N-Cinnamoylated chloroquine analogues as dual-stage antimalarial leads

Article abstract of DOI:10.1021/jm301654b

The control of malaria is challenged by drug resistance, and new antimalarial drugs are needed. New drug discovery efforts include consideration of hybrid compounds as potential multitarget antimalarials. Previous work from our group has demonstrated that hybrid structures resulting from cinnamic acid conjugation with heterocyclic moieties from well-known antimalarials present improved antimalarial activity. Now, we report the synthesis and SAR analysis of an expanded series of cinnamic acid derivatives displaying remarkably high activities against both blood-and liver-stage malaria parasites. Two compounds judged most promising, based on their in vitro activity and druglikeness according to the Lipinski rules and Veber filter, were active in vivo against blood-stage rodent malaria parasites. Therefore, the compounds reported represent a new entry as promising dual-stage antimalarial leads.

Full text of DOI:10.1021/jm301654b

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Article
N-cinnamoylated chloroquine analogues as dual-stage antimalarial leads
Bianca C. Pérez, Cátia Teixeira, Inês S Albuquerque, Jiri Gut, Philip J
Rosenthal, José R. B. Gomes, Miguel Prudêncio, and Paula Gomes
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301654b • Publication Date (Web): 30 Dec 2012
Downloaded from http://pubs.acs.org on January 4, 2013
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N-cinnamoylated chloroquine analogues as dual-stage antimalarial leads
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Bianca C. Pérez,1 Cátia Teixeira,1,2 Inês S. Albuquerque,3 Jiri Gut,4 Philip J. Rosenthal,4 José R.
B. Gomes,2 Miguel Prudêncio,3 Paula Gomes1,*
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1 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, Pꢀ4169ꢀ007 Porto, Portugal
2 CICECO, Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago, 3810ꢀ
193 Aveiro, Portugal
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Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas
Moniz, 1649ꢀ028 Lisboa, Portugal
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Department of Medicine, San Francisco General Hospital, University of California, CA 94143ꢀ0811,
USA
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Abbreviations: 13CꢀNMR, carbonꢀ13 nuclear magnetic resonance; HꢀNMR, proton nuclear
magnetic resonance; ACT, artemisininꢀbased combination therapies; CQ, chloroquine; DCM,
dichloromethane; DIEA, NꢀethylꢀN,Nꢀdiisopropylamine; DMF, N,Nꢀdimethylformamide;
DMSOꢀd6, deuterated dimethylsulfoxide; ESIꢀIT, electrospray ionization and ionꢀtrap
quadrupole detection; HEFLECINs, heterocyclicꢀcinnamic conjugates; HPLCꢀDAD, high
performance liquid chromatography with diode array detection; MS, mass spectrometry; NPP,
new permeation pathways; PQ, primaquine; PRIMACINs, primaquineꢀcinnamic acid conjugates;
RBC, red blood cell; SAR, structureꢀactivity relationship; TBTU, Oꢀ(benzotriazolꢀ1ꢀyl)ꢀ
N,N,N',N'ꢀtetramethyluronium tetrafluoroborate.
* To whom correspondence should be addressed: P. Gomes, Centro de Investigação em Química da Universidade do
Porto, Rua do Campo Alegre, 687, Pꢀ4169ꢀ007, Porto, Portugal; Phone and Fax: +351220402563; Eꢀmail:
pgomes@fc.up.pt.
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ABSTRACT
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The control of malaria is challenged by drug resistance, and new antimalarial drugs are needed. New
drug discovery efforts include consideration of hybrid compounds as potential multiꢀtarget antimalarials.
Previous work from our group has demonstrated that hybrid structures resulting from cinnamic acid
conjugation with heterocyclic moieties from well known antimalarials present improved antimalarial
activity. Now, we report the synthesis and SAR analysis of an expanded series of cinnamic acid
derivatives displaying remarkably high activities against both bloodꢀ and liverꢀstage malaria parasites.
Two compounds judged most promising, based on their in vitro activity and drugꢀlikeness according to
the Lipinski rules and Veber filter, were active in vivo against blood stage rodent malaria parasites.
Therefore, the compounds reported represent a new entry as promising dualꢀstage antimalarial leads.
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Keywords: antimalarials, bloodꢀschizontocidal, chloroquine, cinnamic acid derivatives, dualꢀ
action, liver stage, Plasmodium falciparum, Plasmodium berghei.
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Introduction
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Several concerted efforts to control malaria and achieve its eradication have been made through history.1
Despite initial success, with regional elimination in Southern Europe and some countries in North Africa
and the Middle East, malaria has not been controlled in many other countries due to a number of factors.2
Two main reasons behind malaria remaining such a burden to humanity are the widespread resistance of
the malaria parasite to available drugs and its complex life cycle.2 Human malaria is triggered by
infection by an intracellular Plasmodium parasite, which is transmitted to human hosts via an infected
female Anopheles mosquito bite. Once in the host circulation, Plasmodium sporozoites initiate the asexual
phase of the life cycle by quickly moving to the liver. During this clinically silent liverꢀstage infection,
thousands of merozoites are formed and released into the bloodstream, where they invade red blood cells,
thus starting the pathogenic asexual erythrocytic cycle, resulting in the various clinical manifestations of
malaria.3
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Resistance of malaria parasites to previous generations of medicines, such as chloroquine (1), became
widespread in the 1970s and 1980s, undermining malaria control efforts. During the last decade,
artemisininꢀbased combination therapies (ACTs) have been adopted globally as the first line of treatment.
ACTs include a rapidly acting artemisinin component and a partner drug to improve efficacy and hinder
emergence of resistance to artemisinins.4 However, there are now worrisome signs that malarial parasites
are developing resistance to artemisinins (2),5 and so the dependence of all new drug combinations on
artemisinin derivatives is a matter of concern. While most available antimalarial agents target the bloodꢀ
stage parasites, relatively few drugs are known to inhibit liver stage parasites. Primaquine (3) remains the
only drug in clinical use worldwide that acts against liver stages of all Plasmodium species,6 including P.
vivax and P. ovale hypnozoites, which are latent forms unique to these species that cause relapsing
malaria long after the original infection, complicating control efforts.7, 8 Taking into account concerns of
drug resistance and difficultꢀtoꢀtreat chronic liver stages, optimal therapy should be effective against
multidrugꢀresistant parasite strains and be active against both liver and erythrocytic parasite stages.
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A recent approach in antimalarial drug design characterized as “covalent bitherapy” involves linking two
active molecules, thus packaging dual activity into a single hybrid molecule with potential to enhance
efficacy, improve safety and/or reduce propensity to elicit resistance relative to the parent drugs.9, 10 As
part of our efforts to develop novel hybrids, we previously reported a firstꢀgeneration of heterocyclicꢀ
cinnamic acid conjugates (4 and 5, Figure 1), linking the heteroaromatic ring of known antimalarials to a
transꢀacid cinnamic motif, active in vitro against bloodꢀstage P. falciparum.11 From these studies we
discovered that a spacer between the heterocycle and the cinnamoyl moiety is required for antiplasmodial
activity and that the higher the lipophilicity of conjugates 4, the higher their in vitro antiplasmodial
activity. Based on these results, we developed a secondꢀgeneration of conjugates (7 and 8, Figure 1)
where the cinnamoyl core was linked to the heterocyclic core of known antimalarials through a flexible
and more hydrophobic butylamine chain.12, 13 Compounds 7 (IC50 against cultured malaria parasites: 11 –
59 nM)13 and 8 (IC50: 1.4 – 2.4 ꢁM)12 had higher in vitro potency than their parent compounds, 1 (IC50:
138 nM) and 3 (IC50: 7.5 ꢁM), against bloodꢀstage P. falciparum (W2 strain) and liverꢀstage P. berghei.
These findings established the cinnamoyl core as a valuable pharmacophore to enhance potency of
established antimalarials.
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Building on these compounds, we have performed further SAR studies and herein report the synthesis and
antimalarial assessment of a series of new heterocyclicꢀcinnamic conjugates (in brief, HEFLECINs). In
terms of SAR, the presence of aromaticity in the heterocyclic core, the number of the heteroaromatic
rings, the length of the aliphatic chain, the nature (amide or ester) of the linkage between the heterocycle
and cinnamic core and the nature of the cinnamic substituent were all evaluated (Figure 2). Compounds
were tested in vitro against erythrocytic stages of the human parasite P. falciparum; two of them, 7d and
7h, were further confirmed to be active in vivo against the clinically relevant bloodꢀstage infection in a P.
berghei rodent malaria model. Additionally, our recent findings on the improved liverꢀstage antimalarial
activity of Nꢀcinnamoyl derivatives of primaquine,12 prompted us to evaluate the activity of heterocyclicꢀ
cinnamic conjugates against liverꢀstage P. berghei parasites. Most of the HEFLECINs were structurally
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related to chloroquine, presented outstanding activity against both erythrocytic and liver stages of the
parasite lifeꢀcycle, and were nonꢀcytotoxic against Huh7 human hepatoma cells. To the best of our
knowledge, chloroquineꢀrelated compounds active against both liverꢀ and bloodꢀstage parasites have not
previously been reported. These results bring new insights into the development of dual action
antimalarial agents.
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Figure 1: Chemical structures of chloroquine (1), artemisinin (2), primaquine (3), firstꢀ (4 and 5) and secondꢀ (7 and 8)
generation heterocycleꢀcinnamic acid conjugates.
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Results and Discussion
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Chemistry. For the past decade we have been working on the design, synthesis and evaluation of
potential antimalarial candidates, bearing in mind that synthetic pathways should be kept as cheap and
straightforward as possible. The HEFLECINs reported here are examples of how simple chemistry can
yield promising antimalarial leads (Scheme 1). Compound 8d was obtained through a single amide
coupling step between the parent drug PQ (3) and the desired cinnamic acid, using previously reported
standard peptide coupling conditions12, namely, activation of the cinnamic acid with Oꢀ(benzotriazolꢀ1ꢀ
yl)ꢀN,N,N',N'ꢀtetramethyluronium tetrafluoroborate (TBTU) in the presence of NꢀethylꢀN,Nꢀ
diisopropylamine (DIEA), followed by addition of PQ. Compounds 713 and 9-13 were obtained in two
steps, starting from (i) a classical nucleophilic aromatic substitution reaction between the appropriate
diaminoalkane (1,3ꢀdiaminopropane, 1,4ꢀdiaminobutane or 1,5ꢀdiaminopentane) or 4ꢀaminobutanꢀ1ꢀol (in
the synthesis of 11) and the desired chlorinated heterocycle (4,7ꢀdichoroquinoline, 4ꢀchloroquinoline or 4ꢀ
chloropyridine), followed by (ii) coupling the resulting compounds to the appropriate cinnamic acids,
again conveniently activated by TBTU/DIEA. The synthesis of 14 was the only one requiring three steps,
as (i) morpholine was first reacted with 1ꢀ(Nꢀphthaloyl)aminoꢀ4ꢀbromobutane, followed by (ii)
hydrazinolysis of the phthaloyl aminoꢀprotecting group, to afford the free amine for subsequent (iii)
PyBOP/DIEAꢀmediated coupling to the appropriate cinnamic acid.
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Although not all synthesis yields were high, the rather small number of synthesis steps, cheap starting
materials and straightforward purification techniques (cf. Experimental Section) largely compensate for
low yields. All the target compounds where obtained in high purity, as determined by high performance
liquid chromatography with diode array detection (HPLCꢀDAD), and their structures were confirmed by
proton (1Hꢀ) and carbonꢀ13 (13Cꢀ) nuclear magnetic resonance (NMR), as well as by mass spectrometry
(MS) analysis with electrospray ionization and ionꢀtrap quadrupole detection (ESIꢀIT); relevant data are
presented in the Experimental Section.
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Scheme 1. Synthetic pathways towards heterocycleꢀcinnamic acid conjugates 7, 9-12.
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Biology. Our recent discovery of the potent in vitro activity of CQ analogues 7 against bloodꢀstage CQꢀ
resistant P. falciparum parasites unveiled the key role of the cinnamoyl moiety in reversing parasite
resistance to CQ.13 We have now expanded the family of heterocyclicꢀcinnamic acid conjugates
(HEFLECINs) and tested their in vitro activity against bloodꢀstage P. falciparum (Table 1), in order to (i)
establish relevant SAR (Figure 2), and (ii) select the most promising candidates for in vivo challenge in
the P. berghei rodent malaria model. For some of the compounds we compared activity against
chloroquineꢀsensitive (3D7) and ꢀresistant (W2) P. falciparum parasites. Furthermore, given their
structural analogy to CQ, which acts via inhibition of βꢀhematin formation, selected compounds were
evaluated as in vitro inhibitors of this process (Table 1).14, 15 Finally, based on our latest disclosure of
primaquineꢀcinnamic acid conjugates (PRIMACINs) with improved in vitro activity against liverꢀstage
Plasmodia,12 we also assessed the in vitro activity of a subset of the HEFLECINs against liverꢀstage P.
berghei (Figure 3, Table 1).
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Figure 2: Structural parameters considered in the SAR analysis of the heterocycleꢀcinnamic acid conjugates.
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In vitro activity against intraerythrocytic P. falciparum parasites. Bloodꢀstage activity of HEFLECINs
was determined by methods previously reported by us.16, 17 Close inspection of our data (Table 1, columns
8 and 9) shows the following.
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(i) Remarkably, most of the test compounds assayed against both strains were more active against the
CQꢀresistant (W2) strain than against the CQꢀsensitive (3D7) strain.
(ii) Compounds from the 7, 9 and 10 series with a single substituent (R) on the cinnamoyl group (i.e., all
except 7p) were highly active, with IC50s in the range of 11ꢀ79 nM. These series differ only in the length
of the polymethylene spacer between the chloroquinoline and cinnamoyl cores. Comparison between
homologues from series 7, 9 and 10 (i.e., 7c vs. 9c vs. 10a, or 7h vs. 9h vs. 10c, or 7d vs. 9d vs. 10b)
shows that a butyl spacer is preferred over pentyl and propyl spacers.
(iii) Removal of the 7ꢀchlorine substituent from the chloroquinoline ring leads to a decrease in
antimalarial activity of about one order of magnitude, as shown for compounds 7c (IC50 11 nM) and its
dechlorinated analogue 12 (115 nM).
(iv) Replacement of the amide bond in 7d (IC50 20 nM) by its ester isostere, to afford 11, leads to a sixꢀ
fold decrease in activity (122 nM).
(v) Substitution of the chloroquinoline ring (e.g. 7d, IC50 ~ 20 nM) by another heteroaromatic ring, either
quinolinic (8ꢀaminoꢀ6ꢀmethoxyquinoline ring in 8d), pyridinic (single heteroaromatic pyridine ring in 13)
or nonꢀaromatic (morpholine ring in 14), leads to complete loss of activity.
(vi) Comparison of IC50 values within a given series, e.g. compounds 7 or 9, shows that the cinnamoyl
substituent
R has some effect on activity, but that this effect is not dramatic. The
electrodonating/withdrawing character of R seems to have no significant role in activity, whereas the para
position is clearly preferred over the ortho or meta positions, as shown for 7k-l and 9k-l.
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(vii) Increase in compound lipophilicity and/or the bulkiness of R within a series seems to enhance in
vitro activity. We previously showed that IC50 values for compounds 7 showed good correlation with both
clogP values and Charton parameters.13 For the new 9 series, we observed a good correlation with
Charton parameters, but not clogP values (cf. Table S1 provided as Supporting Information). Series 10
was too small to allow drawing definitive conclusions in this regard.
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(viii) Finally, HEFLECINs bearing the chloroquinoline core were globally more active than CQ, whereas
the two most active compounds, 7c and 7h, had activities comparable to that of artemisinin. Therefore,
these two compounds, as well as 7d, the most active compound within the series satisfying the leadꢀ
likeness,18 Lipinski19 and Veber20 filters, were selected for in vivo studies with P. berghei.
In vitro activity against β-hematin formation. The ability of HEFLECINs to inhibit βꢀhematin
formation to hemozoin, the main mechanism attributed to CQ and related antimalarials, was assessed by
previously reported methods.11 This activity was not fully correlated with in vitro antimalarial activities
(Table 1), suggesting that HEFLECINs owe their antimalarial properties to mechanisms in addition to
inhibition of βꢀhematin crystallization. Nonetheless, we believe that inhibition of βꢀhematin formation
contributes to activity because (i) variation of βꢀhematin inhibition potency amongst homologues, e.g., 7c,
9c and 10a follows the same trend as variation of activities against bloodꢀstage parasites and (ii) the
significant decrease in antimalarial activity upon removal of the 7ꢀchlorine substituent (7c vs. 12) may be
explained by the prior observation that this chlorine atom is essential to ensure effective βꢀhematin
inhibition by CQ and related structures.21, 22
Based on previous and current findings, it is concluded that HEFLECINs do not act against bloodꢀstage
parasites via inhibition of the cysteine protease falcipains.13 Activity may include impairment of the New
Permeation Pathways (NPP) created by Plasmodia in the infected red blood cell (RBC)13 and inhibition of
βꢀhematin crystallization.
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In vivo treatment of P. berghei-infected mice. For the 3 most promising HEFLECINS, in vivo
efficacy was evaluated using a standard P. berghei mouse model. Compounds chosen for in vivo analysis
were 7c and 7h due to their potency against cultured bloodꢀstage P. falciparum and 7d due to its better
drugꢀlikeliness. Mice were infected intraperitoneally (ip) with P. berghei and then treated twice daily with
ip injections of 10, 30 and 100 mg/kg. Chloroquine was used as a positive control at 3, 10 and 30 mg/kg
ip twice daily. All mice died due to malaria or toxicity by day 17 at the doses administered. In order to
compare compound efficacies, survival over time after the initiation of treatment was compared.
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Close inspection of the data leads to the following observations (Figure 3). Compounds 7c and 7h were
highly toxic at 100 mg/kg, and no mice survived until the last day of treatment. At lower dosages, 7h
extended survival compared to untreated controls by 2ꢀ7 days at 30 mg/kg and by 2 days at 10 mg/kg.
Compound 7c did not improve survival compared to untreated controls. Compound 7d did not appear to
be toxic at any tested dosage. It extended survival by 2 days compared to untreated controls at the highest
dosage, but offered no benefit over untreated controls at the lower dosages.
Studies with the rodent malaria model confirmed that 7d and 7h have in vivo antimalarial activity. The
lack of in vivo activity of 7c can be attributed to its high lipophilicity (clogP > 5, see Table S1 on SI),19
but compounds 7d and 7h are only slightly less lipophilic (clogP ~5, see Table S1 on SI) than 7c.
However, in vivo activities for 7d and 7h were lower than might be expected based on their in vitro
activities, possibly due to limitations in bioavailability. Additionally, relatively poor performance of the
compounds in vivo might be explained by biological differences between P. berghei and P. falciparum
and by the high hydrophobicity of the compounds, which might result in extensive binding to plasma
proteins.23
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Figure 3: Survival curves for P.bergheiꢀinfected mice treated with compounds 7c, 7d and 7h, and CQ.
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Table 1. In vitro data on test compounds for βꢀhematin inhibition, and blood and liver stage activity.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pf W2
βꢀHematin
Liver Stage
Pf 3D7
Compound
R3
HET
n
X
R2
IC50 (nM)[b]
IC50 (nM)[b]
IC50 (ꢀM)[c]
inhibition[a]
H
p-Me
p-iPr[c]
p-OMe[c]
p-NH2
m-F
NA
NA
+
46.6 ± 5.5
16.9 ± 1.2
11.0 ± 6.2
20.0 ± 2.6
58.8 ± 1.5
34.1 ± 4.2
19.7 ± 0.4
11.6 ± 0.4
18.2 ± 2.8
38.3 ± 4.2
26.2 ± 5.0
23.5 ± 1.3
25.8 ± 2.1
110.8 ± 13.0
>10000[h]
74.8 ± 2.0
47.4 ± 1.2
38.0 ± 11.2
55.1 ± 2.3
79.8 ± 2.0
73.3 ± 6.8
46.7 ± 3.3
41.6 ± 2.9
38.2 ± 6.9
52.1 ± 4.0
50.3 ± 1.2
23.7 ± 2.1
31.5 ± 3.8
50.3 ± 2.6
25.1 ± 0.2
7a
7b
7c
7d
7e
7f
62.85±3.5
24.23±0.2
15.63±5.4
30.63±9.5
78.12±2.2
50.92±1.0
39.85±3.5
26.21±2.0
28.75±6.3
56.91±5.1
42.06±7.1
48.94±3.2
ND
1.06 ± 0.1
2.36 ± 0.5
2.50 ± 0.2
2.86 ± 0.4
ND[g]
+
NA
+
1.13 ± 0.2
1.42 ± 0.2
1.44 ± 0.3
2.28 ± 0.6
1.09 ± 0.2
ND
p-F
p-Cl [d]
+
7g
7h
7i
Cq
R1 = Cl
2
N
H
+
p-Br
++
+
o-NO2
m-NO2
p-NO2
p-NMe2
m,p-diOMe
7j
NA
++
+
7k
7l
ND
7o
7p
8d
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
ND
NA
ND
ꢀ
4.05 ± 0.2
2.35 ± 0.19[h]
ND
Pq
2
1
N
N
CH3
pꢀOMe
H
NA
NA
NA
NA
NA
NA
NA
+
141.3±9.3
55.85±5.1
50.27±1.0
49.51±5.8
134.85±5.4
137.95±1.2
47.73±1.3
46.47±4.5
49.96±3.5
64.15±6.7
52.73±5.3
52.53±10.8
ND
p-Me
p-iPr
p-OMe
p-NH2
m-F
p-F
p-Cl
p-Br
o-NO2
m-NO2
p-NO2
p-iPr
p-OMe
p-Cl
ND
ND
4.02 ± 0.6
ND
ND
ND
ND
ND
ND
ND
ND
Cq
R1 = Cl
H
NA
+
+
+
+
10a
10b
10c
ND
Cq
R1 = Cl
NA
NA
3
N
H
ND
2.27 ± 0.6
1.62 ± 0.1
ND
Cq
R1 = Cl
Cq
R1 = H
Py
11
12
2
2
O
N
H
H
pꢀOMe
pꢀiPr
+
ND
ND
121.9 ± 5.5
6.47 ± 0.9
NA
ND
114.9 ± 3.0
13
14
ART
CQ
PQ
2
2
N
N
H
H
pꢀOMe
pꢀiPr
NA
NA
>10000
>10000
ND
ND
>10
>10
ND
Mu
ꢀ
9.5 ± 1.9
138[e]
3300[f]
23.5±0.8
52.45
++
15.9 ± 0.0
7.5[h]
[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 (+); ≥75%,
highly active (++); [b] bloodꢀstage antiplasmodial activity was determined against the CQꢀresistant P. falciparum strain W2 and strain 3D7, using
artemisinin (ART) and CQ as reference drugs;[c] IC50 of the most active compounds against liver stage;[d] also tested in vivo;[e] Value taken from
Ref 24
;
[f] Value taken from Ref. 17
,
[g] ND, not determined, [h] Value taken from Ref. 12
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9
In vitro activity against liver-stage P. berghei parasites. Recent findings from our group, demonstrating
that the liverꢀstage activity of PQ is significantly enhanced by conjugation with cinnamic acids, prompted
us to investigate whether HEFLECINs would also exhibit liver stage activity, measured using P. berghei
as previously reported.12, 17, 25 We found that HEFLECINs bearing the chloroquinoline core of CQ (7, 9,
10) were all active against liver stage parasites. This result was surprising, as CQ is not active against
liverꢀstage malaria26. In view of these results, the activities of compounds selected as most promising
based on activity and cytotoxicity, were compared quantitatively (Figure 4, Table 1). Remarkably,
activities were much higher than those observed for either CQ or PQ. Specifically, IC50 values were 5ꢀ15
times lower than those for CQ, and 3ꢀ7 times lower than those for the reference drug PQ. This is, to our
knowledge, the first report of 4ꢀaminoꢀ7ꢀchloroquinolinic (CQꢀrelated) structures displaying in vitro
activity against liverꢀstage Plasmodia greater than that of PQ. This finding, together with the previous
observation that Nꢀcinnamoylation of PQ also enhances its in vitro activity against liverꢀstage
Plasmodia,12 strongly suggests that the Nꢀcinnamoyl moiety is a relevant pharmacophore to boost
antiplasmodial activity in both 8ꢀ and 4ꢀaminoquinolines. Some SAR could be devised from the liverꢀ
stage assays, as follows:
10
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(i) As in bloodꢀstage activity assays, a butyl spacer between the chloroquinoline and the cinnamic cores is
generally preferred over propyl and pentyl spacers, both in terms of activity and toxicity, as inferred from
comparison of homologues from series 7, 9 and 10.
(ii) Unlike what was observed in bloodꢀstage activity assays, substitution of the cinnamoyl aromatic ring
in series 7 led to slightly decreased activity, i.e., the most active compound was 7a, where R = H.
(iii) The presence of electrodonating R groups in the para position led to a decrease in activity.
(iv) An increase of the number of OMe substituents on the cinnamoyl core from one (7d) to two (7p) led
to a decrease in toxicity, but also in activity.
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(v) Removal of the 7ꢀchlorine atom from the chloroquinoline ring led, as observed for bloodꢀstage
activity, to a decrease in liverꢀstage activity.
7
8
9
(vi) Also as observed against bloodꢀstage Plasmodia, replacement of the chloroquinoline ring by pyridine
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or morpholine led to loss of activity against liverꢀstage parasites.
As is the case for PQ, the mechanism of HEFLECINs against liverꢀstage Plasmodia remains to be
elucidated.12 Studies are under way to establish the target(s) of HEFLECINs against liverꢀ and bloodꢀ
stage parasites. The discovery that HEFLECINs bearing the CQ heterocyclic core, unlike their PQ
analogues (PRIMACINs12), combine significant liverꢀstage activity with potent bloodꢀstage activity, is of
great interest. To our knowledge, this is the first example of CQꢀrelated molecules emerging as multiꢀ
stage antimalarial leads.
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Figure 4: Activity of HEFLECINS against P. berghei liver stages. Antiꢀinfective activity (infection scale, yꢀaxis) and toxicity to
hepatoma cells (cell confluency scale, xꢀaxis) are shown. Primaquine (PQ) and chloroquine (CQ) were included for comparison.
The black, red and blue circles represent results for the tested compounds at 10, 5 and 1 ꢁM, respectively. Infection loads of
Huh7 cells, a human hepatoma cell line, were determined by bioluminescence measurements of cell lysates 48 h after infection
with luciferaseꢀexpressing P. berghei parasites.25
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Concluding remarks
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7
8
9
A new family of chloroquine analogues, HEFLECINS, as dualꢀstage antimalarial leads, was
found. These compounds display potent in vitro activity against both liverꢀ and bloodꢀstage Plasmodia,
including chloroquineꢀresistant bloodꢀstage P. falciparum W2 parasites; all of them performed better than
chloroquine itself in vitro on both stages, and the best couple of compounds, 7c and 7h, were equipotent
to artemisinin on bloodꢀstage parasites. Further, all but 11, 13 and 14 were better than primaquine on
liverꢀstage parasites. Relevantly, both bloodꢀ and liverꢀstage activities were lost upon replacement of the
4ꢀaminoꢀ7ꢀchloroquinoline ring with either a nonꢀaromatic (i.e., morpholine) or an aromatic (i.e., 4ꢀ
aminopyridine) heterocycle, while replacement with the primaquine’s 8ꢀaminoꢀ6ꢀmethoxyquinoline
moiety only eliminated bloodꢀstage activity. This demonstrates that the 4ꢀaminoꢀ7ꢀchloroquinoline motif
has a critical role for the display of dualꢀstage antimalarial activity. Two of the most promising
compounds were confirmed to be active against the murine model of malarial infection, even though with
modest in vivo performances as compared to in vitro ones; this is possibly due to bioavailability issues
requiring future optimization of the reported antimalarial leads. Studies on the possible mechanism(s)
underlying dualꢀstage activity displayed by the Nꢀcinnamoylated chloroquine analogues are under way;
falcipain inhibition has been ruled out, whereas blocking of βꢀhematin formation cannot fully account for
the activity levels observed. The role of these antimalarial leads as blockers of ion channels specific to
infected host cells, like the soꢀcalled NPP created in P. falciparumꢀinfected RBC, is presently under
investigation.
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11
12
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Overall, data show that Nꢀcinnamoylation of chloroquine enhances the parent drug’s activity against
both bloodꢀ and liverꢀstage Plasmodia, which is the first example of chloroquineꢀderived molecules with
multiꢀstage antimalarial activity. This finding, along with the recent disclosure of an apparent reꢀ
susceptibilization of P. falciparum to chloroquine in Nigeria,29 may have relevant impact on the
reactivation of chloroquine manufacturers, especially in African countries like Ghana, in which
chloroquine production has been halted due to the wellꢀknown parasite resistance to this drug.30
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Experimental Section
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6
7
Chemistry
8
9
Chemicals and instrumentation. All solvents and common chemicals were from Sigmaꢀ
Aldrich (Spain), whereas Bocꢀprotected amino acids were from NovaBiochem (VWR International,
Portugal) and the coupling agent TBTU was from Bachem (Switzerland). NMR spectra were acquired on
a Bruker Avance III 400 spectrometer from solutions in either deuterated chloroform or deuterated
dimethylsulfoxide (DMSOꢀd6) containing tetramethylsilane as internal reference. Mass spectroscopy
(MS) analyses were run on a Thermo Finnigan LCQ Deca XP Max LC/MSn instrument operating with
electrospray ionization and ionꢀtrap (ESIꢀIT) quadrupole detection. The target compounds were
confirmed to have at least 95% purity, based on peak areas obtained through HPLC analyses that were run
using the following conditions: 30ꢀ100% of B in A (A = H2O with 0.05% of trifluoroacetic acid;
B=acetonitrile) in 22 minutes with a flow rate of 1 mL/min on a MerckꢀHitachi Lachrom Elite instrument
equipped with a diodeꢀarray detector (DAD) and thermostated (Peltier effect) autosampler, using a
Purospher STAR RPꢀ18e column (150 × 4.0 mm; particle size, 5 ꢀM).
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12
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Synthesis of compounds 7-10. Compounds 7a-l and 8 were prepared by previously described
methods, and their analytical and structural data were in perfect agreement with formerly reported data.12,
13. Compounds 7o,p were prepared identically to 7a-l and their spectroscopic data were compatible with
the respective structures, as follows.
4-[N-(p-dimethylamino)cinnamoylaminobutyl]amino-7-chloroquinoline (7o): White solid (14 mg,
8%); mp=176ꢀ180oC; RF (DCM/MeOH 8:2) 0.44; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.6Hz, 2H), 8.32
(d, J=8.8Hz, 1H), 7.95 (m, 1H), 7.79 (d, J=2Hz, 1H), 7.62 (m, 1H), 7.47 (dd, J=8.8, 2.4Hz, 1H), 7.36 (d,
J=8.8Hz, 2H), 7.29 (d, J=16Hz, 1H), 6.70 (d, J=8.8Hz, 2H), 6.53 (d, J=5.6Hz, 1H), 6.35 (d, J=16Hz, 1H),
3.32 (m, 2H), 3.22 (m, 2H), 2.94 (s, 6H), 1.68 (m, 2H), 1.57 (m, 2H); δC (100 MHz, DMSOꢀd6) 165.6,
150.9, 150.7, 150.6, 147.6, 138.8, 133.9, 128.7, 126.3, 124.3, 122.3, 117.1, 116.7, 111.9, 98.6, 42.1, 38.1,
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m
26.9, 25.1; ESIꢀIT MS: /z (M+H+) 423.47, M+ (C24H27ClN4O) requires 422.19; HPLCꢀDAD: rt = 11.7
5
6
min, % area = 96.0.
7
8
9
4-[N-(m,p-dimethoxy)cinnamoylaminopropyl]amino-7-chloroquinoline (7p): White solid (29 mg,
17%); mp=168ꢀ172oC; RF (DCM/MeOH 8:2) 0.44; δH (400 MHz, DMSOꢀd6) 8.41 (d, J=5.6Hz, 1H), 8.36
(d, J=9.2Hz, 1H), 8.06 (m, 1H), 7.81 (m, 2H); 7.50 (dd, J=9.4Hz, 2.4Hz, 1H), 7.34 (d, J=16Hz, 1H), 7.10
(m, 2H), 6.96 (d, J=8.4Hz, 1H), 6.57 (d, J=5.6Hz, 1H), 6.51 (d, J=16Hz, 1H), 3.78 (s, 3H), 3.77 (s, 3H),
3.35 (m, 2H), 3.23 (m, 2H), 1.69 (m, 2H), 1.57 (m, 2H); δC (100 MHz, DMSOꢀd6) 165.2, 151.2, 150.0,
149.8, 148.8, 146.6,138.4,134.3, 127.6, 125.5, 124.5, 124.4, 121.2, 119.9, 116.9, 111.7, 109.9, 98.6, 55.4,
10
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m
55.3, 42.2, 38.2, 26.7, 25.1; ESIꢀIT MS: /z (M+H), M+ (C24H26ClN3O3) requires 439.17; HPLCꢀDAD: rt
= 12.5 min, % area = 100.
Compounds 9 and 10 were synthesized by methods similar to those previously described for compounds
7;13 briefly, 4,7ꢀdichloroquinoline (1 eq, 1.2 mmol) was reacted with the appropriate diaminoalkane (10
eq, 12 mmol) at 100 ºC for 3 h; the mixture was brought to room temperature and then diluted with
dichlorometane (DCM), and the resulting solution was washed with 5% aqueous Na2CO3. The organic
layer was isolated and dried over anhydrous Na2SO4, filtered and evaporated to dryness to afford the
desired 4ꢀ(Nꢀaminoalkyl)aminoꢀ7ꢀchloroquinoline (6, Scheme 1); each compound 6 was then coupled to
the respective cinnamic acid in N,Nꢀdimethylformamide (DMF), using Oꢀ(benzotriazolꢀ1ꢀyl)ꢀN,N,N',N'ꢀ
tetramethyluronium tetrafluoroborate (TBTU) and N,NꢀdiethylꢀNꢀisopropylamine (DIEA) (Scheme 1): a
solution of cinnamic acid (1.1 eq, 0.22 mmol), TBTU (1.1 eq, 0.22 mmol), and 2.2eq of DIEA (68ꢀL) in
DMF was stirred at 0 ºC for 10 min; a solution of the appropriate compound 6 (1 eq, 0.2 mmol) in DMF
was then added, and the reaction allowed to proceed at room temperature for 24 h; the mixture was
diluted with DCM and the resulting solution was washed with 5% aqueous Na2CO3; the organic layer was
separated, dried over anhydrous Na2SO4, filtered and evaporated to dryness, and the crude product was
purified by liquid chromatography on silica using EtOAc/MeOH, 8:2 (v/v) to give the desired compounds
9, 10. In the particular case of 9e, 4ꢀ(Nꢀaminopropyl)aminoꢀ7ꢀchloroquinoline was first coupled to pꢀ(Nꢀ
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tertꢀbutoxycarbonyl)aminocinnamic acid as above, to yield the N-tertꢀbutoxycarbonyl (Boc) protected
precursor (9e’), followed by removal of Boc through acidolysis with neat trifluoroacetic acid (TFA).
Spectral/analytical data on compounds 9,10 follow.
9
4-(N-cinnamoylaminopropyl)amino-7-chloroquinoline (9a). White solid (32 mg, 42%); mp 174ꢀ192oC;
RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.6Hz, 1H), 8.27 (m, 2H), 7.79 (d,
J=2.4Hz, 1H), 7.46 (m, 8H), 6.67 (d, J=15.6Hz, 1H), 6.49 (d, J=5.6Hz, 1H), 3.32 (m, 4H), 1.89 (m, 2H);
δC (100 MHz, DMSOꢀd6) 172.0, 165.1, 151.9, 150.0, 149.0, 138.5, 134.8, 133.3, 129.3, 128.8, 127.4,
10
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m
127.4, 124.0, 122.1, 117.4, 98.6, 40.0, 36.6, 27.8; ESIꢀIT MS: /z 366.47 (M+H+), M+ (C21H20ClN3O)
requires 365.13; HPLCꢀDAD: rt = 13.0 min, % area = 100.
4-[N-(p-methyl)cinnamoylaminopropyl]amino-7-chloroquinoline (9b): White solid (53 mg, 68%); mp
204ꢀ209oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.6Hz, 1H), 8.27 (d,
J=9.2Hz, =1H), 8.22 (m, 1H), 7.79 (d, J=2.4Hz, 1H), 7.40 (m, 5H), 7.20 (d, J=8Hz, 2H), 6.60 (d, J=16Hz,
1H), 6.48 (d, J=5.6Hz, 1H), 3.32 (m, 4H), 2.30 (s, 3H), 1.87 (m, 2H); δC (100 MHz, DMSOꢀd6) 165.2,
151.8, 149.9, 149.0, 139.1, 138.5, 133.3, 132.1, 129.4, 127.4, 127.4, 124.0, 121.0, 117.4, 98.6, 39.4, 36.6,
27.8, 20.8; ESIꢀIT MS: m/z 380.4 (M+H+) M+ (C22H22ClN3O) requires 379.15; HPLCꢀDAD: rt = 14.0 min,
% area =100.
4-[N-(p-isopropyl)cinnamoylaminopropyl]amino-7-chloroquinoline (9c). White solid (28.7 mg, 33%);
mp 173ꢀ178oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.2Hz, 1H), 8.27 (d,
J=9.2Hz, =1H), 8.22 (m, 1H), 7.79 (d, J=2.4Hz, 1H), 7.40 (m, 5H), 7.27 (d, J=8Hz, 2H), 6.59 (d,
J=15.6Hz, 1H), 6.48 (d, J=5.6Hz, 1H), 3.32 (m, 4H), 2.89 (m,1H), 1.86 (m, 2H), 1.19 (d, J=6.8Hz, 6H);
δC (100 MHz, DMSOꢀd6) 165.2, 151.8, 150.0, 149.9, 149.0, 138.5, 133.3, 132.5, 127.5, 127.5, 126.8,
m
124.0, 121.1, 117.4, 98.6, 40.0, 36.6, 33.2, 27.8, 23.6; ESIꢀIT: /z 408.47 (M+H+), M+ (C24H26ClN3O)
requires 407.18; HPLCꢀDAD: rt = 16.0 min, % area =94.9.
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4-[N-(p-methoxy)cinnamoylaminopropyl]amino-7-chloroquinoline (9d). White solid (15 mg, 7%); mp
178ꢀ180oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.6Hz, 1H), 8.28 (d,
J=9.2Hz, =1H), 8.18 (m, 1H), 7.79 (d, J=2.4Hz, 1H), 7.44 (m, 5H), 6.97 (d, J=8.8Hz, =2H), 6.51 (m, 2H),
3.78 (s, 3H), 3.32 (m, 4H), 1.85 (m, 2H); δC (100 MHz, DMSOꢀd6) 172.0, 165.4, 160.2, 151.6, 150.1,
148.7, 138.2, 133.4, 129.0, 127.4, 127.2, 124.0, 119.6, 117.4, 114.2, 98.6, 55.2, 40.0, 36.5, 27.8; ESIꢀIT
MS: m/z 396.53 (M+H+), M+ (C22H22ClN3O2) requires 395.14; HPLCꢀDAD: rt = 13.1 min, % area = 98.0.
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4-{N-[p-(N-tert-butoxycarbonyl)amino]cinnamoylaminopropyl}amino-7-chloroquinoline
(9e’).
White solid (124.5 mg, 41%); mp 218ꢀ220oC; RF (EtOAc/MeOH 8:2) 0.1; δH (400 MHz, DMSOꢀd6) 9.54
(s, 1H), 8.39 (d, J=5.6Hz, 1H), 8.27 (d, J=8.8Hz, =1H), 8.15 (m, 1H), 7.78 (d, J=2.4Hz, 1H), 7.46 (m,
5H), 7.34 (m, 2H), 6.49 (m, 2H), 3.32 (m, 4H), 1.85(m, 2H), 1.48 (s, 9H); δC (100 MHz, DMSOꢀd6)
165.3, 152.5, 151.8, 149.9, 149.0, 140.7, 138.3, 133.3, 128.5, 128.1, 127.4, 124.0, 123.9, 119.9, 118.0,
98.6, 79.3, 40.0, 36.5, 28.0, 27.8; ESIꢀIT: m/z 481.33 (M+H+), M+ (C26H29ClN4O3) requires 480.19;
HPLCꢀDAD: rt = 15.0 min, % area = 100.
4-[N-(p-amino)cinnamoylaminopropyl]amino-7-chloroquinoline (9e). White solid (28.8 mg, 73%);
mp 103ꢀ105oC; RF (EtOAc/MeOH 8:2) 0.1; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.2Hz, 1H), 8.27 (d,
J=9.2Hz, =1H), 8.0 (m, 1H), 7.79 (d, J=2Hz, 1H), 7.44 (dd, J=8.8Hz, 2Hz, 1H), 7.29 (m, 4H), 6.56 (d,
J=8.4Hz, 2H), 6.48 (d, J=5.6Hz, 1H), 6.29 (d, J=15.6Hz, 1H), 5.55 (b, 2H), 3.29 (m, 4H), 1.84 (m, 2H);
δC (100 MHz, DMSOꢀd6) 166.0, 151.8, 150.4, 150.0, 149.0, 139.4, 133.3, 129.0, 127.4, 124.0, 122.1,
m
117.4, 115.7, 113.6, 98.6, 40.0, 36.5, 27.9; ESIꢀIT MS: /z 381.40 (M+H+), M+ (C21H21ClN4O) requires
380.14; HPLCꢀDAD: rt = 8.65 min, % area = 96.0.
4-[N-(m-fluoro)cinnamoylaminopropyl]amino-7-chloroquinoline (9f). White solid (36 mg, 45%); mp
185ꢀ190oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.2Hz, 1H), 8.27 (m, 2H),
7.79 (d, J=2Hz, 1H), 7.41 (m, 6H), 7.20 (m, 1H), 6.67 (d, J=16Hz, 1H), 6.48 (d, J=5.6Hz, 1H), 3.33 (m,
4H), 1.87 (m, 2H); δC (100 MHz, DMSOꢀd6) 172.7, 164.8, 163.6, 161.2, 151.8, 150.0, 149.0, 137.4,
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137.2, 133.3, 130.7, 127.4, 124.0, 123.6, 117.4, 115.9, 113.8, 98.6, 40.0, 36.7, 27.7; ESIꢀIT MS: /z
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384.40 (M+H+), M+ (C21H19ClFN3O) requires 383.12; HPLCꢀDAD: rt = 13.4 min, % area = 100.
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4-[N-(p-fluoro)cinnamoylaminopropyl]amino-7-chloroquinoline (9g). White solid (27 mg, 34%); mp
187ꢀ193oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.2Hz, 1H), 8.28 (m, 2H),
7.78 (d, J=2.4Hz, 1H), 7.61 (m, 2H), 7.42 (m, 2H), 7.23 (m, 2H), 6.59 (d, J=16Hz, 1H), 6.48 (d, J=4.8Hz,
1H), 3.32 (m, 4H), 1.87 (m, 2H); δC (100 MHz, DMSOꢀd6) 172.1, 165.0, 163.8, 161.3, 151.8, 150.0,
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149.0, 137.4, 133.3, 129.6, 127.4, 124.0, 122.0, 117.4, 115.9, 98.6, 40.0, 36.6, 27.8; ESIꢀIT MS: /z
384.47 (M+H+) M+ (C21H19ClFN3O) requires 383.12; HPLCꢀDAD: rt = 13.3 min, % area = 100.
4-[N-(p-chloro)cinnamoylaminopropyl]amino-7-chloroquinoline (9h). White solid (39 mg, 46%); mp
191ꢀ195oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.6Hz, 1H), 8.28 (m, 2H),
7.78 (d, J=2.4Hz, 1H), 7.58 (m, 2H), 7.44 (m, 5H), 6.63 (d, J=15.6Hz, 1H), 6.49 (d, J=5.6Hz, 1H), 3.32
(m, 4H), 1.87 (m, 2H); δC (100 MHz, DMSOꢀd6) 164.9, 151.7, 150.1, 148.8, 137.2, 133.8, 133.4, 129.1,
m
128.9, 127.3, 124.1, 124.0, 122.9, 117.4, 98.6, 40.0, 36.6, 27.8; ESIꢀIT MS: /z 400.40 (M+H+), M+
(C21H19Cl2N3O) requires 399.09; HPLCꢀDAD: rt = 13.4 min, % area = 95.7.
4-[N-(p-bromo)cinnamoylaminopropyl]amino-7-chloroquinoline (9i). White solid (22 mg, 39%); mp
214ꢀ219oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (d, J=5.2Hz, 1H), 8.29 (m, 2H),
7.78 (d, J=2Hz, 1H), 7.60 (m, 2H), 7.51 (m, 2H), 7.40 (m, 3H), 6.65 (d, J=16Hz, 1H), 6.48 (d, J=5.6Hz,
1H), 3.32 (m, 4H), 1.86 (m, 2H); δC (100 MHz, DMSOꢀd6) 164.9, 151.8, 150.0, 149.0, 137.3, 134.1,
m
133.3, 131.8, 129.4, 127.4, 124.0, 123.0, 122.5, 117.4, 98.6, 40.0, 36.6, 27.7; ESIꢀIT MS: /z 446.20
(M+H+) M+ (C21H19ClBrN3O) requires 443.04; HPLCꢀDAD: rt = 14.7, % area = 100.
(4-[N-(o-nitro)cinnamoylaminopropyl]amino-7-chloroquinoline (9j). White solid (61.7 mg, 72%); mp
190ꢀ195oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (m, 2H), 8.29 (d, J=9.2Hz, 1H),
8.03 (d, J=8Hz, 1H), 7.69 (m, 5H), 7.44 (dd, J= 9Hz, 2Hz, 1H), 7.33 (m, 1H), 6.63 (d, J=15.6Hz, 1H),
6.49 (d, J=5.2Hz, 1H), 3.33 (m, 4H), 1.89 (m, 2H); δC (100 MHz, DMSOꢀd6) 164.2, 151.8, 149.9, 149.0,
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148.2, 133.7, 133.5, 133.3, 130.1, 130.0, 128.6, 127.4, 126.8, 124.5, 124.0, 117.4, 98.6, 40.0, 36.7, 27.7;
ESIꢀIT MS: m/z 411.47 (M+H+), M+ (C21H19ClN4O3) requires 410.11; HPLCꢀDAD: rt = 13.0 min, % area
= 100.
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4-[N-(m-nitro)cinnamoylaminopropyl]amino-7-chloroquinoline (9k). Yellow solid (22 mg, 25%); mp
209ꢀ214oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (m, 2H), 8.26 (m, 2H), 8.20 (dd,
J=8Hz, 1.6Hz, 1H), 8.01 (d, J=7.6Hz, 1H), 7.77 (d, J=2Hz, 1H), 7.70 (m, 1H), 7.55 (d, J=15.6Hz, 1H),
7.44 (dd, J=8.8Hz, 2.4Hz, 1H), 7.34 (m, 1H), 6.82 (d, J=15.6Hz, 1H), 6.49 (d, J=5.6Hz, 1H), 3.34 (m,
4H), 1.89 (m, 2H); δC (100 MHz, DMSOꢀd6) 164.5, 151.8, 149.9, 149.0, 148.2, 136.7, 136.2, 133.8,
m
133.3, 130.4, 127.4, 125.0, 123.8, 123.6, 121.4, 117.4, 98.6, 36.7, 27.7; ESIꢀIT MS: /z 411.40 (M+H+),
M+ (C21H19ClN4O3) requires 410.11; HPLCꢀDAD: rt = 13.2 min, % area = 95.0.
4-[N-(p-nitro)cinnamoylaminopropyl]amino-7-chloroquinoline (9l). Yellow solid (42 mg, 49%); mp
200ꢀ205oC; RF (EtOAc/MeOH 8:2) 0.25; δH (400 MHz, DMSOꢀd6) 8.39 (m, 2H), 8.26 (m, 3H), 7.81 (m,
3H), 7.53 (d, J=15.6Hz, 1H), 7.45 (d, J=9Hz, 2.4Hz, 1H), 7.35 (m, 1H), 6.81 (d, J=16Hz, 1H), 6.49 (d,
J=5.6Hz, 1H), 3.34 (m, 4H), 1.88 (m, 2H); δC (100 MHz, DMSOꢀd6) 164.4, 151.7, 150.0, 149.0, 147.4,
m
141.5, 136.2, 133.4, 128.5, 127.3, 126.4, 124.0, 124.0, 117.4, 98.6, 36.8, 27.8; ESIꢀIT MS: /z 411.40
(M+H+) M+ (C21H19ClN4O3) requires 410.11; HPLCꢀDAD: rt = 13.2 min, % area = 96.0.
4-[N-(p-isopropyl)cinnamoylaminopentyl]amino-7-chloroquinoline (10a). White solid (190 mg, 50%);
mp=152ꢀ155oC; RF (DCM/MeOH 8:2) 0.58; δH (400 MHz, DMSOꢀd6) 8.38 (d, J=5.6Hz, 1H), 8.28 (d,
J=9.5Hz, 1H), 8.07 (m, 1H), 7.77 (d, J=2Hz, 1H), 7.36 (m, 7H), 6.56 (d, J=16Hz, 1H), 6.46 (d, J=5.6Hz,
1H), 3.22 (m, 4H), 2.89 (m, 1H), 1.69 (m, 2H), 1.46 (m, 4H), 1.19 (d, J=6.8Hz, 6H); δC (100 MHz,
DMSOꢀd6) 164.9, 151.8, 150.0, 149.8, 148.9, 138.2, 133.3, 132.5, 127.4, 127.3, 126.8, 124.0, 123.9,
121.3, 117.3, 98.5, 42.3, 38.5, 33.2, 28.9, 27.4, 24.0, 23.6; ESIꢀIT MS: m/z (M+H+) 436.47, M+
(C26H30ClN3O) requires 435.21; HPLCꢀDAD: rt = 16.3 min, % area = 98.0.
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4-[N-(p-methoxy)cinnamoylaminopentyl]amino-7-chloroquinoline (10b). White solid (100 mg, 27%);
mp=142.144oC; RF (DCM/MeOH 8:2) 0.58; δH (400 MHz, DMSOꢀd6) 8.38 (d, J=5.6Hz, 1H), 8.28 (d, Jꢀ
9.2Hz, 1H), 8.01 (m, 1H), 7.77 (d, J=2Hz, 1H), 7.39 (m, 4H), 6.96 (d, J=8.8Hz, 2H), 6.47 (m, 2H), 3.78
(s, 3H), 3.22 (m, 4H), 1.69 (m, 2H), 1.47 (m, 4H); δC (100 MHz, DMSOꢀd6) 165.1, 160.1, 151.7, 150.0,
148.9, 138.0, 133.3, 128.9, 127.4, 127.3, 124.0, 123.9, 119.8, 117.3, 114.3, 98.5, 55.1, 42.3, 38.4, 28.9,
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27.4, 24.0; ESIꢀIT MS: /z (M+H+) 424.40, M+ (C24H26ClN3O2) requires 423.17; HPLCꢀDAD: rt = 13.7
min, % area = 100.
(4-[N-(p-chloro)cinnamoylaminopentyl]amino-7-chloroquinoline (10c). Yellowish solid (65 mg,
17%); mp=164ꢀ168oC; RF (DCM/MeOH 8:2) 0.58; δH (400 MHz, DMSOꢀd6) 8.38 (d, J=5.2Hz, 1H), 8.29
(d, J=8.8Hz, 1H), 8.16 (m, 1H), 7.77 (d, J=1Hz, 1H), 7.56 (d, J=8.8Hz, 2H), 7.42 (m, 4H), 7.32 (m, 1H),
6.64 (d, J=16Hz, 1H), 6.44 (d, J=5.2Hz, 1H), 3.22 (m, 4H), 1.68 (m, 2H), 1.51 (m, 2H), 1.41 (m, 2H); δC
m
(100 MHz, DMSOꢀd6); ESIꢀIT MS: /z (M+H+) 428.53, M+ (C23H23Cl2N3O) requires 427.12; HPLCꢀ
DAD: rt = 14.9, % area = 100.
Synthesis of compound 11.
4-[(7-chloroquinolin-4-yl)amino]butan-1-ol (11’). 1 eq of 4,7ꢀdichloroquinoline (0.5g, 2.5mmol) was
mixed with 4 eq of 4ꢀaminobutanꢀ1ꢀol (0.9g, 10mmol) in a round bottom flask, and reaction was allowed
to proceed at 100oC for 4 h. Then, DCM (25 mL) was added, followed by addition of 30% aqueous
Na2CO3 (25 mL), with concomitant precipitation of a light pink solid; the solid was submitted to column
chromatography on silica, using DCM:MeOH (8:2 v/v) as eluent, and pure 11’ was obtained as a white
solid (0.49 g, 78%); mp=171ꢀ174oC; RF=0.26 (DCM/MeOH 8:2); δH (400 MHz, DMSOꢀd6); 8.38 (d,
J=5.2Hz, 1H), 8.27 (d, J=9.2Hz, 1H), 7.77 (d, J=2.4Hz, 1H), 7.42 (dd, J=9.2Hz, 2.4Hz, 1H), 7.32 (m, 1H),
6.44 (d, J=5.6Hz, 1H), 4.51 (b, 1H), 3.46 (m, 2H), 3.26 (m, 2H), 1.69 (m, 2H), 1.54 (m, 2H); δC (100
MHz, DMSOꢀd6) 151.8, 150.0, 149.0, 133.2, 127.4, 124.0, 123.9, 117.4, 98.5, 60.4, 42.2, 30.0, 24.4; ESIꢀ
IT MS: m/z (M+H+) 251.33, M+ (C13H15ClN2O) requires 250.09.
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4-[N-(p-methoxy)cinnamoyloxypropyl]amino-7-chloroquinoline (11). 1 eq of pꢀmethoxycinnamoyl
chloride (0.08 g, 0.4 mmol) was reacted with 1 eq of 11’ (0.1 g, 0.4 mmol) in reluxing DCM (2 mL), in
the presence of 2 eq of triethylamine (112 ꢀL); the solution turned yellow after 10 min and the reaction
ran for 24 h. The organic layer was washed with 5% aqueous Na2CO3 (3 × 2 mL), dried with anhydrous
Na2SO4, filtered, and led to dryness on a rotary evaporator. The residue was submitted to column
chromatography on silica, using DCM:Me2CO (1:1 v/v) as eluent, to yield 11 as a white solid (34 mg,
21%); mp=129ꢀ134oC; RF (DCM/Me2CO 1:1) 0.26; δH (400 MHz, CDCl3) 8.49 (d, J=5.2Hz, 1H), 7.93 (d,
J=2Hz, 1H), 7.71 (d, J=9.2Hz, 1H), 7.63 (d, J=16Hz, 1H), 7.43 (d, J=8.4Hz, 2H), 7.31 (d, J=8.8Hz, 1H),
6.88 (d, J=8.4Hz, 2H), 6.39 (d, J=5.2Hz, 1H), 6.28 (d, J=16Hz, 1H), 5.38 (m, 1H), 4.27 (m, 2H), 3.82 (s,
3H), 3.36 (m, 2H), 1.89 (m, 4H); δC (100 MHz, CDCl3) 167.3, 161.4, 151.7, 149.8, 148.8, 144.7, 134.9,
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129.7, 128.4, 126.9, 125.3, 121.1, 117.1, 115.1, 114.3, 99.0, 63.7, 55.3, 42.9, 26.5, 25.3; ESIꢀIT MS: /z
(M+H+) 411.33, M+ (C23H23ClN2O3) requires 410.14; HPLCꢀDAD: rt = 15.4 min, % area = 97.0.
4-[N-(p-isopropyl)cinnamoylaminopropyl]aminoquinoline (12). Following the experimental procedure
previously described for compounds 9 and 10, and starting from 4ꢀchloroquinoline (0.25 g, 1.5 mmol)
instead of 4,7ꢀdichloroquinoline, 12 was obtained as a beige solid (55 mg, 11%); mp=73ꢀ78oC; RF
(DCM/MeOH 8:2) 0.30; δH (400 MHz, CDCl3) 8.41 (d, J=5.6Hz, 1H), 8.02 (d, J=8.4Hz, 1H), 7.95 (d,
J=8.4Hz, 1H), 7.60 (m, 2H), 7.39 (m, 3H), 7.18 (d, J=8Hz, 2H), 6.52 (m, 1H), 6.46 (d, J=16Hz, 1H), 6.34
(d, J=5.6Hz, 1H), 6.15 (b, 1H), 3.46 (m, 2H), 3.35 (m, 2H), 2.89 (m, 1H), 1.74 (m, 4H), 1.22 (d, J=6.8Hz,
6H) ; δC (100 MHz, CDCl3) 166.7, 150.9, 149.2, 146.6, 141.0, 132.4, 129.7, 128.0, 127.9, 126.9, 125.0,
120.5, 119.7, 118.5, 98.4, 43.1, 39.1, 34.0, 27.8, 25.5, 23.8; ESIꢀIT MS: m/z (M+H+) 388.27, M+
(C25H29N3O) requires 387.23; HPLCꢀDAD: rt = 15.2 min, % area = 99.0.
Synthesis of compound 13.
N1-(pyridin-4-yl)butane-1,4-diamine (13’). 1 eq of 4ꢀchloropyridine (0.5 g, 3.3 mmol) was reacted with
10 eq of butaneꢀ1,4ꢀdiamine (2.9 g, 33 mmol) in a round bottom flask put under reflux for 2 hours; after
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cooling the reaction mixture to room temperature, 25 mL of distilled water were added, and the desired
product was extracted with DCM (3 × 25 mL); the organic layer was dried over anhydrous Na2SO4,
filtered, and led to dryness on a rotary evaporator; this afforded 13’ as a yellow oil (0.22 g, 40%); ESIꢀIT
MS: m/z (M+H+) 166.33, M+ (C9H15O3) requires 165.13. Compound 13’ was used without further
purification in the synthesis of 13.
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4-[N-(p-methoxy)cinnamoylbutyl]aminopyridine (13). In a round bottom flask put at 0 oC, 1.1 eq of pꢀ
methoxycinnamic acid (0.29 g, 1.5 mmol) was dissolved in DMF (2.5 mL) and activated by addition of
1.1 eq of TBTU (0.47 g, 1.5 mmol) and 2 eq of DIEA (454 ꢀL); after 10 min, a solution of 1 eq of 13’
(0.22 g, 1.3 mmol) in DMF (2.5 mL) was added to the previous mixture and the reaction allowed to
proceed for 24 h. Water (25 mL) was added to the reaction mixture and the desired product was extracted
with DCM (3 × 25 mL); the organic extract was then washed with 5% aqueous Na2CO3 (3 × 25mL), dried
over anhydrous Na2SO4, filtered, and taken to dryness on a rotary evaporator; the residue was submitted
to column chromatography on silica, using DCM/MeOH 8:2 (v/v) as eluent, to afford 13 as a white solid
(0.13 g, 30%); mp=132ꢀ136oC; RF=0.13 (DCM/MeOH 8:2); δH (400 MHz, CDCl3) 8.04 (b, 2H), 7.54 (d,
J=16Hz, 1H), 7.33 (d, J=8.8Hz, 2H), 7.28 (m, 1H), 6.77 (d, J=8.8Hz, 2H), 6.36 (m, 3H), 5.14 (m, 1H),
3.73 (s, 3), 3.35 (m, 2H), 3.06 (m, 2H), 1.60 (m, 4H); δC (100 MHz, CDCl3) 166.8, 160.6, 153.7, 149.0,
140.0, 129.1, 127.4, 118.5, 114.1, 107.4, 55.2, 42.1, 39.1, 27.2, 26.0; ESIꢀIT MS: m/z (M+H+) 326.33, M+
(C19H23N3O2) requires 325.18; HPLCꢀDAD: rt = 11.6 min, % area = 100.
Synthesis of compound 14.
N-(N-phthaloyl)aminobutylmorpholine (14’’). 2 eq of morpholine (0.5g, 5.7 mmol) were reacted with 1
eq of 1ꢀ(Nꢀphthaloyl)aminoꢀ4ꢀbromobutane (0.8 g, 2.9 mmol) in refluxing DCM for 24 h, after which the
reaction mixture was washed three times with 5% aqueous Na2CO3, dried over anhydrous Na2SO4,
filtered, and led to dryness in a rotary evaporator; the residue was submitted to column chromatography
on silica, using as eluent DCM:MeOH (8:2 v/v), to afford 14’ as a white solid (0.53 g, 64%); mp=61ꢀ
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63oC; RF=0.67 (DCM/MeOH 8:2); δH (400 MHz, CDCl3) 7.80 (m, 2H), 7.70 (m, 2H), 3.66 (m, 6H), 2.39
(m, 6H), 1.68 (m, 2H), 1.51 (m, 2H); δC (100 MHz, CDCl3) 168.4, 133.8, 132.1, 123.1, 66.9, 58.3, 53.6,
37.8, 26.4, 23.8; ESIꢀIT MS: m/z (M+H+) 289.40, M+ (C16H20N2O3) requires 288.15.
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N-aminobutylmorpholine (14’). 1 eq of 14’’ (0.1 g, 0.30 mmol) was reacted with 50% ethanolic
hydrazine (0.40 mL) for 2 h at room temperature, for removal of the phthaloyl Nꢀprotecting group; water
was then added to the reaction mixture and the desired product was extracted with DCM (3 × 25 mL), the
organic layer was dried over anhydrous sodium sulfate, filtered, and led to dryness on the rotary
evaporator to afford 14’ as a yellowish oil (25 mg, 45%); ESIꢀIT MS: m/z (M+H+) 159.27, M+ (C8H18N2O)
requires 158.14. Compound 14’ was used without further purification in the synthesis of 14.
N-(p-methoxy)cinnamoylaminobutylmorpholine (14). 1 eq of Nꢀaminobutylmorpholine (0.025 g, 0.16
mmol) was immediately used, without further purification, in the coupling with pꢀmethoxycinnamic acid
(0.032 g, 0.17 mmol) carried out as above described for 13 but using PyBOP (0.088 g, 0.17 mmol)
instead of TBTU as coupling reagent, and DCM as a solvent; this yielded 14 as a white solid (32 mg,
61%); mp=92ꢀ94oC; RF (DCM/MeOH 8:2) 0.52; δH (400 MHz, CDCl3) 7.59 (d, J=15.6Hz, 1H), 7.40 (d,
J=8.4Hz, 2H), 7.20 (d, J=8.4Hz, 2H), 6.34 (m, 2H), 3.72 (m, 4H), 3.38 (m, 2H), 2.90 (m, 1H), 2.44 (m,
4H), 2.37 (m, 2H), 1,60 (m, 4H), 1.23 (d, J=6.8Hz, 6H); δC (100 MHz, CDCl3); 160.1, 150.7, 140.7,
m
132.5, 127.7, 126.9, 119.8, 66.9, 58.3, 53.6, 39.5, 34.0, 27.4, 24.0, 23.8; ESIꢀIT MS: /z (M+H+) 331.40,
M+ (C20H30N2O2) requires 330.23; HPLCꢀDAD: rt = 13.2 min, % area = 100.
In vitro assays
Blood stage activity assays. The activity of compounds against cultured P. falciparum was evaluated as
previously reported.11 Briefly, synchronized ringꢀstage W2 strain P. falciparum parasites were cultured
with multiple concentrations of test compounds (added from 1,000× stocks in DMSO) in RPMI 1640
medium supplemented with 0.5% Albumax (Invitrogen, GIBCO) and 100 uM hypoxanthine. After a 48 h
incubation, when control cultures contained new rings, parasites were fixed with 1% formaldehyde in
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PBS, pH 7.4, for 48 h at room temperature and then labeled with 1 nM YOYOꢀ1 (Molecular Probes) in
0.1% Triton Xꢀ100, 100 mM ammonium chloride 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 level of parasitemia of the control relative to inhibitor concentration. In each
case, goodness of curve fit was documented by R2 values of > 0.95.
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Inhibition of β-hematin. The βꢀhematin inhibition assay was performed as previously described.27, 28
Different concentrations (0.1 to 1 mM) of test compounds dissolved in DMSO were added in triplicate to
50 ꢀL hemin chloride dissolved in DMSO (5.2 mg/mL). Negative controls were water and DMSO. βꢀ
hematin formation was initiated by the addition of acetate buffer 0.2M (100 ꢀL, pH 4.4), plates were
incubated at 37ºC for 48 h and they were then centrifuged at 3000 rpm for 15 min (SIGMA 3ꢀ30K). After
discarding the supernatant, the pellet was washed four times with DMSO (200 ꢀL), and finally dissolved
in 0.2 M aq. NaOH (200 ꢀL). The solubilized aggregates were further diluted 1:6 with 0.1 M aq. NaOH
and absorbances were recorded at 405 nm (Biotek Powerwave XS with software Gen5 1.07).
Liver stage infection assays. Inhibition of liver stage infection by test compounds was determined by
measuring the luminescence intensity in Huhꢀ7 cells infected with a firefly luciferaseꢀexpressing P.
berghei line, PbGFPꢀLuccon, as previously described.29 Huhꢀ7 cells, a human hepatoma cell line, were
cultured in 1640 RPMI medium supplemented with 10% v/v fetal calf serum, 1% v/v nonꢀessential amino
acids, 1% v/v penicillin/streptomycin, 1% v/v glutamine and 10 mM 4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineꢀ
ethanesulphonic acid (HEPES), pH 7, and maintained at 37 °C with 5% CO2. For infection assays, Huhꢀ7
cells (1.2 × 104 per well) were seeded in 96ꢀwell plates the day before drug treatment and infection.
Medium in the cells was replaced by medium containing the appropriate concentration of each compound
approximately 1 h prior to infection with sporozoites freshly obtained through disruption of salivary
glands of infected female Anopheles stephensi mosquitoes. Sporozoite addition was followed by
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centrifugation at 1700g for 5 min. At 24 h postꢀinfection, medium was replaced by fresh medium
containing the appropriate concentration of each compound. Inhibition of parasite development was
measured 48 h after infection. The effect of the compounds on the viability of Huhꢀ7 cells was assessed
by the AlamarBlue assay (Invitrogen, UK), using the manufacturer’s protocol.
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In vivo assays
Evaluation of the in vivo antimalarial effects of HEFLECINS. Female Swiss Webster mice (n = 5)
were infected with 106 P. bergheiꢀinfected erythrocytes by ip injection. Two hours later the treatment was
initiated with ip injections of 10, 30 and 100 mg/kg of compounds 7c, 7d, and 7h twice daily for 4 days.
Chloroquine was used as a positive control at 3, 10 and 30 mg/kg ip twice daily. For all murine malaria
experiments, mice were evaluated daily for toxicity and for parasitemia by evaluation of Giemsaꢀstained
blood smears. Animals were sacrificed when significant toxicity was identified or when parasitemias
topped 50%.
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