Structure-Activity Relationship Studies and Plasmodium Life Cycle Profiling Identifies Pan-Active N-Aryl-3-trifluoromethyl Pyrido[1,2- a]benzimidazoles Which Are Efficacious in an in Vivo Mouse Model of Malaria

Article abstract of DOI:10.1021/acs.jmedchem.8b01769

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2-a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Full text of DOI:10.1021/acs.jmedchem.8b01769

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Article
Structure-activity relationship studies and Plasmodium life cycle
profiling identifies pan-active N-aryl-3-trifluoromethyl pyrido[1,2-
a]benzimidazoles which are efficacious in an in vivo mouse model of malaria.
Godfrey Mayoka, Mathew Njoroge, John Okombo, Liezl Gibhard, Margarida Sanches-
Vaz, Diana Fontinha, Lyn-Marie Birkholtz, Janette Reader, Mariëtte van der Watt,
Theresa L. Coetzer, Sonja Lauterbach, Alisje Churchyard, Belinda Bezuidenhout, Timothy
J Egan, Clive Leonard Yeates, Sergio Wittlin, Miguel Prudêncio, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01769 • Publication Date (Web): 18 Dec 2018
Downloaded from http://pubs.acs.org on December 19, 2018
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Structure-activity relationship studies and
Plasmodium life cycle profiling identifies pan-
active N-aryl-3-trifluoromethyl pyrido[1,2-
a]benzimidazoles which are efficacious in an in
vivo mouse model of malaria.
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Godfrey Mayoka†, Mathew Njoroge#, John Okombo†, Liezl Gibhard#, Margarida Sanches-Vaz\,
Diana Fontinha\, Lyn-Marie Birkholtz∞, Janette Reader∞, Mariëtte van der Watt∞, Theresa L
Coetzer∇, Sonja Lauterbach∇, Alisje Churchyard∇, Belinda Bezuidenhout∇, Timothy J. Egan†, T
,
Clive Yeates∥, Sergio WittlinΨ, ‡, Miguel Prudꢀncio\ and Kelly Chibale*†, §, T
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa; #Drug
Discovery and Development Centre (H3D), Division of Clinical Pharmacology, Department of
∞
Medicine, University of Cape Town, Observatory, 7925, South Africa; Department of
Biochemistry, Genetics and Microbiology, Institute for Sustainable Malaria Control, University
of Pretoria, Private Bag X20, Hatfield 0028, South Africa; ∇Wits Research Institute for Malaria,
Faculty of Health Sciences, University of the Witwatersrand and National Health Laboratory
∥
Service, Johannesburg 2193, South Africa; Inpharma Consultancy, 6 Dudley Hill Close,
\
Welwyn, Hertfordshire AL60QQ, U.K.; Instituto de Medicina Molecular João Lobo Antunes,
Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa,
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Portugal; ΨDepartment of Medical Parasitology and Infection Biology, Swiss Tropical and Public
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‡
§
Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland; South African
Medical Research Council, Drug Discovery and Development Research Unit, University of Cape
Town, Rondebosch 7701, South Africa; T Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 7701, South Africa
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Supporting information
ABSTRACT: Structure-activity relationship studies involving N-aryl-3-trifluoromethyl
pyrido[1,2-a] benzimidazoles (PBI) identified several compounds possessing potent in vitro
activities against the asexual blood, liver and gametocyte stages of the Plasmodium parasite with
no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption,
distribution, metabolism and excretion (ADME) profiles were subjected to in vivo proof-of-
concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the
identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with
mean survival days of 12 and 14, respectively, at an oral dose of 4x50 mg/kg. In vivo
pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution and low
clearance profiles. Furthermore, this series displayed low propensity to inhibit the human ether-
a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with
cardiotoxicity.
INTRODUCTION
Drug resistance is a perennial threat to the control of malaria, a disease concentrated mainly in
the tropical and sub-tropical regions of the world, with disproportionate endemicity in Africa.1
Malaria is transmitted by parasites of the Plasmodium genus with five species known to infect
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humans: P. falciparum, P. malariae, P. vivax, P. ovale and P. knowlesi, with infections by P.
falciparum and P. vivax being the most virulent and widespread.2–5 Human malaria infection is
initiated when a female anopheles mosquito deposits sporozoites during a blood meal. These
sporozoites migrate to the liver where they undergo further development into schizonts which
produce merozoites that enter the systemic circulation where they infect red blood cells and cause
the typical symptoms of malaria.6 A subset of the liver stage parasites, however, remain dormant
in a form known as hypnozoites in the case of malaria caused by P. ovale and P. vivax parasites.
These hypnozoites can be re-activated in future to initiate blood stage infections.5 On the other
hand, some of the asexual blood stage parasites transform into male and female gametocytes
which are taken up by a mosquito during a blood meal, fuse within the midgut of the mosquito to
form a zygote which later differentiates into sporozoites. The sporozoites are introduced into
humans during a mosquito bite, thus completing the life cycle of the parasite.7
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The socio-economic and public health impact of malaria is startling: hundreds of thousands of
lives are lost annually, in addition to major indirect losses in terms of productivity and human
capacity development, as assessed by disability- and quality-adjusted life years (DALYS and
QALYS, respectively). Thus, malaria seems to perpetuate cycles of poverty in countries where it
is rife.8 Chemotherapy remains a fundamental component of malaria control, in addition to public
health approaches that focus on vector control and vaccine development.9–13 Unfortunately, the
decreased efficacy of even the most promising antimalarial agents in some regions threatens a
reversal of gains in the malaria elimination agenda.14–16 Furthermore, the treatment, control and
eradication of malaria hinges on targeting all the life cycle stages of Plasmodium; a profile that
most standard antimalarial drugs lack. For example, primaquine is the only antimalarial drug with
good activity against late stage gametocytes17 and, together with tafenoquine, is effective in
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preventing relapse due to Plasmodium vivax malaria infections. Unfortunately, the two drugs are
beset by toxicity concerns in patients with Glucose-6-phosphate dehydrogenase (G6PD) enzyme
deficiency.18 The above described scenarios, therefore, warrant sustained efforts in the discovery
and development of new antimalarial drugs as suitable alternatives to currently existing ones and
having transmission-blocking potential.
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The tricyclic pyridobenzimidazole (PBI) scaffold has emerged as an important medicinal
chemistry substructure with several pharmacological and biological activities reported including
antifungal, antibacterial, antiviral and antitumour.19–21 Our group has previously reported on the
antimalarial properties of PBI derivatives following medicinal chemistry iterations based on
phenotypic whole cell screening hits against the human parasite P. falciparum.22 Although the
mechanism of the antimalarial action of these compounds remain unresolved, targeting the
hemoglobin degradation pathway has been proposed based on structural congruences the PBIs
share with canonical inhibitors of this process such as the aminoquinoline antimalarials. In the
present work antiplasmodium structure-activity relationship (SAR) studies focusing on N-aryl
substitutions on a 3-trifluoromethyl PBI nucleus was evaluated (Figure 1). In line with most drug
discovery programs, analogues from this study were also profiled for their inhibition of the human
ether-a-go-go-related gene (hERG)-encoded potassium ion channels to understand cardiotoxicity
risks that may be associated with this series. Herein, we report the identification of potent, multi-
stage active analogues with an attractive hERG potassium ion channel inhibition profile along
with good in vivo efficacy in a murine model of malaria. The pharmacokinetics profile of a
representative lead compound 10 following oral and intravenous administration in mice is also
reported.
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Figure 1. SAR strategy around the 3-trifluoromethyl PBI scaffold.
RESULTS AND DISCUSSION
Chemistry: Target compounds were prepared as previously described (Scheme 1).23 Briefly,
compounds devoid of substitution on the left-hand side were synthesized from commercially-
sourced 2-benzimidazole acetonitrile (IIa). For other targets, the corresponding substituted
diamino benzene starting material (I) was condensed with ethyl-2-cyanoacetate to form the
equally substituted benzimidazole acetonitrile derivative (IIb and IIc). Subsequently, the
benzimidazole acetonitrile and ethyl 4,4,4-trifluoro-3-oxobutanoate were condensed in the
presence of ammonium acetate to provide the tricyclic hydroxyl intermediate (III), which is
converted to the penultimate chloro intermediate (IV) using phosphoryl trichloride (POCl3).
Finally, amination of IV using the relevant amine either in the presence of triethylamine in a
microwave reactor or the palladium-catalyzed Buchwald-Hartwig aromatic amination reaction
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using a suitable ligand and base were employed to give the final compounds V: 1, 3-7, 10-15, 21-
30, 32-38, 40-53, 57-61 and 2, 8, 9, 16-20, 31, 39, 54-56, respectively. Oxidation of the chloro
intermediate IVa using concentrated sulfuric acid produced the amide-containing intermediate
VIa, which was subsequently aminated as in reaction iv to furnish the target compounds 58-60.
On the other hand, the use of the starting material IId, containing a methyl ester group (R) instead
of a nitrile, afforded the target compound 61.
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Scheme 1. General scheme for synthesis of the PBI compounds.
Reagents and conditions: (i) Ethyl cyanoacetate, DMF, 160 ˚C, 2 h, 61-76%; (ii) Ethyl 4,4,4-trifluoro-3-
oxobutanoate, NH4OAc, 145 ˚C, 2h, 27-66% ; (iii) POCl3, (20eq), 130 ˚C, 3h, 63-94%; (iv) Relevant amine (RNH2),
Et3N, microwave, 80 ˚C, 150 W, 20-60 min (9-86%) or the appropriate amine, Pd2(dba)3, BINAP or Brettphos,
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K2CO3 or Cs2CO3, toluene or 1,4-dioxane or tert-butanol, 100-120 ˚C, 12-16 h (6-54% ); (v) concentrated sulfuric
acid, 85 ˚C, 2h, 89%. X, Y, Z and R are as described in Tables 1-3.
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Biology: Antiplasmodium activity against Asexual Blood Stage parasites. With the aim of
defining the structure-activity relationship pattern around the PBI scaffold, synthesized analogues
were first tested for their in vitro activity against the drug-susceptible P. falciparum NF54 strain.
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SAR1 Targets: Structural modifications at SAR1 (Figure 1) produced compounds that may be
classified broadly into either N-linked (n=0; aniline-type) or N-methylene-linked (n=1;
benzylamine-type) series (Figure 1). In the first series, the side group moiety comprises a phenyl
ring directly linked to the PBI central core by a nitrogen. The electronic and lipophilic features
of substitutions around the aromatic ring appeared to influence the antiplasmodium activity
observed. For instance, unlike hydrophilic substituents, lipophilic groups on the aromatic side
chains favored antiplasmodium activity. Active analogues in the low [3 (IC50 = 1.16 µM), 4 (IC50
= 1.01 µM), 10 (IC50 = 0.17 µM) and 13 (IC50 = 1.59 µM)] to moderate [1 (IC50 = 2.14 µM) and
5 (IC50 = 2.26 µM)] micromolar range were identified (Table 1). The most active derivative in
the series was 10 (IC50 = 0.17 µM), which had a trifluoromethoxy (-OCF3) substituent that
combines both lipophilic and electron-withdrawing properties.
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Table 1. Antiplasmodium potency of SAR1 Targets against chloroquine-susceptible (PfNF54)
and resistant (PfK1) strains of P. falciparum.
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aAntiplasmodium
activity
Compound
Resistance
R
IC50 (µM)
PfNF54 PfK1
Index
Number
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2.14
2.36
1.16
1.01
2.26
5.62
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b>10
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aAntiplasmodium
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activity
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Resistance
Index
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IC50 (µM)
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PfNF54
PfK1
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>2.36
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>2.32
0.17
10
0.15
0.23
0.88
0.58
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12
0.39
0.87
0.62
0.71
13
14
1.59
>2.83
15
16
>2.21
>2.82
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aAntiplasmodium
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activity
Compound
Number
Resistance
Index
R
IC50 (µM)
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PfNF54
PfK1
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18
>2.52
>2.82
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20
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22
>2.69
4.42
2.98
1.27
0.89
0.7
23
>2.72
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25
6.11
3.18
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aAntiplasmodium
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activity
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Resistance
Index
R
IC50 (µM)
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PfNF54
PfK1
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27
28
0.92
12.58
>2.68
0.84
0.91
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2.60
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31
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34
1.16
>2.72
>2.72
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1.64
1.41
1.49
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activity
Compound
Number
Resistance
Index
R
IC50 (µM)
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PfNF54
PfK1
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>2.36
>2.28
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10.20
2.40
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40
4.80
10.03
aValues are a mean of n≥2 [3H]-hypoxanthine incorporation assays. IC50 values generally differed
less than 2-fold; those that varied above 3-fold were excluded from the analysis. b> IC50 value is
above the highest concentration used in the assay. Chloroquine and artesunate were used as
controls whose established standard IC50 values from n≥10 determinations in our lab are 0.016
µM (PfNF54); 0.194 µM (PfK1) and 0.004 µM (PfNF54); 0.003 µM (PfK1), respectively.
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Introduction of a methylene linker (n = 1; Figure 1) into the side group to extend the SAR from
aniline-type to benzylamine-type series afforded analogues with antiplasmodium activities in the
low micromolar range: 22 (IC50 = 1.27 µM), 26 (IC50 = 0.92 µM) and 34 (IC50 = 1.49 µM) [Table
1]. As with the aniline-type series, lipophilic electron-withdrawing groups were equally favored
in this series.
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Isosteric replacement of the phenyl ring with various heteroaromatic groups produced diverse
compounds including pyridyl (17, 30, 31, 32), pyrimidyl (16, 19, 39), pyrazinyl (29 and 20), and
thiazolyl (18) analogues (Table 1). Among them, only the pyridyl ring in the benzylamine series
produced an active compound 30 (IC50 = 1.16 µM). Interestingly, there seems to be a stringent
requirement with regards to the positioning of the nitrogen on the pyridyl moiety, with only the
2 (ortho)-position (30; IC50 = 1.16 µM) permitted for potency, while the 3 (meta)- and 4 (para)-
positions produced compounds with lower activities, as observed for compounds 32 and 31,
respectively. On the other hand, replacement of the bulky aromatic side groups with the more
polar, heterocyclic groups like piperazine was detrimental to activity, as seen with compound 40
(IC50 = 10.03 µM) [Table 1].
The introduction of a methyl substituent on the nitrogen bridging the PBI core and the aromatic
appendage (R, Figure 1) led to a decrease in activity, as illustrated by compound 23 (IC50 >2.72
µM), which bears a tertiary nitrogen, compared to its analogue 30 (IC50 = 1.16 µM), in which this
nitrogen is secondary. This observation may suggest unfavorable steric interaction in the case of
a tertiary nitrogen or the importance of hydrogen-bonding interactions, in the case of a secondary
nitrogen, as in 30, for suitable interactions with the target to take place, leading to the observed
biological activity.
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SAR2 Analogues: Target compounds in this subset were generated by modifications on the left-
hand side (LHS) of the PBI core, as shown in Table 2. Following this modification, there was a
general trend towards retention of, or improvement in, potency. For instance, 44 (IC50 = 1.42 µM)
is approximately equipotent to 30 (IC50 = 1.16 µM; Table 1), while 43 (IC50 = 0.44 µM) and 45
(IC50 = 0.40 µM) are two to four-fold as potent as their corresponding analogues lacking the
dichloro modification, 26 (IC50 = 0.84 µM; Table 1) and 34 (IC50 = 1.49 µM; Table 1),
respectively. Similarly, a nearly 5-fold improvement in potency was observed with 42 (IC50 =
0.26 µM; Table 2), the dichloro substituted analogue of 22 (IC50 = 1.28 µM; Table 1).
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Table 2. Antiplasmodium potency of SAR2 Targets against chloroquine-susceptible (PfNF54)
and resistant (PfK1) strains of P. falciparum.
aIn vitro activity
Compound
Resistance
IC50 (µM)
X
Y
Z
R
Index
Number
PfNF54
PfK1
41
42
Cl
H
H
Cl
Cl
0.31
0.44
0.21
0.68
0.64
Cl
0.28
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aIn vitro activity
5
6
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8
Compound
Number
Resistance
Index
IC50 (µM)
X
H
Y
Z
R
9
PfNF54
PfK1
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
43
Cl
Cl
0.44
44
45
46
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
1.42
0.40
b>1.97
47
48
H
H
Cl
Cl
Cl
Cl
0.44
0.31
0.23
0.14
0.52
0.45
49
50
H
H
Cl
Cl
Cl
Cl
0.17
0.19
0.14
0.15
0.82
0.79
51
52
H
H
Cl
Cl
Cl
Cl
8.11
5.34
5.61
1.05
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8
Compound
Number
Resistance
Index
IC50 (µM)
X
Y
Z
R
9
PfNF54
PfK1
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
53
54
H
H
Cl
Cl
Cl
Cl
4.97
5.64
6.73
>6
55
56
H
H
Cl
Cl
Cl
Cl
5.85
0.87
aValues are a mean of n≥2 [3H]-hypoxanthine incorporation assays. IC50 values generally differed less than
2-fold; those that varied above 3-fold were excluded from the analysis. b> IC50 value is above the highest
concentrationused in the assay. Chloroquine and artesunate were used as controls whose established standard
IC50 values from n≥10 determinations in our lab are 0.016 µM (PfNF54); 0.194 µM (PfK1) and 0.004 µM
(PfNF54); 0.003 µM (PfK1), respectively.
Further SAR iterations within this series revealed that the phenyl ring at SAR1 (R1, Figure 1)
could be replaced with smaller heteroaromatic systems, such as furanyl 47 (IC50 = 0.44 µM) and
thiophene 48 (IC50 = 0.31 µM) with retention of activity when accompanied by dichloro
modifications at SAR2 (Y, Z = Cl, Table 2). However, replacement of the bulky aromatic side
groups with more polar, heterocyclic groups like piperazine, resulted in diminished activity, as
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seen with 51 (IC50 = 8.11 µM). Poor antiplasmodium activity was observed among analogues in
which the methylene linker (n = 1, SAR1, Figure 1) is substituted by the more polar sulfonyl
group (-SO2-), as observed with 55 (IC50 = 6.73 µM) vs 43 (IC50 = 0.44 µM), respectively (Table
2).
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
When comparing the position of the chloro groups on the LHS of the scaffold, the 3,5- dichloro
analogue 41 (IC50 = 0.21 µM) and the 4,5-dichloro compound 42 (IC50 = 0.26 µM) were
equipotent, indicating a negligible impact of regio-isomerism on this portion of the scaffold. On
the contrary, regio-isomerism appears to be more important in the side groups at SAR1. For
example, moving the position of the substituents on the aromatic side group between the ortho,
meta and para positions resulted in 50 (meta, IC50 = 0.19 µM) and 49 (ortho, IC50 = 0.17 µM)
being equipotent, and about twice as potent as analogue 42 (para, IC50 = 0.44 µM) [Table 2].
SAR3 Targets: To explore effects on antiplasmodium activity by initiating structural changes
around SAR3, analogues of previously active targets were prepared in which the nitrile at this
position was replaced with other small groups, resulting in compounds 57 (H; IC50 = 4.24 µM),
58, (CONH2; IC50 > 6 µM), 59 (CONH2, IC50 = 5.16 µM), 60 (CONH2, IC50 >10 µM) and 61
(COOMe, IC50 = 5.52 µM) [Table 3]. The loss of antiplasmodium potency of these compounds
compared with that of compounds 10, 22, 26 and 30, suggests that a nitrile group at this site is
required for activity.
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Table 3. Antiplasmodium potency of SAR3 Targets against chloroquine-susceptible (PfNF54)
and resistant (PfK1) strains of P. falciparum.
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
aIn vitro activity
Compound
Resistance
IC50 (µM)
R1
R
Index
Number
PfNF54
PfK1
3.36
0.79
57
58
H
4.24
b>6
7.59
>10
1.47
59
5.16
60
61
>10
5.52
aValues are a mean of n≥3 determinations. IC50 values generally differed less than 2-fold; those that varied above 3-
fold were excluded from the analysis. b> IC50 value is above the highest concentration used in the assay. Chloroquine
and artesunate were used as controls whose established standard IC50 values from n≥10 determinations in our lab are
0.016 µM (PfNF54); 0.194 µM (PfK1) and 0.004 µM (PfNF54); 0.003 µM (PfK1), respectively.
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Compounds with potent activity against the chloroquine-susceptible (CQS) PfNF54 parasite
strain (IC50 ≈ 1µM) were also evaluated against the chloroquine-resistant (CQR) PfK1 strain.
Cross-resistance of these selected compounds with chloroquine was assessed by determining the
fold change in the IC50 between the drug-sensitive and drug-resistant strains, expressed as the
resistance index (RI), calculated as the ratio of IC50PfK1 to IC50PfNF54. Generally, activity trends
were maintained across the two parasite strains (Tables 1-3). These results suggest that these
compounds are unlikely to demonstrate cross-resistance with chloroquine (RI=12.1), as indicated
by a less than 2-fold IC50 potency shift between the CQS and CQR parasite strains among the
compounds.
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
Inhibition of β- hematin (BH) formation. Besides the ability to adopt a planar conformation,
PBIs also have a rich electron density and protonatable nitrogen centers – features notably shared
by aminoquinolines like CQ. This flat conformation, as demonstrated in CQ, is essential in
facilitating pi-pi interactions with heme molecules leading to inhibition of hemozoin (Hz)
formation in P. falciparum.24 The library of compounds generated in this study were evaluated
for their ability to inhibit formation of beta-hematin (synthetic Hz) in an in vitro set up designed
to mimic the conditions of P. falciparum digestive vacuole (DV), the site of action of CQ.25
Most of the compounds displayed weak β-hematin inhibition (BHI) activity (IC50 > 100 µM)
(Table S1, Supporting Information). Only five compounds, 15 (IC50 = 50 µM); 44 (IC50 = 59 µM);
46 (IC50 = 32 µM); 53 (IC50 = 32 µM) and 55 (IC50 = 78 µM) demonstrated BHI activity in the
desired range (IC50 < 100 µM), with 46 and 53 displaying the greatest potencies (IC50 = 32 µM).
In contrast, chloroquine, used as a positive control, displayed a BHI IC50 of 18 µM.
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The dichloro substitution on the LHS improved the BHI potency 53 (IC50 = 32 µM) of the
previously inactive 28 (IC50 > 100 µM) analogue, a pattern also observed for 44 (IC50 = 59 µM)
vs 30 (IC50 > 100 µM). A sulfonyl linker between the nitrogen attached to the PBI core and the
aromatic portion of the side chain in the stead of a methylene group was detrimental to the
potential to disrupt BH formation, as observed in the case of 56 (IC50 > 100 µM) vs 44 (IC50 = 59
µM). In addition, the chiral compound 53 (IC50 = 32 µM) was more potent than its achiral
analogue 52 (IC50 > 100 µM), suggesting the role of the methyl substitution in offering additional
beneficial interactions or facilitating suitable compound orientation for BHI.
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
There was no correlation between the antiplasmodium activity and the ability of this series to
inhibit BH formation (R2 = 0.04, p-value = 0.19). Whereas the planar architecture of these
compounds would be expected to impart BHI capabilities, as previous observed with analogous
compounds,23,26 it appears that the structural diversity pursued in this study introduced features
that distorted the ability for appropriate binding to heme, leading to low BHI activities for most
compounds. Nonetheless, owing to the presence of protonatable centers on these molecules, it is
conceivable that intracellular DV accumulation might occur to sufficient concentrations that lead
to inhibition of Hz formation. On the other hand, it is possible that these compounds elicit
antiplasmodium activity via another mechanism independent from Hz inhibition. In fact,
inhibition of β-1, 6-glucan synthase in fungi,27 the lytic activity of pore-forming perforin28 and
cytochrome b in trypanosomes29 by other PBI derivatives has previously been reported.
Plasmodium life cycle activity profiling of frontrunner compounds. To assess the ability of
these compounds to potentially block malaria transmission as well as offer chemoprotection, the
compounds displaying the highest potency against the parasite’s asexual blood stages, were
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evaluated for their activity against the liver stages of the rodent P. berghei parasite and against P.
falciparum gametocyte stage. With reference to the liver stage activity profile, all tested
compounds (Table 4) demonstrated in vitro sub-micromolar potency (IC50 = 0.06 - 0.89 µM)
which was greater than that observed for primaquine (IC50 = 6.25 µM), the control drug used in
this assay. Among the compounds, 47, 49 and 50 demonstrated especially high activity with IC50
potencies of less than 100 nM. This is the first report of the liver stage activity of this series which
indicates their prospects as antimalarial chemoprophylactic drug discovery leads. With
tafenoquine having received US FDA approval only recently, primaquine is presently the only
clinical compound widely used for radical cure of P. vivax and P. ovale-induced malaria.30
However, despite the low in vitro activity observed with primaquine, the drug displays potent in
vivo activity, which is attributed to its biotransformation to the active metabolite.31 An accurate
extrapolation of the in vivo potency of these compounds is therefore not possible based on direct
comparisons with primaquine but will, rather, require animal experimentation in a model
recapitulating the liver stage infection.
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
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Table 4. aLiver and gametocyte stage activity of selected compounds.
PfNF54 Gametocyte activity
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
IC50 Fold
Pb
Compound
Final
Change
(EG:LG/
LG:EG) b
Liver stage IC50
Number
Late Stage IC50
(M)
(M)
10
22
26
41
42
43
44
45
47
48
49
0.11
0.89
0.48
3.32
0.74
0.69
0.21
2.48
0.60
2.00
1.67
0.37
3.0/0.3
0.19
0.2/6.0
0.06
0.14
0.05
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50
0.07
6.25
0.27
1.2/0.5
Primaquine
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
Methylene blue
DHA
0.9
14
aValues are the average of n≥3 determinations. IC50 values generally differed less than 2-fold; those
that varied above 3-fold were excluded from the analysis. EG: Early stage gametocyte; LG: Late
stage gametocyte. Final late stage IC50 represents the lowest of each value obtained for the luciferase
reporter or ATP bioluminescence assay.
b
Based on the asexual blood stage activity observed for the compound library generated in this
study, selected compounds were prioritized for the evaluation of their gametocytocidal potential.
Ten analogues were subsequently tested against late stage gametocytes (>95% stage IV/V). Six
compounds (41, 42, 43, 45, 49 and 50) showed sub-micromolar IC50 values (Table 4) with 43
(IC50 = 0.21 µM) the most potent, followed by 50 (IC50 = 0.27 µM), 49 (IC50 = 0.37 µM), 45 (IC50
= 0.60 µM), 42 (IC50 = 0.69 µM) and 41 (IC50 = 0.74 µM). None of the compounds, except 45
displayed increased activity towards the early stage gametocytes when compared to the late stages
(Table 4). This is the first report of the gametocytocidal activity of this series, thereby reflecting
the pan-active potential of the N-aryl-3-trifluoromethyl pyrido[1,2-a] benzimidazole scaffold.
Cumulatively, in view of the sub-micromolar potency of these compounds across the life cycle
stages, these PBI derivatives are promising leads towards the identification of pan-active agents
capable of preventing transmission and enabling chemoprevention. From the current antimalarial
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arsenal, over 90% of the drugs do not show potent activity towards late stage gametocytes and
only primaquine and atovaquone have liver stage activities.23
7
8
9
ADME and cytotoxicity: To select compounds for in vivo evaluation, compounds with the most
potent in vitro asexual blood stage antiplasmodium activity were evaluated in terms of their
cytotoxicity profile in the Chinese Hamster Ovarian cell line (Table 5). Generally, good
selectivity indices (SI) were obtained in both the aniline-type and benzylamine-type series
without manipulations on the LHS. In both subsets, all the compounds had a SI above 30 except
for 34, having a methyl substituent, that showed lower selectivity (SI = 17). Compounds 10, 30
and 26 displayed the best safety profiles (SI > 100). Interestingly, it appears that introducing a
methylene bridge in the aromatic side chain of 10 (SI = 103) to give 26 (SI > 241) improves
selectivity. Within the benzylamine series, better selectivity was achieved with 26 (SI > 241),
which has the more lipophilic trifluoromethoxy substituent, compared to 22 (SI = 35) bearing a
fluoro substituent. On the other hand, the methyl-substituted analogue 34 displayed the least
attractive safety profile (SI = 17).
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
Notably, although the LHS modification whereby dichloro substitutions were performed
improved the potency of many of the compounds, it also increased the potential for inducing
cytotoxicity. Consequently, 22 (SI = 35), from which this modification is absent, displayed a
better safety profile than both 41 (SI = 5) and 42 (SI = 30), which have reduced selectivity,
whereas 34 (SI = 17) and 45 (SI = 15) have about the same selectivity profiles. Similarly, the
excellent safety profile of 30 (SI > 234) was completely lost when dichloro groups were
introduced at the LHS to give 44 (SI = 0.5).
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Regio-isomerism of the LHS seems to impact on the selectivity profiles of these compounds since
41 (SI = 5), with a 3,5 dichloro substitution pattern, is less selective than its 4, 5 dichloro-
substituted analogue 42 (SI = 30). In contrast, regio-isomerism with respect to the position of
substituents on the aromatic side group appears inconsequential to selectivity, as 42 (para-F, SI
= 30), 49 (ortho-F, SI = 25) and 50 (meta-F, SI = 37) displayed similar selectivity indices. Overall,
two out of the 14 compounds, 41 and 44, failed to meet our defined criteria for compounds with
good selectivity (SI > 10).
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
In general, the compounds showed moderate to high metabolic stability as assessed in mouse and
human liver microsomes, MLM and HLM, respectively (Table 5). Compounds incorporating
heteroaromatic moieties tended to be more labile, as exemplified by 44, which had less than 75%
of compound remaining after the 30-minute incubation period in both HLM and MLM. Moreover,
these compounds seem to be more efficiently metabolized in mouse than human microsomes,
except for 10, which was equally stable in both species. Overall, compounds with the highest
microsomal stability in both species, with over 75% remaining after an incubation period of 30
minutes were 10, 49 and 50.
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Table 5. In vitro metabolic stability and cytotoxicity profile of frontrunner analogues.
5
6
7
in vitro
8
9
% rem. after
IC50
30min (t
)
½min
Compound
Code
Selectivity
Index
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
Structure
(µM)
CHO PfNF54
MLM
HLM
>99
>99
17.58
0.17
103
10
(>150)
(>150)
30
22
26
34
-
-
-
-
-
-
-
-
>272
45
1.16
1.28
0.92
1.49
>234
35
>222
25
>241
17
76
82
41
42
1.66
13
0.31
0.44
5
(75)
(105)
102
72
30
(>150)
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in vitro
IC50
% rem. after
30min (t
)
½min
Compound
Code
Selectivity
Index
Structure
(µM)
CHO PfNF54
MLM
HLM
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
(63)
82
>99
43
44
45
5.02
0.75
6.06
0.44
1.42
0.4
11
0.5
15
(>150)
(105)
31
72
(63)
91
(18)
69
(56)
66
(>150)
100
47
48
49
4.84
8.48
4.30
0.44
0.31
0.17
10
27
25
(>150)
(50)
-
-
84
97
(119)
(>150)
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in vitro
IC50
% rem. after
30min (t
)
½min
Compound
Code
Selectivity
Index
Structure
(µM)
CHO PfNF54
MLM
HLM
100
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
84
50
7.03
0.19
37
(119)
(>150)
MLM: Mouse Liver Microsomes; HLM: Human Liver Microsomes; CHO: Chinese Hamster Ovarian Cell lines;
%rem: Percent compound remaining after 30 minutes incubation; t½min: Projected half-life in minutes. SI:
selectivity index is the ratio of CHO IC50: PfNF54IC50.
hERG inhibition profiling: Drugs that inhibit the hERG-encoded potassium ion channel can
cause perturbations in myocardial function and lead to potentially fatal cardiac arrhythmias
known as torsade de pointes.32,33 For this reason, drug discovery programs often profile lead
compounds against this target to establish any potential risk associated with the inhibition of this
protein. To understand the potential for cardiotoxicity risk arising from possible off-target
interactions with the hERG potassium ion channel, we profiled representative in vitro active
compounds (10, 47, 48, 49 and 50) in the hERG assay using the IonWorks patch-clamp
electrophysiology.34 Experimental procedures for this assay are described in Supporting
Information (Section F) with data for the tested compounds summarized in Table 6.
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Table 6. hERG inhibition activity of selected compounds.
5
6
7
8
% inhibition (SD) at
Compound number
Chemical structure
aIC50, µM
0.3 µM
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
10
7.51 (2.85)
>10
47
48
6.82 (2.81)
6.13 (5.14)
>10
>10
49
50
5.49 (3.90)
6.53 (4.15)
>10
>10
aCompounds showed less than 40% inhibition at the highest tested concentration of 10 µM. All values
represent the mean of 3 results obtained from experiments performed as biological and technical triplicates.
This series showed a promising hERG potassium ion channel inhibition profile as depicted by the
high IC50 values (>10 µM) [Table 6]. The positive control drug used in the experiment, verapamil
hydrochloride, inhibited the target potently, with an IC50 between 0.2 and 0.8 µM.
29
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