New pentasubstituted pyrrole hybrid atorvastatin-quinoline derivatives with antiplasmodial activity

Article abstract of DOI:10.1016/j.bmcl.2016.03.027

Cerebral malaria is caused by Plasmodium falciparum. Atorvastatin (AVA) is a pentasubstituted pyrrole, which has been tested as an adjuvant in the treatment of cerebral malaria. Herein, a new class of hybrids of AVA and aminoquinolines (primaquine and chloroquine derivatives) has been synthesized. The quinolinic moiety was connected to the pentasubstituted pyrrole from AVA by a linker group (CH₂)_n = 2-4 units. The activity of the compounds increased with the size of the carbons chain. Compound with n = 4 and 7-chloroquinolinyl has displayed better activity (IC₅₀ = 0.40 μM) than chloroquine. The primaquine derivative showed IC₅₀ = 1.41 μM, being less toxic and more active than primaquine.

Full text of DOI:10.1016/j.bmcl.2016.03.027

Accepted Manuscript
New Pentasubstituted Pyrrole Hybrid Atorvastatin-Quinoline Derivatives with
Antiplasmodial Activity
Rita C.C. Carvalho, Wagner A. Martins, Tayara P. Silva, Carlos R. Kaiser,
Mônica M. Bastos, Luiz C.S. Pinheiro, Antoniana U. Krettli, Núbia Boechat
PII:
S0960-894X(16)30247-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.027
BMCL 23670
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
22 January 2016
7 March 2016
8 March 2016
Please cite this article as: Carvalho, R.C.C., Martins, W.A., Silva, T.P., Kaiser, C.R., Bastos, M.M., Pinheiro, L.C.S.,
Krettli, A.U., Boechat, N., New Pentasubstituted Pyrrole Hybrid Atorvastatin-Quinoline Derivatives with
Antiplasmodial Activity, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2
016.03.027
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Graphical Abstract
New Pentasubstituted Pyrrole Hybrid
Atorvastatin-Quinoline Derivatives with
Antiplasmodial Activity
Leave this area blank for abstract info.
Rita C. C. Carvalho, Wagner A. Martins, Tayara P. Silva, Carlos R. Kaiser, Mônica M. Bastos, Luiz C. S.
Pinheiro, Antoniana U. Krettli and Núbia Boechat*

Bioorganic & Medicinal Chemistry Letters
New Pentasubstituted Pyrrole Hybrid Atorvastatin-Quinoline Derivatives with Antiplasmodial
Activity
a,b
a
c
b
a
Rita C. C. Carvalho , Wagner A. Martins , Tayara P. Silva , Carlos R. Kaiser , Mônica M. Bastos , Luiz
a
c
a*
C. S. Pinheiro , Antoniana U. Krettli and Núbia Boechat
a
Departamento de Síntese de Fármacos, Instituto de Tecnologia em Fármacos, Farmanguinhos - FIOCRUZ, Rua Sizenando Nabuco 100, Manguinhos, Rio de
Janeiro, RJ, 21041-250, Brazil.
Programa de Pós Graduação em Química, PGQu Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
b
c
Laboratório de Malária, Centro de Pesquisas René Rachou, CPqRR - FIOCRUZ, Belo Horizonte, MG, 30190-002, Brazil.
*
Corresponding author boechat@far.fiocruz.br
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Cerebral malaria is caused by Plasmodium falciparum. Atorvastatin (AVA) is a
pentasubstituted pyrrole, which has been tested as an adjuvant in the treatment of
cerebral malaria. Herein, a new class of hybrids of AVA and aminoquinolines
(primaquine and chloroquine derivatives) has been synthesized. The quinolinic moiety
was connected to the pentasubstituted pyrrole from AVA by a linker group (CH )
2 n=2-4
units. The activity of the compounds increased with the size of the carbons chain.
Compound with n = 4 and 7-chloroquinolinyl has displayed better activity (IC50 = 0.40
µM) than chloroquine. The primaquine derivative showed IC50=1.41 µM, being less
Keywords:
Malaria;
Atorvastatin;
Pentasubstituted Pyrrole;
Chloroquine;
Primaquine
toxic and more active than primaquine. 2009 Elsevier Ltd. All rights
reserved
.
1
2
Malaria is a parasitic disease caused by protozoa of the
Plasmodium genus and transmitted by Anopheles mosquitoes.
falciparum
and to promote a synergistic effect with
13,14
1
dihydroartemisinin (DHA) activity.
Drug resistance, documented in the species P. falciparum, and P.
vivax, has been one of the main obstacles in the fight against
malaria. In 2013, 584,000 people died due to cerebral malaria,
2
the most severe manifestation that is caused by P. falciparum.
Of these deaths, 78% were among African children under five
2
years old. It is recommended that patients diagnosed with
cerebral malaria should be treated with quinine, quinidine and
one of the artemisinin derivatives, preferably by intravenous
3
artesunate.
Pyrrole is a simple aromatic heterocycle commonly found in
4
,5
nature that shows varied biological activity.
pyrroles have shown antiplasmodial activity
Substituted
6
-11
(Fig. 1). An
isoquine analog containing tetrasubstituted pyrrole 1 was shown
6
in vitro to have IC50 = 42.5 nM. The trisubstituted pyrrole 2
inhibited the entry of Plasmodium sporozoites with in vitro and
7,8
in vivo IC50 values of 10 µM and < 100 nM, respectively,
blocked the development of erythrocytic schizonts in vitro and
was also partially effective against the erythrocyte stages of P.
9
berghei in vivo. The disubstituted pyrrole 3 showed high activity
10,11
Figure 1: Known pyrroles with antiplasmodial activity.
in vitro against P. falciparum (IC50 = 4.6 nM),
which was
probably potentialized by a moiety of artemisinin derivative in its
chemical structure.
AVA has shown significant pleiotropic effects such as
15
Atorvastatin (AVA, Fig. 1) is one of the leading pyrrole drugs.
Employed in hypercholesterolemia treatment for its inhibition of
the HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA
reductase) enzyme, AVA has been shown to be active against P.
neuroprotective and anti-inflammatory activities. AVA is
considered to be an outstanding adjuvant in the treatment of
14,16-21
cerebral malaria
, having been shown to inhibit the increased
plasma and cerebrospinal fluid levels of interferon gamma

induced protein 10 (CXCL10), which is a chemokine secreted by
several cell types in response to IFN-γ in fatal human cerebral
16-18
The aminoquinoline moiety was included because it is present
in chloroquine and in the primaquine, as 7-chloroquinolin-4-yl
and 6-methoxyquinolin-8-yl parts, respectively. In the proposed
hybrids, the quinoline moiety was connected to the
malaria.
Unfortunately, no clinical trials in cerebral malaria
using AVA as adjuvant of an artemisinin-based combination
therapy have been yet performed. Studies have demonstrated an
absence of cross-resistance in vitro between AVA and
antimalarials, suggesting different modes of action and indicating
pentasubstituted pyrrole by a linker group containing 2–4 CH
2
12-13, 19-22
units (Fig. 2).
that AVA can be potent against malaria.
The synthesis of the new pyrrole-atorvastatin derivatives 4a-d
Scheme 1) was achieved through the Paal-Knorr reaction,
Studies using in silico and in vitro tests with atorvastatin were
carried out with lactate dehydrogenase of P. falciparum
35
(
between 1,4-diketone 5 and aminoquinolines 6a-d at 80-100°C in
(PfLDH), a crucial enzyme for Plasmodium’s anaerobic lifestyle
35,36
2
6-91 hours,
and the process was not optimized. Toluene can
and survival during the parasite erythrocytic cycle in the
be used as the solvent, but a 1:1 mixture of tetrahydrofuran and
cyclohexane has shown better solubility. Acetic acid has not been
very successful as a catalyst, but pivalic acid has shown good
results. To remove the water from the reaction medium, a Dean-
Stark apparatus was used, and anhydrous sodium sulfate was
added occasionally.
vertebrate host. Atorvastatin inhibited the enzyme function and is
23
likely to act as therapeutic agent targeting parasite.
Combination therapies are recommended by the WHO
because the use of monotherapy for decades contributed to the
emergence of strains resistant to excellent medicines such as
2
4
cloroquine and mefloquine.
Currently, the molecular
Products were purified by column chromatography using a
hybridization of two pharmacophoric groups in the same
2
5-
solvent gradient of CHCl
3
:MeOH or hexane:AcOEt, with yields
molecule has become an important tool in medicinal chemistry
27
of 18-64%. All compounds were fully characterized by FTIR,
and is the main goal of the present work. Hybridizations may
1
3
MS and 1D and 2D NMR. For example, the C NMR spectra
showed the absence of both signals for carbonyl groups and the
presence of an alkyl chain and aminoquinolinyl aromatic ring,
proving the formation of the desired products.
improve pharmacological drug profiles by dual or new
mechanisms of action, making it possible to overcome the
25-27
parasite resistance mechanisms.
In our previous search for new anti-malaria drugs, the
2
8
effectiveness of hybrid salts of mefloquine and artesunate,
29
primaquine
and
artesunate,
2-
3
0
(trifluoromethyl)[1,2,4]triazolo[1,5-a]pyrimidine,
1H-1,2,3-
31
32
triazole-quinoline, and quinoline-sulfonamide hybrids against
P. falciparum was demonstrated.
Chloroquine is an inexpensive and safe 4-aminoquinoline that
has been a standard in malaria chemotherapy for decades because
it is highly efficient against blood schizonts of all Plasmodium
species. Malaria parasites grow within the red blood cells of host,
and is believed that the efficacy of chloroquine is due its ability
33
of blocking the hematin detoxification. Its use as a single drug
has been abandoned in the case of P. falciparum due to the
33
emergence of resistant strains.
Primaquine is an 8-
aminoquinoline and the only drug available to cure the late
relapses caused by the P. vivax and P. ovale liver stages termed
hypnozoites. However primaquine causes hemolysis in patients
with G6PD deficiency. The search for new analogues or
3
4
substitutes for chloroquine or primaquine has been a challenge.
6
Reagents and conditions: (i) pivalic acid, THF:C H12, 80-100°C, 18-64%.
Using the molecular hybridization tool, the objective of this
study was to synthesize and test a new class of hybrid derivatives
with AVA and aminoquinolines in the structure. AVA was
chosen based on its proven activity against malaria parasites and
also because it is a highly substituted pyrrolic compound (Fig. 2).
Scheme 1: Synthesis of the new pyrrole-atorvastatin analogues 4a-d.
Figure 2: New pentasubstituted pyrrole hybrid atorvastatin-quinoline derivatives.

The diamino-7-chloroquinolinyl derivatives 6a-c were
synthesized by reflux for 4 hours of 7 and the corresponding
untreated cells and the minimum lethal dose for 50% of the cells
(MLD50) was determined.
37,38
diamine-alkanes of interest,
). Primaquine was obtained by the simple neutralization of
primaquine diphosphate with 10% aqueous NaOH solution at
with yields of 91-96% (Scheme
The results of the antiplasmodial activity and cytotoxicity
assays of four new compounds (4a-d) are summarized in Table 1.
All compounds synthesized showed activity against a W2-
chloroquine resistant P. falciparum clone, with IC50 values
ranging from 0.40 to 1.41 μM in the SYBR test, in a similar
range to chloroquine (IC50 = 0.59 μM); in addition, all were
better than primaquine (IC50 = 1.89 μM) and than atorvastatin
2
39
room temperature.
(IC50 = 10.3 μM). Compounds 4a, 4b and 4c were the most
active, with IC50 values of 0.99, 0.65 and 0.40 μM, respectively.
All compounds were also evaluated regarding cytotoxicity
against a BGM cell line. Compounds 4a and 4d presented the
lowest toxicity measured at doses up to 1,000 μg/mL with SI >
Reagents and conditions: (i) appropriate diamine, reflux, 4 h, 91-96%.
Scheme 2: Obtaining of intermediary aminoquinolines6a-c.
1,677 and 1,107, respectively.
Table 1. Evaluation in vitro of antiplasmodial activity against
Plasmodium falciparum W2 clone (chloroquine resistant), cytotoxicity
against a monkey kidney cell line (BGM), and drug selectivity index (SI) of
compounds 4a–d, 8, chloroquine and primaquine.
P. falciparum parasites were cultured in human red blood cells
40
under conditions established by Trager & Jensen (1976), with
41
minor modifications. Briefly, parasites were cultured in Petri
dishes (Corning, Santa Clara, CA, USA) with 5% hematocrit in
RPMI culture medium supplemented with 1% (v / v) albumax II
Compounds
IC50 (µM)
0.99 ± 0.3
0.65 ± 0.5
0.40 ± 0.02
1.41 ± 0.14
6.39 ± 0.98
0.59 ± 0.03
1.89 ± 0.2
10.3 ± 1.2
MDL50 (µM)
> 1,661 ± 0.0
136.4 ± 14
187.3 ± 13
> 1,562 ± 0.0
> 2,202
SI (MDL50/IC50
> 1,677
210
)
(
Gibco, USA). Plates were maintained at 37°C, using the candle
4
4
a
jar method. Daily exchanges of the culture medium were carried
out and parasitaemia monitored in Giemsa-stained smears. The
parasites were synchronized by sorbitol solution as described by
b
42
Lambros & Vandenberg (1979) to get predominantly ring
forms, diluted and incubated in 96 well plates containing the test
and control compounds, or culture medium with 0.5% DMSO,
used as a positive control of parasite growth.
4c
468
4
d
> 1,107
> 345
2,066
8
The SYBR test was used as previously described by
43
Chloroquine
Primaquine
Atorvastatin
1,219
Smilkstein et. al. (2004) with some modifications, as follows.
Briefly, the test compounds, in serial dilutions, were incubated
with the parasite suspensions (0.5% parasitaemia and 2%
hematocrit) in 96-wells plates ("U" bottom). After 48 h at 37°C,
the culture supernatant was removed replaced by 100µL of lysis
buffer solution [Tris (20 mM; pH 7.5), EDTA (5 mM), saponin
451.7
239
77
7.5
IC50: inhibitory concentration for 50% of parasite growth, evaluated in
two to four different experiments for each test; MDL50: minimal dose lethal
concentration for 50% of BGM cells; SI: selectivity index. The MDL of
atorvastatin is based in one experiment.
(
0.008%; wt/vol), and Triton X-100 (0.08%; vol/vol)] followed
by addition of 0.2 μL/mL Sybr Safe (Sigma-Aldrich, Carlsbad,
CA, USA). The plate content was transferred to a flat bottom
plate, then incubated in the dark for 30 minutes. The reading was
made in a fluorometer (Synergy H4 Hibrid Reader, BioteK) with
excitation 485 nm and emission of 535 mm.
The literature indicates that a side chain length of 3 or 4
carbons generally has better activity against P. falciparum, but
the activity also depends on the structure of the terminal
32,46
amine.
We have recently evaluated quinoline–sulfonamide
For the test of cytotoxicity, a monkey kidney cell line (BGM),
originally received from the Federal University of Minas Gerais
hybrids, which have a 7-chloroquinoline moiety connected by a
32
44
linker group to arylsulfonamide moieties . The most active
hybrids assayed against P. falciparum had four methylene groups
as linkers. Herein, the activity of the compounds increased with
the size of the carbon chain. Compound 4c (n=4) displayed better
activity even though the terminal amine is a tertiary amine with
the electron lone pair impaired by the aromaticity of the pyrrole
ring. Despite being the least active of the series, primaquine
derivative 4d (IC50=1.41) was significantly less toxic and more
active than primaquine, encouraging us to proceed in new
searches for a substitute for this toxic drug.
was used and the test performed as described. Cells were
2
cultured in 75 cm plates with RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS) and gentamicine 40 mg/L, at
% CO atmosphere and 37°C. When the cell monolayer was
2
5
confluent it was trypsinized, washed with culture medium,
5
distributed in a flat-bottomed 96-well plate (1×10 cells/mL) and
incubated for 18 h at 37°C to ensure cell adherence. The cells
were properly diluted and incubated with 20 µL of the
compounds at different concentrations (1,000-1 µg/mL) for 24 h
45
2
in a 5% CO and air atmosphere at 37°C. The neutral red assay
was used to evaluate cell viability by the accumulation of dye in
the viable cell lysosome. 200 µL of neutral red solution (4
mg/mL), were added and the plates and incubated for 3 h. The
supernatant was carefully removed, followed by the addition of
Additionally, to evaluate the importance of the pyrrolic moiety
in the hybrid compounds, compound 8 was obtained (Fig. 3)
using the same method as for the series 4a-d. Compound 8 is a
pentasubstitued pyrrole without the aminoquinolinyl moiety. No
toxicity was found in vitro with doses up to 1,000 μg/mL (>
47
2
200 µL of formaldehyde (0.5% v/v) and CaCl (1%) solution.
After 5 min, the supernatant was removed, then 100 µL of an
alcohol- acetic acid (50-1%) solution were added to extract the
dye. The absorbance was read at 540 nm on an ELISA reader
2
,202 μM), and its activity (IC50 = 6.39 μM) is higher than that
found for AVA (IC50 = 10.3 μM). This result suggests that the
pentasubstituted pyrrole might be the pharmacophoric group of
AVA. Comparing these new results for 4a, 4b, 4c and 4d with
384
(
SpectraMax340PC , Molecular Devices). Cell viability was
expressed as the percentage of control absorbance obtained in

the results found for AVA our compounds were 10, 16, 26 and 7-
fold more active than AVA, respectively, showing the
significance of the aminoquinolinyl attached to AVA’s pyrrolic
moiety connected by a linker with four methylenes.
11. Posner, G. H.; Woo, S. H.; Ploypradith, P.; Parker, M. H.;
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Baret, E.; Mosnier, J.; Briolant, S.; Fusai, T.; Rogier, C.
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3. Savini, H.; Souraud, J. B.; Briolant, S.; Baret, E.; Amalvict, R.;
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4. Dormoi, J.; Briolant, S.; Pascual, P.; Desgrouas, C.; Travaillé, C.;
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Figure 3: Structure of compound 8, a pentasubstituted pyrrole synthesized
with no aminoquinoline moiety.
In summary, four new pyrrolic hybrids of AVA with
aminoquinolines were synthesized and assayed against P.
falciparum. None of these compounds was significantly toxic to
BGM cells, although one of them (4b) was more toxic than the
others and thus had the lowest selectivity index. All compounds
showed anti-P. falciparum activity ranging from 0.40 to 1.41 µM
in the SYBR test; they were almost 5-fold more active than
primaquine. Compounds 4c and 4d (IC50 = 0.40 and 1.41 µM,
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potency and solubility, and will also be assayed for cerebral
antimalarial activity to clarify the importance of AVA in this
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The authors thank the Coordination for the Improvement of
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Brazil (CNPq) for the fellowships granted. We also thank the
Foundations for Research of the State of Rio de Janeiro
2
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(
FAPERJ), the Foundations for Research of the State of Minas
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analogues (4a-d and 8): Diaminoquinolines (6a-d) (1.8 to 2.7
mmol)
and
1,4-diketone
2-(2-(4-fluorophenyl)-2-oxo-1-
(5) (1.5
phenylethyl)-4-methyl-3-oxo-N-phenylpentanamide
mmol) were solubilized in a mixture 1:1 of cyclohexane and dry
anhydrous THF. Pivalic acid (1.5 to 4.5 mmol) was added to the
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with a gradient of CHCl :MeOH or hexane:AcOEt.
2
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3
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3
3
8. General procedure for the preparation of N-(7-chloro-4-quinolyl)-
,n-diaminoalkanes (6a-c): A mixture of 4,7-dichloroquinoline (5
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mmol) (7) and 3 mL of diaminoalkane was heated to reflux for 4
h. The mixture was then poured into crushed ice, and the resulting
pale-yellow precipitate was filtered under vacuum and used
without further purification.
9. Procedure for the preparation of free base primaquine (6d):
Primaquine diphosphate (4 mmol) was dissolved in the minimum
amount of distilled water with constant magnetic stirring, then it
was covered with aluminum foil. An aqueous solution of 10%
sodium hydroxide was added slowly until the reaction medium
reached pH = 10. The reaction was left under stirring and shielded
from light at room temperature for 2 hours. The mixture was
extracted with dichloromethane and dried over anhydrous sodium
sulfate to give a brown oil.
4
0. Trager, W.; Jensen, J. B. Science. 1976. 193, 673.