational
model of the enzymes derived from the X-ray structures of FP2
(PDB code: 3BPF) and FP3 (PDB code: 3BWK) co-crystalized with
E64 and K11017, respectively.
zoin inhibitory activity with an IC50 ꢃ 20
mM against FP2; however,
such was not translated into the highest anti-plasmodial activity
being observed for 8e.
Taken together, data from in vitro falcipain inhibition and
parasite development assays suggest that the dipeptide spacer in
HEDICINs 8 promotes uptake into infected RBCs. This hypothesis
could explain why HEDICINs perform better than HECINs as anti-
plasmodials, despite the observation that HECINs were better
falcipain-inhibitors than the HEDICINs. In addition, lipophilicity
could have a role in compound uptake and anti-plasmodial action,
The computational results suggest that all test compounds,
belonging to both series (8 and 9), are not able to fit into the FP3
catalytic site as efficiently as into FP2 subsites. Fig. 3 represents the
preferred docking conformation of the most active inhibitor for
each series (compounds 8a and 9j) into the FP2 and FP3 catalytic
sites. As we can observe, the vinyl group in 8a is placed in the S2
subsite of FP2 (Fig. 3A) while in FP3 it is located at the S20 cavity
Fig. 3. Preferred docked binding mode of HEDICIN 8a (A and B) and HECIN 9j (C and D) into falcipain-2 and falcipain-3 binding sites, respectively. The inhibitors are shown in ball
and stick representation while residues forming the “oxyanion hole” of parasitic cysteine proteases are in CPK representation. Enzyme subsites are shown on panels A and C.

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B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
(Fig. 3B), which severely hinders an attack on the catalytic Cys
thiolate. Similarly, the putative site of nucleophilic attack of HECINs
9 is placed far away from the catalytic Cys of FP3 (Fig. 3D). Indeed,
the docked binding mode of molecule 9j is entirely outside the
FP3 binding pocket, and this is probably because the area
between the subsites is too narrow to accommodate two bulky
(7-chloroquinolyl and cinnamoyl) groups so close to each other. The
remaining test compounds displayed docking conformations in the
FP3 active site similar to their respective analogs, which likely
explains why none of the compounds inhibited FP3.
potential falcipain inhibitors. This conclusion is supported by
comparative evaluation of HEDICIN and HECIN ligand efficiencies,
as follows. Enzyme inhibitor candidates can be profiled regarding
their ligand efficiencies by use of simple techniques, originally
developed to provide a fast and simple way of assessing the quality
of hits in high-throughput screening efforts [34]. As ligand effi-
ciency is expectedly lower for smaller compounds, given the fewer
contact points with the target enzyme, ligand size is a key
parameter in calculating ligand quality [35]. Among current tools to
rank ligand efficiencies is the Binding Efficiency Index (BEI), which
divides the pIC50 (derived from M units) by the molecular weight
(kDa units) of the compound [35]. This estimates how a ligand
optimally binds a receptor relative to other ligands of any size. We
have applied this methodology to compare the efficiency of HEDI-
CINs 8 and HECINs 9 as falcipain ligands. BEI values ranged between
11.6 for 9h and 14.1 for 9b, and between 7.2 for 8g to 8.1 for 8a,
consistent with the conclusion that HECINs 9 have higher falcipain-
binding efficiencies than HEDICINs 8.
Computational results also suggested
a preferred binding
conformation for HEDICINs 8 into the FP2 binding site. All HEDICINs
docked in an orientation similar to that previously found for 8f
(Fig. S1B), by placing the 7-chloroquinolyl, homo-phenylalanyl,
leucyl, and cinnamoyl groups into the S20, S10, S1, and S2 receptor
subsites, respectively. Docking also suggested that, in general, the
7-chloroquinoline ring of all HEDICINs established
pep interac-
tions with Trp206 and that its aromatic nitrogen formed
a hydrogen bond with the backbone eNH of Gln36. The only
exception to this binding mode was found for compound 8c that,
due to the bulky para-isopropyl group in the aryl ring, was not able
to dock into the FP2 catalytic site. No preferred docking confor-
mation was evident for this molecule but, in all conformations, the
cinnamoyl moiety was far away from the catalytic Cys. Thus, it was
not surprising that compound 8c showed negligible activity against
FP2. For all the other HEDICINs, the vinyl bond was placed within
w4.5 Å of the enzyme’s Cys thiolate, suggesting that this large
distance between the enzyme’s nucleophile and the ligand’s elec-
trophile may explain the modest inhibitory activity shown by
compounds 8.
By analyzing the docked binding modes of all HECINs, we
observed a preference for the positioning of the cinnamoyl group
at the S2 cavity (Fig. 3C), except for compounds 9c and 9i (Fig. S2),
which presented an upside-down orientation because their cin-
namoyl groups could not fit into the S2 subsite. Despite the
existence of two different binding conformations for this series,
both orientations placed the ligand’s vinyl group within 2.8e3.3 Å
of the catalytic Cys thiolate, which could explain why HECINs
were generally more active than HEDICINs as FP2 inhibitors
(Table 1).
2.3.2. Fukui indices of the vinyl carbons in HECINs
As in all docked binding modes of HECINs 9, vinyl groups were in
close proximity to the catalytic Cys (within 2.8e3.3 Å of the thio-
late), we investigated the influence of different aryl substituents on
the electrophilic character of the HECINs’ vinylic carbons. This
study was based on the assumption that the vinyl bond would act
as the Michael acceptor in the ligand’s irreversible binding to the
falcipain catalytic Cys. Atomic Fukui indices (fþ), derived from
Natural Bond Order (NBO) population analysis of the neutral
compound and its respective cation [36], were used to quantify
electrophilicity at particular atomic sites of the test compounds.
Based on the rationale underlying this work, we considered only
Table 2
Calculated Fukui indices (fþ) used to predict the electrophilicity of atomic sites in
HECINs 9. The value in brackets corresponds to the ratio between the fþ values of
the most reactive vinyl carbon (highlighted in bold) and the other vinyl carbon. This
can be used as a measure of the double bond activation. The fþ values in bold
indicate the putative atomic site of nucleophilic attack for each inhibitor.
Another factor behind the overall better performance of HECINs
9 as FP2 inhibitors, as compared to HEDICINs 8, concerns the
ability of compounds to establish strong non-covalent interactions
within the enzyme’s binding pocket. Indeed, after accommodation
of any ligand into the falcipain catalytic site, the ligand will need to
establish a minimum of contact points with the enzyme so it can
remain tightly bound to it. In other words, a high ligand efficiency
must be ensured, otherwise the ligand will leave the cavity
spontaneously or upon competition with the natural substrate,
even before it reacts with the catalytic Cys. The relevance of tight
ligand-enzyme non-covalent interactions is consistent with the
observation that two HECINs bearing a NO2 substituent (9iej)
were active against FP2, whereas unsubstituted 9a was not.
Indeed, comparing the stability of FP2 complexes with molecules
9a and 9j by molecular dynamics, we observed that after 500 ps
the distance between the catalytic Cys and the putative site of
nucleophilic attack of compound 9a increased to w6 Å (Fig. S3),
suggesting that the inhibitor left the binding site. In contrast,
compound 9j had its double bond mainly within 3.5e4.0 Å of the
thiolate, suggesting that this molecule creates more contact points
with the enzyme and, consequently, remains for a longer time in
the binding site.
Compound
R
Vinyl carbon
fþ
9a (2.12)
H
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
0.054
0.114
0.049
0.114
0.055
0.096
0.042
0.116
0.069
0.116
0.069
0.116
0.061
0.110
0.064
0.102
0.065
0.020
0.058
0.026
0.092
0.037
9b (2.35)
9c (1.76)
9d (2.75)
9e (1.73)
9f (1.69)
9g (1.79)
9h (1.60)
9i (3.29)
9j (2.25)
9k (2.48)
p-Me
p-iPr
p-OMe
m-F
p-F
p-Cl
p-Br
o-NO2
m-NO2
p-NO2
Both in vitro and in silico data suggest that smaller HECINs 9
constitute better templates than HEDICINs 8 for development of
second-generation cinnamic acid/4-aminoquinoline conjugates as

B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
893
proteins [39] and found to be directly implicated in drug-
resistance mechanisms [40,41];
c) increasedpermeabilityof dipeptidederivatives due tothe new
permeationpathways created in P. falciparum-infected RBCs to
promote uptake of nutrients such as sugars or nucleosides
[42], as well as amino acids and oligopeptides [31];
the two vinylic carbons in HECINs (atoms 1 and 2 in Table 2). The
calculated Fukui indices showed that the greater the magnitude of
fþ, the greater the change in electron density near the atoms of
interest, and thus the higher reactivity of a molecule at that atomic
site towards nucleophilic attack. Similarly, the ratio between the
Fukui values of the two vinyl carbons was used as a measure of the
activation, i.e., electron density imbalance in the double bond. Not
II. Alternatively, a difference in the respective mechanism of
action (MOA) may be operating in addition, or in alternative,
to a difference in uptake of HEDICINs versus HECINs:
a) due to their dipeptide moiety, HEDICINs might be able to
inhibit plasmodial cytosolic proteases like aminopepti-
dases PfA-M1 and PfA-M17, which function in regulating
the intracellular pool of amino acids required for parasite
growth and development inside the red blood cell
[43,44];
b) HEDICINs anti-plasmodial activity could otherwise be
related to the cinnamoyl moiety, as it has been shown that
cinnamic acid derivatives inhibit the growth of intra-
erythrocytic P. falciparum in culture by inhibiting mono-
carboxylate (e.g. lactate) transport and translocation of
other nutrients, as carbohydrates and amino acids required
for parasite nourishment [45];
c) finally, HEDICINs anti-plasmodial activity could as well be
due to inhibition of the new permeation pathways;
meaning that HEDICINs could eventually block the new
permeation pathways created in infected RBC (see above,
I.c), hampering the uptake of molecules essential to the
parasite; it is interesting to notice that amongst our most
active molecules as antiplasmodials are those bearing
a nitro-aryl moiety, also present in potent new permeation
pathways inhibitors such as 5-nitro-2-(phenylalkylamino)
benzoic acid derivatives [31].
surprisingly, the
b-carbon of the a,b-unsatured carbonyl moiety
was the preferred site of nucleophilic attack (highlighted in bold,
Table 2) for almost all HECINs 9 (Table 2). The only exceptions were
for nitro derivatives 9iek. This result likely arises from the high
electron-withdrawing character of the nitro substituent, favoring
electron delocalization towards the aryl instead of the carbonyl
group. Also, we observed that compounds 9iej were among those
with higher activation of the double bond (Fukui index ratios of
3.29 and 2.25 for 9i and 9j, respectively) and also were among the
most potent FP2 inhibitors in the HECIN series (Table 2). This is
consistent with data for 9b, which had the third highest fþ(C2 or
C1)/fþ(C1 or C2) ratio (2.35) and also the greatest inhibition of FP2
among HECINs (Table 1).
These data suggest that there is a threshold fþ(C2)/fþ(C1) or
fþ(C1)/fþ(C2) ratio equal to 2 for HECINs, above which FP2 inhi-
bition should be observed; 9a, 9d and 9k apparently escape this
rule. Regarding compound 9a, this fact can be ascribed to the lack
of an adequate aryl substituent able to stabilize this ligand within
the enzyme’s ligand cavity, as previously shown by MD calcula-
tions (Fig. S3). Concerning molecules 9d and 9k, data suggest that
bulky aryl substituents (9d, p-OMe and 9k, p-NO2) in the para
position are unfavorable for activity possibly by establishing steric
clashes with S2 subsite residues that eventually will compel the
groups to leave the cavity. Of note, compound 9j (m-NO2)
accommodates into S2 cavity and remains within, as seen earlier
by MD calculations (Fig. S3). This suggests that the aryl substituent
could be slightly bulky in depth but not in length, which matches
the limitation of S2 cavity as already proposed by a previous
study [37].
3. Concluding remarks
In summary, we have found compounds able to inhibit hemo-
zoin formation and/or FP2 activity, but data obtained do not allow
establishing a correlation between these inhibitory capacities and
compound’s anti-plasmodial activity. In other words, results
available do not allow establishing why HEDICINs 8 are active
against blood-stage Plasmodium falciparum while HECINs 9 are
completely devoid of such activity; likely explanations are that
either HECINs are not efficiently uptaken by intra-erythrocytic
parasites, and/or that HEDICINs exert their anti-plasmodial activi-
ties through a mechanism other than inhibition of hemozoin
growth or of FP2 activity.
The combined experimental and computational work under-
taken, involving more than 20 novel compounds, led to the
following observations.
1. Both families of compounds studied (HEDICINs 8 and HECINs 9)
do not properly fit the narrow catalytic site of FP3.
2. HECINs 9 are better FP2 inhibitors than are HEDICINs 8, likely
because the former are substantially smaller and have their
vinyl bonds in closer vicinity to the FP2 catalytic Cys.
3. Among HECINs (9), the best FP2 inhibitors are those in which
(a) the vinyl bond is activated, i.e., where the ratios between
Fukui indices calculated for the two vinylic carbons are greater
than 2, and (b) the aryl ring has a suitable substituent for the
stabilization of the ligand-enzyme complex through non-
covalent interactions.
4. Most HEDICINs 8 are able to block hemozoin formation in vitro,
whereas HECINs 9 are not, which suggests that the spacer
between the 4-aminoquinoline and the cinnamoyl moieties has
a key role in mediating this activity.
I. A higher uptake of HEDICINs 8, as compared to HECINs 9,
could arise from:
a) a more efficient transport by passive diffusion, as HEDICINs
are more lipophilic than HECINs; globally, HEDICINs and
HECINs have similar acidebase properties (average esti-
mated pKa w 4 for deprotonation of the quinolinic
nitrogen), but differ by almost two units in their estimated
logP values (4.2 < logP < 5.1 for HECINs; 5.8 < log P < 7.9
for HEDICINs; cf. Table 1) [33], with the most lipophilic, 8c,
being also the most active anti-plasmodial; amino acid and
dipeptide esters are known to cross cell membranes by
passive diffusion [38], so it is possible that the presence of
the dipeptide spacer in HEDICINs augments cell perme-
ation of these compounds by such process;
5. Unlike HECINs (9), most HEDICINs (8) are active in vitro against
blood-stage P. falciparum parasites, suggesting that the dipep-
tide spacer also has a key role in mediating this activity.
Overall, we can conclude that it is possible to create cinnamic
acid/4-aminoquinoline conjugates with promising antimalarial and
FP-inhibitory activities, at levels comparable to those of recently
reported chalcone-based hybrids [46,47]. Such conjugates consti-
tute promising leads for future development of novel anti-
plasmodials targeted at blood-stage malaria parasites.
b) recognition of the dipeptide motif by specific transporters
from the P. falciparum ABC transporter super-family, many
of which have been taken as putative drug transporting

894
B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
4. Experimental section
(d, J ¼ 16 Hz,1H), 4.69 (m,1H), 4.57 (m,1H), 2.72 (m, 2H), 2.32 (s, 3H),
2.10 (m, 2H), 1.61 (m, 3H), 0.90 (d, J ¼ 16 Hz, J ¼ 6.4 Hz, 6H); dC
(100 MHz, DMSO-d6) 172.7, 171.7, 165.0, 152.2, 149.0, 141.4, 141.0,
139.2,134.9,134.1,132.0,129.5,128.3,128.2,127.9,127.4,126.3,125.8,
124.5, 120.8, 119.3, 112.4, 53.4, 51.6, 40.8, 32.9, 31.4, 24.8, 22.9, 21.6,
20.9; ESI-IT MS: m/z (M þ Hþ) 597.60 (C35H37ClN4O3 requires
596.26); HPLC-DAD: tr ¼ 18.3 min (% area ¼ 98%).
4.1. Chemistry
All solvents and common chemicals were from SigmaeAldrich
(Spain), whereas Boc-protected amino acids were from Nova-
Biochem (VWR International, Portugal) and the coupling agentTBTU
was from Bachem (Switzerland). NMR spectra were acquired on
a Bruker Avance III 400 spectrometer from solutions in either
deuterated chloroform or deuterated dimethylsulfoxide (DMSO-d6)
containing tetramethylsilane as internal reference. MS spectra were
obtained on a Thermo Finnigan LCQ Deca XP Max LC/MSn instru-
ment operating with electrospray ionization and ion-trap (ESI-IT)
quadrupole detection. HPLC analyses were run for target
compounds (8 and 9) using the following conditions: 30e100% of B
in A (A ¼ H2O with 0.05% of trifluoroacetic acid; B ¼ acetonitrile) in
22 min with a flow rate of 1 mL/min on a MerckeHitachi Lachrom
Elite instrument equipped with a diode-array detector (DAD) and
thermostated (Peltier effect) autosampler, using a Purospher STAR
4.5. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-2-((E)-3-(4-iso-propylphenyl)acrylamido)-4-
methylpentanamide (8c)
White solid (23 mg, 47%); mp 238e240 ꢀC; RF (DCM/Me2CO 6:1)
0.44; dH (400 MHz, DMSO-d6) 10.34 (s, 1H), 8.83 (d. J ¼ 5.2 Hz, 1H),
8.56 (d, J ¼ 6.8 Hz,1H), 8.31 (m, 2H), 8.05 (m, 2H), 7.67 (d, J ¼ 8.8 Hz,
1H), 7.42 (m, 4H), 7.28 (m, 6H), 6.73 (d, J ¼ 16 Hz, 1H), 4.69 (m, 2H),
2.81 (m, 3H), 2.09 (m, 2H), 1.61 (m, 3H), 1.20 (d, J ¼ 6.8 Hz, 6H), 0.90
(dd, J ¼ 16.6 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.7, 171.7,
165.0,152.1,150.0,149.0,141.4,141.0,138.9,134.1,132.4,128.3,128.2,
127.9, 127.5, 126.8, 126.3, 125.8, 124.5, 120.9, 119.36, 112.4, 53.4, 51.0,
40.8, 33.2, 33.0, 31.4, 24.8, 23.6, 22.9, 21.6; ESI-IT MS: m/z (M þ Hþ)
625.36 (C37H41ClN4O3 requires 624.29); HPLC-DAD: tr ¼ 14.5 min
(% area ¼ 93%).
RP-18e column (150 ꢄ 4.0 mm; particle size, 5
mM).
4.2. General procedure for the synthesis of compounds 8aed, 8fel
The relevant cinnamic acid (1.1 eq), PyBOP (1.1 eq), DIEA (2 eq.)
and DCM (2 mL) were mixed in a round bottom flask and put under
stirring for 20 min. Then, a solution of 7 in DCM (2 mL) was added
and the reaction allowed to proceed for three days. Precipitation
was observed and the precipitate was collected by suction filtration,
washed with ice-cold DCM, dried and identified as the pure target
compound, except in the case of 8l (p-nitrocinnamic acid deriva-
tive), which was further submitted to liquid chromatography on
silica, using DCM/Me2CO 6:1 (v/v) as eluent. The synthesis details,
analytical and spectroscopic data of intermediate compounds 3 to 7
and compound 10a are given in detail in SI.
In the particular case of compound 8e, derived from p-amino-
cinnamic acid, the N-Boc-protected precursor of this cinnamic acid
derivative was coupled to 7 as above described, and the resulting
Boc-protected compound (8e0) was treated with neat TFA as
described for the synthesis of 5 and 7 (see SI), to give the final
unprotected compound, 8e. Analytical and spectroscopic data for
8ael and 8e0 are given below.
4.6. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-2-((E)-3-(4-methoxyphenyl)acrylamido)-4-
methylpentanamide (8d)
White solid (38 mg, 47%); mp 223e241 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.34 (s, 1H), 8.83 (d. J ¼ 5.2 Hz, 1H),
8.54 (d, J ¼ 7.6 Hz, 1H), 8.32 (d, J ¼ 8.8 Hz, 1H), 8.24 (d, J ¼ 8 Hz, 1H),
8.05 (m, 2H), 7.66 (d, J ¼ 9.2 Hz, J ¼ 2 Hz,1H), 7.50 (d, J ¼ 8.8 Hz, 2H),
7.38 (d, J ¼ 16 Hz, 1H), 7.25 (m, 5H), 6.98 (d, J ¼ 8.8 Hz, 2H), 6.63
(d, J ¼ 15.6 Hz, 1H), 4.70 (m, 1H), 4.56 (m, 1H), 3.79 (s, 3H), 2.72
(m, 2H), 2.10 (m, 2H), 1.61 (m, 3H), 1.20 (d, J ¼ 6.8 Hz, 6H), 0.90 (dd,
J ¼ 16.8 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.8,171.7,165.2,
160.3, 152.1, 149.0, 141.4, 141.0, 138.7, 134.1, 129.0, 128.7, 128.2, 127.9,
127.3,126.3,125.8,124.5,119.4,114.3,112.4, 55.2, 53.4, 51.0, 40.8, 33.0,
31.4, 24.2, 22.9, 21.6; ESI-IT MS: m/z (M þ Hþ) 613.73 (C35H37ClN4O4
requires 612.25); HPLC-DAD: tr ¼ 12.3 min (% area¼ 91%).
4.7. tert-Butyl (4-((E)-3-(((R)-1-(((R)-1-((7-chloroquinolin-4-yl)
amino)-1-oxo-4-phenylbutan-2-yl)amino)-4-methyl-1-oxopentan-
2-yl)amino)-3-oxoprop-1-en-1-yl)phenyl)carbamate (8e0)
4.3. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-4-methyl-2-((E)-3-acrylamido)pentanamide (8a)
White solid (29.5 mg, 66%); mp 241e245 ꢀC; RF (DCM/Me2CO
6:1) 0.36; dH (400 MHz, DMSO-d6) 10.36 (s, 1H), 8.84 (d, J ¼ 5.2 Hz,
1H), 8.59 (d, J ¼ 7.6 Hz, 1H), 8.34 (m, 2H), 8.05 (m, 2H), 7.67
(dd, J ¼ 9.28 Hz, J ¼ 2 Hz,1H), 7.56 (b, 2H), 7.41 (m, 4H), 7.24 (m, 5H);
6.79 (d, J ¼ 15.6, 1H); 4.69 (m, 1H), 4.58 (m, 1H), 2.71 (m, 2H), 2.09
(m, 2H), 1.66 (m, 1H), 1.54 (m, 2H), 0.90 (d, J ¼ 16 Hz, J ¼ 6.4 Hz, 6H);
dC (100 MHz, DMSO-d6); 172.7, 171.8, 164.9, 152.2, 149.1, 141.5, 141.1,
139.0, 134.8, 134.2, 129.5, 128.9, 128.3, 128.3, 128.0, 127.5, 126.4,
125.9, 124.5, 121.9, 119.4, 112.4, 53.5, 51.1, 40.9, 33.0, 31.5, 24.2, 23.0,
21.6; ESI-IT MS: m/z (M þ Hþ) 583.47 (C34H35ClN4O3 requires
582.24); HPLC-DAD: tr ¼ 12.4 min (% area ¼ 96%).
Yellow solid (10 mg,13%); mp (dec.) 238 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.33 (s, 1H), 9.55 (s, 1H), 8.83
(d, J ¼ 4.8 Hz,1H), 8.53 (d, J ¼ 7.2 Hz,1H), 8.32 (d, J ¼ 9.2 Hz,1H), 8.26
(d, J ¼ 8 Hz, 1H), 8.05 (m, 2H), 7.66 (dd, J ¼ 9.2 Hz, J ¼ 2 Hz, 1H), 7.47
(m, 4H), 7.34 (d, J ¼ 16 Hz,1H), 7.23 (m, 5H), 6.65 (d, J ¼ 15.6 Hz,1H),
4.69 (m, 1H), 4.55 (m, 1H), 2.72 (m, 2H), 2.08 (m, 2H), 1.61 (m, 3H),
1.48 (s, 9H), 0.90 (dd, J ¼ 17.2 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-
d6) 172.8, 171.8, 165.2, 152.5, 152.2, 149.0, 141.4, 141.0, 140.8, 138.7,
134.1, 128.5, 128.3, 128.2, 128.1, 127.1, 126.4, 125.9, 124.5, 119.7, 119.4,
118.0,112.0, 79.3, 53.4, 51.1, 40.7, 33.0, 31.4, 28.0, 24.2, 22.9, 21.6; ESI-
IT MS: m/z (M þ Hþ) 698.47 (C39H44ClN5O5 requires 697.30); HPLC-
DAD: tr ¼ 16.5 min (% area ¼ 97%).
4.4. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-4-methyl-2-((E)-3-(4-tolyl)acrylamido)
pentanamide (8b)
4.8. (R)-2-((E)-3-(4-Aminophenyl)acrylamido)-N-((R)-1-((7-
chloroquinolin-4-yl)amino)-1-oxo-4-phenylbutan-2-yl)-4-
methylpentanamide (8e)
White solid (30.1 mg, 76%); mp 230e235 ꢀC; RF (DCM/Me2CO
6:1) 0.24; dH (400 MHz, DMSO-d6) 10.35 (s, 1H), 8.83 (d. J ¼ 5.2 Hz,
1H), 8.55 (d, J ¼ 7.6 Hz, 1H), 8.32 (m, 2H), 8.05 (m, 2H), 7.66
(dd, J ¼ 9 Hz, J ¼ 2 Hz, 1H), 7.42 (m, 4H), 7.24 (m, 6H), 6.75
Yellow solid (12 mg, 78%); mp 240e242 ꢀC; RF (DCM/Me2CO 6:1)
0.11; dH (400 MHz, DMSO-d6) 10.32 (s, 1H), 8.83 (d, J ¼ 5.2 Hz, 1H),
8.49 (d, J ¼ 7.6 Hz, 1H), 8.32 (d, J ¼ 9.2 Hz, 1H), 8.08 (d, J ¼ 8 Hz, 1H),

B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
895
8.05 (m, 2H), 7.66 (dd, J ¼ 9 Hz, J ¼ 2 Hz, 1H), 7.23 (m, 8H), 6.56
(d, J ¼ 8.4 Hz, 2H), 6.43 (d, J ¼ 16z,1H), 5.57 (S, 2H), 4.69 (m,1H), 4.52
(m,1H), 2.71 (m, 2H), 2.08 (m, 2H),1.62 (m, 3H), 0.90 (dd, J ¼ 18.4 Hz,
J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.9, 171.8, 165.9, 152.1,
150.5,149.0,141.4,141.1,139.9,134.1,129. 0,128.3,128.2,127.9,126.4,
125.8,124.5,122.0,119.4,115.4,113.6,112.4, 53.4, 51.2, 40.7, 33.0, 31.4,
24.2, 22.9, 21.6; ESI-IT MS: m/z (M þ Hþ) 598.30 (C34H36ClN5O3
requires 597.25); HPLC-DAD: tr ¼ 16.5 min (% area ¼ 97%).
(d, J ¼ 16 Hz, 1H), 4.69 (m, 1H), 4.57 (m, 1H), 2.71 (m, 2H), 2.09 (m,
2H), 1.63 (m, 3H), 0.90 (dd, J ¼ 16 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz,
DMSO-d6) 172.6, 171.7, 164.6, 152.2, 149.0, 141.4, 141.0, 137.7, 134.1,
131.8,129.4,128.3,128.2,127.9,126.3,125.8,124.4,122.8,122.6,119.4,
112.4, 53.4, 51.1, 40.8, 33.0, 31.4, 24.2, 22.9, 21.6; ESI-IT MS: m/z
(M þ Hþ) 663.40 (C34H34BrClN4O3 requires 660.15); HPLC-DAD:
tr ¼ 18.6 min (% area ¼ 98%).
4.13. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-4-methyl-2-((E)-3-(2-nitrophenyl)acrylamido)
pentanamide (8j)
4.9. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-2-((E)-3-(3-fluorophenyl)acrylamido)-4-
methylpentanamide (8f)
White solid (25 mg, 40%); mp 260e265 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.38 (s, 1H), 8.83 (d, J ¼ 5.2 Hz, 1H),
8.62 (d, J ¼ 6 Hz, 1H), 8.49 (d, J ¼ 9.2 Hz, 1H), 8.32 (d, J ¼ 7.2 Hz, 1H),
8.05 (m, 3H), 7.73 (m, 5H), 7.25 (m, 5H), 6.79 (d, J ¼ 15.6 Hz, 1H),
4.71 (m, 1H), 4.60 (m, 1H), 2.73 (m, 2H), 2.10 (m, 2H), 1.62 (m, 3H),
0.90 (dd, J ¼ 14.4 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.5,
171.8, 163.9, 152.2, 149.0, 148.3, 141.4, 141.0, 134.1, 133.8, 133.7, 130.2,
129.8, 128.5, 128.3, 128.2, 127.9, 126.6, 126.3, 125.8, 124.6, 124.5,
119.4, 112.4, 53.5, 51.0, 40.9, 33.0, 31.5, 24.2, 22.9, 21.6; ESI-IT MS:
m/z (M þ Hþ) 628.47 (C34H34ClN5O5 requires 627.22); HPLC-DAD:
tr ¼ 17.8 min (% area ¼ 98%).
White solid (28 mg, 56%); mp 249e254 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.36 (s, 1H), 8.83 (d, J ¼ 4.8 Hz, 1H),
8.58 (d, J ¼ 7.2 Hz, 1H), 8.34 (m, 2H), 8.05 (m, 2H), 7.67 (d, J ¼ 9.2 Hz,
J ¼ 2 Hz 1H), 7.43 (m, 4H), 7.24 (m, 6H), 6.83 (d, J ¼ 16 Hz, 1H), 4.70
(m, 1H), 4.59 (m, 1H), 2.72 (m, 2H), 2.09 (m, 2H), 1.61 (m, 3H), 0.90
(dd, J ¼ 15.4 Hz, J ¼ 6.8 Hz, 6H); dC (100 MHz, DMSO-d6) 172.6, 171.7,
164.5, 163.6, 161.1, 152.2, 149.0, 141.4, 141.0, 137.6, 137.4, 134.1, 130.9,
128.3,128.2,127.9,126.3,125.8,124.5,123.5,119.4,116.1,113.8,112.4,
53.5, 51.1, 40.8, 33.0, 31.5, 24.2, 22.9, 21.6; ESI-IT MS: m/z (M þ Hþ)
601.40 (C34H34ClFN4O3 requires 600.23); HPLC-DAD: tr ¼ 11.2 min
(% area ¼ 100%).
4.14. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-4-methyl-2-((E)-3-(3-nitrophenyl)acrylamido)
pentanamide (8k)
4.10. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-2-((E)-3-(4-fluoro-phenyl)acrylamido)-4-
methylpentanamide (8g)
White solid (26 mg, 50%); mp 202e221 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.36 (s, 1H), 8.83 (d, J ¼ 4.8 Hz, 1H),
8.60 (d, J ¼ 7.2 Hz,1H), 8.40 (m, 2H), 8.33 (d, J ¼ 8.8 Hz,1H), 8.21 (dd,
J ¼ 8.2 Hz, J ¼ 1.2 Hz, 1H), 8.05 (m, 3H), 7.68 (m, 2H), 7.56
(d, J ¼ 15.6 Hz, 1H), 7.19 (m, 5H), 6.99 (d, J ¼ 16 Hz, 1H), 4.71 (m, 1H),
4.60 (m, 1H), 2.71 (m, 2H), 2.09 (m, 2H), 1.62 (m, 3H), 0.90 (dd,
J ¼ 14.4 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.5, 171.7,
164.3, 152.2, 149.0, 148.2, 141.4, 141.0, 136.7, 136.6, 134.1, 133.6,
130.4, 128.3, 128.2, 127.9, 126.3, 125.9, 124.8, 124.5, 123.7, 121.4,
119.4, 112.4, 53.5, 51.1, 40.8, 33.0, 31.5, 24.2, 23.0, 21.6; ESI-IT MS:
m/z (M þ Hþ) 628.53 (C34H34ClN5O5 requires 627.22); HPLC-DAD:
tr ¼ 11.1 min (% area ¼ 100%).
White solid (39 mg, 78%); mp 246e249 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.35 (s, 1H), 8.83 (d, J ¼ 5.2 Hz, 1H),
8.56 (d, J ¼ 7.2 Hz, 1H), 8.32 (d, J ¼ 9.2 Hz, 2H), 8.05 (m, 2H), 7.63
(m, 3H), 7.44 (d, J ¼ 15.6 Hz,1H), 7.23 (m, 7H), 6.73 (d, J ¼ 15.6 Hz,1H),
4.69(m,1H), 4.58(m,1H), 2.72(m, 2H), 2.09(m, 2H),1.61(m, 3H), 0.90
(dd, J ¼ 16 Hz, J ¼ 6.4 Hz, 6H); dC (100 MHz, DMSO-d6) 172.7, 171.7,
164.8, 152.2, 149.0, 141.4, 141.0, 137.8, 134.1, 131.4, 129.6, 128.3, 128.2,
127.9, 126.3, 125.8, 124.5, 121.8, 119.4, 116.0, 115.7, 112.4, 53.5, 51.0,
40.8, 33.0, 31.5, 24.2, 22.9, 21.6; ESI-IT MS: m/z (M þ Hþ) 601.47
(C34H34ClFN4O3 requires 600.23); HPLC-DAD: tr
(% area ¼ 85%).
¼
12.5 min
4.11. (R)-2-((E)-3-(4-Chlorophenyl)acrylamido)-N-((R)-1-((7-
chloroquinolin-4-yl)amino)-1-oxo-4-phenylbutan-2-yl)-4-
methylpentanamide (8h)
4.15. (R)-N-((R)-1-((7-chloroquinolin-4-yl)amino)-1-oxo-4-
phenylbutan-2-yl)-4-methyl-2-((E)-3-(4-nitrophenyl)acrylamido)
pentanamide (8l)
White solid (25 mg, 59%); mp 249e260 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.35 (s, 1H), 8.83 (d, J ¼ 4.8 Hz, 1H),
8.57 (d, J ¼ 7.6 Hz, 1H), 8.34 (M, 2H), 8.05 (m, 2H), 7.66 (dd, J ¼ 9 Hz,
J ¼ 2, 1H), 7.58 (m, 2H), 7.48 (m, 2H), 7.42 (d, J ¼ 16 Hz, 1H), 7.22
(m, 5H), 6.79 (d, J ¼ 15.6 Hz, 1H), 4.70 (m, 1H), 4.58 (m, 1H), 2.71
(m, 2H), 2.09 (m, 2H),1.61 (m, 3H), 0.90 (dd, J ¼ 16 Hz, J ¼ 6.4 Hz, 6H);
dC (100 MHz, DMSO-d6) 172.6, 171.7, 164.7, 152.2, 149.0, 141.4, 141.0,
137.6,134.1,133.8,133.7,129.1,128.9,128.3,128.2,127.9,126.3,125.8,
124.5, 122.7, 119.4, 112.4, 53.5, 51.1, 40.8, 33.0, 31.5, 24.2, 22.9, 21.6;
ESI-IT MS: m/z (M þ Hþ) 617.23 (C34H34Cl2N4O3 requires 616.20);
HPLC-DAD: tr ¼ 18.5 min (% area ¼ 100%).
Yellowish solid (11 mg, 17%); mp 252e257 ꢀC; RF (DCM/Me2CO
6:1) 0.35; dH (400 MHz, DMSO-d6) 10.34 (s, 1H), 8.82 (d, J ¼ 5.2 Hz,
1H), 8.54 (m, 2H), 8.28 (m, 2H), 8.04 (m, 3H), 7.81 (m, 2H), 7.66 (dd,
J ¼ 9.2 Hz, J ¼ 2 Hz, 1H), 7.53 (d, J ¼ 15.6, 1H), 7.22 (m, 5H), 6.96
(d, J ¼ 16 Hz, 1H), 4.69 (m, 1H), 4.58 (m, 1H), 2.72 (m, 2H), 2.08
(m, 2H), 1.60 (m, 3H), 0.90 (dd, J ¼ 15.6 Hz, J ¼ 6.4 Hz, 6H); dC
(100 MHz, DMSO-d6) 172.6, 171.8, 164.3, 152.3, 149.1, 147.6, 141.5,
141.4, 141.1, 136.8, 134.3, 128.6, 128.4, 128.3, 128.0, 126.5, 126.0,
124.6, 124.2, 122.8, 119.5, 112.6, 53.4, 51.2, 40.7, 33.0, 31.4, 24.2, 22.9,
21.6; ESI-IT MS: m/z (M þ Hþ) 628.73 (C34H34ClN5O5 requires
627.22); HPLC-DAD: tr ¼ 18.0 min (% area ¼ 92%).
4.12. (R)-2-((E)-3-(4-Bromophenyl)acrylamido)-N-((R)-1-((7-
chloroquinolin-4-yl)amino)-1-oxo-4-phenylbutan-2-yl)-4-
methylpentanamide (8i)
4.16. General procedure for the synthesis of compounds 9aek
The relevant cinnamic acid (1.1 eq), PyBOP (1.1 eq), DIEA (2 eq.)
and DCM (2 mL) were mixed in a round bottom flask and put under
stirring for 20 min. Then, a solution of 3 (0.250 g, 1.40 mmol) in
DMF (2 mL) was added and the reaction allowed to proceed for one
day at room temperature. Following, the reaction mixture was
diluted with 14 mL of DCM and sequentially washed with 1% aq.
White solid (31 mg, 70%); mp 240e246 ꢀC; RF (DCM/Me2CO 6:1)
0.35; dH (400 MHz, DMSO-d6) 10.35 (s, 1H), 8.83 (d, J ¼ 4.8 Hz, 1H),
8.57 (d, J ¼ 7.2 Hz, 1H), 8.34 (m, 2H), 8.05 (m, 2H), 7.64 (m, 3H), 7.51
(d, J ¼ 8.4 Hz, 2H), 7.40 (d, J ¼ 15.6 Hz, 1H), 7.24 (m, 5H), 6.80

896
B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
HCl (3 ꢄ 18 mL) and 5% aq. Na2CO3 (3 ꢄ 18 mL). Finally, the organic
layer was dried with anhydrous Na2SO4, filtered, and evaporated to
dryness In all cases, the crude product had to be purified by column
liquid chromatography on silica, using DCM/Me2CO 6:1 v/v as
eluent. Target compounds were isolated as solids, with analytical
and spectral data given below.
4.22. (E)-N-(7-chloroquinolin-4-yl)-3-(4-fluorophenyl) acrylamide (9f)
White solid (19 mg, 4%); mp 210e212 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.42 (s, 1H), 8.85 (d, J ¼ 4.8 Hz, 1H),
8.48 (d, J ¼ 9.2 Hz, 1H), 8.32 (d, J ¼ 4.8 Hz, 1H), 8.06 (d, J ¼ 2 Hz, 1H),
7.73 (m, 4H), 7.33 (m, 2H), 7.21(d, J ¼ 15.6 Hz, 1H); dC (100 MHz,
DMSO-d6) 164.7, 152.2, 149.1, 141.7, 140.7, 134.1, 131.2, 130.1, 128.0,
126.3, 124.4, 121.4, 119.0, 116.1, 115.9, 111.5; ESI-IT MS: m/z (M þ Hþ)
327.11 (C18H12ClFN2O requires 326.06); HPLC-DAD: tr ¼ 8.50 min
(% area ¼ 89%).
4.17. N-(7-chloroquinolin-4-yl)cinnamamide (9a)
White solid (62 mg, 18%); mp 170e176 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.42 (s, 1H), 8.85 (d, J ¼ 5.2 Hz, 1H),
8.48 (d, J ¼ 9.2 Hz, 1H), 8.33 (d, J ¼ 5.2 Hz, 1H), 8.05 (d, J ¼ 2.4 Hz,
1H), 7.69 (m, 4H), 7.47 (m, 3H), 7.27 (d, J ¼ 15.6 Hz, 1H); dC
(100 MHz, DMSO-d6) 164.8, 152.2, 149.1, 141.9, 141.7, 134.5, 134.1,
130.1, 129.0, 128.0, 127.9, 126.3, 124.4, 121.5, 119.0, 111.5; ESI-IT MS:
m/z (M þ Hþ) 309.33 (C18H13ClN2O requires 308.07); HPLC-DAD:
tr ¼ 8.10 min (% area ¼ 85%).
4.23. (E)-3-(4-Chlorophenyl)-N-(7-chloroquinolin-4-yl)acrylamide (9g)
White solid (44 mg, 21%); mp 240e250 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.44 (s, 1H), 8.85 (d, J ¼ 5.2 Hz, 1H),
8.47 (d, J ¼ 9.2 Hz, 1H), 8.32 (d, J ¼ 4.8 Hz, 1H), 8.06 (d, J ¼ 2 Hz, 1H),
7.72 (m, 4H), 7.55 (d, J ¼ 8.4 Hz, 2H), 7.26(d, J ¼ 16 Hz, 1H); dC
(100 MHz, DMSO-d6) 164.6, 152.2, 149.1, 141.7, 140.5, 134.5, 134.1,
133.5, 129.5, 129.1, 128.0, 126.3, 124.3, 122.3, 119.0, 111.6; ESI-IT MS:
m/z (M þ Hþ) 343.33 (C18H12Cl2N2O requires 342.03); HPLC-DAD:
tr ¼ 8.30 min (% area ¼ 96%).
4.18. (E)-N-(7-chloroquinolin-4-yl)-3-(4-tolyl)acrylamide (9b)
Beige solid (11 mg, 2%); mp 209e218 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.38 (s, 1H), 8.85 (d, J ¼ 5.2 Hz, 1H),
8.49 (d, J ¼ 9.2 Hz, 1H), 8.33 (d, J ¼ 5.2 Hz, 1H), 8.05 (d, J ¼ 2 Hz, 1H),
7.70 (m, 2H), 7.58 (d, J ¼ 8.4 Hz, 2H), 7.29 (d, J ¼ 8 Hz, 2H), 7.21
(d, J ¼ 15.6 Hz,1H), 2.35 (s, 3H); dC (100 MHz, DMSO-d6) 164.9,152.2,
149.1, 141.9, 141.8, 140.0, 134.1, 131.8, 129.6, 128.0, 127.9, 126.3, 124.4,
120.4,119.0,111.5, 20.9;ESI-ITMS:m/z (Mþ Hþ) 323.40 (C19H15ClN2O
requires 322.09); HPLC-DAD: tr ¼ 9.30 min (% area ¼ 85%).
4.24. (E)-3-(4-Bromophenyl)-N-(7-chloroquinolin-4-yl)acrylamide (9h)
Green solid (38 mg, 7%); mp (dec.) 190 ꢀC; RF (DCM/Me2CO 3:1)
0.56;dH (400MHz, DMSO-d6)10.44(s,1H), 8.85(d,J¼ 5.2Hz,1H),8.47
(d, J ¼ 8.8 Hz,1H), 8.32 (d, J ¼ 4.8 Hz,1H), 8.08 (d, J ¼ 2.4 Hz,1H), 7.70
(m, 6H), 7.27 (d, J ¼ 16 Hz, 1H); dC (100 MHz, DMSO-d6) 164.6, 152.2,
149.1, 141.7, 140.5, 134.1, 133.8, 132.0, 129.8, 128.0, 126.3, 124.3, 123.3,
122.4, 118.9, 111.6; ESI-IT MS: m/z (M þ Hþ) 387.27 (C18H12BrClN2O
requires 385.98); HPLC-DAD: tr ¼ 8.60 min (% area ¼ 97%).
4.19. (E)-N-(7-chloroquinolin-4-yl)-3-(4-isopropylphenyl)
acrylamide (9c)
Beige solid (17 mg, 3%); mp 187e204 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.40 (s, 1H), 8.84 (d, J ¼ 5.2 Hz, 1H),
8.49 (d, J ¼ 9.2 Hz, 1H), 8.33 (d, J ¼ 5.2 Hz, 1H), 8.05 (d, J ¼ 2.4 Hz,
1H), 7.70 (m, 2H), 7.61 (d, J ¼ 8.4 Hz, 2H), 7.35 (d, J ¼ 8 Hz, 2H), 7.23
(d, J ¼ 15.6 Hz, 1H), 2.93 (s, 1H), 1.22 (d, J ¼ 6.8 Hz, 6H); dC (100 MHz,
DMSO-d6) 164.9, 152.2, 150.8, 149.1, 141.9, 141.8, 134.1, 132.1, 128.0,
127.0, 126.7, 126.3, 124.4, 120.8, 118.9, 111.5, 33.3, 23.5; ESI-IT MS:
m/z (M þ Hþ) 351.27 (C21H19ClN2O requires 350.12); HPLC-DAD:
tr ¼ 11.4 min (% area ¼ 85%).
4.25. (E)-N-(7-chloroquinolin-4-yl)-3-(2-nitrophenyl)acrylamide (9i)
Yellow solid (36 mg, 7%); mp 168e200 ꢀC; RF (DCM/Me2CO 3:1)
0.56;dH (400MHz,DMSO-d6)10.56(s,1H),8.87(d, J¼ 5.2Hz,1H), 8.47
(d, J ¼ 8.8 Hz,1H), 8.32 (d, J ¼ 4.8 Hz,1H), 8.12 (d, J ¼ 8 Hz,1H), 8.07 (d,
J¼ 2 Hz,1H), 8.01(d, J ¼ 15.6Hz,1H), 7.87 (m, 2H), 7.72(m, 3H), 7.25(d,
J ¼ 15.6 Hz, 1H); dC (100 MHz, DMSO-d6) 163.9, 152.2, 149.0, 148.2,
141.4, 137.0, 134.1, 133.9, 130.7, 129.8, 128.8, 128.0, 126.4, 126.1, 124.8,
124.3, 119.0, 111.7; ESI-IT MS: m/z (M þ Hþ) 354.33 (C18H12ClN3O3
requires 353.06); HPLC-DAD: tr ¼ 7.90 min (% area ¼ 97%).
4.20. (E)-N-(7-chloroquinolin-4-yl)-3-(4-methoxyphenyl)
acrylamide (9d)
4.26. (E)-N-(7-chloroquinolin-4-yl)-3-(3-nitrophenyl)acrylamide (9j)
Beige solid (83 mg, 18%); mp 240e242 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.33 (s, 1H), 8.83 (d, J ¼ 4.8 Hz, 1H),
8.49 (d, J ¼ 8.8 Hz, 1H), 8.33 (d, J ¼ 9.2 Hz, 1H), 8.05 (d, J ¼ 2 Hz, 1H),
7.67 (m, 4H), 7.12 (d, J ¼ 15.6 Hz, 1H), 7.04 (m, 2H), 3.82 (s, 3H); dC
(100 MHz, DMSO-d6) 165.1, 160.8, 152.2, 149.1, 141.9, 141.7, 134.0,
129.6, 127.9, 127.1, 126.2, 124.4, 118.9, 118.9, 114.5, 111.3, 55.3; ESI-IT
MS: m/z (M þ Hþ) 339.33 (C19H15ClN2O2 requires 338.08); HPLC-
DAD: tr ¼ 6.80 min (% area ¼ 97%).
White solid (11 mg, 2%); mp (dec.) 240e247 ꢀC; RF (DCM/Me2CO
3:1) 0.56; dH (400 MHz, DMSO-d6) 10.49 (s, 1H), 8.86 (d, J ¼ 4.8 Hz,
1H), 8.53 (d, J ¼ 2 Hz, 2H), 8.34 (d, J ¼ 5.2 Hz, 1H), 8.27 (dd,
J ¼ 8.2 Hz, J ¼ 1.2 Hz, 1H), 8.11(d, J ¼ 8 Hz, 1H), 8.07 (d, J ¼ 2 Hz, 1H),
7.84 (d, J ¼ 16 Hz, 1H), 7.76 (m, 2H), 7.46 (d, J ¼ 16 Hz, 1H); dC
(100 MHz, DMSO-d6) 164.2, 152.2, 149.1, 148.2, 141.5, 139.4, 136.3,
134.5, 134.2, 130.6, 128.0, 126.4, 124.4, 124.3, 121.5, 118.9, 111.5; ESI-
IT MS: m/z (M þ Hþ) 354.53 (C18H12ClN3O3 requires 353.06); HPLC-
DAD: tr ¼ 7.20 min (% area ¼ 100%).
4.21. (E)-N-(7-chloroquinolin-4-yl)-3-(3-fluorophenyl)acrylamide (9e)
4.27. (E)-N-(7-chloroquinolin-4-yl)-3-(4-nitrophenyl)acrylamide
(9k)
Yellow solid (4 mg, 1%); mp 181e190 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.45 (s, 1H), 8.85 (d, J ¼ 5.2 Hz, 1H),
8.47 (d, J ¼ 8.8 Hz, 1H), 8.32 (d, J ¼ 5.2 Hz, 1H), 8.06 (dd, J ¼ 2 Hz,
1H), 7.71(m, 2H), 7.52 (m, 3H), 7.29 (m, 2H); dC (100 MHz, DMSO-d6)
164.5, 163.6, 152.2, 149.1, 141.7, 140.5, 137.1, 134.1, 131.0, 128.0, 126.4,
124.3, 124.0, 123.1, 119.0, 116.6, 114.4, 111.6; ESI-IT MS: m/z (M þ Hþ)
327.33 (C18H12ClFN2O requires 326.06); HPLC-DAD: tr ¼ 8.70 min
(% area ¼ 98%).
Beige solid (9 mg, 5%); mp desc. 205 ꢀC; RF (DCM/Me2CO 3:1)
0.56; dH (400 MHz, DMSO-d6) 10.58 (s, 1H), 8.87 (d, J ¼ 5.2 Hz, 1H),
8.48 (d, J ¼ 9.2 Hz, 1H), 8.33 (m, 3H), 8.07 (d, J ¼ 2.4 Hz, 1H), 7.94 (d,
J ¼ 8.8 Hz, 2H), 7.83 (d, J ¼ 16 Hz,1H), 7.74 (dd, J ¼ 9 Hz, J ¼ 2 Hz,1H),
7.44 (d, J ¼ 15.6 Hz, 1H); dC (100 MHz, DMSO-d6) 164.1, 152.2, 149.1,
147.8, 141.5, 141.0, 139.3, 134.2, 128.9, 128.0, 126.4, 125.8, 124.3,

B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
897
124.2, 119.0, 111.7; ESI-IT MS: m/z (M þ Hþ) 354.27 (C18H12ClN3O3
requires 353.06); HPLC-DAD: tr ¼ 7.20 min (% area ¼ 92%).
independent runs of the docking algorithm with each compound,
using the default genetic algorithm (GA) search parameters and the
GoldScore scoring function. The automated assignment of protein
and ligand atom types was checked to ensure a correct treatment of
the potential hydrogen bond donors and acceptors. The binding site
was defined as 15 Å radius from the catalytic amino acid Cys42 and
Cys51 for FP2 and FP3, respectively. Previously, the docking
protocol was tested to verify if GOLD was able to reproduce the
experimental conformation of the ligands bound to the enzymes.
4.28. In vitro assays
4.28.1. Inhibition of heme polymerization
The heme polymerization assay was performed as previously
described [28,29]. Briefly, different concentrations (0.1e1 mM) of
test compounds dissolved in DMSO were added in triplicate to
50
m
L hemin chloride dissolved in DMSO (5.2 mg/mL). Controls
-hematin formation
L, pH
contained equal volumes of water (or DMSO.
b
4.29.2. Molecular dynamics simulations
was initiated by the addition of acetate buffer 0.2 M (100
m
A topological file and a coordinate set was prepared for each
complex using the leap module of AMBER 10 with the ff03 and GAFF
[55] force field parameters for the protein and ligands, respectively.
Partial atomic charges were determined with the restrained elec-
trostatic potential (RESP) [56], after ab initio calculations using the
Gaussian 03 [57] software at the HF/6-31G(d) level, and then were
assigned to atoms using the Antechamber module. Each complex
was solvated by placing a periodic box of TIP3P water up to
a distance of 8 Å from the protein and the system was neutralized by
adding Naþ ions. After a careful equilibration of the water box, the
full system was gently heated through six 5 ps molecular dynamic
(MD) runs where backbone restraints were gradually reduced from
10 to 5 kcal/mol, while the temperature was raised from 0 to 300 K.
The heating was followed by a 100 ps run in NVT ensemble and four
100 ps runs in NpT ensemble where backbone restraints will be
gradually reduced from 5 to 1 kcal/mol. After minimization,
a subsequent 3 ns production run (NpT conditions) was performed.
4.4), plates were incubated at 37 ꢀC for 48 h and they were
centrifuged at 3000 rpm for 15 min (SIGMA 3-30K). After dis-
carding the supernatant, the pellet was washed four times with
DMSO (200 mL), and finally dissolved in 0.2 M aq. NaOH (200 mL).
The solubilized aggregates were further diluted 1:6 with 0.1 M aq.
NaOH and absorbances recorded at 405 nm Biotek Powerwave XS
with software Gen5 1.07).
4.28.2. Antiplasmodial activity assays
The activity of compounds against cultured P. falciparum was
evaluated as previously reported [48]. Briefly, synchronized ring-
stage W2 strain parasites were cultured with multiple concentra-
tions of test compounds (added from 1000 ꢄ stocks in DMSO) in
RPMI 1640 medium with 10% human serum or 0.5% Albumax serum
substitute. After a 48 h incubation, when control cultures contained
new rings, parasites were fixed with 1% formaldehyde in PBS, pH 7.4,
for 48 h at room temperature and then labeled with YOYO-1 (1 nM;
Molecular Probes) in 0.1% Triton X-100 in PBS. Parasitemias were
determined from dot plots (forward scatter vs. fluorescence)
acquired on a FACSort flow cytometer using CELLQUEST software
(Becton Dickinson). IC50s for growth inhibition were determined
with GraphPad Prism software from plots of percentages of the
control parasitemia relative to inhibitor concentration. In each case,
goodness of curve fit was documented by R2 values of >0.95.
4.29.3. Fukui indices
To obtain some information about the favored position for the
nucleophilic attack of the catalytic thiolate over the double bond of
the compounds, the local Fukui indices were calculated according
to Eq. (1).:
fAþ ¼ PAðN þ 1Þ ꢁ PAðNÞ
(1)
where fAþ stands for the electrophilicity of atom A in a molecule and
P for the population of atom A in a molecule with N or Nþ1 elec-
trons. The populations for the neutral (Nþ1) and cationic (N)
species were obtained from Natural Bond Order (NBO) population
analysis [58] for the optimized structures of the compounds in the
gaseous phase. The calculations considered the hybrid density
functional B3LYP [59,60] approach and the 6-31 þ G(d) basis set as
included in the Gaussian 03 suite [57], taking as starting structures
the docked configurations.
4.28.3. FP inhibition assays
Recombinant FP2 and FP3 were expressed as previously
described [49,50] and assays of inhibition of the enzymes were
performed as previously reported [9]. In brief, equal amounts (1 nM)
of recombinant FP2 and FP3 were incubated with different
concentrations of test compounds (added from 10 mM stocks in
DMSO) in 100 mM sodium acetate, pH 5.5, 10 mM dithiothreitol for
30 min at room temperature before addition of the substrate ben-
zoxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin (final concen-
tration 25
mM). Fluorescence was continuously monitored for
Acknowledgments
30 min at room temperature in a Labsystems Fluoroskan II spec-
trofluorometer and IC50s were determined from plots of activity
over enzyme concentration with GraphPad Prism software.
This work was mainly supported by Fundação para a Ciência e
a Tecnologia (FCT, Portugal) through projects PTDC/QUI/65142/
2006 and PTDC/QUI-QUI/116864/2010 coordinated by PG, and
through strategic projects PEst-C/QUI/UI0081/2011 and PEst-C/
CTM/LA0011/2011 awarded to CIQUP and CICECO, respectively.
Thanks are due to FCT also for both the LC-MS and NMR facilities,
respectively funded through projects CONC-REEQ/275/QUI and
REDE/1517/RMN/2005. CT thanks FCT for post-doctoral grant SFRH/
BPD/62967/2009. JRBG is a holder of a Ciência 2007 position of the
Portuguese Ministry of Science. PJR is a Distinguished Clinical
Scientist of the Doris Duke Charitable Foundation.
4.29. In silico studies
4.29.1. Docking of inhibitors
Docking was performed with GOLD [51] version 5.0.1, allowing
full flexibility for the ligand while keeping the protein fixed. The X-
ray structure of FP2 and FP3 (PDB code: 3BPF [7] and 3BWK [16],
respectively) were used and prepared for docking. The original
ligand, ions and solvent molecules were removed and the proteins
were protonated using the Hþþ server [52] assuming a pH of 5.5
and a salinity of 0.15 mol/L. The proteins were then minimized with
the AMBER program [53] by 500 steps of steepest descent followed
by 2000 steps of conjugate gradient to remove bad contacts using
a generalized-Born solvent model. The biomolecular force field ff03
[54] was used. The docking exploration consisted of 500
Appendix A. Supplementary information
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.05.022.

898
B.C. Pérez et al. / European Journal of Medicinal Chemistry 54 (2012) 887e899
[29] A. Barazarte, G. Lobo, N. Gamboa, J.R. Rodrigues, M.V. Capparelli, A. Alvarez-
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