Antimalarial Benzimidazole Derivatives Incorporating Phenolic Mannich Base Side Chains Inhibit Microtubule and Hemozoin Formation: Structure-Activity Relationship and in Vivo Oral Efficacy Studies

Article abstract of DOI:10.1021/acs.jmedchem.1c00354

A novel series of antimalarial benzimidazole derivatives incorporating phenolic Mannich base side chains at the C2 position, which possess dual asexual blood and sexual stage activities, is presented. Structure-activity relationship studies revealed that the 1-benzylbenzimidazole analogues possessed submicromolar asexual blood and sexual stage activities in contrast to the 1H-benzimidazole analogues, which were only active against asexual blood stage (ABS) parasites. Further, the former demonstrated microtubule inhibitory activity in ABS parasites but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/kg. Generally, the compounds were noncytotoxic to mammalian cells.

Full text of DOI:10.1021/acs.jmedchem.1c00354

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Article
Antimalarial Benzimidazole Derivatives Incorporating Phenolic
Mannich Base Side Chains Inhibit Microtubule and Hemozoin
Formation: Structure−Activity Relationship and In Vivo Oral Efficacy
Studies
Godwin Akpeko Dziwornu, Dina Coertzen, Meta Leshabane, Constance M. Korkor, Cleavon K. Cloete,
Mathew Njoroge, Liezl Gibhard, Nina Lawrence, Janette Reader, Mariëtte van der Watt, Sergio Wittlin,
Lyn-Marie Birkholtz, and Kelly Chibale*
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ABSTRACT: A novel series of antimalarial benzimidazole
derivatives incorporating phenolic Mannich base side chains at
the C2 position, which possess dual asexual blood and sexual stage
activities, is presented. Structure−activity relationship studies
revealed that the 1-benzylbenzimidazole analogues possessed
submicromolar asexual blood and sexual stage activities in contrast
to the 1H-benzimidazole analogues, which were only active against
asexual blood stage (ABS) parasites. Further, the former
demonstrated microtubule inhibitory activity in ABS parasites
but more significantly in stage II/III gametocytes. In addition to being bona fide inhibitors of hemozoin formation, the 1H-
benzimidazole analogues also showed inhibitory effects on microtubules. In vivo efficacy studies in Plasmodium berghei-infected mice
revealed that the frontrunner compound 41 exhibited high efficacy (98% reduction in parasitemia) when dosed orally at 4 × 50 mg/
kg. Generally, the compounds were noncytotoxic to mammalian cells.
efforts at an early stage that considers all stages of the parasite’s
life cycle as potential druggable targets to combat and eradicate
the disease.5,6
Complemented by its binding possibilities to a wide range of
biological targets, the benzimidazole scaffold has been
extensively explored in drug discovery. Several reviews and
research articles have reported the diverse biological properties
of benzimidazole derivatives.7−10 Within the context of
malaria, the antimalarial properties of benzimidazole deriva-
tives have been reported.11−17
Like the benzimidazole substructure, the biological useful-
ness of Mannich bases is evident in some current drugs on the
market including amodiaquine, the first Mannich base used to
treat malaria. Notwithstanding its success as an antimalarial
drug, the rapid oxidative metabolism of the 4-aminophenol
moiety of amodiaquine to electrophilic 1,4-quinoneimine and
aldehyde 1,4-quinoneimine metabolites, highly reactive to
INTRODUCTION
■
Efforts in the fight against malaria saw a decline in incidence
rates from 2010 to 2015, which have since plateaued out with
sub-Saharan Africa still carrying the heaviest disease burden.
Artemisinin-based combination therapy (ACT), the current
first-line chemotherapy regimen for the treatment of malaria
caused by Plasmodium falciparum, has been successful in most
parts of the world, although artemisinin resistance has been
reported in Cambodia, Lao People’s Democratic Republic, and
Vietnam.1 More recently, genetic markers of resistance to
artemisinins, namely, a validated mutation in the Pfkelch13
gene (K13 R561H), have been identified in Rwanda, which is
an alarming sign for a possible evolution toward treatment
failures as witnessed in South East Asia during the last two
decades.2 Apart from the artemisinin-based endoperoxides,
current drugs belong mainly to the aminoquinoline,
sulfonamides, and amino alcohol chemical classes, which also
have significant resistance rates, negating their use as
monotherapies.3
Received: February 26, 2021
Published: April 12, 2021
Undoubtedly, there is a need for structurally differentiated
novel antimalarials, regardless of mode of action, to overcome
current and emerging drug resistance challenges.4 Further, the
advent and periodic optimization of target candidate and
product profiles (TCPs and TPPs) for new malaria medicines
has provided a more definitive roadmap for drug discovery
https://doi.org/10.1021/acs.jmedchem.1c00354
© 2021 American Chemical Society
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Article
intracellular glutathione and other protein molecules, has been
associated with hepatotoxicity and agranulocytosis.18−20 Other
antimalarial phenolic Mannich bases include WR-194,965,
JPC-2997, and MK-4815, which is still in preclinical studies
(Figure 1).21−23
The phenolic Mannich bases (11a−11m) were obtained as
hydrochloride salts by first subjecting the respective amino
phenols (8a−8i) to an acetylation reaction with acetic
anhydride in THF to protect the amino group as the
acetamides (9a−9i). These were then subjected to a Mannich
condensation reaction with formaldehyde in the presence of
N,N-diethylamine, N-ethylamine, or N-tert-butylamine in
EtOH. Last, acid hydrolysis afforded the Mannich bases
11a−11m. The target compounds 12−51 were finally
synthesized via nucleophilic aromatic substitution of the 2-
chloro benzimidazoles 1−7 with the phenolic Mannich bases
11a−11m in the presence of KH2PO4 as the base and n-BuOH
as the solvent. Meanwhile, acid cleavage of the p-methox-
ybenzyl (PMB) protecting group of intermediate III afforded
52.
Multistage Antiplasmodium Activity. The synthesized
benzimidazole analogues were evaluated for their in vitro
multistage antiplasmodium activity against the asexual blood
stage (ABS) drug-sensitive (NF54) and multidrug-resistant
(K1) strains and early-stage (stage II/III) and late-stage (stage
IV/V) gametocytes of P. falciparum (Table 1). Regarding SAR
1 exploration, based on the Mannich base used, 5-methyl-3-
((N,N-diethylamino)methyl)-2-hydroxy aniline (R′ = D, Table
1) showed superior activity (IC50 Pf NF54/K1 = [0.19−0.65]/
[0.06−0.46] μM) against both strains, followed by its parent 3-
((N,N-diethylamino)methyl)-2-hydroxy aniline (R′ = C; IC50
Pf NF54/K1 = [0.36−1.00]/[0.13−0.77] μM). For example,
among the synthesized 1H-benzimidazole series, compounds
14 and 15 displayed submicromolar potency against both
NF54 and K1 strains with IC50 Pf NF54/K1 = 0.56/0.77 μM
and IC50 Pf NF54/K1 = 0.23/0.42 μM, respectively, with no
evidence of cross-resistance (RI values, 1.38 and 1.83).
Compounds 12 and 16 showed good activity (IC50 < 1 μM)
against the NF54 strain, but the latter displayed moderate
activity (IC50 = 1.62 μM) against K1 parasites, while 12 lost
activity (RI value, 9.09). Compound 13 was the least active of
the 1H-benzimidazole analogues, showing the largest cross-
resistance formation (IC50 Pf NF54/K1 = 1.88/>10 μM).
All analogues belonging to the N-benzyl benzimidazole
series displayed submicromolar activity against the NF54
Figure 1. Examples of antimalarial Mannich bases.
The abovementioned presence of benzimidazole and
phenolic Mannich base moieties in antimalarial agents
provided a rational basis for attaching phenolic Mannich
base side chains to a benzimidazole core. Two structure−
activity relationship (SAR) studies were undertaken in the
present work (Figure 2). SAR 1 focused on exploring 2-, 3-,
and 4-amino phenol Mannich bases at the C2 position of the
benzimidazole core, along with investigation of the contribu-
tions of para-substituted benzyl groups at the N1 position. The
diethylaminomethylene Mannich base side chain was kept
constant in SAR 1. Subsequently, SAR 2 was undertaken to
optimize the frontrunner compound from SAR 1 by derivatiz-
ing the phenolic Mannich base while introducing fluoro- and
chloro-substituents on the benzimidazole core.
RESULTS AND DISCUSSION
strain, except compounds 26 (IC50 = 2.88 μM) and 31 (IC50
=
■
Chemistry. The synthesis of the target compounds is
described in Scheme 1. Briefly, 2-chloro-1H-benzimidazole (1)
was reacted with 4-cyanobenzyl bromide or 4-
(trifluoromethyl)benzyl bromide in the presence of K2CO3
to obtain the respective substituted 1-benzyl-2-chlorobenzimi-
dazole (2 and 3). Meanwhile, compounds 5 and 6 were
synthesized via chlorination of 1,3-dihydro-2-oxo-benzimida-
zole intermediates I and II in the presence of phosphoryl
oxychloride. Compound 7 was obtained from 5 as previously
described above.
2.27 μM) (Table 1). Generally, 1-((4-trifluoromethyl)benzyl)
(22−26, IC50 Pf NF54/K1 = [0.19−0.47]/[0.06−1.69] μM)
displayed superior activity to the 1-(4-fluorobenzyl)-
benzimidazole analogues (27−31, IC50 Pf NF54/K1 =
[0.36−0.68]/[0.18−1.95] μM) and the 1-(4-cyanobenzyl)-
benzimidazole analogues (17−21, IC50 Pf NF54/K1 = [0.37−
0.87]/[0.46−1.97] μM). All N-benzyl analogues of the 3-
((N,N-diethylamino)methyl)-4-hydroxy aniline Mannich base
were the least active in each series against the K1 strain (IC50
Pf K1 = 1.69−1.97 μM, Table 1). Consequently, compound
Figure 2. Summary of SAR exploration.
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Scheme 1. Reagents and Reaction Conditions: (i) Appropriate Benzyl Bromide, K2CO3, CH3CN, 75 °C, 2 h; (ii) Appropriate
1,2-Diaminobenzene, Urea, THF, 130 °C, MW, 30 min; (iii) POCl3, 130 °C, 3 h; (iv) Acetic Anhydride, THF, 60 °C, 1 h (for
9a−9i); (v) Appropriate Amine, HCHO, EtOH, 85 °C, 2 h; (vi) 6 N HCl, 100 °C, 2 h (for 11a−11m); (vii) KH2PO4, n-BuOH,
100 °C, 8 h (for 12−51); (viii) p-Methoxybenzyl Amine, HCHO, EtOH, 85 °C, 16 h (for III); (ix) TFA, DCM, 25 °C, 48 h (for
52)
25, which bears 1-((4-trifluoromethyl)benzyl) benzimidazole
incorporating 5-methyl-3-((N,N-diethylamino)methyl)-2-hy-
droxy aniline, was the most potent against the ABS parasites
(IC50 Pf NF54/K1 = 0.19/0.06 μM), with notable activity
against the K1 strain, with no cross-resistance formation.
Subsequent SAR 2 studies focused on compound 25 as the
frontrunner. Synthesized compounds for SAR 2 studies
maintained submicromolar activity against both strains, except
compound 38 (IC50 Pf NF54/K1 = 1.84/0.38 μM, Table 2).
From a SAR perspective, compounds containing the
diethylaminomethylene Mannich base side chain were more
active than those containing the corresponding monoethyla-
minomethylene and t-butylaminomethylene Mannich base side
chains. This is specifically evident in the activities of 25 (IC50
Pf NF54/K1 = 0.19/0.06 μM), 32 (IC50 Pf NF54/K1 = 0.78/
0.30 μM), 33 (IC50 Pf NF54/K1 = 0.40/0.23 μM), 34 (IC50
Pf NF54/K1 = 0.15/0.05 μM), 35 (IC50 Pf NF54/K1 = 0.28/
0.11 μM), and 36 (IC50 Pf NF54/K1 = 0.28/0.06 μM). The
activity of 34 (IC50 Pf NF54/K1 = 0.15/0.05 μM) was
comparable to 25 (IC50 Pf NF54/K1 = 0.19/0.06 μM),
suggesting that the methyl group can be replaced with a
chloro group on the phenol moiety. Similarly, potency was
maintained with the fluoro substituent, as evident in 37 (IC50
Pf NF54/K1 = 0.17/0.19 μM), although a decrease in activity
against the K1 strain was observed. On the contrary, the loss in
activity of 38 (IC50 Pf NF54/K1 = 1.84/0.38 μM), a positional
isomer of 34 (IC50 Pf NF54/K1 = 0.15/0.05 μM), suggests
that the C5′ position of the phenol moiety is optimal for
activity. Furthermore, a comparison of the activities of 39 (IC50
Pf NF54/K1 = 0.26/0.13 μM) and 40 (IC50 Pf NF54/K1 =
0.25/0.15 μM) to 32 (IC50 Pf NF54/K1 = 0.78/0.30 μM) and
33 (IC50 Pf NF54/K1 = 0.40/0.23 μM), respectively, seems to
suggest that fluoro substitution on the benzimidazole core
scaffold potentiates activity. The 1-benzylbenzimidazole
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Table 1. In Vitro Antiplasmodium Activity against Sensitive (NF54) and Multidrug-Resistant Strains (K1) and Cytotoxicity of
SAR 1 Compounds
a
ABS , IC50 (μM)
gametocytes, % inhibition (5 μM/1 μM) cytotoxicity IC50 (μM)
b
c
d
e
f
g
compound
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
R
R′
Pf NF54
Pf K1
RI
EG
IC50 (μM)
LG
CHO
SI
H
H
H
H
H
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
A
B
C
D
E
0.80
1.88
0.56
0.23
0.86
0.47
0.37
1.00
0.65
0.87
0.39
0.47
0.44
0.19
2.88
0.68
0.64
0.36
0.55
2.27
0.01
7.27
>10
0.77
0.42
1.62
1.97
0.76
0.66
0.46
0.83
1.69
0.87
0.13
0.06
0.94
1.95
1.28
0.18
0.21
1.32
0.42
9.09
>5.32
1.38
1.83
1.88
4.19
2.05
0.66
0.71
0.95
4.33
1.85
0.30
0.32
0.33
2.87
2.00
0.50
0.38
0.58
42.00
9/30
28/0
>50
>50
>50
>50
>50
>50
>62
>26
>89
>217
>58
>106
105.51
>50
21.83
>57
44.87
88.29
>113
154.26
11.76
17.63
71.54
>136
34.00
>22
0/3
34/0
44/1
32/3
37/34
31/34
35/9
(4-CN)C6H4CH2
(4-CN)C6H4CH2
(4-CN)C6H4CH2
(4-CN)C6H4CH2
(4-CN)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-F)C6H4CH2
23/20
44/38
69/47
98/39
96/19
48/34
41/36
93/71
97/91
90/18
59/33
15/12
91/65
97/81
24/14
21/4
39.04
>50
14.19
>50
17.50
41.50
>50
29.31
33.88
11.99
45.79
>50
43/19
52/15
22/0
45/31
24/2
56/35
62/41
43/1
39/22
36/29
58/11
58/31
39/36
1.45
0.11
28
29
30
31
(4-F)C6H4CH2
(4-F)C6H4CH2
(4-F)C6H4CH2
(4-F)C6H4CH2
2.23
0.88
18.70
>50
chloroquine
emetine
methylene blue
0.02
0.14
h
h
93
0.19
90
a
b
ABS, asexual blood stage; IC50’s represent means from n values of ≥2 independent experiments. RI, resistance index (ratio of IC50 [K1]:IC50
c
d
e
f
[NF54]). EG, early-stage gametocytes (stage I/III). IC50’s against early gametocytes. LG, late-stage gametocytes (stage IV/V). CHO, Chinese
g
h
hamster ovarian cell line. SI, selectivity index (ratio of IC50[CHO]:IC50[NF54]). %Inhibition at 1 μM; dual-point values were determined for one
biological repeat (n = 1) in technical triplicates. IC50 values are means from at least three independent biological repeats (n ≥ 3) performed in
technical triplicates. Blank spaces, not tested.
analogues 32−42 maintained better potency against the K1
strain than the NF54 strain of P. falciparum, as observed for
their SAR 1 congeners above.
performed at concentrations of 1 and 5 μM (Table 1) against
early-stage (50% stage II/50% stage III) and late-stage
gametocytes (40% stage IV/60% stage V). Compounds
exhibiting good potency (>70% inhibition at 5 μM and
>50% inhibition at 1 μM) were progressed to dose-dependent
IC50 determinations.
The 1H-benzimidazole analogues 43−51 exhibited similar
SAR trends as described for the 1-benzylbenzimidazole
analogues, showing comparable submicromolar IC50 values
(Pf NF54/K1 = [0.15−0.30]/[0.25−0.88] μM, Table 2). This
seems to suggest that the benzyl moiety does not significantly
contribute to antiplasmodium activity. It is also evident from
this series of compounds that the C5′ tert-butyl group, as in 46
and 50, makes a positive contribution to activity, albeit has the
drawback of increasing lipophilicity. In contrast to the 1-
benzylbenzimidazole analogues, the SAR 2 1H-benzimidazole
derivatives, like their SAR 1 congeners, were more potent
against the NF54 than the K1 strain of P. falciparum with an
average RI of 2.75.
From Table 1, the 1-benzylbenzimidazole series showed
better potency against early-stage gametocytes than to later
stages. Analogues based on the ortho-hydroxy aniline Mannich
base (R′ = C, Table 1) and its derivatives (R′ = D and E)
displayed good activity (>70% inhibition) against early-stage
but not late-stage gametocytes at the 5 μM drug concentration.
Generally, the 1-((4-trifluoromethyl)benzyl) and 1-(4-fluoro-
benzyl) analogues were more potent (>90% inhibition at 5
μM) than their corresponding 1-(4-cyanobenzyl) congeners,
which displayed moderate activity. The frontrunner compound
25 maintained >90% inhibition at 1 μM and recorded an IC50
value of 0.11 μM toward early-stage gametocytes. Compounds
24, 29, and 30 also showed good IC50 values of 1.45, 2.23, and
0.88 μM, respectively. Similarly, the SAR 2 congeners of 25
exhibited <1 μM IC50 values, except 38. Moreover, 34 and 37
The effect of the synthesized compounds on P. falciparum
gametocyte viability was investigated using the transgenic
parasite line NF54-Pfs16-GFP-Luc in a luciferase reporter
assay,24 allowing for stage-specific determination of game-
tocytocidal action. Dual-point screens of each compound were
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Table 2. In Vitro Antiplasmodium Activity against Sensitive (NF54) and Multidrug-Resistant Strains (K1) and Cytotoxicity of
SAR 2 Compounds
cytotoxicity, IC50
a
ABS , IC50 (μM)
gametocyte, % inhibition (5 μM/1 μM)
(μM)
b
c
d
compound
25
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
X
R
R′
PfNF54 Pf K1
RI
EG
IC50 (μM)
LG
IC50 (μM) CHO
SI
H
H
H
H
H
H
H
H
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
(4-CF3)C6H4CH2
D
F
G
J
K
L
M
N
F
G
K
L
G
K
L
0.19
0.78
0.40
0.15
0.28
0.28
0.17
1.84
0.26
0.25
0.19
0.19
0.20
0.30
0.27
0.08
0.26
0.25
0.26
0.15
0.22
5.47
0.42
0.06
0.30
0.23
0.05
0.11
0.06
0.19
0.38
0.13
0.15
0.08
0.09
0.79
0.88
0.63
0.25
0.83
0.78
0.53
0.28
0.40
0.32
0.39
0.58
0.33
0.39
0.21
1.12
0.21
0.5
97/91
95/30
96/45
89/69
91/66
92/44
90/74
59/7
94/86
96/60
91/62
70/14
47/0
23/0
0/0
56/30
31/0
35/0
16/0
0/0
0/0
38/20
0.11
0.99
0.92
0.056
0.43
0.59
0.097
3.06
0.21
0.52
0.46
0.69
62/41
14/0
62/0
10/0
8/2
0/0
10/0
1/0
29.31
18.80
30.03
>50
33.81
36.28
>50
154.26
24.10
75.07
>333
120.75
129.57
>294
>27
1.90
>5
>5
>50
5,6-F
5,6-F
5,6-F
5,6-F
H
24/5
73/0
0/0
2.33
>5
2.04
7.84
0.6
10.04
>50
40.16
>263
>263
166.5
>166
>185
>380
64.03
>200
152.84
227.66
117.18
10.45
0.42
0.47
3.95
2.93
2.33
3.125
3.19
3.12
2.40
1.87
1.81
1/0
>50
H
H
H
H
H
H
H
H
39/31
31/22
0/0
0/0
0/0
0/0
0/0
0/0
0/0
33.30
>50
H
H
H
>50
P
30.46
16.65
>50
39.74
34.15
25.78
47.56
47
48
49
50
51
52
5,6-F
5,6-F
5,6-F
5,6-F
4,6-Cl
H
G
K
L
P
H
G
Q
0.01
(4-CF3)C6H4CH2
0/0
chloroquine
emetine
methylene blue
42.00
0.02
e
e
93
0.19
90
0.14
a
b
ABS, asexual blood stage; IC50’s represent means from n values of ≥2 independent experiments. RI, resistance index (ratio of IC50 [K1]:IC50
c
d
e
[NF54]). CHO, Chinese hamster ovarian cell line. SI, selectivity index (ratio of IC50[CHO]:IC50[NF54]). %Inhibition at 1 μM; dual-point
values were determined for one biological repeat (n = 1) in technical triplicates. IC50 values are means from at least three independent biological
repeats (n ≥ 3) performed in technical triplicates. Blank spaces, not tested.
demonstrated potent activity with IC50 values of 0.056 and
0.097 μM, comparable to 25 (IC50 = 0.11 μM), corroborating
the earlier finding in ABS activity (for SAR 2) above that the
C5′ methyl group on the phenol unit can be replaced by the
chloro and fluoro groups with retention of activity.
Compounds with early-stage gametocytocidal activity of
IC50 < 0.5 μM were prioritized for IC50 determinations against
late-stage gametocytes. In this regard, only 34 (IC50 = 1.90
μM) and 39 (IC50 = 2.33 μM) showed moderate activities
(Table 2). Key compounds (24, 25, 34, 35, 37, 39, and 40)
were further screened for their potential transmission blocking
activity against male gametes at 2 μM, but the compounds
showed <50% inhibition of male gamete exflagellation. The
poor activity of these compounds toward the transmissible
gamete stages correlates with the lack of potency observed
against the late-stage gametocytes, confirming their specificity
toward early-stage gametocytes.
To summarize, Figure 3 depicts a structure−activity
landscape evaluation showing a clear chemically distinct profile
between the synthesized 1-benzylbenzimidazole (17−42, and
52) and 1H-benzimidazole (12−16 and 43−51) analogues
with respect to antiplasmodium activity. That is, the 1-
benzylbenzimidazoles showing ABS and early-stage gameto-
cyte dual activities from both SAR 1 and SAR 2 clustered
separately from the less active 1H-benzimidazoles. Within the
1-benzylbenzimidazole cluster, from SAR 1, compounds
containing either 3-((N,N-diethylamino)methyl)-2-hydroxy
aniline (R′ = C, Table 1) or its 5-methyl congener (R′ = D,
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Figure 3. Structure−activity landscape index (SALI) plot of the 1-benzylbenzimidazoles and 1H-benzimidazole analogues. Pairwise structural
feature (SkelSphere) of asexual blood stage (ABS) and late-stage (LG, IV/V) analysis superimposed with early-stage (EG, I-III) cliff analysis (Osiris
DataWarrior) at a similarity threshold of 86%. Activity inclusions include hit IC50 (μM) of asexual blood stage (ABS) strains and early-stage (EG)
and late-stage (LG) gametocytes. Clusters indicate structurally related compounds, with compounds indicated as nodes and structural connectivity
indicated through the edges. Node size reflects IC50 values against EG, and node color and shadow (clouds) indicate ABS and LG activities,
respectively.
Table 1) Mannich bases derivatized with either 1-((4-
trifluoromethyl)benzyl)benzimidazole (as in 24 and 25) or
1-(4-fluorobenzyl)benzimidazole (as in 29 and 30) groups
showed the most potent ABS and early-stage gametocyte (EG)
activities. Subsequent SAR 2 analogues (32−42) based on
compound 25 maintained their respective ABS and EG dual
activities with IC50’s < 1 μM, as they clustered closely with 25
(Figure 3). Meanwhile, all other Mannich base side chains
(that is, R′ = A, B, and E; Table 1) and analogues derived from
the 1-(4-cyanobenzyl)benzimidazole scaffold constituted a less
active cluster. Furthermore, Figure 3 also confirms the strong
selectivity of the present benzimidazole compounds toward
early-stage gametocytes compared to late-stage gametocytes,
with only two compounds (34 and 39) showing moderate late-
stage gametocyte activity (IC50’s between 1 and 5 μM, Table
2).
Cytotoxicity. The toxicity of the synthesized compounds
to mammalian cells was assessed against the Chinese hamster
ovarian (CHO) cell line. The compounds were relatively
noncytotoxic, exhibiting acceptable selectivity indices (SI >
10), except 39 (Tables 1 and 2). Meanwhile, representative
compounds were evaluated for toxicity against the Caucasian
hepatocellular carcinoma cells, HepG2 (Table S1, Supporting
Information).
25 was, however, much more rapidly metabolized compared to
32 and 33.
Table 3. In Vitro Microsomal Stability and Physicochemical
Properties of Selected Analogues
metabolic stability (%
remaining after 0.5 h)
a
compound MLM RLM HLM cLogP
solubility (μM), pH 6.5
25
32
33
35
36
39
40
41
42
44
48
<7
52
62
73
36
93
>99
86
94
97
98
<7
51
41
66
9
77
89
98
94
98
>99
36
98
99
94
81
99
90
>99
89
7.0
6.0
6.7
6.3
7.0
6.2
7.0
6.6
7.3
3.9
4.2
<5
35
50
12
<5
16
<5
<5
<5
200
12
93
>99
a
Predicted from ChemDraw Professional, version 19.0.
Metabolite identification studies on 25 in MLM revealed
that it underwent two main metabolic processes involving
oxidation of either the benzimidazole core or the phenol unit
and deethylation of the aminoalkylmethylene moiety (Figure
4A). Analyses of the relative proportions of the metabolites,
however, revealed that the N-desethyl metabolite of 25 (the
equivalent of 32) is rapidly formed and is hence a major
contributor to the loss of 25 (Figure 4B), which is congruent
to the metabolism of amodiaquine to its active metabolite,
desethyl amodiaquine.25 A substantial proportion of the
oxidized metabolite (P+16) was formed, which is comparable
to the P-12 metabolite, most likely formed after oxidation of
Metabolic Stability in Liver Microsomes and Phys-
icochemical Properties. Compounds 25, 32, and 33 were
assessed for metabolic stability in mouse, rat, and human liver
microsomes (MLM, RLM, and HLM, respectively). Com-
pounds with >70% remaining after 30 min of incubation with
liver microsomes were adjudged to be metabolically stable.
The compounds showed species-specific metabolism with
respect to rodents and humans (Table 3). The compounds
were more stable in HLM than in MLM and RLM. Compound
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Figure 4. (A) Identified metabolites from the metabolism of the parent (P) compounds 25 and 33 in mouse liver microsomes (MLM); (B) XIC
plot showing the relative abundances of the identified metabolites from 25; (C) XIC plot showing the relative abundance of 33 and its only
metabolite (P+16) in MLM.
the N-desethyl metabolite (P-28 [32]). Like desethyl
amodiaquine, the antiplasmodium activity of 32 (IC50
Pf NF54/K1 = 0.78/0.30 μM, Table 2) has useful pharmaco-
logical significance. The low relative abundance of the
dideethylation metabolite (P-56), formed either directly from
the parent molecule or after further deethylation of P-28,
suggests that its formation is not a rapid process (Figure 4B).
However, it is noteworthy that this metabolite (P-56 [52, IC50
Pf NF54 = 5.47 μM], Table 2) was about 6- and 24-fold less
active than the desethyl (P-28, 32) and diethyl (25) congeners,
respectively. Further, in contrast to the rapid metabolism of the
Mannich base side chain of 25 and 32, the N-t-butyl moiety of
33 was stable to metabolism; thus, only the oxidized
metabolite was observed (Figure 4C).
From Figure 4, selected compounds were assessed for
further metabolic stability studies in MLM, RLM, and HLM
with the aim to investigate and mitigate the formation of the
oxidized metabolite (P+16). The compounds were selected
based on the hypothesis that the oxidation arose from
hydroxylation of either the benzene unit of the benzimidazole
core or the methyl group on the phenol unit. In this regard, all
the selected compounds showed improved stability in liver
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microsomes in all the three species compared to the parent
compounds (32 and 33, Table 3). That is, fluoro substitution
on the benzimidazole core (as in 39 and 40) resulted in higher
stability than the replacement of the methyl group on the
phenol unit with a chloro group (as in 35 and 36). Further,
substitutions at both sites, as in compounds 41 and 42,
afforded the most metabolically stable analogues. The results
suggest that oxidation on the benzimidazole core is more
detrimental to stability in liver microsomes than on the phenol
unit. However, from the results of 44 and 48, which lack the
benzyl moiety on the benzimidazole core, it is apparent that
the presence of the benzyl group makes the benzimidazole core
scaffold more susceptible to metabolism via oxidation. In this
regard, the possibility of the oxidation happening at the
benzylic position rather than on the benzimidazole core cannot
be overlooked.
Table 5. Intravenous (i.v.) and Oral (p.o.) Mouse
Pharmacokinetics Parameters for 41 and 48
a
41
48
route
i.v.
3
p.o.
10
0.4
i.v.
p.o.
10
0.026
0.7
nominal dose (mg/kg)
Cmax (μM)
3
Tmax (h)
2.8
8.3
t1/2 terminal (h)
clearance CLb (mL/min/kg)
Vd (L/kg)
6.3
12.6
6.9
ND
151
96
ND
Vss (L/kg)
5.8
58
AUC0−t (min·μmol/L)
F (%)
443
321
24
65
11
5
a
Blank spaces, not determined.
Compounds selected for microsomal metabolic stability
studies were also profiled for aqueous solubility at a pH of 6.5
(Table 3). As expected, fluorination of the benzimidazole core
scaffold and substitution of the 5′-methyl group with the
chloro group on the phenol moiety led to poor aqueous
solubility. Compound 44, which lacks the benzyl and fluoro
groups on the benzimidazole core, was highly soluble (200
μM) compared to 41 (<5 μM) and 48 (12 μM), thus
highlighting the expected negative contributions of these
groups to solubility.
In Vivo Efficacy Studies. Six compounds with good
antiplasmodium activity and MLM stability were evaluated for
in vivo efficacy in a Plasmodium berghei-infected NMRI mouse
model (Table 4). Compounds were dosed orally at 4 × 50 mg/
Figure 5. Pharmacokinetics profile of 41 and 48 following oral
administration. Each data point represents the mean from three mice,
with error bars indicating the standard deviation.
Table 4. In Vivo Antimalarial Efficacy after 1 Oral Dose of
50 mg/kg on 4 Consecutive Days in P. berghei-Infected Mice
suggests extrahepatic routes of clearance or the involvement of
active transporters in the clearance of 48.
Permeability Studies. To further investigate the con-
tribution of physicochemical parameters to the pharmacoki-
netics profiles of the compounds, the permeability of model
compounds 35 and 44 was investigated using the parallel
artificial membrane assay (PAMPA, Table 6). It was not
Pf NF54, IC50
(μM)
MLM,
(%)
solubility (μM),
%
compound
33
35
41
42
pH 6.5
activity
0.40
0.28
0.19
0.19
0.30
0.25
62
73
85
94
97
98
50
12
<5
<5
200
12
<40
<40
98
<40
<40
<40
99.9
44
Table 6. In Vitro Permeability Studies for 35 and 44
48
a
chloroquine
compound
permeability (LogPapp)
a
Oral dose, 4 × 30 mg/kg.
35
44
−5.1
−6.2
kg. Parasitemia reduction was expressed as a percentage
relative to the control. The only exception was compound 41,
which achieved an encouraging parasitemia reduction of 98%.
Interestingly, no apparent correlation could be deduced
between the observed in vivo efficacy results and the in vitro
ABS activity, hepatic microsomal stability, and solubility of the
test compounds (Table 4).
Pharmacokinetics Studies. To ascertain the possible
underlying reasons for the lack of in vivo efficacy, such as drug
absorption, permeation, and exposure-related shortfalls,
pharmacokinetics studies were carried out in healthy Balb/c
mice using two representative compounds, 41 and 48 (Table
5). A higher oral exposure was measured for 41 as compared to
48 (Figure 5). Given that the antiplasmodium activity is
comparable between the two compounds, this difference in
exposure could explain the lower efficacy of 48. The clearance
of 41 is also markedly lower than the clearance of 48, with the
latter clearance being higher than hepatic blood flow. This
possible to get reproducible data for the same compounds for
which pharmacokinetics studies had been performed due to
their poor solubility under the assay conditions.
As expected from the cLogP data (Table 3), 35 was more
permeable than 44, with the two compounds falling into the
moderate and low permeability classes, respectively. Improved
permeability is associated with better passive permeability
through the body and with a reduced contribution by active
transport processes to both absorption and clearance.26,27 If
the same trends hold across the series, then 48 would be
expected to be less permeable than 41 and thus more prone to
active transport contributions to its clearance, thus explaining
lower exposure.
Mechanistic Studies. Antimalarial benzimidazole deriva-
tives have recently been shown to inhibit hemozoin
formation.16,28,29 Thus, inhibition of hemozoin formation as
a potential mode of action of the synthesized analogues in ABS
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Figure 6. Heme species in synchronized control and drug-treated PfNF54 parasites. Plots (A) and (B) represent free heme and hemozoin,
respectively, represented in terms of iron (Fe) measured in fg/cell. Asterisks indicate statistical significance relative to the control, where *p < 0.05,
**p < 0.01, and ***p < 0.001 (Figures S2−S5, Supporting Information).
parasites was first evaluated in vitro by investigating their ability
to inhibit the formation of β-hematin, the synthetic equivalent
of hemozoin, in a cell-free assay. Most compounds passed a
discriminatory cut-off of β-hematin inhibition activity (βHIA)
at IC50 < 100 μM and were therefore classified as good
inhibitors of β-hematin formation (Table S1, Supporting
Information).
To validate the above, a cellular heme fractionation assay
was further carried out to determine if these compounds are
true inhibitors of hemozoin formation. In this regard, four
compounds, 35, 41, 44, and 48, having βHIA IC50 values of
33.31, 18.14, 12.71, and 7.15 μM, respectively, compared to
amodiaquine (IC50 βHIA = 13.16 μM), were chosen for
investigation in this assay, which measures the free heme and
hemozoin levels when synchronized ring-stage parasites are
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treated with increasing concentrations of the compounds. For
true inhibitors of hemozoin formation, a dose-dependent
increase in free heme is observed with a concomitant decrease
in hemozoin.30 From Figure 6, compounds 44 and 48 were
identified as bona fide hemozoin inhibitors. However, 35 and
41, despite their ability to inhibit β-hematin in the cell-free
assay, showed no significant concentration-dependent effect on
the heme and hemozoin levels in the cellular fractionation
assay (Figure 6).
Taken together, the results suggest hemozoin inhibition as
one of the mechanisms by which 44 and 48 act. However,
inhibition of hemozoin formation is not the sole mode of
action of the current compounds since 35 and 41 do not
directly act on this pathway.
Interestingly, colchicine inhibited microtubule polymerization
from stage I gametocyte development, with 41 and 44
treatment only showing little or no apparent decrease in
Tubulin Tracker signal intensity in stage I gametocytes
compared to untreated parasites. However, all three com-
pounds decreased tubulin formation from stage II and III
gametocytes, among which 41 showed the most significant
decrease in Tubulin Tracker intensity, with the signal only
limited to the periphery of the parasite (Figure 7B). Between
41 and 44, these results support the high in vitro early-stage
gametocytocidal activity (Tables 1 and 2) of the 1-
benzylbenzimidazole series.
Although potent microtubule destabilizers, colchicine (IC50
= 10 μM) and colchicine-site binders, such as albendazole
(IC50 = 30 μM), mebendazole (IC50 = 200 μM), and
nocodazole (IC50 = 17 μM), are less active against P.
falciparum ABS and show low therapeutic indices (0.001−
0.01) relative to mammalian cells.34 The poor antiplasmodium
activity of these benzimidazole drugs is due to poor binding to
β-tubulin (the presence of Met rather than Val or Ile in P.
falciparum β-tubulin position 316).32 Further, P. falciparum
gametocytes are insensitive to colchicine,36 although in
Plasmodium yoelii nigeriensis gametocytes, colchicine was
shown to inhibit the polymerization of cytoplasmic-axonemal
and intranuclear mitotic microtubules, but male and female
gametocytes remained unchanged at inhibitory concentrations
of the drug.37 On the contrary, however, coupled with
microtubule activity (Figure 7), the benzimidazoles presented
here show potent ABS activity (Tables 1 and 2) and the 1-
benzylbenzimidazole series exhibits activity as low as IC50 = 56
nM (compound 34) against stage II/III gametocytes, with
good selectivity margins (SI >10). Furthermore, the lack of
microtubule inhibition in stage I gametocytes by the present
benzimidazoles contrary to colchicine (Figure 7B), and yet a
significant microtubule activity in stage II/III gametocytes,
clearly suggests that the former affect gametocyte microtubule
structures in a different manner to colchicine, halting
polymerization and/or decreasing tubulin population, possibly
by inducing microtubule catastrophe, from stage I gametocyto-
genesis onward, which could arrest further development. In
effect, it would be worth investigating in much more detail the
inhibitory effects of the potent benzimidazoles presented here
against ABS and early-stage gametocyte microtubules, bearing
in mind that the functions of these cytoskeletal proteins are not
only limited to their role in cell division. If the current
benzimidazoles are bona fide inhibitors of microtubule
formation, then the lack of late-stage gametocytocidal activity
could be related to disassembled microtubules, especially in
stage V gametocytes.33
In further mechanistic studies, compounds 41 and 44 were
investigated for the potential to inhibit microtubule formation
in P. falciparum ABS and early-stage gametocytes, with
colchicine as a positive control (Figure 7). This was prompted
Figure 7. Inhibition of microtubule formation in (A) asexual
trophozoites and (B) stage I−III gametocytes. Live-cell confocal
imaging of asexual trophozoites and early-stage gametocytes (20%
stage I/60% stage II/20% stage III) labeled with Tubulin Tracker
(TT, green) and Hoechst 3324 nuclear stain (blue). Bright-field
images are shown on the right side of each image. UT, untreated.
Scale bar represents 5 μm.
by knowledge of anthelminthic benzimidazole drugs, which
bind to the colchicine site and inhibit microtubule assembly
between α-tubulin and β-tubulin through interaction with the
β-tubulin subunit.31,32 Microtubules are present during all
asexual blood stages, and in gametocytes, microtubules play
key roles in the morphological changes associated with parasite
development from stages I to IV but disassemble in stage V.33
For this reason, these cytoskeletal proteins have long been
known as a potential drug target for malaria treatment,
although not extensively explored.32−35 This further prompted
us to investigate the inhibition of microtubule formation as a
potential contributing mechanism.
Live-cell confocal microscopy of treated ABS parasites and
early-stage gametocytes stained with Tubulin Tracker showed
a marked decrease in tubulin density in ABS parasite stages
(Figure 7A,B and Figures S6 and S7, Supporting Information),
similar to that observed for colchicine, indicating that the
compounds are able to inhibit tubulin polymerization.
CONCLUSIONS
■
Novel benzimidazole analogues incorporating phenolic Man-
nich base side chains were synthesized and evaluated as
potential antimalarial agents. Most of the analogues showed
antiplasmodium activity against the NF54 and K1 strains of P.
falciparum with IC50 values <1 μM, as well as acceptable
selectivity margins with respect to cytotoxicity. The 1-
benzylated benzimidazole analogues exhibited early-stage
gametocytocidal activity with IC50 values <1 μM. One
analogue (41) showed 98% activity in P. berghei-infected
mice after 1 oral dose of 50 mg/kg on 4 consecutive days. The
synthesized analogues showed inhibitory activity against
hemozoin formation and microtubule polymerization (in
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BuOH was heated at 100 °C in the presence of excess KH2PO4 as a
base. Upon completion (TLC, 8−16 h), the reaction mixture was
allowed to cool down and diluted with water, and the solvent was
removed under reduced pressure. The residue was dissolved in a
mixture of DCM:MeOH (1:1, v/v) and completely dried in the
Genevac to remove all traces of n-BuOH. The target compounds
(12−51) were obtained after flash column chromatography using
mixtures of DCM and MeOH as a mobile phase.
trophozoites and early-stage II/III gametocytes). The strategy
and plans for a future lead optimization campaign to improve
potency, maintain favorable ADME-PK properties, limit
cytotoxicity, and improve efficacy will include SAR expansion
around the benzimidazole scaffold, benzylic group, and
Mannich base side chain.
EXPERIMENTAL SECTION
2-(3-((N,N-Diethylamino)methyl)-4-hydroxyanilino)-1H-benzo-
■
1
[d]imidazole (12). Brown viscous oil (82 mg, 41% yield); H NMR
The starting reagents, 2-chloro benzimidazole, 1-(4-fluorobenzyl)-2-
chlorobenzimidazole, and the amino phenols, were purchased from
commercial vendors. Unless otherwise stated, all solvents used were
anhydrous. Reactions were monitored using aluminum silica pre-
coated thin-layer chromatography (TLC) plates (Silica gel 60 F254
(Merck KGaA, Darmstadt, Germany)). TLC spots were visualized
under ultraviolent light (UV) at 254 nm. The 1H NMR and 13C NMR
spectra were acquired on either a Bruker AV 400 (1H 400.0, 13C 100.6
MHz) or Bruker Ascend 600 (1H 600.0, 13C 151 MHz) spectrometer.
An Agilent LC−MS instrument comprising an Agilent 1260 Infinity
Binary Pump, Agilent 1260 Infinity Diode Array Detector, Agilent
1290 Infinity Column Compartment, Agilent 1260 Infinity
Autosampler, Agilent 6120 Quadrupole MS, and Peak Scientific
Genius 1050 Nitrogen Generator and fitted with an X-bridge (C18,
2.5 μm, 3.0 mm (ID) × 50 mm length) column maintained at either
35 or 40 °C was used to monitor the progress of reactions including
percent purity determinations. Target compounds were confirmed to
have >95% purity.
(400 MHz, DMSO-d6) δ 7.69 (d, J = 2.7 Hz, 1H), 7.46 (dd, J = 3.2,
5.9 Hz, 2H), 7.35 (dd, J = 2.7, 8.7 Hz, 1H), 7.21 (dd, J = 3.2, 5.9 Hz,
2H), 7.13 (d, J = 8.6 Hz, 1H), 4.25 (s, 2H), 3.14 (q, J = 7.1 Hz, 4H),
1.31 (t, J = 7.1 Hz, 6H); 13C NMR (101 MHz, DMSO-d6) δ 154.5,
148.3, 130.8 (2C), 127.9, 126.8, 125.6, 122.7 (2C), 117.1, 116.8,
111.8 (2C), 49.5, 46.0 (2C), and 8.3 (2C); LC−MS (ESI): m/z 311.1
[M + H]+; purity (LC−MS): 98%, tR = 0.38 min.
2-(4-((N,N-Diethylamino)methyl)-3-hydroxyanilino)-1H-benzo-
1
[d]imidazole (13). Brown viscous oil (71 mg, 35% yield); H NMR
(600 MHz, MeOH-d4) δ 7.59 (br s, 2H), 7.46 (d, J = 8.1 Hz, 1H),
7.16 (dd, J = 3.1, 5.9 Hz, 2H), 6.91 (d, J = 2.1 Hz, 1H), 6.88 (dd, J =
2.2, 8.1 Hz, 1H), 4.31 (s, 2H), 3.26 (q, J = 7.2 Hz, 4H), 1.40 (t, J =
7.2 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 159.2, 152.5, 142.2,
135.1 (2C), 123.4 (2C), 113.9 (2C), 112.3 (2C), 108.1 (2C), 52.5,
48.7 (2C), and 9.3 (2C); LC−MS (ESI): m/z 311.1 [M + H]+; purity
(LC−MS): 98%, tR = 0.33 min.
2-(3-((N,N-Diethylamino)methyl)-2-hydroxyanilino)-1H-benzo-
1
[d]imidazole (14). Brown semisolid (81 mg, 40% yield); H NMR
General Synthetic Procedure I (2 and 3). A mixture of 2-
chloro-1H-benzimidazole (1 equiv) and the corresponding benzyl
bromide (1.1 equiv) in acetonitrile was refluxed at 75 °C in the
presence of K2CO3 (1.1 equiv) as a base. Upon completion (TLC, 2
h), the reaction mixture was allowed to cool down, diluted with
DCM, and filtered, and the filtrate was removed in vacuo to obtain a
white amorphous solid residue. The residue was triturated with
diethyl ether to remove unreacted benzyl bromide. The benzylated
benzimidazole intermediates (2 and 3) were used for the next
reaction without any further purification.
General Synthetic Procedure II (5 and 6). A mixture of urea
and the corresponding 1,2-benzenediamine in THF was irradiated at
130 °C under microwave conditions for 30 min. The precipitate
formed was triturated with diethyl ether and the amorphous solid (5
or 6) was used in the subsequent reaction without any further
purification.
General Synthetic Procedure III (7). Compound 7 was
synthesized from 5 according to the general synthetic procedure I.
General Synthetic Procedure IV (11a−11m). A mixture of the
corresponding hydroxyaniline (8a−8i, 1.0 equiv) and acetic
anhydride (1.5 equiv) in THF was heated at 60 °C for 1 h. After
completion of reaction (TLC and LCMS), the solvent was removed
under reduced pressure and the residue was triturated with diethyl
ether to obtain N-(hydroxyphenyl) acetamides (9a−9i). The
acetamides (1 equiv) were dissolved in EtOH (10 mL) and heated
(80 °C) with N,N-diethylamine, N-ethylamine, or N-tert-butylamine
(1.5 equiv) and formaldehyde (1.5 equiv) until completion (TLC, 2
h). The solvent was removed in vacuo. The residue was dissolved in
DCM (20 mL) and acidified with 1 M HCl (15 mL). The aqueous
layer was separated through a separating funnel and basified with a
saturated solution of NaOH to a pH of 8−10. This was followed by
extraction of the target compound with DCM (2 × 20 mL). The
combined organic phase was dried over anhydrous Na2SO4. After
filtration, the solvent was removed under reduced pressure to obtain
compounds 10a−10m. Compounds (2 mmol) were refluxed in 6 N
HCl (2 mL) at 100 °C for 2 h (TLC). The solvent was removed
under reduced pressure. The residue was dissolved in EtOH (2 × 15
mL) and the solvent was removed in vacuo to obtain the
corresponding N-deacetylated Mannich base products 11a−11m (as
HCl salt).
(600 MHz, MeOH-d4) δ 7.86 (dd, J = 1.5, 7.9 Hz, 1H), 7.28 (dd, J =
3.3, 6.6 Hz, 2H), 7.03 (dd, J = 3.3, 6.6 Hz, 2H), 6.77 (t, J = 7.7 Hz,
1H), 6.71 (dd, J = 1.5, 7.7 Hz, 1H), 3.84 (s, 2H), 2.68 (q, J = 7.1 Hz,
4H), 1.13 (t, J = 7.1 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ
153.0, 149.3, 129.2 (2C), 123.1 (2C), 122.8, 122.5 , 121.8 (2C),
119.5 (2C), 118.7, 57.7, 47.5 (2C), and 11.5 (2C); LC−MS (ESI):
m/z 311.1 [M + H]+; purity (LC−MS): 98%, tR = 0.36 min.
2-(3-((N,N-Diethylamino)methyl)-2-hydroxy-5-methylanilino)-
1H-benzo[d]imidazole (15). Yellow semisolid (110 mg, 52% yield);
1H NMR (600 MHz, MeOH-d4) δ 7.69 (d, J = 1.9 Hz, 1H), 7.29 (dd,
J = 3.1, 5.8 Hz, 2H), 7.03 (dd, J = 3.1, 5.8 Hz, 2H), 6.52 (d, J = 1.9
Hz, 1H), 3.77 (s, 2H), 2.65 (q, J = 7.1 Hz, 4H), 2.27 (s, 3H), 1.12 (t,
J = 7.1 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 153.0, 146.6,
129.1 (2C), 128.8, 123.6 (2C), 122.6, 121.8 (3C), 119.2 (2C), 57.7,
47.5 (2C), 21.0, and 11.6 (2C); LC−MS (ESI): m/z 325.2 [M + H]+;
purity (LC−MS): 98%, tR = 0.60 min.
2-(3-((N,N-Diethylamino)methyl)-2-hydroxy-6-methylanilino)-
1H-benzo[d]imidazole (16). Brown viscous oil (106 mg, 50% yield);
1H NMR (600 MHz, MeOH-d4) δ 7.18 (dd, J = 3.1, 5.8 Hz, 2H),
6.96 (dd, J = 3.1, 5.8 Hz, 2H), 6.90 (d, J = 7.7 Hz, 1H), 6.71 (d, J =
7.7 Hz, 1H), 3.84 (s, 2H), 2.69 (q, J = 7.1 Hz, 4H), 2.24 (s, 3H), 1.11
(t, J = 7.1 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 155.7, 155.6,
138.5, 137.2, 127.8 (2C), 126.1, 121.6 (2C), 121.5, 121.4 (2C),
113.0, 57.2, 47.4 (2C), 18.2, and 11.3 (2C); LC−MS (ESI): m/z
325.2 [M + H]+; purity (LC−MS): 98%, tR = 0.34 min.
1-(4-Cyanobenzyl)-2-(3-((N,N-diethylamino)methyl)-4-
hydroxyanilino)benzo[d]imidazole (17). Pink semisolid (150 mg,
1
94% yield); H NMR (600 MHz, MeOH-d4) δ 7.70 (d, J = 8.1 Hz,
2H), 7.63 (d, J = 2.6 Hz, 1H), 7.42−7.39 (m, 1H), 7.39−7.35 (m,
3H), 7.20−7.16 (m, 2H), 7.11 (td, J = 7.6, 1.0 Hz, 1H), 6.97 (d, J =
8.6 Hz, 1H), 5.60 (s, 2H), 4.32 (s, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.39
(t, J = 7.2 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 155.0, 152.5,
142.8, 138.9, 134.2, 133.8 (2C), 132.2, 128.6 (2C), 127.6, 127.0,
123.9, 122.9, 119.4, 118.2, 117.4, 115.9, 112.7, 110.1, 52.5, 48.5 (2C),
46.4, and 9.1 (2C); LC−MS (ESI): m/z 426.2 [M + H]+; purity
(LC−MS): 98%, tR = 2.38 min.
1-(4-Cyanobenzyl)-2-(4-((N,N-diethylamino)methyl)-3-
hydroxyanilino)benzo[d]imidazole (18). Brown semisolid (87 mg,
1
55% yield); H NMR (600 MHz, MeOH-d4) δ 7.63 (d, J = 8.2 Hz,
2H), 7.43−7.39 (m, 1H), 7.26 (d, J = 7.8 Hz, 2H), 7.14−7.07 (m,
2H), 7.05−7.01 (m, 1H), 6.94−6.84 (m, 3H, 5.49 (s, 2H), 3.73 (s,
2H), 2.63 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H); 13C NMR
General Synthetic Procedure V (12−51). A mixture of the
corresponding benzimidazole (1−7) (1.0 equiv) in n-BuOH and the
respective phenolic Mannich base (11a−11m) (1.1 equiv) in n-
5208
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J. Med. Chem. 2021, 64, 5198−5215

Journal of Medicinal Chemistry
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Article
(151 MHz, MeOH-d4) δ 160.0, 152.4, 143.7, 142.3, 134.9, 133.7
(2C), 131.6, 130.1, 128.5 (2C), 123.1, 122.0, 119.4, 117.9, 117.5,
112.4, 111.3, 109.6, 108.1, 57.1, 47.3 (2C), 46.3, and 11.6 (2C); LC−
MS (ESI): m/z 426.2 [M + H]+; purity (LC−MS): 98%, tR = 2.41
min.
(101 MHz, MeOH-d4) δ 152.4, 150.4, 142.6, 142.0, 135.1, 131.1,
129.3, 128.6 (2C), 126.8 (2C), 125.5, 123.8, 122.9, 122.5, 121.9,
120.0, 119.3, 117.4, 109.4, 57.6, 47.4 (2C), 46.3, and 11.3 (2C); LC−
MS (ESI): m/z 469.2 [M + H]+; purity (LC−MS): 98%, tR = 2.65
min.
1-(4-Cyanobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-
1-((4-Trifluoromethyl)benzyl)-2-(3-((N,N-diethylamino)methyl)-
hydroxyanilino)benzo[d]imidazole (19). Brown semisolid (119 mg,
2-hydroxy-5-methylanilino)benzo[d]imidazole (25). Brown solid
1
1
75% yield); H NMR (400 MHz, MeOH-d4) δ 7.74 (dd, J = 2.9, 6.5
(120 mg, 46% yield); H NMR (400 MHz, MeOH-d4) δ 7.65−7.53
Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.44−7.40 (m, 1H), 7.36 (d, J =
8.3 Hz, 2H), 7.19−7.15 (m, 1H), 7.11 (td, J = 7.7, 1.3 Hz, 1H), 7.05
(td, J = 7.7, 1.3 Hz, 1H), 6.76−6.69 (m, 2H), 5.47 (s, 2H), 3.82 (s,
2H), 2.67 (q, J = 7.2 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H); 13C NMR
(101 MHz, MeOH-d4) δ 152.4, 150.5, 143.3, 142.6, 135.0, 133.8
(2C), 129.3, 128.9 (2C), 123.9, 123.0, 122.6, 122.0, 120.2, 119.4,
119.2, 117.5, 112.7, 109.3, 57.6, 47.4 (2C), 46.4, and 11.3 (2C); LC−
MS (ESI): m/z 426.2 [M + H]+; purity (LC−MS): 98%, tR = 2.46
min.
(m, 3H), 7.46−7.42 (m, 1H), 7.40−7.34 (m, 2H), 7.21−7.16 (m,
1H), 7.11 (td, J = 1.4, 7.6 Hz, 1), 7.05 (td, J = 1.2, 7.6 Hz, 1H), 6.59−
6.49 (m, 1H), 5.44 (s, 2H), 3.75 (s, 2H), 2.63 (q, J = 7.2 Hz, 4H),
2.23 (s, 3H), 1.07 (t, J = 7.2 Hz, 6H); 13C NMR (101 MHz, MeOH-
d4) δ 152.4, 147.5, 142.6, 142.0, 135.1, 131.2, 128.9, 128.8, 128.6
(2C), 126.8 (2C), 125.5, 124.3, 123.0, 122.5, 121.9, 120.6, 117.4,
109.3, 57.5, 47.5 (2C), 46.3, 21.0, 11.4 (2C); LC−MS (ESI): m/z =
483.2 [M + H]+; purity (LC−MS): 98%, tR = 2.68 min.
1-((4-Trifluoromethyl)benzyl)-2-(3-((N,N-diethylamino)methyl)-
2-hydroxy-6-methylanilino)benzo[d]imidazole (26). Brown semi-
solid (96 mg, 62% yield); 1H NMR (600 MHz, MeOH-d4) δ 7.61 (d,
J = 8.2 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.26−7.23 (m, 1H), 7.08−
7.05 (m, 1H), 7.02 (td, J = 7.6, 1.2 Hz, 1H), 6.97 (td, J = 7.5, 1.1 Hz,
1H), 6.86 (d, J = 7.7 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H), 5.49 (s, 2H),
3.82 (s, 2H), 2.68 (q, J = 7.1 Hz, 4H), 2.11 (s, 3H), 1.09 (t, J = 7.1
Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 155.2, 154.5, 142.5,
142.4, 137.3, 135.4, 130.7, 128.6 (2C), 127.6, 126.6 (2C), 125.5,
124.7, 122.6, 121.5, 121.2, 121.1, 116.4, 109.1, 57.1, 47.4 (2C), 46.2,
18.3, and 11.2 (2C); LC−MS (ESI): m/z 483.2 [M + H]+; purity
(LC−MS): 98%, tR = 2.64 min.
1-(4-Cyanobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-hydroxy-
5-methylanilino)benzo[d]imidazole (20). Yellow semisolid (145 mg,
1
88% yield); H NMR (400 MHz, MeOH-d4) δ 7.64 (d, J = 8.0 Hz,
2H), 7.55 (d, J = 1.9, Hz, 1H), 7.45−7.40 (m, 1H), 7.35 (d, J = 7.8
Hz, 2H), 7.19−7.14 (m, 1H), 7.11 (td, J = 7.6, 1.3 Hz, 1H), 7.05 (td,
J = 8.1, 1.3 Hz, 1H), 6.54 (d, J = 1.9, Hz, 1H), 5.46 (s, 2H), 3.76 (s,
2H), 2.67 (q, J = 7.4 Hz, 4H), 2.23 (s, 3H), 1.09 (t, J = 7.1 Hz, 6H);
13C NMR (101 MHz, MeOH-d4) δ 152.4, 147.7, 143.3, 142.6, 135.0,
133.8 (2C), 128.9, 128.8 (2C), 128.7, 124.4, 123.0, 122.5, 122.0,
120.8, 119.4, 117.4, 112.7, 109.3, 57.5, 47.5 (2C), 46.4, 21.0, and 11.4
(2C); LC−MS (ESI): m/z 440.2 [M + H]+; purity (LC−MS): 98%,
tR = 2.54 min.
1-(4-Fluorobenzyl)-2-(3-((N,N-diethylamino)methyl)-4-
hydroxyanilino)benzo[d]imidazole (27). Pink semisolid (146 mg,
1-(4-Cyanobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-hydroxy-
1
91% yield); H NMR (600 MHz, MeOH-d4) δ 7.64 (d, J = 2.6 Hz,
6-methylanilino)benzo[d]imidazole (21). Brown semisolid (98 mg,
1
1H), 7.46−7.44 (m, 1H), 7.43 (dd, J = 2.6, 8.6 Hz, 1H), 7.36 (dd, J =
5.2, 8.5 Hz, 2H), 7.35−7.32 (m, 1H), 7.26 (td, J = 7.7, 1.4 Hz, 1H),
7.23 (td, J = 7.6, 1.3 Hz), 7.11 (t, J = 8.7 Hz, 2H), 7.07 (d, J = 8.6 Hz,
1H), 5.55 (s, 2H), 4.34 (s, 2H), 3.27 (q, J = 7.2 Hz, 4H), 1.40 (t, J =
7.2 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 163.9, 156.8, 151.2,
133.6, 132.9, 132.0, 130.1 (2C), 129.9, 129.8, 129.0, 124.9, 124.3,
118.9, 117.9, 116.9 (2C), 114.3, 111.3, 52.2, 48.6 (2C), 46.6, and 9.1
(2C); LC−MS (ESI): m/z 419.2 [M + H]+; purity (LC−MS): 98%,
tR = 2.41 min.
60% yield); H NMR (600 MHz, MeOH-d4) δ 7.67 (d, J = 8.1 Hz,
2H), 7.42 (d, J = 8.1 Hz, 2H), 7.26−7.23 (m, 1H), 7.07−7.03 (m,
2H), 6.97 (td, J = 7.5, 1.1 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.67 (d, J
= 7.7 Hz, 1H), 5.49 (s, 2H), 3.80 (s, 2H), 2.66 (q, J = 7.1 Hz, 4H),
2.11 (s, 3H), 1.08 (t, J = 7.1 Hz, 6H); 13C NMR (151 MHz, MeOH-
d4) δ 155.3, 154.6, 143.8, 142.5, 137.2, 135.4, 133.6 (2C), 128.9 (2C),
127.5, 126.8, 122.7, 121.5, 121.3, 121.2, 119.5, 116.5, 112.4, 109.0,
57.3, 47.3 (2C), 46.2, 18.3, and 11.4 (2C); LC−MS (ESI): m/z 440.2
[M + H]+; purity (LC−MS): 98%, tR = 2.45 min.
1-(4-Fluorobenzyl)-2-(4-((N,N-diethylamino)methyl)-3-
1-((4-Trifluoromethyl)benzyl)-2-(3-((N,N-diethylamino)methyl)-
4-hydroxyanilino)benzo[d]imidazole (22). Pink semisolid (127 mg,
hydroxyanilino)benzo[d]imidazole (28). Brown semisolid (102 mg,
1
1
63% yield); H NMR (600 MHz, MeOH-d4) δ 7.41−7.38 (m, 1H),
84% yield); H NMR (600 MHz, MeOH-d4) δ 7.67 (d, J = 8.2 Hz,
7.16 (dd, J = 5.3, 8.5 Hz, 2H), 7.13−7.10 (m, 1H), 7.08 (td, J = 7.7,
1.1 Hz, 1H), 7.03 (td, J = 7.7, 1.1 Hz, 1H), 7.00 (t, J = 8.7 Hz, 2H),
6.95−6.84 (m, 3H), 5.37 (s, 2H), 3.74 (s, 2H), 2.63 (q, J = 7.2 Hz,
4H), 2.10 (s, 3H), 1.10 (t, J = 7.1 Hz, 6H); 13C NMR (151 MHz,
MeOH-d4) δ 163.6, 160.0, 152.3, 142.5, 134.9, 133.8, 131.6, 130.1,
129.7 (2C), 122.9, 121.9, 117.8, 117.3, 116.5 (2C), 111.2, 109.8,
108.1, 57.1, 47.3 (2C), 46.0, and 11.6 (2C); LC−MS (ESI): m/z
419.2 [M + H]+; purity (LC−MS): 98%, tR = 2.52 min.
2H), 7.63 (d, J = 2.6 Hz, 1H), 7.46−7.42 (m, 3H), 7.40 (dd, J = 2.7,
8.7 Hz, 1H), 7.28−7.25 (m, 1H), 7.23 (td, J = 7.6, 1.2 Hz, 1H), 7.18
(td, J = 7.6, 1.1 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 5.63 (s, 2H), 4.33
(s, 2H), 3.26 (q, J = 7.3 Hz, 4H), 1.39 (t, J = 7.3 Hz, 6H); 13C NMR
(151 MHz, MeOH-d4) δ 155.9, 152.0, 141.1, 136.3, 133.6, 131.2,
131.0, 128.4 (2C), 126.9 (2C), 125.5, 126.4, 124.6, 124.5, 123.7,
118.6, 117.6, 115.2, 110.6, 52.4, 48.6 (2C), 46.6, and 9.1 (2C); LC−
MS (ESI): m/z 469.2 [M + H]+; purity (LC−MS): 98%, tR = 2.59
min.
1-(4-Fluorobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-
hydroxyanilino)benzo[d]imidazole (29). Brown semisolid (112 mg,
1-((4-Trifluoromethyl)benzyl)-2-(4-((N,N-diethylamino)methyl)-
1
70% yield); H NMR (400 MHz, MeOH-d4) δ 7.77 (dd, J = 2.2, 7.3
3-hydroxyanilino)benzo[d]imidazole (23). Brown semisolid (75 mg,
1
Hz, 1H), 7.44−7.40 (m, 1H), 7.27 (dd, J = 5.2, 8.6 Hz, 2H), 7.25−
7.21 (m, 1H), 7.13−7.06 (m, 2H), 7.03 (t, J = 8.7 Hz, 2H), 6.77−
6.69 (m, 2H), 5.36 (s, 2H), 3.84 (s, 2H), 2.69 (q, J = 7.2 Hz, 4H),
1.12 (t, J = 7.2 Hz, 6H); 13C NMR (101 MHz, MeOH-d4) δ 163.8,
152.2, 150.1, 142.6, 135.1, 133.4, 130.0 (2C), 129.4, 123.6, 122.8,
122.5, 121.9, 119.5, 119.3, 117.4, 116.7 (2C), 109.4, 57.6, 47.5 (2C),
46.1, and 11.3 (2C); LC−MS (ESI): m/z 419.2 [M + H]+; purity
(LC−MS): 98%, tR = 2.55 min.
50% yield); H NMR (600 MHz, MeOH-d4) δ 7.59 (d, J = 8.1 Hz,
2H), 7.45−7.42 (m, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.17−7.09 (m,
4H), 7.07 (td, J = 7.7, 1.1 Hz, 1H), 6.93 (dd, J = 2.2, 8.2 Hz, 1H),
5.53 (s, 2H), 4.04 (s, 2H), 2.97 (q, J = 7.2 Hz, 4H), 1.25 (t, J = 7.2
Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 159.1, 151.9, 144.3,
142.4, 134.9, 132.3, 131.7, 130.9, 128.2, 128.1 (2C), 126.7 (2C),
125.5, 123.2, 122.3, 117.6, 111.4, 109.9, 107.1, 54.5, 48.0 (2C), 46.3,
and 10.1 (2C); LC−MS (ESI): m/z 469.2 [M + H]+; purity (LC−
MS): 96%, tR = 2.61 min.
1-((4-Trifluoromethyl)benzyl)-2-(3-((N,N-diethylamino)methyl)-
2-hydroxyanilino)benzo[d]imidazole (24). Brown semisolid (105
mg, 70% yield); 1H NMR (400 MHz, MeOH-d4) δ 7.77 (dd, J = 2.2,
7.2 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.44−7.40 (m, 1H), 7.38 (d, J
= 8.0 Hz, 2H), 7.19−7.15 (m, 1H), 7.11 (td, J = 7.6, 1.3 Hz, 1H),
7.05 (td, J = 7.5, 1.1 Hz, 1H), 6.77−6.67 (m, 2H), 5.46 (s, 2H), 3.81
(s, 2H), 2.64 (q, J = 7.2 Hz, 4H), 1.09 (t, J = 7.1 Hz, 6H); 13C NMR
1-(4-Fluorobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-hydroxy-
5-methylanilino)benzo[d]imidazole (30). Yellow semisolid (140 mg,
1
84% yield); H NMR (400 MHz, MeOH-d4) δ 7.61 (d, J = 2.0 Hz,
1H), 7.45−7.41 (d, J = 7.5 Hz, 1H), 7.24 (dd, J = 5.3, 8.6 Hz, 2H),
7.22−7.19 (m, 1H), 7.14−7.06 (m, 2H), 7.03 (t, J = 8.7 Hz, 2H),
6.53 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H), 3.77 (s, 2H), 2.65 (q, J = 7.2
Hz, 4H), 2.24 (s, 3H), 1.09 (t, J = 7.2 Hz, 6H); 13C NMR (101 MHz,
MeOH-d4) δ 163.8, 152.2, 147.2, 142.5, 135.1, 133.4, 130.0 (2C),
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1
129.0, 128.9, 124.2, 122.8, 122.4, 121.9, 120.2, 117.4, 116.7 (2C),
109.4, 57.5, 47.5 (2C), 46.1, 21.0, and 11.4 (2C); LC−MS (ESI): m/
z 433.2 [M + H]+; purity (LC−MS): 98%, tR = 2.61 min.
(37). Brown solid (45 mg, 35% yield); H NMR (600 MHz, MeOH-
d4) δ 7.73 (dd, J = 3.0, 10.6 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.51
(d, J = 7.8 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H),
7.19−7.15 (m, 1H), 7.15−7.10 (m, 1H), 6.49 (dd, J = 2.9, 8.7 Hz,
1H), 5.52 (s, 2H), 3.83 (s, 2H), 2.70 (q, J = 7.2 Hz, 4H), 1.13 (t, J =
7.2 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 157.6, 151.4, 145.5,
141.9, 135.0, 131.2, 128.6 (2C), 127.9, 126.9 (2C), 125.5, 123.1
(2C), 122.3 (2C), 117.7, 109.5, 108.8, 106.1, 57.3, 47.6 (2C), 46.3,
11.3 (2C); LC−MS (ESI): m/z = 487.2 [M + H]+; purity (LC−MS):
98%, tR = 2.53 min.
1-(4-Fluorobenzyl)-2-(3-((N,N-diethylamino)methyl)-2-hydroxy-
6-methylanilino)benzo[d]imidazole (31). Brown semisolid (112 mg,
1
68% yield); H NMR (600 MHz, MeOH-d4) δ 7.31 (dd, J = 5.3, 8.6
Hz, 2H), 7.24−7.21 (d, J = 7.8 Hz, 1H), 7.10−7.07 (d, J = 8.1 Hz,
1H), 7.03 (t, J = 8.7 Hz, 2H), 7.00 (td, J = 7.5, 1.3 Hz, 1H), 6.96 (td,
J = 7.5, 1.1 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.67 (d, J = 7.7 Hz,
1H), 5.37 (s, 2H), 3.81 (s, 2H), 2.66 (q, J = 7.2 Hz, 4H), 2.10 (s,
3H), 1.09 (t, J = 7.1 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ
163.7, 155.2, 154.5, 142.4, 137.1, 135.5, 133.9, 130.0 (2C), 127.4,
126.7, 122.4, 121.5, 121.3, 121.1, 116.5 (2C), 116.4, 109.2, 57.3, 47.3
(2C), 45.9, 18.3, and 11.4 (2C); LC−MS (ESI): m/z 433.2 [M +
H]+; purity (LC−MS): 98%, tR = 2.51 min.
3-Chloro-2-((N, N-diethylamino)methyl)-6-((1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
1
(38). Brown solid (40 mg, 34% yield); H NMR (600 MHz, MeOH-
d4) δ 7.82 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.48−7.43
(m, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.25−7.20 (m, 1H), 7.15 (td, J =
1.2, 7.6 Hz, 1H), 7.10 (td, J = 1.1, 7.6 Hz, 1H), 6.77 (d, J = 8.7 Hz,
1H), 5.51 (s, 2H), 4.15 (s, 2H), 2.84 (q, J = 7.1 Hz, 4H), 1.19 (t, J =
7.2 Hz, 6H); 13C NMR (151 MHz, MeOH-d4) δ 153.7, 152.0, 142.4,
142.0, 135.0, 130.9, 129.0, 128.5 (2C), 127.7, 126.9 (2C), 125.5,
123.0, 122.1, 120.2, 118.4, 118.3, 117.4, 109.4, 54.4, 47.8 (2C), 46.3,
10.8 (2C); LC−MS (ESI): m/z = 503.1 [M + H]+; purity (LC−MS):
98%, tR = 2.59 min.
2-((N-Ethylamino)methyl)-4-methyl-6-((1-(4-(trifluoromethyl)-
benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol (32). Brown solid
1
(56 mg, 34% yield); H NMR (600 MHz, MeOH-d4) δ 7.62 (d, J =
8.1 Hz, 2H), 7.55−7.51 (m, 1H), 7.46−7.37 (m, 3H), 7.17−7.21 (m,
1H), 7.12 (td, J = 1.2, 7.6 Hz, 1H), 7.06 (td, J = 1.1, 7.6 Hz, 1H),
6.64−6.59 (m, 1H), 5.51 (s, 2H), 3.96 (s, 2H), 2.78 (q, J = 7.2 Hz,
2H), 2.24 (s, 3H), 1.18 (t, J = 7.2 Hz, 3H); 13C NMR (151 MHz,
MeOH-d4) δ 152.5, 149.8, 142.5, 142.1, 135.1, 130.9, 129.8, 128.5
(2C), 127.4, 126.9 (2C), 125.5, 125.1, 123.0, 122.5, 121.9, 120.9,
117.2, 109.3, 51.6, 46.2, 43.3, 20.9, 13.6; LC−MS (ESI): m/z = 455.2
[M + H]+; purity (LC−MS): 98%, tR = 2.54 min.
2-((N-tert-Butylamino)methyl)-4-methyl-6-((1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
(33). Brown solid (58 mg, 38% yield); 1H NMR (400 MHz, MeOH-
d4) δ 7.63 (d, J = 8.1 Hz, 2H), 7.45−7.36 (m, 3H), 7.18 (dd, J = 1.1,
8.1 Hz, 1H), 7.12 (td, J = 1.3, 7.6 Hz, 1H), 7.06 (td, J = 1.2, 7.6 Hz,
1H), 6.75 (d, J = 2.1 Hz, 1H), 5.53 (s, 2H), 4.06 (s, 2H), 2.25 (s,
3H), 1.36 (s, 9H); 13C NMR (101 MHz, MeOH-d4) δ 152.7, 149.6,
142.2, 142.1, 135.1, 130.9, 130.0, 128.4 (2C), 128.3, 126.9 (2C),
126.0, 125.5, 123.1, 122.5, 122.2, 122.0, 117.0, 109.5, 55.7, 46.3, 45.0,
26.9 (3C), 20.8; LC−MS (ESI): m/z = 483.2 [M + H]+; purity (LC−
MS): 98%, tR = 2.58 min.
2-((5,6-Difluoro-1-(4-(trifluoromethyl)benzyl)-1H-benzo[d]-
imidazol-2-yl)amino)-6-((N-ethylamino)methyl)-4-methylphenol
1
(39). Brown solid (47 mg, 56% yield); H NMR (600 MHz, MeOH-
d4) δ 7.64 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 2.1 Hz, 1H), 7.39 (d, J =
8.0 Hz, 2H), 7.22 (dd, J = 7.2, 10.8 Hz, 1H), 7.14 (dd, J = 7.0, 10.2
Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H), 5.48 (s, 2H), 4.01 (s, 2H), 2.84 (q,
J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H); 13C NMR
(151 MHz, MeOH-d4) δ 154.1, 149.7, 148.3, 147.6, 146.8, 138.0,
131.3, 130.7, 129.3, 128.4 (2C), 128.0, 126.9 (2C), 126.2, 125.5,
122.4, 122.3, 105.0, 98.3, 50.9, 46.5, 43.4, 20.8, 13.1; LC−MS (ESI):
m/z = 491.1 [M + H]+; purity (LC−MS): 98%, tR = 2.57 min.
2-((N-tert-Butylamino)methyl)-6-((5,6-difluoro-1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)-4-
1
methylphenol (40). Brown solid (57 mg, 47% yield); H NMR (600
MHz, CDCl3) δ 7.81 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.33 (m, 3H),
6.84 (dd, J = 6.8, 9.7 Hz, 1H), 6.48 (s, 1H), 5.37 (s, 2H), 3.90 (s,
2H), 2.16 (s, 3H), 1.28 (s, 9H); 13C NMR (151 MHz, CDCl3) δ
151.3, 147.3, 146.4, 143.8, 138.9, 136.8, 130.6, 128.9, 128.0, 126.2
(2C), 126.1 (2C), 125.5, 124.7, 123.1, 122.9, 121.5, 105.2, 96.5, 52.9,
46.0, 45.1, 27.8 (3C), 20.9; LC−MS (ESI): m/z = 519.2 [M + H]+;
purity (LC−MS): 98%, tR = 2.60 min.
4-Chloro-2-((5,6-difluoro-1-(4-(trifluoromethyl)benzyl)-1H-
benzo[d]imidazol-2-yl)amino)-6-((N-ethylamino)methyl)phenol
(41). Brown solid (45 mg, 36% yield); 1H NMR (600 MHz, CDCl3) δ
8.09 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.15 (m, 3H), 6.65 (dd, J = 6.9,
9.4 Hz, 1H), 6.39 (s, 1H), 5.02 (s, 2H), 3.71 (s, 2H), 2.51 (q, J = 7.2
Hz, 2H), 0.96 (t, J = 7.2 Hz, 3H); 13C NMR (151 MHz, CDCl3) δ
150.2, 147.4, 146.5, 144.8, 138.5, 137.1, 130.8, 128.9, 127.1 (2C),
126.5, 126.3 (2C), 125.5, 123.9, 121.8, 120.9, 116.7, 105.7, 96.3, 51.5,
46.1, 43.0, 14.4; LC−MS (ESI): m/z = 511.2 [M + H]+; purity (LC−
MS): 98%, tR = 2.60 min.
4-Chloro-2-((N, N-diethylamino)methyl)-6-((1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
(34). Brown solid (40 mg, 32% yield); 1H NMR (600 MHz, MeOH-
d4) δ 7.81 (s, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 7.7 Hz, 1H),
7.39 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 7.8 Hz, 1H), 7.15 (t, J = 7.4 Hz,
1H), 7.10 (t, J = 7.5 Hz, 1H), 6.80 (s, 1H), 5.52 (s, 2H), 3.94 (s, 2H),
2.82 (q, J = 7.2 Hz, 4H), 1.18 (t, J = 7.2 Hz, 6H); 13C NMR (151
MHz, MeOH-d4) δ 151.7, 149.5, 141.9, 141.7, 134.9, 131.1 (2C),
128.5 (3C), 126.9 (2C), 125.5, 123.7, 123.2, 122.7, 122.3, 119.9,
117.3, 109.6, 56.4, 47.8 (2C), 46.3, 10.7 (2C); LC−MS (ESI): m/z =
503.1 [M + H]+; purity (LC−MS): 98%, tR = 2.59 min.
4-Chloro-2-((N-ethylamino)methyl)-6-((1-(4-(trifluoromethyl)-
benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol (35). Brown solid
1
(40 mg, 35% yield); H NMR (600 MHz, CDCl3) δ 7.99 (s, 1H),
7.52 (dd, J = 7.7, 15.8 Hz, 3H), 7.30 (d, J = 7.9 Hz, 2H), 7.20−7.12
(m, 1H), 7.12−7.02 (m, 2H), 6.59 (s, 1H), 5.33 (s, 2H), 3.89 (s, 2H),
2.76 (q, J = 7.1 Hz, 2H), 1.18 (t, J = 6.8 Hz, 3H); 13C NMR (151
MHz, CDCl3) δ 149.6, 145.6, 140.6, 139.4, 133.5, 130.2, 129.7, 127.1
(2C), 126.7, 126.0 (2C), 125.5, 122.2, 122.1, 121.5, 121.2, 118.6,
116.9, 108.0, 50.2, 45.6, 42.8, 13.3; LC−MS (ESI): m/z = 475.1 [M +
H]+; purity (LC−MS): 98%, tR = 2.56 min.
2-((N-tert-Butylamino)methyl)-4-chloro-6-((5,6-difluoro-1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
(42). Brown solid (44 mg, 37% yield); 1H NMR (600 MHz, CDCl3) δ
8.36−8.23 (m, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.37 (dd, J = 8.6, 24.1
Hz, 3H), 6.86 (t, J = 8.2 Hz, 1H), 6.62 (d, J = 2.5 Hz, 1H), 5.26 (s,
2H), 3.91 (s, 2H), 1.24 (s, 9H); 13C NMR (151 MHz, CDCl3) δ
150.3, 147.4, 146.5, 144.9, 138.6, 130.8, 129.1, 128.9 (2C), 127.2
(2C), 126.2 (2C), 125.5, 124.7, 123.9, 122.9, 122.6, 105.6, 96.4, 52.2,
46.2, 45.3, 28.1 (3C); LC−MS (ESI): m/z = 539.1 [M + H]+; purity
(LC−MS): 98%, tR = 2.61 min.
2-((N-tert-Butylamino)methyl)-4-chloro-6-((1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
(36). Brown solid (40 mg, 36% yield); 1H NMR (600 MHz, MeOH-
d4) δ 7.99 (d, J = 2.6 Hz, 1H), 7.65−7.60 (m, 2H), 7.50−7.47 (m,
1H), 7.42−7.38 (m, 2H), 7.23−7.20 (m, 1H), 7.14 (td, J = 1.2, 7.6
Hz, 1H), 7.08 (td, J = 1.1, 7.6 Hz, 1H), 6.78 (d, J = 2.6 Hz, 1H), 5.52
(s, 2H), 4.04 (s, 2H), 1.35 (s, 9H); 13C NMR (151 MHz, MeOH-d4)
δ 153.7, 151.5, 142.6, 142.0, 134.9, 132.4, 131.4, 128.4 (2C), 126.9
(2C), 125.5, 123.2, 123.1, 122.1, 121.0, 120.2, 118.3, 117.5, 109.3,
55.9, 46.2, 45.1, 26.6 (3C); LC−MS (ESI): m/z = 503.1 [M + H]+;
purity (LC−MS): 98%, tR = 2.54 min.
2-((1H-Benzo[d]imidazol-2-yl)amino)-6-((N-tert-butylamino)-
methyl)-4-methylphenol (43). Brown solid (22 mg, 43% yield); H
1
NMR (600 MHz, MeOH-d4) δ 7.29 (dd, J = 3.2, 5.8 Hz, 2H), 7.11−
7.09 (m, 1H), 7.05 (dd, J = 3.2, 5.9 Hz, 2H), 6.97−6.95 (m, 1H), 4.16
(s, 2H), 2.28 (s, 3H), 1.46 (s, 9H); 13C NMR (151 MHz, MeOH-d4)
δ 153.6, 146.9, 138.2, 130.7, 129.7, 128.1, 124.9, 122.9, 122.3 (4C),
113.5, 58.0, 43.5, 26.1 (3C), 20.5; LC−MS (ESI): m/z = 325.2 [M +
H]+; purity (LC−MS): 98%, tR = 2.06 min.
2-((N, N-D ie thylamino)methyl)- 4- fluo ro-6-((1-(4-
(trifluoromethyl)benzyl)-1H-benzo[d]imidazol-2-yl)amino)phenol
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2-((1H-Benzo[d]imidazol-2-yl)amino)-4-chloro-6-((N-
ethylamino)methyl)phenol (44). Brown solid (38 mg, 50% yield);
1H NMR (600 MHz, MeOH-d4) δ 7.34 (d, J = 2.5 Hz, 1H), 7.31 (dd,
J = 3.2, 5.9 Hz, 2H), 7.12 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 3.1, 5.9
Hz, 2H), 4.19 (s, 2H), 3.11 (q, J = 7.3 Hz, 2H), 1.35 (t, J = 7.3 Hz,
3H); 13C NMR (151 MHz, MeOH-d4) δ 152.8, 148.1, 137.6, 131.5,
126.6, 124.9, 123.9, 123.3, 122.6 (4C), 113.7, 48.1, 43.8, 11.5; LC−
MS (ESI): m/z = 317.1 [M + H]+; purity (LC−MS): 98%, tR = 0.67
min.
the presence of formaldehyde (1.2 equiv). The reaction lasted for 16 h
for complete consumption of starting materials, followed by an acid
work-up (1 M HCl) to obtain a crude product, which was purified by
flash chromatography. Compound 52 was obtained by cleavage of the
PMB group in the presence of trifluoroacetic acid in DCM at room
temperature for 48 h, followed by purification by flash chromatog-
raphy. Brown solid (13 mg, 28% yield); 1H NMR (600 MHz, MeOH-
d4) δ 7.59 (d, J = 7.5 Hz, 2H), 7.39 (m, 3H), 7.34 (d, J = 7.8 Hz, 1H),
7.14 (d, J = 7.5 Hz, 1H), 7.10 (t, J = 7.4 Hz, 1H), 7.04 (t, J = 7.5 Hz,
1H), 6.72 (m, 1H), 5.47 (s, 2H), 3.97 (s, 2H), 2.24 (s, 3H). 13C
NMR (151 MHz, MeOH-d4) δ 152.8, 149.4, 142.5, 142.1, 135.1,
131.0, 130.1, 128.3 (2C), 126.8 (2C), 126.4, 125.7, 125.6, 124.6,
123.1, 122.0, 121.9, 117.0, 109.5, 55.7, 46.2, 20.8; LC−MS (ESI): m/
z = 427.1 [M + H]+; purity (LC−MS): 98%, tR = 2.45 min.
Antiplasmodium Activity Evaluation. Compounds were
screened against multidrug-resistant (K1) and sensitive (NF54)
strains of P. falciparum in vitro using the modified [3H]-hypoxanthine
incorporation assay.38 In vivo efficacy was assessed as previously
described,39 with the modification that mice (n = 3) were infected
with a GFP-transfected P. berghei ANKA strain (donated by A. P.
Waters and C. J. Janse, Leiden University, The Netherlands), and
parasitemia was determined using standard flow cytometry
techniques. The detection limit was 1 parasite in 1000 erythrocytes
(that is, 0.1%). The activity was calculated as the difference between
the mean percent parasitemia for the control and treated groups
expressed as a percent relative to the control group. Compounds were
dissolved or suspended in a vehicle consisting of 70% Tween-80 and
30% ethanol, followed by a 10-fold dilution in H2O and oral
administration as four consecutive daily doses (4, 24, 48, and 72 h
after infection). Blood samples for the quadruple-dose regimens were
collected on day 4 (96 h after infection).
Gametocytocidal Activity Evaluation. The luciferase reporter
assay24 was established to enable accurate, reliable, and quantifiable
investigations of the stage-specific action of gametocytocidal
compounds for the early and late gametocyte marker cell line
NF54-PfS16-GFP-Luc. Drug assays were set up on days 5 and 10
(representing >90% of either early stage II/III or mature stage IV/V
gametocytes, respectively). In each instance, assays were set up using
a 2−3% gametocytemia and 1.5% hematocrit culture and 48 h drug
pressure in a gas chamber (90% N2, 5% O2, and 5% CO2) at 37 °C.
The luciferase activity was determined in 30 μL of parasite lysates by
adding 30 μL of luciferin substrate (Promega Luciferase Assay
System) at room temperature and detection of resultant bio-
luminescence at an integration constant of 10 s with the GloMax
Explorer Detection System with Instinct Software. Methylene blue (5
μM) was used as a reference compound in the in vitro gametocyte
luciferase reporter assay as previously reported.24 Dual-point analysis
was performed for a single biological replicate in technical triplicates.
The IC50 was again determined using a nonlinear curve fit (GraphPad
Prism V6.0) normalized to maximum and minimum inhibition and
data are from at least three independent biological replicates, each
performed in technical triplicates.
2-((1H-Benzo[d]imidazol-2-yl)amino)-6-((N-tert-butylamino)-
1
methyl)-4-chlorophenol (45). Brown solid (13 mg, 25% yield); H
NMR (600 MHz, MeOH-d4) δ 7.76 (d, J = 2.6 Hz, 1H), 7.31 (dd, J =
3.2, 5.8 Hz, 2H), 7.05 (dd, J = 3.2, 5.9 Hz, 2H), 6.87 (d, J = 2.6 Hz,
1H), 4.09 (s, 2H), 1.39 (s, 9H); 13C NMR (151 MHz, MeOH-d4) δ
152.6, 151.3, 138.4, 132.1, 123.7, 122.7, 122.1 (5C), 119.4, 113.6,
56.1, 44.7, 26.7 (3C); LC−MS (ESI): m/z = 345.1 [M + H]+; purity
(LC−MS): 98%, tR = 2.18 min.
2-((1H-Benzo[d]imidazol-2-yl)amino)-4-(tert-butyl)-6-((N-tert-
butylamino)methyl)phenol (46). Brown solid (20 mg, 39% yield);
1H NMR (600 MHz, MeOH-d4) δ 7.30 (d, J = 2.4 Hz, 1H), 7.29 (dd,
J = 3.2, 5.8 Hz, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.05 (dd, J = 3.1, 5.9
Hz, 2H), 4.21 (s, 2H), 1.47 (s, 9H), 1.33 (s, 9H); 13C NMR (151
MHz, MeOH-d4) δ 153.7, 146.9, 144.2, 138.2, 129.3, 124.7, 122.6,
122.3 (4C), 121.6, 131.5, 58.0, 43.9, 35.0, 31.9 (3C), 26.1 (3C); LC−
MS (ESI): m/z = 367.2 [M + H]+; purity (LC−MS): 98%, tR = 2.35
min.
2-((N-tert-Butylamino)methyl)-6-((5,6-difluoro-1H-benzo[d]-
imidazol-2-yl)amino)-4-methylphenol (47). Brown solid (20 mg,
1
35% yield); H NMR (600 MHz, MeOH-d4) δ 7.19 (d, J = 2.1 Hz,
1H), 7.16−7.12 (m, 2H), 6.91 (d, J = 2.0 Hz, 1H), 4.13 (s, 2H), 2.28
(s, 3H), 1.43 (s, 9H); 13C NMR (151 MHz, MeOH-d4) δ 155.0, 147
(3C), 134.0, 130.1, 129.4, 127.6 (2C), 124.3, 122.8, 101.7, 101.6,
57.3, 43.9, 26.4 (3C), 20.6; LC−MS (ESI): m/z = 361.1 [M + H]+;
purity (LC−MS): 98%, tR = 2.33 min.
4-Chloro-2-((5,6-difluoro-1H-benzo[d]imidazol-2-yl)amino)-6-
((N-ethylamino)methyl)phenol (48). Brown solid (33 mg, 39%
1
yield); H NMR (600 MHz, MeOH-d4) δ 7.47 (d, J = 2.5 Hz, 1H),
7.23−7.17 (m, 2H), 7.12 (d, J = 2.4 Hz, 1H), 4.21 (s, 2H), 3.12 (q, J
= 7.3 Hz, 2H), 1.37 (t, J = 7.3 Hz, 3H); 13C NMR (151 MHz,
MeOH-d4) δ 154.2, 148.2, 148.1 (2C), 147.1, 133.6, 131.3, 126.3
(2C), 124.9, 123.7, 122.9, 101.9, 48.3, 43.8, 11.6; LC−MS (ESI): m/z
= 353.1 [M + H]+; purity (LC−MS): 98%, tR = 2.30 min.
2-((N-tert-Butylamino)methyl)-4-chloro-6-((5,6-difluoro-1H-
benzo[d]imidazol-2-yl)amino)phenol (49). Brown solid (21 mg,
1
37% yield); H NMR (600 MHz, MeOH-d4) δ 7.86 (d, J = 2.6 Hz,
1H), 7.19−7.13 (m, 2H), 6.81 (d, J = 2.5 Hz, 1H), 4.06 (s, 2H), 1.36
(s, 9H); 13C NMR (151 MHz, MeOH-d4) δ 154.0, 152.2, 148.2 (2C),
134.2, 131.9, 123.4 (2C), 122.2, 121.3, 118.7 (2C), 101.7, 55.7, 45.1,
26.8 (3C); LC−MS (ESI): m/z = 381.1 [M + H]+; purity (LC−MS):
98%, tR = 2.39 min.
4-(tert-Butyl)-2-((tert-butylamino)methyl)-6-((5,6-difluoro-1H-
benzo[d]imidazol-2-yl)amino)phenol (50). Brown solid (19 mg,
1
34% yield); H NMR (600 MHz, MeOH-d4) δ 7.35 (d, J = 2.4 Hz,
Male Gamete Exflagellation Assay. The male gamete
exflagellation inhibition assay (EIA)40 was performed by treating
mature (>90% stage V) gametocytes with 2 μM compound (10 mM
stock solutions in 100% DMSO (w/v), Sigma-Aldrich) in complete
culture media (final DMSO concentration of <0.1% (v/v)) for 48 h at
37 °C under 90% N2, 5% CO2, and 5% O2 hypoxic conditions.
Following treatment, gametogenesis was induced with 100 μM
xanthurenic acid (Sigma-Aldrich) in ookinete medium (complete
RPMI 1640 with 25 mM HEPES, 0.2% sodium bicarbonate (pH 8.0),
and 20% human serum) at room temperature for 15 min.
Exflagellating centers were detected by video microscopy (Carl
Zeiss NT 6V/10W Stab480 microscope with a MicroCapture camera,
10× magnification) in a Neubauer chamber at RT and semiautomati-
cally quantified after 20 min from 15 randomly located fields from
which videos of 8−10 s each were taken at 30 s intervals. The total
exflagellating centers per treatment were quantified using ICY (open-
source imaging software GPLv3) normalized to an untreated control.
Assays were performed for a single biological repeat.
1H), 7.18 (d, J = 2.4 Hz, 1H), 7.16−7.12 (m, 2H), 4.18 (s, 2H), 1.45
(s, 9H), 1.32 (s, 9H); 13C NMR (151 MHz, MeOH-d4) δ 155.2,
148.0 (2C), 147.6, 144.0, 134.0, 128.9, 124.7 (2C), 122.5, 121.6
(2C), 101.7, 57.6, 44.0, 35.1, 31.9 (3C), 26.3 (3C); LC−MS (APCI/
ESI): m/z = 403.2 [M + H]+; purity (LC−MS): 98%, tR = 2.48 min.
2-((N-tert-Butylamino)methyl)-6-((4,6-dichloro-1H-benzo[d]-
imidazol-2-yl)amino)-4-methylphenol (51). Brown solid (22 mg,
1
36% yield); H NMR (600 MHz, MeOH-d4) δ 7.28−7.26 (m, 1H),
7.21 (d, J = 1.8 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 2.1 Hz,
1H), 4.11 (s, 2H), 2.27 (s, 3H), 1.41 (s, 9H); 13C NMR (151 MHz,
MeOH-d4) δ 154.9, 148.1, 139.7, 135.8, 129.7, 129.2, 127.4, 127.2,
123.9, 122.9, 121.6, 119.0, 112.0, 57.0, 44.1, 26.4 (3C), 20.6; LC−MS
(ESI): m/z = 393.1 [M + H]+; purity (LC−MS): 98%, tR = 2.50 min.
2-(Aminomethyl)-4-methyl-6-((1-(4-(trifluoromethyl)benzyl)-1H-
benzo[d]imidazol-2-yl)amino)phenol (52). Intermediate III was
synthesized from 3 and 2-amino-4-methylphenol (8d) (Scheme 1)
according to general synthetic procedure V, followed by a Mannich
reaction with 4-methoxy benzylamine (PMB, 1.2 equiv) in ethanol in
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SALI Plot Analysis. Structure−activity landscape index (SALI)
was performed using Osiris Datawarrior V 5.2.1 software (www.
openmolecules.org). Compound similarity/activity cliff analysis was
performed based on compound SMILES, taking stereochemistry into
account, and separation was carried out based on compound neighbor
at a similarity threshold of 86%.
and evaporated using a Genevac system. Phosphate buffer (pH 6.5)
was then added to the wells and the plate was incubated for 24 h at 25
°C with shaking. At the end of this incubation, the samples were
centrifuged at 3500g for 15 min and then transferred to an analysis
plate. A calibration curve in DMSO for each sample between 10 and
220 μM was prepared and included in the analysis plate. Analysis was
then performed by HPLC-DAD (Agilent Rapid Resolution HPLC
with Diode array detector) and the solubility of each sample was
determined from the corresponding calibration curve. Reserpine,
hydrocortisone, and MMV390048 were used as assay controls.
Pharmacokinetics. Male Balb/c mice were used for animal
pharmacokinetics evaluations. All animal work was carried out in
accordance with the University of Cape Town’s ethics policies using
protocols approved by the research ethics committee (AEC017/026).
Compounds were administered intravenously as a bolus of DMA/
PEG/PPG (10/30/60) and orally in 0.5% (w/v) HPMC with 0.2%
Tween-80 aqueous solution to male Balb/c mice (n = 3 for each
group for 41 and for the oral group for 48 and n = 5 for the
intravenous group for 48). Mice were not fasted overnight and were
allowed to eat ad libitum. Animals were permitted access ad libitum to
water.
PK Sampling. Blood samples were collected from the tail vein of
the mice into heparinized microcentrifugation tubes at predetermined
sampling times (0.17, 0.5, 1, 3, 5, 7, 9, and 24 h for intravenous
dosing; 0.5, 1, 3, 5, 7, 9, and 24 h for oral dosing). All samples were
stored at −80 °C until extraction.
Bioanalytical Method. Frozen whole blood was thawed at room
temperature. Twenty microliters of samples was extracted by protein
precipitation using 100 μL of acetonitrile containing a 10 ng/mL
internal standard (MMV394902) and centrifuged. Calibration
standards and quality controls were extracted following the same
procedure. Supernatants were injected onto the column for LC−MS/
MS analysis. The analytical limit of quantitation (LOQ) was 2 ng/mL.
The accuracy, precision, and recovery for each study were within
acceptable limits (Table S2, Supporting Information).
Cytotoxicity Evaluation. The in vitro cytotoxicity of the
synthesized compounds was evaluated against the Chinese hamster
ovarian (CHO) cancer cell line using the MTT (3-(4,5-dimethylth-
iazolyl-2)-2,5-diphenyltetrazolium bromide) assay, which is a
colorimetric assay based on assessment of the cell metabolic activity.41
The synthesized compounds were assayed in triplicate. Stock
solutions of 2 mg/mL test samples in DMSO were prepared with
poorly soluble samples being tested as suspensions. The compounds
were kept at −20 °C until required. In all experiments, emetine was
used as a reference drug. Starting from an initial concentration of 100
μg/mL, 10-fold serial dilutions were made in complete medium to
give six concentrations to the lowest concentration of 0.001 μg/mL.
The cell viability was not affected by the highest concentration of the
solvent to which the cells were exposed. The full dose−response
curves were plotted using a nonlinear dose−response curve fitting
analysis via GraphPad Prism V4 software. By this, the minimum
concentrations required for 50% inhibition (IC50) were determined
for each compound.
Caucasian hepatocellular carcinoma cells (HepG2) were main-
tained as previously described.42 Cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) (HyClone) supplemented with
5% fetal bovine serum (heat-inactivated) and 1% penicillin/
streptomycin (Sigma-Aldrich) at 37 °C at 5% CO2. Cell viability
was monitored microscopically with 0.2% Trypan Blue. Cells were
trypsinized with 1× Trypsin−EDTA (Sigma) and ∼100,000 cells
were plated in 96-well plates and grown for 24 h. The cells were
treated with the relevant compound and incubated for 48 h at 37 °C
and 5% CO2. Cytotoxicity was determined using the CytoSelect LDH
Cytotoxicity Assay Kit (Cell Biolabs Inc., CBA-241), which was added
to the supernatant. Absorbance was measured at 450 nm. Data
obtained were analyzed in Excel and experiments were performed in
technical duplicates for three biological repeats (n = 3).
In Vitro Hepatic Microsomal Stability. Metabolic stability
studies were conducted in human, mouse, and rat liver microsomes
using a single-point (30 min) study design.43 Briefly, 1 μM of the
compound was incubated with 0.4 mg/mL microsomes in 0.1 M
phosphate buffer (pH 7.4). The reactions were quenched by the
addition of ice-cold acetonitrile containing an internal standard
(carbamazepine, 0.0236 μg/mL). The samples were centrifuged, and
the supernatant was analyzed by means of LC−MS/MS (Agilent
Rapid Resolution HPLC, AB SCIEX 4500 MS). The percentage
compound remaining was calculated by using the internal standard
corrected peak areas at 30 min, with those of the 0 min incubation.
Propranolol, midazolam, and MMV390048 were used as assay
controls.
Metabolite Identification Studies. The test compounds (10
μM) were incubated at 37 °C in a solution containing 1 mg/mL
microsomes, magnesium chloride (5 mM), and NADPH (1 mM) in
potassium phosphate buffer (100 mM, pH 7.4) for 60 min while
shaking. The samples were then prepared by ice-cold acetonitrile
precipitation, centrifuged, and filtered for LC−MS analysis. Controls
containing all the sample constituents (not incubated), and in which
NADPH, microsomes, or the test compound were individually
excluded, were also prepared and handled similarly to the test sample.
Verapamil (10 μM) was incubated concomitantly as a positive
control. Metabolites formed in microsomal incubations were
identified by comparison of the T60 chromatograms with chromato-
grams at T0 using Lightsight 2.3. The tentative identity of the
metabolites was deduced by comparison of the product ion spectra of
the [M + H]+ ions of the metabolites with that of the parent
compound using Analyst 1.6.3.
Calculation of Pharmacokinetics Parameters. PK parameters
were calculated by noncompartmental analysis using PK Solutions 2.0
(Summit Research Services, Montrose, CO, USA) with a method
based on curve stripping.
In Vitro Permeability. The PAMPA (Parallel Artificial Membrane
Assay) assay was performed in triplicate in 96-well MultiScreen filter
plates (Millipore, 0.4 μm PCTE membrane). Membrane filters were
precoated with 5% hexadecane in hexane and allowed to dry prior to
the assay. A membrane integrity marker, Lucifer yellow, was added to
the apical wells of the precoated MultiScreen plate donor/drug
solutions containing the test compound. Phosphate buffer (pH 7.4)
was added to the 96-well acceptor plate. An amount of 10 mM test
compound was used to spike (1 μM) the donor buffer at
physiologically relevant pH values (4 and 6.5), and the donor plate
was slotted into the acceptor plate and incubated (4 h at room
temperature) with gentle shaking (40−50 rpm). Following
incubation, samples from the acceptor wells and theoretical
equilibrium wells were transferred to the analysis plate and matrix-
matched with blank donor buffer. Acetonitrile containing an internal
standard (carbamazepine, 0.0236 μg/mL) was added to all samples,
and they were analyzed by LC−MS/MS (Agilent Rapid Resolution
HPLC, AB SCIEX 4500 MS). The normalized (analyte/internal
standard) peak areas were used to calculate the apparent permeability
(Papp). The membrane integrity was assessed by calculating the Papp of
Lucifer yellow (acceptable values, <50 nm/s) using a Modulus
microplate reader.46,47
β-Hematin Inhibition. The inhibition of β-hematin formation
assay was conducted to investigate the possible mechanism of
antiplasmodium activity of the synthesized benzimidazole analogues
as inhibitors of heme polymerization. This noncell assay evaluates the
ability of the test compounds to prevent the synthetic dimerization of
hematin (β-hematin) in lipid medium.
Kinetic Solubility. Solubility was measured at pH 6.5 using an
adapted miniaturized shake-flask method in 96-well plate format.44,45
Briefly, 4 μL of a 10 mM stock in DMSO was added to a 96-well plate
Hundred microliters of a solution containing water/305.5 μM NP-
40/DMSO at a v/v ratio of 70%/20%/10% was added to every well in
columns 2−12 of a 96-well plate. To column 1 were added 140 μL of
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pubs.acs.org/jmc
Article
water and 40 μL of 305.5 μM NP-40. Twenty microliters of the test
compounds (20 mM), that is, the synthesized compounds and
control, was added to column 1 in duplicate. One hundred microliters
of the test solution was diluted to column 11, with column 12 left as a
blank (0 μM of compound). A 178.8 μL aliquot of hematin (hemin)
stock was suspended in 20 mL of 1 M acetate buffer (pH 4.8) and 100
μL of this hematin suspension was added into each well. Plates were
then incubated for ∼5 h at 37 °C after which 32 μL of pyridine
solution (20% water, 20% acetone, 10% 2 M HEPES buffer (pH 7.4),
and 50% pyridine) was added followed by addition of 60 μL of
acetone to all wells. Plates were again read at 405 nm on a Thermo
Scientific Multiscan Go Microplate Spectrophotometer. The IC50
values were determined using GraphPad Prism 6.
Cell-Based Heme Assay. Target validation was carried out
through a cellular fractionation assay, optimized to a multiwell
colorimetric assay for determining heme species in P. falciparum as
previously described.48 The cellular fractionation allows the direct
quantification of heme species in isolated trophozoites such as the
freely exchangeable heme and hemozoin.
A two-tailed t-test (95% CI) was used for determination of
statistical significance of differences in measurements relative to
controls. The data represent a minimum of three repeats with
standard deviations calculated for each of the average results. All
analyses were done using GraphPad Prism version 6.0.0 software.
Microtubule Inhibition. Asexual Pf NF54 parasites were
maintained and early-stage gametocytes were induced and maintained
as described above. Parasites were treated with 41 and 44 at 600 nM
against asexual stages (∼1% parasitemia and 5% hematocrit, >95% D-
sorbitol synchronized ring-stage culture) and 2400 nM against early
stage (∼2% gametocytemia and 2% hematocrit 20% I/60% II/20%
III) and incubated for 24 h at 37 °C under 90% N2, 5% CO2, and 5%
O2 hypoxic conditions (final DMSO concentration of <0.1% (v/v)).
Colchicine (20 μM) was used as a positive control with treated
asexual parasites and early-stage gametocytes incubated for 24 h at 37
°C under 90% N2, 5% CO2, and 5% O2 hypoxic conditions. Following
incubation, parasites were stained with 250 nM Tubulin Tracker
Oregon Green 488, Taxol bis-acetate (Invitrogen) for 30 min to
visualize tubulin formation and costained with the nuclear dye
Hoechst 33324 (100 nM) for 10 min at 37 °C under 90% N2, 5%
CO2, and 5% O2 hypoxic conditions in the dark. After staining, cells
were washed three times with prewarmed PBS (137 mM NaCl, 2.7
mM KCl, and 10 mM phosphate (pH 7.4)) and visualized as live cells
using a Zeiss LSM 510 Meta Confocal Microscope with Zeiss ZEN
Black software (Zeiss, Germany).
African Medical Research Council Drug Discovery and
Development Research Unit, University of Cape Town,
Rondebosch 7701, South Africa; orcid.org/0000-0002-
1327-4727; Phone: +27-21-6502553;
Email: kelly.chibale@uct.ac.za
Authors
Godwin Akpeko Dziwornu − Department of Chemistry,
University of Cape Town, Rondebosch 7701, South Africa
Dina Coertzen − Department of Biochemistry, Genetics and
Microbiology, University of Pretoria Institute for Sustainable
Malaria Control, University of Pretoria, Hatfield 0028, South
Africa
Meta Leshabane − Department of Biochemistry, Genetics and
Microbiology, University of Pretoria Institute for Sustainable
Malaria Control, University of Pretoria, Hatfield 0028, South
Africa
Constance M. Korkor − Department of Chemistry, University
of Cape Town, Rondebosch 7701, South Africa;
orcid.org/0000-0001-6925-8905
Cleavon K. Cloete − Department of Chemistry, University of
Cape Town, Rondebosch 7701, South Africa
Mathew Njoroge − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, Department of
Medicine, University of Cape Town, Cape Town 7925, South
Africa
Liezl Gibhard − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, Department of
Medicine, University of Cape Town, Cape Town 7925, South
Africa
Nina Lawrence − Drug Discovery and Development Centre
(H3D), Division of Clinical Pharmacology, Department of
Medicine, University of Cape Town, Cape Town 7925, South
Africa
Janette Reader − Department of Biochemistry, Genetics and
Microbiology, University of Pretoria Institute for Sustainable
Malaria Control, University of Pretoria, Hatfield 0028, South
Africa
Mariëtte van der Watt − Department of Biochemistry,
Genetics and Microbiology, University of Pretoria Institute for
Sustainable Malaria Control, University of Pretoria, Hatfield
0028, South Africa
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00354.
■
Sergio Wittlin − Swiss Tropical and Public Health Institute,
Basel 4002, Switzerland; University of Basel, Basel 4003,
Switzerland
sı
Lyn-Marie Birkholtz − Department of Biochemistry, Genetics
and Microbiology, University of Pretoria Institute for
Sustainable Malaria Control, University of Pretoria, Hatfield
0028, South Africa; orcid.org/0000-0001-5888-2905
1D-NMR and corresponding HPLC−MS spectra of
representative target compounds; β-hematin inhibition
activity (βHIA) and cytotoxicity (HepG2) of the
selected compounds; LC−MS/MS analytical conditions
for PK analysis; heme species in synchronized control
and compound 35-treated, compound 41-treated,
compound 44-treated, and compound 48-treated
Pf NF54 parasites; live-cell confocal imaging of asexual
trophozoites and stage I−III gametocytes of P.
falciparum (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00354
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Biological results (XLSX)
Molecular formula strings (CSV)
The University of Cape Town, South African Medical
Research Council, and South African Research Chairs Initiative
of the Department of Science and Innovation, administered
through the South African National Research Foundation
(NRF; to K.C., UID: 64767; to L.-M.B., UID: 84627) and a
NRF Community of Practice on “Evaluating Malaria Control
Interventions” (UID: 110666), are gratefully acknowledged for
AUTHOR INFORMATION
Corresponding Author
Kelly Chibale − Department of Chemistry, Institute of
Infectious Disease and Molecular Medicine, and South
■
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Article
Ballell-Pages, L.; Sanz, L. M.; Castan
̃
eda-Casado, P.; Jiménez-Díaz, M.
support. At Swiss TPH, we thank Sibylle Sax, Rahel Buehler,
and Ursula Lehmann for assistance in performing antimalarial
in vitro and in vivo assays.
B.; Martínez-Martínez, M. S.; Viera, S.; Kiuru, P.; Calderón, F.; Yli-
Kauhaluoma, J. A Developability-Focused Optimization Approach
Allows Identification of in Vivo Fast-Acting Antimalarials: N -[3-
[(Benzimidazol-2-Yl)Amino]Propyl]Amides. J. Med. Chem. 2015, 58,
4573−4580.
ABBREVIATIONS
■
(15) Attram, H. D.; Wittlin, S.; Chibale, K. Incorporation of an
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Bezuidenhout, B. C.; Lauterbach, S. B.; Liebenberg, D.; Birkholtz, L.-
ABS, asexual blood stage; EG, early-stage gametocytes; LG,
late-stage gametocytes; TCP, target candidate profile; TPP,
target product profile; RI, resistance index; SI, selectivity index;
CHO, Chinese hamster ovarian cells; MLM, mouse liver
microsomes; RLM, rat liver microsomes; HLM, human liver
microsomes; βHIA, β-hematin inhibition assay; SALI,
structure−activity landscape index
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