Synthesis and in vitro and in vivo pharmacological evaluation of new 4-aminoquinoline-based compounds

Article abstract of DOI:10.1021/ml400311r

A new class of 4-aminoquinolines was synthesized and evaluated in vitro for antiplasmodial activity against both the chloroquine-sensitive (3D7) and -resistant (K1 and W2) strains. The most active compounds 3c-3e had acceptable cytotoxicity but showed strong inhibition toward a panel of cytochrome P450 enzymes in vitro. Pharmacokinetic studies on 3d and 3e in mice showed that they had moderate half-life (4-6 h) and low oral bioavailability. The front runner compound 3d exhibited moderate inhibition of the malaria parasite on P. berghei infected mice following oral administration (5 mg/kg), achieving reduction of parasitemia population by 47% on day 7.

Full text of DOI:10.1021/ml400311r

Letter
pubs.acs.org/acsmedchemlett
Synthesis and in Vitro and in Vivo Pharmacological Evaluation of
New 4‑Aminoquinoline-Based Compounds
Matshawandile Tukulula,†,○ Mathew Njoroge,† Efrem T. Abay,‡,§ Grace C. Mugumbate,†
Lubbe Wiesner,∥ Dale Taylor,∥ Liezl Gibhard,∥ Jennifer Norman,∥ Kenneth J. Swart,§ Jiri Gut,⊥
Philip J. Rosenthal,⊥ Samuel Barteau,# Judith Streckfuss,# Jacques Kameni-Tcheudji,#
and Kelly Chibale*,†,∇
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Department of Chemistry (48), University of the Free State, Bloemfontein 339, South Africa
§PAREXEL International Clinical Research Organization, Private Bag X09, Brandhof 9324, Bloemfontein 339, South Africa
∥Department of Pharmacology, University of Cape Town, Medical School, Observatory 7925, South Africa
⊥Department of Medicine, San Francisco General Hospital, University of San Francisco, San Francisco, California 94143, United
States
#Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, Basel CH-4057, Switzerland
∇Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: A new class of 4-aminoquinolines was synthesized and evaluated in vitro for antiplasmodial activity against both
the chloroquine-sensitive (3D7) and -resistant (K1 and W2) strains. The most active compounds 3c−3e had acceptable
cytotoxicity but showed strong inhibition toward a panel of cytochrome P450 enzymes in vitro. Pharmacokinetic studies on 3d
and 3e in mice showed that they had moderate half-life (4−6 h) and low oral bioavailability. The front runner compound 3d
exhibited moderate inhibition of the malaria parasite on P. berghei infected mice following oral administration (5 mg/kg),
achieving reduction of parasitemia population by 47% on day 7.
KEYWORDS: Aminoquinolines, antiplasmodial activity, pharmacokinetics, plasma protein binding
hloroquine (1) (Figure 1) exerts its activity against the
erythrocytic forms of all plasmodial species and is
mitigating the emergence and spread of antimalarial drug
resistance; (i) use of combination therapies, (ii) use of parasite-
resistance reversers, (iii) drug-repositioning, and (iv) rationally
designing analogues of the currently administered drugs and
using them in such a way that cross-resistance is circum-
vented.3,4 With regards to the last point, various studies have
shown that lengthening or shortening the chloroquine alkyl
side-chain leads to compounds that are effective against drug-
resistant strains of P. falciparum.5 However, Roepe et al.6
recently concluded that these observations only hold for
quinoline derivatives that contain a diethyl substituent on the
terminal nitrogen. Although, there is still some debate over the
effects of modifying the length of the alkyl side-chain, there is a
consensus that such modifications do not improve the
C
Figure 1. Chloroquine.
believed to function by inhibiting formation of hemozoin in
the Plasmodium falciparum digestive vacuole (DV).1 The
attractive features of chloroquine (CQ) are its mild side-effects
and affordability.2
However, the clinical use of this drug is limited by the
widespread development of resistant-strains. The Roll Back
Malaria (RBM) consortium devised the following strategies in
Received: August 9, 2013
Accepted: October 1, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
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metabolic stability, especially the N-dealkylation of the terminal
tertiary amino group that normally occurs during metabolism of
CQ and amodiaquine.2,7 This N-dealkylation reduces lipid
solubility of these drugs and their derivatives, an effect that is
suggested to result in an increase in the potential for cross-
resistance with CQ.8 As a direct consequence of this
observation, O’Neill et al.9 and Guy et al.10 showed that the
use of bulkier substituents (such as piperidyl, pyrrolidinyl, and
morpholinyl rings) attached to the terminal amino group
increased the in vivo efficacy and simultaneously decreased the
potential for cross-resistance, presumably by circumventing
metabolic N-dealkylation.
aldehydes in the presence of tert-butyl isocyanide and
trimethylsilane azide (TMSN3) using a previously described
procedure to give compounds 3a−g.11−13 Subsequent, cleavage
of the tert-butyl group gave rise to compounds 4a−g in low to
moderate yields after purification by column chromatography.
All the synthesized target compounds were fully characterized
by analytical and spectroscopic techniques with their HPLC
purity greater than 95% (Supporting Information).
The protocols followed for antiplasmodial evaluation of the
target compounds were the same as those described
previously.11 Furthermore, because of the poor activity of
4a−g in the K1 strain compared to 3a−g, we did not find it
necessary to do further testing, against the 3d7 and W2 strains.
Compounds 3c−3g were all more efficacious than CQ on all
the tested P. falciparum strains; more specifically, against the
chloroquine-resistant K1 strain, 6 (3a and 3c−3g) out of the 14
compounds showed greater activity than chloroquine (CQ),
with compound 3e (IC50 = 1.0 nM) exhibiting a 36-fold greater
activity (Table 1).
Inspired by the work of O’Neill et al.9 and Guy et al.,10 we
have rationally designed our target compounds largely around
circumventing metabolic N-dealkylation by incorporating
bulkier substituents such as aromatic and tetrazole rings while
varying the length of the alkyl side-chain (Figure 2). The
incorporation of the tetrazole ring is based on the rationale that
was highlighted previously.11−13
Table 1. In Vitro Antiplasmodial Activity and Solubility of
Target Compounds
b
IC50 (μM)
solubility (μM)
a
entry
3D7
K1
W2
RI
pH 2.0 pH 7.4
3a
3b
3c
3d
3e
3f
0.005
3.821
0.001
0.0004
0.002
0.002
0.002
nd
0.038
0.069
0.046
0.0311
0.0305
0.0255
0.039
0.020
7.6
>200
nd
111
nd
0.155
0.002
0.008
0.001
0.027
0.008
0.041
2
127
194
>200
nd
39.6
25
Figure 2. Rationale for design of 4-aminoquinoline-based target
compounds.
20
0.5
13.5
4
6.88
nd
3g
4a
4b
4c
4d
4e
4f
nd
nd
Thus, in this letter, we describe the synthesis and
pharmacological evaluation of new 4-aminoquinolines. Briefly,
the synthesis commenced with the preparation of key quinoline
diamines (2a−c) following known literature procedures
(Scheme 1).14
These diamines (2a−c) were then treated under multi-
component reaction (MCR) conditions with various aromatic
c
d
14.9
nd
nd
nd
c
nd
13.2
nd
nd
nd
c
nd
17.4
nd
nd
nd
c
nd
4.39
nd
191
>200
nd
70.1
164
nd
c
nd
2.38
nd
c
c
nd
14.26
6.849
nd
4g
CQ
nd
nd
nd
nd
c
e
e
0.0052
0.036 (0.220 )
0.059
6.9
>200
>200
a
Scheme 1
a
b
Resistance index IC50 (KI)/IC50 (3D7). Kinetic solubility.
Antiplasmodial testing done at the Swiss Tropical and Public Health
Institute (STPH). nd: not determined. Chloroquine tested as a
diphosphate salt.
c
d
e
Similarly, against another CQ-resistant W2 strain, the
majority of the tested compounds were more active than CQ,
with the exception of 3a that had comparable activity to CQ. In
terms of structure−activity relationships, the compound with
the shortest ethylene spacer, 3a, was less efficacious than the
compounds with longer ethylene spacers, 3f and 3g. Moreover,
compounds 3a, 3c, and 3g, with resistance index (RI) values of
7.6, 2, and 4, respectively, show a low-level cross-resistance
potential with CQ. At this juncture, it is worth noting that
compounds 3b and 3e have RI values that are lower than 1, an
indication of a reduced likelihood to develop cross-resistance
with CQ. On the basis of in vitro antiplasmodial activity, the in
vitro ADME characterization of the selected compounds, 3c−e,
was assessed. The kinetic solubility of these selected
compounds was generally poorer than that of CQ diphosphate
at both pH regions (Table 1). In terms of cytotoxicity,
compounds 3c−3e exhibited acceptable cytotoxicity against the
a
Reagents and conditions: (i) TMSN3, MeOH, 40 °C, 24 h; (ii) neat
32% HCl(aq), 120 °C, 4 to 8 h.
B
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Chinese Hamster Ovarian (CHO) cell-lines, as indicated by
their IC50 values (Table 2).
compounds were rapidly metabolized in both species (EH >
0.8, see Supporting Information). LC−MS/MS was used to
tentatively identify the metabolites of these compounds in
microsomal and hepatic incubations (Supporting Information).
The major metabolites were shown to result from N-oxidation
of the quinoline ring and hydroxylation at the tert-butyl group.
Details of the metabolite identification are presented in the
Supporting Information.
Table 2. Cytochrome P450 Inhibition and Cytotoxicity
Evaluation of Compounds 3c−e
CYP450 IC50
b
cytotoxicity
entry
2C8
2C9 2C19 2D6
3A4
(IC50) (μM)
The in vivo pharmacokinetics (PK) of compounds 3d and 3e
were evaluated in healthy male C57/BL6 mice following oral
(at 40 and 20 mg/kg doses) and intravenous (at 5 and 2.5 mg/
kg doses) routes, respectively (Table 3). Both compounds
displayed a relatively moderate systemic plasma clearance and
high volume of distribution with elimination half-lives ranging
from 1.5 to 6 h, an indication that these compounds might be
slightly protein bound.
The plasma protein binding affinity studies revealed that
these compounds were moderately bound to the plasma
protein (see Supporting Information). In addition, the oral
bioavailability of both compounds ranged from 16 to 31%.
Compound 3d was selected for in vivo antimalarial efficacy
evaluation against Plasmodium berghei ANKA infected male
C57/BL6 mice. This compound was administered orally at four
different concentrations (20, 10, 5, and 1 mg/kg dose; each
dose group consists of 5 mice) once a day for four days. Blood
smears were collected on days 3, 5, and 7, and the percentage
parasitemia determined using light microscopy. On day 7,
compound 3d reduced the parasitemia load by up to 47% even
though this activity was found to plateau above 5 mg/kg (Table
4). This plateau, which is also noticeable in the in vivo PK data,
possibly indicates solubility- or dissolution-limited absorption at
higher doses.
In conclusion, we have successfully synthesized a series of
new 4-aminoquinoline derivatives of CQ that showed greater
antiplasmodial activity than the reference drug. Moreover, the
front-runner compounds 3c−3e also exhibited acceptably
cytotoxicity, albeit with strong inhibition toward a panel of
CYP3A4 enzymes in vitro. The in silico CYP3A4 docking
studies implicated the aminoquinoline ring’s orientation relative
to the heme as the major cause of this inhibition. These
compounds also displayed a relatively low oral bioavailability in
mice. Compound 3d exhibited 47% reduction in parasitemia
load on day 7.
a
3c
3d
3.98
nd
10.3
nd
>20
nd
9.88
nd
0.17
nd
185.1
10.5
a
3e
12.7
8.91
12.4
4.0
0.08
97.8
emetine
0.129
a
b
IC50 was >20 μM for CYP1A2, 2A6, 2B6 and 2E1. Chinese Hamster
ovarian cell-lines.
Cytochrome P450 (CYP450)-mediated drug−drug inter-
action potential of compounds 3c and 3e was assessed by
determining their inhibition potentials against a panel of
CYP450s in human liver microsomes. Both these compounds
showed moderate inhibition of P450 2C8, 2C9, 2C19, and 2D6
and relatively strong inhibition of CYP3A4. Since CQ is not a
known inhibitor of CYP3A4,15 in silico docking studies were
undertaken in an attempt to elucidate the molecular basis of
this inhibition. The interpretation of results was based on the
proximity of the ligands to the heme and the associated binding
energies. The lowest energy conformations of both compounds
and CQ were docked onto a crystal structure of CYP3A4 (PDB
code 2V0M) using Glide software (www.schrodinger.com).16
One preferred ligand orientation was identified for each of 3c,
3e, and CQ. Compounds 3c and 3e adopt a conformation
where their aminoquinoline rings are aligned parallel and closer
to the heme, while for CQ the ring is aligned almost
perpendicular to the heme (Figure 3). In addition, the binding
affinities of the two molecules are higher than those of CQ, and
their binding energies are comparable to that of ketoconazole, a
strong CYP3A4 inhibitor (see Supporting Information). Thus,
these findings suggest that the derivatives orient differently in
the active site of the enzyme and that their interactions with the
active site of the enzyme and near residues (e.g., in addition to
hydrophobic interactions, 3c forms a hydrogen bond with
SER119) lead to more stable bound conformations, which
increase their propensity for inhibition.
The metabolic stability of compounds 3d and 3e was
assessed in human and mouse liver microsomes. Both
Figure 3. Lowest binding energy conformations for each of (A) 3c (pink), (B) 3e (yellow), and (C) chloroquine (magenta) showing the molecular
surface of selected binding site residues at a radius of 3 Å and heme (red).
C
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ACS Medicinal Chemistry Letters
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Table 3. Pharmacokinetic Parameters of Compounds 3d and 3e Following Intravenous and Oral Administration on Male C57/
a
BL6 Mice
3d
3e
b
b
b
b
parameters
iv
oral
iv
oral
nominal dose (mg/kg)
apparent t1/2 (h)
plasma CLtotal (mL/min/kg)
Vd (L/kg)
5.0
2.3
44.8
8.9
9.1
2.5
1.9
48.9
7.9
8.7
40
20
5.0
2.5
1.6
51.2
7.0
6.5
40
20
3.9
6.0
2.5
4.0
3.6
51.0
10.3
12.6
Vss (L/kg)
Cmax (μM)
0.79
1.4
0.54
1.4
2.81
1.0
0.94
0.8
Tmax (h)
AUC0‑∞ (min· μmol/L)
bioavailability (%)
222
104
287
16.2
256
30.8
221
107
541
30.6
222
25.9
a
b
Empty cells indicate that the value was not measured or was not relevant. Values are means of five animals.
cytotoxicity assays. In addition, M.N. is grateful for an
internship at the Drug Metabolism and Pharmacokinetics
department, Novartis Institutes for BioMedical Research, Basel
(Switzerland), organized by the Novartis Diversity and
Inclusion office. M.N. also acknowledges the assistance of
Robert Nufer, Anja Heubi, Michael Houliet, and the rest of the
Drug Metabolism and Pharmacokinetics Department (Novar-
tis) for their assistance in the acquisition and analysis of the
ADME data. The authors would also like to thank the ADME
group of the University of Cape Town (UCT) Drug Discovery
and Development Centre (H3D) for their assistance in the
technical aspects of the in vitro ADME assays.
Table 4. Antimalarial Efficacy Using a Single Dose of
Compound 3d in the P.berghei Mouse Model on Day 7
% reduction of
compound
dose (mg/kg)
1 × 20
1 × 10
1 × 5
1 × 1
1 × 15
parasitemia
3d
44.5
41.4
46.9
22.7
90.6
positive control
(chloroquine)
negative control (placebo) blank formulation
solution
0.0
ASSOCIATED CONTENT
* Supporting Information
Details regarding compound characterization and biological
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
REFERENCES
■
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AUTHOR INFORMATION
Corresponding Author
*(K.C.) E-mail: kelly.chibale@uct.ac.za. Tel: +27 21 650 2553.
Fax: +27 21 650 5195.
■
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Present Address
○(M.T.) Encapsulation and Delivery Research Group,
Polymers and Composites, Material Sciences and Manufactur-
ing Unit, Council for Scientific and Industrial Research (CSIR),
Pretoria 0001, South Africa.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Funding
We thank the UCT Department of Chemistry Equity
Development Program scholarship and the South African
National Research foundation (NRF) for financial support. The
UCT, South African Medical Research Council, and South
African Research Chairs Initiative of the Department of Science
and Technology administered through the NRF is also
gratefully acknowledged (to K.C.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Marcel Kaiser (Swiss Tropical and Public
Health Institute) and Dr. Susan Little (London School of
Hygiene and Tropical Medicine) for antiplasmodial and
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