Optimization of 4-aminoquinoline/clotrimazole-based hybrid antimalarials: Further structure-activity relationships, in vivo studies, and preliminary toxicity profiling

Article abstract of DOI:10.1021/jm300802s

Despite recent progress in the fight against malaria, the emergence and spread of drug-resistant parasites remains a serious obstacle to the treatment of infections. We recently reported the development of a novel antimalarial drug that combines the 4-aminoquinoline pharmacophore of chloroquine with that of clotrimazole-based antimalarials. Here we describe the optimization of this class of hybrid drug through in-depth structure-activity relationship studies. Antiplasmodial properties and mode of action were characterized in vitro and in vivo, and interactions with the parasites 'chloroquine resistance transporter' were investigated in a Xenopus laevis oocyte expression system. These tests indicated that piperazine derivatives 4b and 4d may be suitable for coadministration with chloroquine against chloroquine-resistant parasites. The potential for metabolism of the drugs by cytochrome P450 was determined in silico, and the lead compounds were tested for toxicity and mutagenicity. A preliminary pharmacokinetic analysis undertaken in mice indicated that compound 4b has an optimal half-life.

Full text of DOI:10.1021/jm300802s

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Article
Optimization of 4-Aminoquinoline/Clotrimazole-Based Hybrid
Antimalarials: Further Structure-Activity Relationships,
in vivo Studies, and Preliminary Toxicity Profiling
Sandra Gemma, Caterina Camodeca, Salvatore Sanna Coccone, Bhupendra P Joshi, Matteo Bernetti, Vittoria
Moretti, Simone Brogi, María-Cruz Bonache, Luisa Savini, Donatella Taramelli, Nicoletta Basilico, Silvia
Parapini, Matthias Rottmann, Reto Brun, Stefania Lamponi, Silvio Caccia, Giovanna Guiso, Robert Summer,
Rowena Martin, Simona Saponara, Beatrice Gorelli, Ettore Novellino, Giuseppe Campiani, and Stefania Butini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300802s • Publication Date (Web): 12 Jul 2012
Downloaded from http://pubs.acs.org on July 16, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Optimization of 4-Aminoquinoline/Clotrimazole-
Based Hybrid Antimalarials: Further Structure-
Activity Relationships, in vivo Studies, and
Preliminary Toxicity Profiling
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†,‡,
√
†,‡,
√
†,‡,√
Sandra Gemma,
Caterina Camodeca,
Salvatore Sanna Coccone,
Bhupendra P.
†
,‡,
√
†,‡,√
†,‡,√
†,‡,√
Joshi,
Matteo Bernetti,
Vittoria Moretti,
Simone Brogi,
Maria Cruz Bonache de
†,‡,
√
†,‡,
√
†,#,
√
†,
∇,√
†,#,√
Marcos,
Luisa Savini,
Donatella Taramelli,
Nicoletta Basilico,
Silvia Parapini,
×
Matthias Rottmann, Reto Brun, Stefania Lamponi,
×
†,‡,√
†,∫
∫
Silvio Caccia, Giovanna Guiso,
&
&
†,∞
⊥
Robert Summers, Rowena Martin, Simona Saponara, Beatrice Gorelli, Ettore Novellino,
⊥
,
†,‡,√
†,‡,√
Giuseppe Campiani,*
and Stefania Butini
†
‡
European Research Centre for Drug Discovery and Development (NatSynDrugs) and Dip.
√
Farmaco Chimico Tecnologico, Università di Siena, Via A. Moro, 53100 Siena, Italy; CIRM
#
Centro Interuniversitario di Ricerche sulla Malaria, Università di Torino, Torino, Italy; Dip. di
∇
Scienze Farmacologiche e Biomolecolari, Via Balzaretti 9, 20133 Milan, Italy; Dip. di Scienze
Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Via Pascal 36,
×
2
0133 Milan, Italy; Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel,
∫
Switzerland and University of Basel, CH-4003 Basel, Switzerland; Istituto di Ricerche
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⊥
Farmacologiche “Mario Negri”, Via la Masa 19, 20156 Milano, Italy; Research School of
&
Biology, The Australian National University, Canberra 0200, Australia, and Dip. di
∞
Neuroscienze, University of Siena, via A. Moro, 53100, Siena, Italy; Dip. di Chimica
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Farmaceutica e Tossicologica, Università di Napoli Federico II, Via D. Montesano 49, 80131
Napoli, Italy
*
To whom correspondence should be addressed. Tel.: +390577234172, Fax: +390577234333,
E-mail: giuseppe.campiani@unisi.it
ABSTRACT. Despite recent progress in the fight against malaria, the emergence and spread of
drug-resistant parasites remains a serious obstacle to the treatment of infections. We recently
reported the development of a novel antimalarial drug that combines the 4-aminoquinoline
pharmacophore of chloroquine with that of clotrimazole-based antimalarials. Here we describe
the optimization of this class of hybrid drug through in depth structure-activity relationship
studies. Antiplasmodial properties and mode of action were characterized in vitro and in vivo,
and interactions with the parasite’s ‘chloroquine resistance transporter’ were investigated in a
Xenopus laevis oocyte expression system. These tests indicated that piperazine-derivatives 4b
and 4d may be suitable for co-administration with chloroquine against chloroquine-resistant
parasites. The potential for metabolism of the drugs by cytochrome P450 was determined in
silico and the lead compounds were tested for toxicity and mutagenicity. A preliminary
pharmacokinetic analysis undertaken in mice indicated that compound 4b has an optimal half-
life.
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Introduction
Malaria morbidity and mortality has decreased in most malaria endemic countries over the last
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decade. A key factor contributing to this reduction in the malaria burden has been the
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deployment of artemisinin-based combination therapies (ACTs ) against increasingly drug-
resistant populations of the parasite. However, the efficacy of these peroxide-based antimalarials
is now threatened by the emergence in Southeast Asia of parasites with reduced susceptibility to
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artemisinin.
The development and spread of artemisinin-resistant parasites would be
disastrous, since no drugs are available to replace the ACTs as the frontline antimalarial
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treatment. For these reasons, a number of novel antimalarial strategies are currently being
explored, including combination therapies that are based on agents which reverse resistance to
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-8
chloroquine (CQ, 1, Chart 1).
Chart 1. Structures of Chloroquine (1), CLT-Based Antimalarials (2), Hybrid Derivatives (3a,b)
and Site of Modifications to the Title Compounds (4a-g, 5-8, 9a-m, 10)
During the intra-erythrocytic stage of its life cycle, the parasite takes up host haemoglobin into
a specialized acidic organelle called the digestive vacuole (DV). Proteolysis of the haemoglobin
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results in the release of toxic heme monomers, which are then converted into the inert crystal
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hemozoin.
CQ exerts its antimalarial effects through the inhibition of hemozoin
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biocrystallization and the build up of free heme monomers.
This mode of action is highly
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effective, and because the target cannot be mutated, the development of resistance is not a feat
easily achieved by the parasite. The molecular determinants of CQ resistance have been
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,17
identified and the mechanism of resistance characterised.
CQ-resistant (CQR) parasites
accumulate much less CQ in their DV than CQ-sensitive (CQS) parasites, and there is now a
general consensus that this decrease in CQ accumulation is due to the efflux of the drug via
mutant forms of the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT;
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17,18
located on the DV membrane).
The wild-type form of this protein does not transport CQ.
Compounds that compete with CQ transport via mutant PfCRT, or that block PfCRT-mediated
transport, partially restore the parasite’s sensitivity to CQ in vitro. This phenomenon of ‘CQ
resistance reversal’ provides a rational basis for the study and implementation of CQ-based
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combination therapies for the treatment of drug-resistant malaria.
Our approach to the development of novel antimalarials has been to design compounds that
interact with the iron centre of free heme within the acidic environment of the DV (pH ~5.5), and
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which produce putative radical species that are selectively toxic to the parasite.
A further
requirement is the ability to accumulate within the DV via weak-base trapping (the mechanism
by which CQ and other quinoline antimalarials reach high concentrations inside this
compartment). Following this rationale, we previously developed a novel class of antimalarials
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7,28
based on the structure of clotrimazole (CLT), typified by the trityl derivative 2 (Chart 1).
This class of CLT-based antimalarial was further optimized using a pharmacophore
hybridization approach in which the polyarylmethyl system of 2 was hybridized with the 4-
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aminoquinoline iron-complexing moiety of CQ, leading to 3a,b as the representative compounds
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of this class. The in vitro and in vivo antimalarial activities of 3a,b as well as their mode of
action, toxicity, and preliminary pharmacokinetic properties have been evaluated with promising
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results. Here we extend our previous findings by characterizing this novel class of drug in
greater detail. A set of 3a,b analogues were synthesized by modifying the protonatable
heterocycles at the benzhydryl moiety (4a-g, 5, 8, Chart 1 and Tables 1,2), the secondary amine
tether (6, 7, Table 2), and by introducing electron-withdrawing or -releasing groups at the
benzhydryl aromatic ring (in ortho position relative to the protonatable side-chain; 9a-m, 10,
Table 3). The antiplasmodial activities of the resulting compounds were measured in both CQS
and CQR parasites. The most promising compounds were then evaluated in a mouse malaria
model and tested for the ability to inhibit PfCRT-mediated CQ transport in a heterologous
expression system. The toxicity profile of the lead compounds was explored and a preliminary
pharmacokinetic evaluation of 4b undertaken.
Chemistry
The synthesis of compounds 4a-g, bearing different protonatable heterocycles at the
benzhydryl system is reported in Scheme 1. Benzyl bromides 11a,b, prepared as previously
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7,28
described,
were used to alkylate imidazole (pre-treated with sodium hydride) to afford 12, or
N-Boc-piperazine to afford intermediates 14a,b. Reduction of 12 and 14a,b with sodium
borohydride was followed by activation of the resulting alcohols 13 and 15a,b into the
corresponding chlorides. These latter compounds were reacted with the appropriate 4-
aminoquinoline intermediates to furnish 4a and, after deprotection with hydrochloric acid in
methanol, 4b-d. Compound 4b was submitted to a reductive amination protocol using
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formaldehyde, benzaldehyde or ferrocenyl aldheyde to afford N-alkylpiperazine derivatives
4e,f,g, respectively.
a
Scheme 1. Synthesis of Compounds 4a-g
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a
Reagents and conditions: (a) NaH, imidazole, N,N-DMF, 25 °C, 1 h, then 11a, 25 °C, 1 h; (b)
NaBH , EtOH, 0 °C, 1 h; (c) i. SOCl , CH Cl , 0 °C 20 min, then 45 °C, 4 h; ii. 7-chloro-4-
aminoquinoline, Et N, MeCN, 80 °C, 6 h; (d) N-(t-butoxycarbonyl)piperazine, Et N, 0 °C, 1 h;
e) i. SOCl , CH Cl , 0 °C, 20 min, then 45 °C, 4 h; ii. 7-chloro-4-aminoquinoline or 6-methoxy-
4-aminoquinoline, Et N, MeCN, 80 °C, 6 h; iii. AcCl, MeOH, 25 °C 2 h; (f) from 4b, CH O 37%
in water, 1% AcOH, EtOH, 25 °C, 6 h; (g) from 4b, benzaldehyde (for 4f) or ferrocenyl aldehyde
for 4g), 1% AcOH, EtOH, 25 °C, 6 h.
4
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N,N’-Disubstituted piperazino derivative 5 was prepared as described in Scheme 2. The benzyl
bromide 11a was treated with piperazine to afford dimeric benzophenone intermediate 16. This
latter compound was converted into the corresponding chloride 17 through
reduction/chlorination reactions and, following the procedure described in Scheme 1, reacted
with 7-chloro-4-aminoquinoline to furnish the final compound 5.
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Scheme 2. Synthesis of dimeric derivative 5
a
Reagents and conditions: (a) piperazine, Et N, 0 °C, 1 h; (b) i. NaBH , EtOH, 0 °C, 1 h; ii.
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SOCl , CH Cl , 0 °C, 20 min, then 45 °C, 4 h; (c) 7-chloro-4-aminoquinoline, Et N, MeCN, 80
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°C, 6 h.
Compounds 6 and 7, possessing a piperazine tether were prepared as reported in Scheme 3.
Carbinols 18a,b were converted in their corresponding chlorides and immediately reacted with
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4
-piperazino-7-chloroquinoline 19, in the presence of triethylamine in acetonitrile heated under
reflux to afford final compounds 6 and 7.
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a
Scheme 3. Synthesis of Compounds 6,7
a
Reagents and conditions: (a) i. SOCl , CH Cl , 0 °C, 20 min, then 45 °C, 4 h; ii. Et N, MeCN,
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80 °C, 6 h.
The key intermediates for the synthesis 8 and 9a-m (Scheme 4) were benzhydrylamines 24a-g,
which were obtained following a modification of the previously reported methodology. Reaction
of Grignard’s reagents obtained from arylbromides 21a,b with excess aldehydes 20a,b afforded
benzophenones 22a-d. Benzophenone 22e was obtained by reacting p-chlorophenylmagnesium
bromide with aldehyde 26, in turn obtained from the corresponding ester 25 through standard
procedures. Benzophenones 22a-e were functionalized through a radical-promoted bromination
followed by alkylation of pyrrolidine or N-Boc-piperazine furnishing intermediates 23a-f.
Ketones 23a,b,d-g, containing pyrrolidine or morpholine basic moieties, were converted into the
corresponding amines 24a,b,d-g through conversion to oximes followed by reduction with zinc
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dust. N-Boc-piperazine derivative 23c was converted to 24c by treatment with ammonia/Ti(IV)
isopropoxide followed by NaBH -mediated reduction.
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Scheme 4. Synthesis of Compounds 8 and 9a-m
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Reagents and conditions: (a) i. 21a,b, Mg turnings, THF, 80 °C, 1 h, then 20a,b, 80 °C, 4 h;
(b) i. NBS, AIBN, CCl , reflux, 4 h; ii. amine, Et N, MeCN, 0 °C, 1 h; (c) from 23a,b,d-g: i.
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NH OH·HCl, BaCO , MeOH, reflux, 18 h; ii. Zn, AcOH, 50 °C, 18 h; from 23c: 7 M NH in
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MeOH, Ti(O-i-Pr) , EtOH, 25 °C, 6 h; then NaBH , 25 °C, 3 h; (d) 4-chloroquinolines,
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pyridinium chloride, 2-ethoxyethanol, 135 °C, 18 h; (e) i. LiAlH , THF, 25 °C, 1h; ii. MnO ,
4
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THF, 85 °C, 2h; (f) p-chlorophenylmagnesium bromide, THF, 85 °C, 6 h.
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Reaction of benzhydrylamines 24a,b,d-g with the appropriate 4-chloroquinolines in
ethoxyethanol in the presence of pyridinium chloride resulted in the formation of final
compounds 9a-m. Under the same reaction conditions, 24c gave rise to compound 8 as the sole
reaction product, probably resulting from the in situ deprotection of the Boc-piperazine moiety.
Finally, trityl derivative 10 was prepared as described in Scheme 5. Reaction of the Grignard
reagent prepared in situ from arylbromide 21a was reacted with benzoic acid methyl ester 27 to
afford carbinol 28. The carbinol was submitted to a bromination reaction in the presence of N-
bromosuccinimide (NBS) and a catalytic amount of AIBN to afford the corresponding bis-benzyl
bromide, which was in turn reacted with pyrrolidine. The resulting alcohol 29 was converted into
the corresponding chloride by exposure to thionyl chloride and reacted with 7-chloro-4-
aminoquinoline to afford compound 10.
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a
Scheme 5. Synthesis of Compound 10
a
Reagents and conditions: (a) 21a, Mg, THF, 25 °C, 2 h, then 27, THF, 25 °C, 18 h; (b) i. NBS,
AIBN, CCl , 85 °C, 2 h; ii. pyrrolidine, Et N, MeCN, 0 °C to 25 °C, 3 h; (c) i. SOCl , CH Cl , 0
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C, 20 min, then 45 °C, 4 h; ii. 7-chloro-4-aminoquinoline, Et N, MeCN, reflux, 6 h.
3
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Results and Discussion
. In vitro Antiplasmodial Activity and Structure-Activity Relationships (SARs). All of the
1
synthesized compounds were tested in vitro as racemates against the P. falciparum CQS strains
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D10 and NF54, and the CQR strains W2 and K1. Antimalarial activity (IC , nM) was quantified
5
0
as inhibition of parasite growth, measured by the production of parasite lactate dehydrogenase
3
(
pLDH; D10 and W2 strains) or the incorporation of [ H]hypoxanthine into parasite DNA (NF54
and K1 strains). The results are presented in Tables 1-3.
2
6
In our previous studies we hypothesized that, within the acidic environment of the DV the
quinoline endocyclic nitrogen of compounds such as 3a,b coordinates the iron of free heme,
eventually leading to an electron-transfer reaction to form putative radical intermediates.
Consistent with this view, we previously observed that the electron density, the quinoline
endocyclic nitrogen, and the dipole moment of the quinoline ring system all appeared to affect
2
6
the heme-quinoline interaction/reactivity. We also found that the protonatability of the
2
5-28
molecule contributes significantly to its ability to kill the parasite.
CQ and other weak bases
diffuse into the DV in their neutral form, whereupon they become protonated in the acidic
environment and these charged species, unable to cross the membrane unassisted, accumulate to
high levels within the DV. With this putative mode of action in mind, and in light of our previous
findings, the SARs of the analogues described here focused on the effect of structural
modifications on the protonatability of the molecule, and on the electron-distribution of the
benzhydryl and quinoline systems.
(a) Modification of the protonatable side chain at the benzhydryl system. The
antiplasmodial activities of a series of compounds (4a-g) with different protonatable heterocycles
at the benzhydryl system are presented in Table 1. We used heterocycles such as imidazole and
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9
piperazines since these moieties are protonatable at pH 5.5 and are able to coordinate metals such
as iron (a property that was previously observed to increase the activity of our molecules against
25,30
CQR strains
). When the protonatable heterocycle at the benzhydryl system was coupled to
10
11
12
13
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49
50
51
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53
54
55
56
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59
60
the 7-chloro-4-aminoquinoline moiety (4a,b,d-f), the resulting compounds were predicted to
share similar protonation states at both DV and blood pH values (Table 1). However, subtle
differences in their antiplasmodial activities were observed. The imidazole derivative 4a and its
pyrrolidine analogue 3a both appeared to exhibit low-level cross-resistance with CQ, particularly
when the D10 and W2 strains were compared; 4a and 3a have resistance index (RI) values of
~
1.9 and 1.6, respectively. The RI is the IC value in the CQR strain divided by the IC value in
50 50
the CQS strain. Hence, a RI close to one indicates that the antiplasmodial activity of the
compound is comparable between the CQS and CQR strains. The piperazine analogue 4b was
slightly less active than 3a against the D10 and W2 strains, but had an RI of ~1, indicating that
the CQ-resistance mechanism has little effect on the antiplasmodial activity of this compound.
Compound 4c also contains an un-substituted piperazine moiety, but showed consistent cross-
resistance with CQ (RIs of 2.4 and 2.8). This indicates that both the quinoline moiety and the
heterocyclic side chain play a role in determining whether a compound displays cross-resistance
with CQ. Furthermore, compounds 4f (in which the piperazine moiety presents a N4-benzyl
substituent), 4g (which contains the ferrocenylmethyl-substituted piperazine) and 5 (a dimeric
analogue of 4b) are less active than the other analogues. It is worth noting that compounds 4g
and 5 have RI values lower than one. This suggests that certain modifications to the benzhydryl
side chain can yield compounds that are considerably more active against the CQR strains W2
and K1 than the CQS strains D10 and NF54. Given this apparent trade-off between cross-
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
resistance and absolute level of activity, 4b and 4d appear to have the best balance of these traits
within this piperazine series.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Ionic Forms and Antiplasmodial Activity of Compounds 4a-g, 5, 3a,b and CQ (1).
a
b
c
b
c
%
Ionic forms D10 W2
NF54 K1
d
RI
e
RI
Cpd R1
R2
R3
f
f
pH 7.4 pH 5.5 IC (nM)
IC (nM)
5
0
50
4
4
4
a
b
c
4-Cl 7-Cl
4-Cl 7-Cl
4-Cl 6-OMe
3-Cl 7-Cl
4-Cl 7-Cl
4-Cl 7-Cl
4-Cl 7-Cl
DP 1
P 20
N 79
DP 70 30
P 28
N 2
57
58
1.9 18
0.93 17
22 1.2
22 1.3
58 2.8
23 1.2
DP 4
P 94
N 2
DP 75 62
P 25
N 0
DP 59 DP 99 67
P 39
N 1
163 2.4 21
P 1
N 0
4d
DP 3
P 94
N 3
DP 74 25
P 26
N 0
33
42
1.4 19
2.0 nt
4
4
4
e
DP 2
P 64
N 34
DP 74 21
P 26
N 0
nt
nt
nt
-
-
-
f
DP 2
P 36
N 62
DP 72 163 790 4.8 nt
P 27
N 1
g
nc
nc
93
33
0.35 nt
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5
-
-
-
TP 0
DP 2
P 31
N 67
TP 53 190 98
DP 39
P 8
N 0
0.5 89
56 0.6
3a
-
-
-
-
-
-
-
-
-
DP 3
P 96
N 1
DP 73 46
P 27
N 0
72
22
1.5 13
1.0 12
14 1.1
16 1.3
154 22
10
11
12
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60
3
b
DP 3
P 96
N 1
DP 73 21
P 27
N 0
1
DP 8
P 92
DP 87 22
P 12
280 12
7.0
a
Percentage of ionic form in parentheses. DP = diprotonated form. P = monoprotonated form.
N = neutral form (ACD/pKa DB, version 11.00, software, Advanced Chemistry Development,
b
c
d
Inc., Toronto, Canada). CQS clone. CQR clone. Resistance index = W2-IC /D10-IC .
5
0
50
e
f
Resistance index = K1-IC /NF54-IC . IC values are the mean of at least three
5
0
50
50
determinations. Standard errors were all within 10% of the mean.
(b) Piperazine-tethered derivatives. We briefly investigated the antiplasmodial activity of
compounds 6 and 7 (Table 2) – hybrid derivatives that combine the 7-chloro-4-piperazinyl
moiety of piperaquine with the polyarylmethyl system of the previously described CLT-based
antimalarials. Compounds 6 and 7 showed poor activity against all of the strains examined. This
finding is consistent with the idea that for this class of compounds, the conjugation between the
polyarylmethyl system and the iron coordinating and electron transfer moiety (4-aminoquinoline)
2
6,27
is necessary for optimal activity.
c) Effect of a third electron-withdrawing or -releasing substituent at the benzhydryl
system. In the previously reported pharmacokinetic characterization of 3b, the formation of at
(
2
6
least three metabolites was observed. This led us to undertake an in silico approach to identify
potential cytochrome P450 (CYP)-mediated sites of metabolism in the lead compounds 3a and
3b. From these analyses, which are described in the next section, the pyrrolidinylmethylene
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5
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5
6
moiety and the aromatic ring bearing the protonatable side chain emerged as potential soft spots
for the CYP3A4 isoform.
0
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3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
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9
0
Table 2. Antiplasmodial Activity of 6-8 and CQ (1).
a
b
a
b
D10 W2 NF54 K1
Cpd
c
IC (nM)
5
0
6
7
8
1
631 735 88
277 582 91
156
92
104 58
83
10
22 280 7.0
154
a
b
c
CQS clone. CQR clone. IC values are the mean of at least three determinations. Standard
50
errors were all within 10% of the mean.
We therefore tested whether these potential sites of metabolism could be masked by the
introduction of additional electron-withdrawing or -releasing groups into the benzhydryl side
chain. The biological activity of the resulting compounds (9a-m) was evaluated in D10, W2,
NF52, and K1 parasites (Table 3). Introduction of halogens (Cl and F, substituent X in Table 3)
into the parent compounds 3a and 3b to obtain compounds 9a,b,h,i had little effect on
antiplasmodial activity, and in the case of compounds 9b and 9h, resulted in improved potency
against D10 parasites, albeit with a concomitant increase in cross-resistance with CQ.
The introduction of F, Cl, or a OMe-group is not predicted to influence the protonatability of
the neighbouring basic chain (e.g. 9a,b vs 3b; 9h,i,m vs 3a). Compounds possessing a
pyrrolidine proved to be more potent than compounds carrying a less basic morpholine in the
protonatable side chain (9c vs 9a or 9f vs 9d). Moreover, the 7-chloro-4-aminoquinoline moiety
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9
(
9a,b,h,i,m) was found to impart greater antiplasmodial activity than the 6-methoxy- or the 7-
trifluoromethyl-4-aminoquinoline moieties (9d,e,j,k, and 9g,l, respectively).
10
11
12
13
14
15
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51
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53
54
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57
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59
60
Table 3. Ionic Forms and Antiplasmodial Activity of Compounds 9a-m, 10, 3a,b and CQ (1).
a
b
c
b
c
%
Ionic forms
D10 W2 NF54 K1
Cpd R1
R2
R3
X
d
pH 7.4 pH 5.5
IC (nM)
50
9a
3-Cl
7-Cl
Cl
DP 3
P 96
N 1
DP 69 57
P 31
N 0
50
25
13
nt
19
nt
9
b
c
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
7-Cl
7-Cl
F
DP 3
P 96
N 1
DP 69 9.0
P 31
N 0
9
Cl
DP 0
P 7
N 93
DP 54 442 277 47
P 40
N 6
107
34
9d
6-OMe Cl
6-OMe F
6-OMe Cl
DP 53 DP 99 56
P 46
N 1
90
27
P 1
N 0
9e
DP 53 DP 99 41
P 46
N 1
101 nt
nt
P 1
N 0
9f
DP 2
P 55
N 43
DP 77 198 229 81
P 23
N 0
114
46
9g
7-CF
3
Cl
DP 0
P 98
N 2
DP 26 74
P 74
N 0
111 34
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
h
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
-
7-Cl
7-Cl
Cl
F
DP 3
P 96
N 1
DP 70 8.4
P 30
N 0
28
45
76
69
76
48
72
22
52
nt
nt
9i
DP 3
P 96
N 1
DP 70 59
P 30
N 0
13
nt
20
nt
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
9
j
6-OMe Cl
6-OMe F
DP 54 DP 99 24
P 45
N 1
P 1
N 0
k
DP 54 DP 99 58
P 45
N 1
25
20
nt
33
33
nt
P 1
N 0
9l
7-CF
3
F
DP 0
P 98
N 2
DP 99 51
P 1
N 0
9m
7-Cl
OMe DP 3
P 96
DP 73 31
P 26
N 0
N 1
3a
-
-
-
-
-
-
-
-
-
-
-
DP 3
P 96
N 1
DP 73 46
P 27
N 0
13
12
9.5
14
16
8.4
154
3
1
1
b
-
DP 3
P 96
N 1
DP 73 21
P 27
N 0
0
-
TP 1
DP 96 DP 53
P 3 P 0
TP 47 26
-
22
280 7.0
a
Percentage of ionic form in parentheses. DP = diprotonated form. P = protonated form. N =
neutral form (ACD/pKa DB, version 11.00, software, Advanced Chemistry Development, Inc.,
b
c
d
Toronto, Canada). CQS clone. CQR clone. IC values are the mean of at least three
5
0
determinations. Standard errors were all within 10% of the mean.
Some differences in antiplasmodial potency were observed between the o-Cl and o-F
analogues (substituent X of Table 3). For example, o-Cl derivative 9a was less potent than the
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corresponding o-F analogue 9b, but the reverse was true for compounds 9h and 9i. Yet these
compounds have similar protonation states (at both pH 7.4 and 5.5) and similar iron interacting
groups. We previously found that both the electron density at the endocyclic quinoline nitrogen
and the polarization of the quinoline ring are also important determinants of potency against
10
11
12
13
14
15
16
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18
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53
54
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60
2
6
malaria parasites. We therefore performed ab initio calculations to predict the electron
distribution of compounds 9a,b,h,i, (Figure 1 of the Main Text and Figure S1 of the Supporting
Information).
3
1
Figure 1. ESP charges from HF/6-31G++* calculations performed in vacuum by Jaguar for
compounds 9a,b,h,i. The compounds are represented by tube (pictures were generated by
3
2
Maestro ).
While all four of the compounds possessed comparable electron-densities at the quinoline
endocyclic nitrogen, the most potent compounds (9b and 9h, Figure 1) were more polarised
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
along the 4-aminoquinoline moiety (from the exocyclic nitrogen to the endocyclic nitrogen)
relative to 9a. This effect was most evident for 9i (Figure 1), for which the polarization of the 4-
aminoquinoline ring is reversed compared with 9b and 9h (Figure 1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2. In silico prediction of potential sites of metabolism. Oral administration of 3b in rats
2
6
resulted in at least three putative metabolite peaks in the plasma chromatograms, which
indicates that this compound undergoes extensive first-pass biotransformation. We therefore
performed an in silico study of the potential sites of metabolism (soft spots) for our lead
compounds, with the aim of synthesizing new derivatives with functionalities that mask the
33
identified soft spots. We used the P450 Site of Metabolism prediction workflow (implemented
3
2
in Maestro suite 2011 ) to predict metabolism mediated by isoforms CYP3A4, CYP2C9 and
3
4
CYP2D6. The prediction for isoforms CYP2C9 and CYP2D6 combined induced-fit docking
(which determines accessibility of the compounds to the CYP reactive centre) with a rule-based
3
5
approach to intrinsic reactivity. In the case of CYP3A4, which has a highly flexible binding
site, only intrinsic reactivity was used to predict soft spots. In silico predictions were performed
for each enantiomer of the most promising compounds identified in this study (see Table S1 of
the Supporting Information). We focused on addressing the potential soft spots located at the
benzhydryl system and at the protonatable side chain, as even small modifications of the 4-
aminoquinoline ring can be detrimental to antimalarial activity. Putative CYP3A4-mediated soft
spots in the benzhydryl system were mainly localized to the protonatable chain (A,B) and the
ortho position of the aromatic ring bearing the side chain (C) (e.g., 3b (Figure 2) and 3a (Figure
S2 of the Supporting Information). Predicted CYP2C9 and CYP2D6-mediated soft spots were
also mainly localized to the benzylic (A) and pyrrolydine (B) rings for both 3a and 3b.
Moreover, 3b contained a further soft spot (D) on the second aromatic ring.
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Structures of (R)- and (S)-enantiomers of compounds 3b and 9a, annotated on the basis
of predicted CYP-mediated soft spots (A-D). The number of rays indicates the value of the Fe-
accessibility from IFD. Each full ray represents a unit of accessibility, with the length of the final
ray representing the decimal portion. Size of the circles: overall SOM score; the color of the
circle indicates the atomic intrinsic reactivity – the redder the circle, the higher the intrinsic
reactivity; a blue ring around the circle indicates that the atom passed through the CYP filtering
stage. No ring indicates that the atom failed the CYP filtering stage.
Introduction of a masking functionality in B led to compounds 9a and 9h, respectively. The
presence of the Cl atom in 9a was predicted to reduce CYP3A4-mediated metabolism and lead to
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2
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2
2
2
2
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3
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3
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3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
more stable analogues (Figure 2). Similar results were obtained for 9h and the F-substituted
analogues 9b,i (Table S1 of the Supporting Information).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Representative binding poses of compounds 3b (panel A) and 9a (panel B) in the
2C9 binding site. The distance of pyrrolidine carbons from the iron center is shown in magenta.
The references compounds and heme group were represented by ball and stick (pictures were
3
2
generated by Maestro ).
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9
The presence of the Cl atom also affected the accessibility of positions A and B of both
enantiomers of 9a and 9h to the iron centre of the CYP 2C9 binding site. Representations of 3b
and 9a inside CYP2C9 (Figure 3) depict how the Cl atom at position C is expected to reduce the
accessibility of positions A and B to the iron centre.
10
11
12
13
14
15
16
17
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30
31
32
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34
35
36
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41
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3. Inhibition of β-Hematin formation. Interaction with free heme is predicted to be key to the
mode of action of these antimalarials. In order to confirm this activity, compound 4b – one of the
most potent compounds identified in this study – was tested using the β-hematin inhibitory
activity (BHIA) assay. As expected, compound 4b exhibited dose-dependent inhibition of β-
hematin formation (Table 4), with an IC value of 0.67. 4b was found to be more potent than 3b
5
0
in the BHIA assay, yet these two compounds possess similar levels of antiplasmodial activity.
However, previous studies of related compounds have observed that activities measured in the
2
4
BHIA assay do not always match those measured in parasite growth assays. Aside from subtle
differences in the pharmacokinetic properties of 4b and 3b, the data presented here, together with
28
previous findings on similar compounds, indicate that an interaction with free heme is
necessary for antiplasmodial activity, but the data also suggests that an additional mode of action
may contribute to the activity of this class of compound.
Table 4. β-Hematin Inhibitory Activity Assay of Compounds 3b, 4b and CQ (1).
a
Cpd
IC50
4
3
1
b
0.67
1.33
1.69
b
, CQ
a
The IC50 represents the molar equivalents of compound, relative to hemin, that inhibit β-
hematin formation by 50%. Data are the mean of three different experiments in triplicate.
Standard errors were all within 10% of the mean.
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3
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3
3
3
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4
4
4
4
4
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5
5
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5
5
5
5
5
6
4
. In vivo antimalarial activity against Plasmodium berghei. The in vivo antimalarial
activities of compounds 4b and 4d were evaluated using a P. berghei mouse model. Groups of
five mice were infected with P. berghei and treated orally with compounds 4b,d, 9h and 10,
using a range of dosage regimens (summarized in Table 5). The key measurements were (1) the
percentage of parasitemia reduction compared to the untreated mice and (2) the prolongation of
survival of the infected mice. All compounds drastically reduced parasitemia (>99%). Notably
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
b and 4d prolonged mouse survival substantially with a single oral dose of 30 mg/kg. Partial
cure was achieved with several doses (e.g., 3x30 mg/kg), and administration of 3 or 4 doses of
d extended the survival of all of the mice until day 30. However, for reasons that are currently
4
unclear, full cure was not observed.
Table 5. In vivo efficacy in a P. berghei rodent malaria model
Cured/
Dosage
%
Average mice
Cpd
a
b
c
Treated
Mice
(mg/kg) Activity survival days
d
4
b
1x100
99.5
99.7
99.8
99.8
99.8
99.6
99.7
99.8
99.8
99.9
99.5
28.8
15.8
29.6
29.0
30.0
29.6
24.0
30.0
30.0
30.0
14.0
2/5
0/5
3/5
1/5
0/5
3/5
1/5
2/5
3/5
3/5
0/3
1
3
3
4
x30
x50
x30
x30
4d
1x100
1x30
3x50
3x30
4x30
1x100
e
9h
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Page 25 of 72
Journal of Medicinal Chemistry
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2
3
4
5
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9
1
x30
1x100
x30
99.4
99.3
99.4
7.7
0/3
0/3
0/3
e
10
21.3
7.0
1
a
compounds were formulated in Ethanol/Tween 80/water 3/7/90. Data are shown as means
with n= 5 mice. % Activity = average parasitemia reduction; Survival of control animals: 6-7
days; Cure = no parasites present at day 30; n=3 mice
10
11
12
13
14
15
16
17
18
19
20
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22
23
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30
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60
b
c
d
e
5
. Inhibition of CQ transport via PfCRT by benzhydryl quinolines. To determine whether
members of this series of compounds interact with a resistance-conferring form of PfCRT
CQR
(PfCRT ), we tested a representative set of derivatives (2, 3a,b, 4b,d,e,g, 6, 7, 9b,h,i and 10),
for the ability to inhibit CQ transport via this protein. Expression of PfCRT in Xenopus oocytes
CQR
enables direct measurements of PfCRT -mediated CQ transport, and thus its inhibition by
potential resistance-reversers or quinoline-based antimalarials. The direction of CQ transport in
the PfCRT expression system is from the mildly acidic extracellular medium (pH 6.0) into the
oocyte cytosol (pH 7.2), which corresponds to the efflux of CQ from the acidic DV (pH 5.5) into
the parasite cytosol (pH 7.3). A key advantage of the oocyte system is that it allows interactions
with PfCRT to be studied directly and in isolation, without confounding effects such as the
binding of drugs to heme or interactions of the compound with other targets or transporters. In an
initial experiment in which the derivatives were tested at an extracellular concentration of 100
CQR
µM, all twelve compounds inhibited CQ transport via PfCRT
(Figure 4 A; P < 0.001,
ANOVA). None of the compounds affected the diffusion of CQ into oocytes expressing
CQS
CQR
PfCRT
(P > 0.05, ANOVA). The fact that compounds 3a,b, and 9b,h,i inhibited PfCRT
-
mediated CQ transport to a similar extent (70-80% reduction in CQ uptake) indicates that
changes to the benzhydryl aromatic ring or the distal chlorinated benzyl group have little effect
CQR
on the ability of these compounds to interact with PfCRT . For comparison, the CQ resistance-
CQR
reverser verapamil reduced CQ transport via PfCRT
by ~74%. The trityl derivatives 6, 7 and
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Journal of Medicinal Chemistry
Page 26 of 72
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0 decreased CQ uptake by 58, 42, and 72%, respectively, which suggests that the substrate
CQR
binding-site of PfCRT
can tolerate large, bulky residues. Compounds 4b and 4d abolished
CQR
CQ uptake via PfCRT
when present at 100 µM; under these conditions, CQ accumulation in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CQS
CQR
oocytes expressing PfCRT
was not significantly different from that measured in PfCRT
-
expressing oocytes (P > 0.05, ANOVA). The ability of the piperazine derivatives (4b,d,e,g) to
CQR
inhibit PfCRT
appeared to decrease as the bulkiness of the terminal amine substituent was
increased. Hence, this position may play an important role in influencing the affinity of
CQR
CQR
interactions with PfCRT . An analysis of the concentration-dependent inhibition of PfCRT
by 4b and 4d (Figure 4 B and C) yielded IC50 values of 10.3 ± 0.9 and 10.5 ± 1.0 µM,
respectively. These values are significantly lower than that measured previously for verapamil
(30 ± 1 µM; P < 0.005, unpaired t-test) and are similar to the IC50 value obtained for the
CQR
antiretroviral drug saquinavir (13 ± 0.9 µM) – the most potent PfCRT
inhibitor reported to
3
6
date. It should be noted that the micromolar concentrations of the compounds used here to
CQR
inhibit PfCRT
are physiologically relevant, given that dibasic compounds are expected to
accumulate to high micromolar, and even millimolar, concentrations within the acidic
environment of the parasite’s DV via weak-base trapping. The ability of 4b and 4d to block
CQR
PfCRT -mediated transport, coupled with their intrinsic antiplasmodial activity, makes these
compounds good candidates for use in combination with CQ against CQR malaria. Since
halogenation of the benzhydryl group did not affect the extent to which the compounds inhibit
CQR
PfCRT
(c.f. 3a and 3b with 9b, 9h and 9i; Figure 4A), it is possible that the same
modification would improve the metabolic stability of 4b and 4d without diminishing their
CQR
ability to block PfCRT
.
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Page 27 of 72
Journal of Medicinal Chemistry
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43
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51
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53
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60
CQR
3
Figure 4. Inhibition of PfCRT -mediated CQ transport by benzhydryl quinolines (A) [ H]CQ
CQR
CQS
uptake into oocytes expressing Dd2 PfCRT
presence of 100 µM each of the eleven antimalarials tested. All compounds caused significant
reductions in CQ uptake via PfCRT
resistance-reverser verapamil is represented as a solid line with s.e.m. from n = 4 represented by
grey dashed lines. Concentration-dependent effects of 4b (B) and 4d (C) on the uptake of
[ H]CQ into oocytes expressing Dd2 PfCRT
The IC values derived from these data for 4b and 4d (10.3 ± 0.9 µM and 10.5 ± 1.0 µM (mean
s.e.m; n = 5), respectively) were obtained by least-squares fit of the equation Y = Y + [(Ymax
min
(black bars) or D10 PfCRT
(grey bars) in the
CQR
(P < 0.001; ANOVA). The level of inhibition by the CQ
3
CQR
CQS
(closed circles) or D10 PfCRT
(open circles).
5
0
±
–
C
CQR
Y )/(1 + ([inhibitor]/IC ) ], where Y is PfCRT
the minimum and maximum values of Y, and C is a constant. PfCRT -mediated CQ transport
mediated CQ transport, Y and Ymax are
min
50
min
CQR
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Journal of Medicinal Chemistry
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CQS
was calculated by subtracting the uptake measured in oocytes expressing D10 PfCRT
from
CQR
that in oocytes expressing Dd2 PfCRT . In all panels uptake is shown as the mean ± s.e.m.
from 4-5 separate experiments, within which measurements were made from 10 oocytes per
CQS
3
treatment. Note that non-injected oocytes and oocytes expressing PfCRT
take up [ H]CQ to
similar (low) levels via simple diffusion of the neutral species; this represents the “background”
level of CQ accumulation in oocytes (refer to Ref. 17 for full data and a detailed discussion).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
. In vitro toxicity assays. Cytotoxicity assays were performed on the lead compounds to
investigate their effects on cell viability in vitro. IC50 values against both human fibroblast
NIH3T3) and hepatic (HepG2) cell lines are presented in Table 6. The data indicate that all of
(
the compounds tested have low cytotoxic potential, although the IC50 values obtained with
hepatocytes are for the most part lower than those obtained with fibroblasts.
Table 6. Cytotoxic activity against 3T3 and HepG2 cell lines of 4a,b,d, 9d,i,l,h, 10 and
reference compounds 3b, CQ and CLT.
3
T3
HepG2
Cpd
TC (ꢀM)
5
0
4
4
4
9
9
a
b
d
d
i
35
1.8
16
24
25
34
16
15
9.0
22
1.8
< 4
12
12
9l
19
9h
10
3b
4.0
6.1
4.3
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Page 29 of 72
Journal of Medicinal Chemistry
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9
CQ
>80
>48
31
CLT
>48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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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
7. Mutagenicity assay: Ames test. As summarized in Table 7, compounds 4a,b,d, 9d,i,l were
tested in vitro for mutagenic potential. None of the compounds were mutagenic in the S.
typhimurium strains TA98 and TA100 at any of the concentrations tested. The number of S.
typhimurium revertants that arose at each concentration of the test compound was expressed as a
percentage of the number of revertants that arose in the positive control.
Table 7. Ames test on compounds 4a,b,d, 9d,i,l,h, 10 and reference compounds 3b and CQ
4a
% R±S.D.
TA98
5±5
4b
% R±S.D.
TA98
4d
% R±S.D.
TA98
7±6
9d
% R±S.D.
TA98
9i
% R±S.D.
TA98
a
a
a
a
a
b
C
b
b
b
b
C
C
C
C
TA100
5±4
TA100
20±20
19±24
22±18
13±10
17±26
27±26
28±9
TA100
3±4
TA100
4±1
TA100
7±13
12±1
10±3
10±3
11±3
11±1
11±4
0
2
4
9
0
3±4
0
1
2
4
8
0
8±5
0
6±4
0
6±3
1.5 2±3
9±8
6±3
1.5 3±3
3±0
1.5 4±0
7±3
6±3
3
6
4±4
6±3
7±7
5±2
3
6
0
0
4±1
3
6
5±5
3±4
0
7±10
5±2
4±4
3±3
4±1
18 3±6
35 3±3
12 4±8
24 4±4
16±10
5±1
12 3±5
24 3±3
4±1
13 3±2
2±2
17 11±8
7±8
5±0
25 11±5
7
0
2±3
100±9
9
2±2
48 4±0
34 4±8
2±2
48 3±3
10±4
50 8±4
c
c
c
c
c
+
100±44
+
100±31 100±55
+
100±13 100±11
+
100±14 100±10
+
100±11 100±25
l
9h
10
3b
CQ (1)
a
a
a
a
a
%
R±S.D.
% R±S.D.
% R±S.D.
% R±S.D.
% R±S.D.
b
C
b
C
b
C
b
C
b
C
TA98
6±4
TA100
6±6
TA98
10±0
17±6
TA100
6±6
TA98
3±4
TA100
4±4
TA98
5±4
TA100
5±2
TA98
10±11
TA100
30±31
36±21
0
2
0
2
0
7
0
1
0
1±2
4±2
4±2
1±2
4±3
2±4
5±9
2.5 24±8
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