Multistage Antiplasmodium Activity of Astemizole Analogues and Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action

Article abstract of DOI:10.1021/acsinfecdis.8b00272

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC₅₀s) of <0.1 μM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 (PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues (PfNF54-IC₅₀ < 0.1 μM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC₅₀: 1-5 μM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of β-hematin formation by the AST derivatives and their antiplasmodium IC₅₀s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Full text of DOI:10.1021/acsinfecdis.8b00272

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Article
Multistage Antiplasmodium Activity of Astemizole Analogues and
Inhibition of Hemozoin Formation as a Contributor to Their Mode of Action
Malkeet Kumar, John Okombo, Dickson Mambwe, Dale Taylor, Nina Lawrence, Janette
Reader, Mariëtte van der Watt, Diana Fontinha, Margarida Sanches-Vaz, Belinda
C Bezuidenhout, Sonja Lauterbach, Dale Liebenberg, Lyn-Marie Birkholtz, Theresa
L. Coetzer, Miguel Prudêncio, Timothy J Egan, Sergio Wittlin, and Kelly Chibale
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00272 • Publication Date (Web): 11 Dec 2018
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Multistage Antiplasmodium Activity of Astemizole Analogues and
Inhibition of Hemozoin Formation as a Contributor to Their Mode of
Action
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Malkeet Kumar1, John Okombo1, Dickson Mambwe1, Dale Taylor2, Nina Lawrence2, Janette
Reader3, Mariëtte van der Watt3, Diana Fontinha4, Margarida Sanches-Vaz4, Belinda C
Bezuidenhout5, Sonja B Lauterbach5, Dale Liebenberg5, Lyn-Marie Birkholtz3, Theresa L
Coetzer5, Miguel Prudêncio4, Timothy J. Egan1,6, Sergio Wittlin7,8, Kelly Chibale* 1,6,9
1Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.
2Drug Discovery and Development Centre (H3D), Division of Clinical Pharmacology, Department
of Medicine, University of Cape Town, Observatory 7925, South Africa.
3Department of Biochemistry, Genetics and Microbiology, Institute for Sustainable Malaria
Control, University of Pretoria, Private Bag X20, Hatfield 0028, South Africa.
4Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas
Moniz, 1649-028 Lisboa, Portugal.
5Department of Molecular Medicine and Haematology, School of Pathology, Faculty of Health
Sciences, University of the Witwatersrand and National Health Laboratory Service, Johannesburg
2193, South Africa
6Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
7701, South Africa.
7Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
8University of Basel, 4003 Basel, Switzerland.
9South African Medical Research Council Drug Discovery and Development Research Unit,
Department of Chemistry University of Cape Town, Rondebosch 7701, South Africa.
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*Corresponding Author: Kelly Chibale
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Email: Kelly.Chibale@uct.ac.za: Phone: +27-21-6502553. Fax: +27-21-6505195.
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A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine
drug whose antimalarial activity was previously identified in a high-throughput screen. The
multistage activity potential against Plasmodium parasite’s life cycle of the subsequent analogues
was examined by evaluating against the parasite asexual blood, liver and gametocyte stages. In
addition, the previously reported contribution of heme detoxification to the compound’s mode of
action was interrogated. Ten of the seventeen derivatives showed IC50s < 0.1 µM against the
chloroquine (CQ)-sensitive Plasmodium falciparum NF54 (PfNF54) strain while maintaining
submicromolar potency against the multidrug resistant strain, PfK1, with most showing low
likelihood of cross-resistance with CQ. Selected analogues (PfNF54- IC50 < 0.1 µM) were tested
for cytotoxicity on CHO cells and found to be highly selective (selectivity index > 100). The first
ever gametocytes screening of AST and its analogues revealed their moderate activity (IC50: 1 – 5
µM) against late stage P. falciparum gametocytes, while the evaluation of activity against P.
berghei liver stages identified one compound (3) with three-fold greater activity than the parent
AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of β-
haematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of
intracellular inhibition of hemozoin formation within the parasite further yielded signatures
attributable to a possible perturbation of the heme detoxification machinery.
Key Words: Astemizole, Repositioning, Plasmodium falciparum, β-haematin, Gametocytes
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The latest statistics from the World Health Organization estimate that there were 216 million cases
and 445,000 deaths due to malaria in 2016.1 While these numbers mirror a trend of declining
malaria burden over the past 15 years due to control strategies, which comprise indoor residual
spraying, insecticide-treated nets, intermittent preventive treatments and adoption of artemisinin
combinations as first-line therapy, the disease still remains a major public health concern. In fact,
the recent evolution and dissemination of artemisinin-tolerant Plasmodium falciparum strains and
suboptimal response to insecticides used for management of the Anopheles vector threaten the
ultimate success of these control efforts.2,3 To minimize the likelihood of rapid development of
drug resistance, there is a clear need for innovation in order to identify safe and effective
antimalarial compounds that can preferably inhibit multiple stages of the parasite’s life cycle,
which includes the asexual erythrocytic blood stage, the transmissible sexual gametocytic stage
and a hepatocytic liver stage . One attractive approach to novel antimalarial drug design that can
significantly reduce the pitfalls inherent to drug development is the repositioning of already-
approved pharmacotherapies – a strategy whose application in malaria and neglected tropical
diseases has been reviewed elsewhere.4
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In the search for new drugs to treat malaria, researchers at Johns Hopkins University (JHU), in
2006, screened a chemical library comprising 2 687 Food and Drug Administration (FDA)-
approved drugs for potential inhibitors of P. falciparum proliferation. This led to the discovery of
antiplasmodium properties of astemizole (AST) (Figure 1), a second-generation antihistamine,
which was withdrawn from market due to cardiotoxicity caused by its hERG channel blocking
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activity and association with arrhythmias.5 In that study, AST exhibited submicromolar half-
maximal inhibitory concentrations (IC50s) against three P. falciparum strains with different levels
of chloroquine (CQ) sensitivity.5 In humans, AST is rapidly absorbed from the gastrointestinal
tract and undergoes extensive first-pass metabolism to the pharmacologically active
desmethylastemizole (DM-AST) and other minor metabolites, including nor-astemizole (Nor-
AST) (Figure 1).6 Incidentally, these metabolites also exhibited antiplasmodium activity in the
JHU screen, with DM-AST showing two- to twelve-fold higher potency than AST, depending on
the parasite strain (Figure 1).5 Further evaluation revealed their antimalarial potential, with 80%
and 81% suppression of parasitaemia in P. vinckei-infected mice treated for four days with AST
(30 mg/m2/day) and DM-AST (15 mg/m2/day), respectively.5
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N
N
N
H
H
N
N
N
NH
N
O
R
R = CH3; Astemizole
R = H; Desmethylastemizole
Nor-Astemizole
F
F
3D7/ Dd2/ ItG
Pf Pf
Pf
AST-IC50 (µM) = 0.23/0.46/0.73
DM-AST-IC50 (µM) = 0.12/0.11/0.06
Nor-AST-IC50 (µM) = 4.48/3.60/2.23
CQ-IC50 (µM) = 0.03/0.08/0.11
Figure 1: Chemical structures and antiplasmodium activity of AST and its metabolites as reported
by Chong et al (2006) against Pf3D7, PfDd2 and PfItG strains.5
Encouragingly, the antimalarial potential of AST has been recently investigated by other
researchers. Employing a conjugated approach that hybridized CQ and AST structural motifs,
Musonda et al. reported on CQ-AST hybrid compounds with in vitro activity against CQ-resistant
(CQR) strains and in vivo activity in a murine malaria model.7 Similarly, Roman and colleagues
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reported on potent AST derivatives bearing several modifications to the AST structure against the
CQR P. falciparum strain, ItG.8 More recently, Tian and co-workers identified AST derivatives
that showed good activity against the CQS strain, Pf3D7, and exhibited reduced hERG channel
inhibition potency.9 Various known chemical modification strategies can be employed to mitigate
hERG channel inhibition activity in new AST analogues. These include (a) reduction of
lipophilicity via removal of aromatic groups as already demonstrated in case of AST9, reduction
in the clogP via incorporation of polar moieties and heteroaromatic replacement of aromatic
groups; (b) attenuation of pKa via modulation of the basicity of a nitrogen atom; (c) incorporation
of three dimensional character and reduction in π-π stacking interactions. While these studies
focused solely on the inhibition of asexual blood stage parasite proliferation, Derbyshire and co-
workers further reported on the effectiveness of AST in a high-throughput phenotypic screen
developed to systematically identify molecules with liver-stage efficacy.10
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The basic premise of the drug repositioning approach involves further re-engineering of the
original molecule for optimization into a new chemical entity with improved potency,
physicochemical profile and/or safety. Although, the findings from the seminal JHU screen have
been pursued by others to highlight the antimalarial potential of AST, to our knowledge no
medicinal chemistry effort to date has focused on probing the multistage antiplasmodium activity
of AST analogues across liver, asexual blood and gametocytic life-cycle stages. As an extension
to our group’s efforts aimed at identifying multistage active antiplasmodium compounds, this
article explores structural modifications around the core AST pharmacophore (Figure 2) in
anticipation of developing analogues with favourable solubility and cytotoxicity indices, as well
as activity against different stages of the parasite’s life cycle. This involved i) amending the central
part of AST through replacement of the 4-aminopiperidine moiety with a piperazine; ii) modifying
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the left hand side (LHS) of AST by a) complete removal of the benzyl group and replacing it with
a methyl or b) replacing the fluoro moiety with, methyl ketone, cyano, acid, amide, sulfonyl and
pyridyl nitrogen or c) regio-isomers of the fluoro- and cyano-substituted benzyl group; and iii)
regio-isomerization of the methoxy substituent or replacement of the 4-methoxyphenyl with 4-
hydroxyphenyl on the right hand side (RHS) of AST. Some of the analogues with modifications
on LHS of AST such as complete removal of the benzyl group and its replacement with a methyl
group as well as replacement of fluoro substituent on benzyl moiety with amide, sulfonyl, acid and
pyridyl groups were also aimed at reducing lipophilicity (cLogP < 4.5 vs AST cLogP of 5.84;
Supplementary Information Table S1).
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During Plasmodium blood stage infection, host haemoglobin (Hb) is degraded into globin as a
nutrient source, and heme is released as a by-product of this obligate catabolism. However, in its
free state, heme is toxic as it can induce free radical formation as well as membrane damage.11,12
To bypass this toxicity, P. falciparum converts heme into insoluble hemozoin (Hz) crystals - a
detoxification process known to be inhibited by quinolines such as CQ and one that is tractable
using in vitro models which mimic the digestive vacuole (DV) milieu. In line with previous
findings showing that AST and DM-AST concentrate within the parasite’s DV, inhibit heme
crystallization and co-purify with Hz in drug-sensitive and resistant parasites,5 we also queried the
contribution of inhibition of heme detoxification as at least one of the possible modes of action
(MoA) of AST.
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Replacement of the 4-
aminopiperidine with
piperazine
Regioisomerization of the methoxy
substituent or replacement of the 4-
methoxyphenyl with 4-hydroxyphenyl
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N
N
H
N
N
OMe
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a.
Removal
or
Central part
Right Hand Side
(RHS)
replacement of benzyl
group with a methyl
F
b. Replacing fluoro with,
methyl ketone, cyano,
acid, amide, sulfonyl and
Left Hand Side
(LHS)
pyridyl
nitrogen.
c. Regio-isomers of the
fluoro and cyano
substituted benzyl group.
Figure 2: Structural modifications around AST to design various derivatives
CHEMISTRY: The synthesis of AST analogues is summarized in Scheme 1. AST analogue 3
with the piperazinyl moiety replacing AST’s amino piperidinyl moiety was synthesized in two
steps, a reaction of commercially available 1b with piperazine followed by 1-(2-bromoethyl)-4-
methoxybenzene. The synthesis of compounds 7-16 commenced with the benzylation of
commercially available 2-chlorobenzimidazole (1a) using K2CO3 in acetone to afford
intermediates 4a-j, which were subsequently reacted with 1-(4-methoxyphenethyl)piperidin-4-
amine in a neat reaction at 170 С to obtain AST analogues 7-16. AST was obtained by direct
reaction of 1b with 1-(4-methoxyphenethyl)piperidin-4-amine while the demethylation of AST
using HBr afforded DM-AST.
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The synthesis of compounds 17-20 began with the reaction of 1a, b with protected
4-aminopiperidines to afford intermediate 5a, b (Scheme 1). The benzylation of 5a with 4-
(bromomethyl)benzonitrile followed by deprotection using 4M HCl in dioxane afforded 6a, while
Nor-AST was obtained by direct deprotection of 5b. The reaction of 6a and Nor-AST with the
respective phenyl ethyl bromide using K2CO3 in acetonitrile at reflux resulted in compounds 17-
20 (Scheme 1). The AST analogue 5c was obtained by direct reaction of 1a with 1-(4-
methoxyphenethyl)piperidin-4-amine. On the other hand, 1a was first N-methylated and then
reacted with 1-(4-methoxyphenethyl)piperidin-4-amine to afford analogue 5d.
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Scheme 1: Synthesis of AST analogues
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8
N
H
N
N
H
N
vii
N R2
NH
N
N
6a: R1 = 4-cyanobenzyl
R1
R1
6
5
9
5a: R1 = H; R2 = Boc
Nor-AST: R1 = 4-Fluorobenzyl
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5b: R1 = 4-fluorobenzyl; R2 = Ethylcarboxylate
5c: R1 = H; R2 = 4-methoxyphenylethyl
5d: R1 = Me; R2 = 4-methoxyphenylethyl
viii
vi
v
N
N
H
N
N
iv
N
iii
DM-AST
N
N
Cl
Cl
R4
N
R1
AST, 7-20
R3
R3
1
X
4
X
1a: R1 = H
4a: R3 = 3-F
4b: R3 = 2-F
4c: R3 = 4-CN
4d: R3 = 3-CN
4e: R3 = 2-CN
4f: R3 = 4-COOMe
4g: R3 = 4-CONH2
4h: R3 = 4-SO2Me
4i: R3 = 4-COMe
4j: X = N; R3 = H
1b: R1 = 4-Fluorobenzyl
i
N
X = CH
N
NH
N
2
F
ii
N
N
N
N
O
3
F
Reagents and conditions: (i) 1b, piperazine, Et3N, t-BuOH, 120 С, 48 h, 87%; (ii) 1-(2-
bromoethyl)-4-methoxybenzene, toluene, reflux, 48 h, 50%; (iii) 1a, appropriate benzyl halide,
K2CO3, acetone, 23 С, 4-6 h, 70-85% (4a-j); (iv) 1-(4-methoxyphenethyl)piperidin-4-amine, 170
С, 6-12 h, 15-74% (AST, 7-16); (v) AST, HBr, reflux (120 С), 6 h, 48%; (vi) 1a, 4-amino-N-
boc-piperidine, TEA, 150 С, 2 h, 60% (5a); 1b, ethyl 4-aminopiperidine-1-carboxylate, 170 С,
4 h, 98% (5b); 1-(4-methoxyphenethyl)piperidin-4-amine, 170 С, 4 h, 48% (5c); (a) 1a, methyl
iodide, K2CO3, acetone, 23 С, 30 min, 68%; (b) 1-(4-methoxyphenethyl)piperidin-4-amine, 170
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С, 8 h, 30% (5d); (vii) (a) 5a, 4-(2-bromomethyl)benzonitrile, K2CO3, DMF, 70 С, 3 h, 88%;
(b) 4N HCl in dioxane, 18 С, 2 h, 80% (6a); 5b, HBr, reflux (120 С), 6 h, 85% (Nor-AST);
(viii) 6a, 4-(2-bromoethyl)phenol, K2CO3, ACN, 79 C, reflux (85 С), 5 h, 36%; (17); Nor-AST,
1-(2-bromoethyl)-3-methoxybenzene, ACN, reflux (85 С), 16 h, 78% (18); Nor-AST, 1-(2-
bromoethyl)-2-methoxybenzene, ACN, reflux (85 С), 24 h, 71% (19); Nor-AST, (2-
bromoethyl)benzene, ACN, reflux (85 С), 12 h, 79% (20).
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RESULTS & DISCUSSION:
Blood Stage Antiplasmodium Activity, Cytotoxicity and Solubility:
The antiplasmodium activity of AST, DM-AST, Nor-AST and all newly synthesized analogues
was evaluated against the CQ sensitive PfNF54 strain. Compounds with sub-micromolar potency
against PfNF54 were further tested against the multidrug resistant PfK1 strain. Additionally,
solubility of all the compounds was determined while analogues with IC50 ≤ 0.1 µM against
PfNF54 were also tested for cellular toxicity against CHO cells (Table 1).
Replacing the amino piperidine in the central core of the AST structure with piperazine (3) did not
increase antiplasmodium activity (PfNF54-IC50 = 1.9 µM), as the resulting compound exhibited
comparably less activity than AST (PfNF54-IC50 = 0.086 µM). The structural changes on the LHS
of the molecule showed that the benzyl group is not important for the activity, as the debenzylated
analogue 5c (PfNF54-IC50 = 0.047 µM) showed enhanced activity compared to AST. However,
methylation of the benzimidazole nitrogen (5d) led to a 7.5-fold reduction in activity (PfNF54-
IC50 = 0.35 µM) as compared to 5c (PfNF54-IC50 = 0.047 µM), demonstrating the plausible
contribution of the free benzimidazole nitrogen towards the potency of 5c. Additionally, the
antiplasmodium activity of 5c (PfK1-IC50 = 1.7 µM) was ablated in the PfK1 drug-resistant strain
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(PfK1-IC50 = 1.7 uM), and this compound showed a high propensity for cross-resistance with CQ
(RI = 35). Regarding the substituents replacing the 4-fluoro group on the benzyl motif, electron
withdrawing substituents such as cyano (9; PfNF54-IC50 = 0.078 µM), sulfonyl (14; PfNF54-IC50
= 0.23 µM) and methyl ketone (15; PfNF54-IC50 = 0.12 µM) were tolerated and showed
comparable activity to AST. On the other hand, amide (13; PfNF54-IC50 = 0.41 µM), acid (12;
PfNF54-IC50 = 1.5 µM) and pyridyl (16; PfNF54-IC50 = 1.7 µM) replacements reduced potency
approximately by five-, seventeen- and nineteen-fold, respectively. The flouro (7; PfNF54-IC50 =
0.068 µM, and 8; PfNF54-IC50 = 0.075 µM) and two of the cyano (9; PfNF54-IC50 = 0.078 µM,
and 10; PfNF54-IC50 = 0.055 µM) regio-isomers showed comparable activity to AST, while the
2-cyano analogue (11; PfNF54-IC50 = 0.033 µM) showed more than two-fold enhanced potency
compared to AST. This demonstrated that substituent positional change on the benzyl group is
tolerated and may deliver compounds with enhanced potency.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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50
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54
55
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Regarding changes to the RHS, the DM-AST congener, 17, bearing a 4-cyano benzyl group was
three-fold more potent than AST (PfNF54-IC50 = 0.086 µM) and showed slightly better activity
than DM-AST (PfNF54-IC50 = 0.04 µM vs 0.028 µM). The methoxy regio-isomers 18 (PfNF54-
IC50 = 0.089 µM), 19 (PfNF54-IC50 = 0.077 µM) and the unsubstituted derivative 20 (PfNF54-
IC50 = 0.089 µM) were equipotent and had comparable activity to AST.
A selected number of analogues (5c, 7-11 and 18-20; PfNF54-IC50 ≤ 0.1 µM) were advanced for
evaluation of cytotoxicity in CHO cells, exhibiting high selectivity indices (selectivity index, SI >
100; Table 1). All the analogues except 11 and 15 also exhibited generally good aqueous solubility
(Supplementary Information Table S1). There was no discernible improvement in solubility
among the derivatives relative to AST, except when the benzyl on the LHS of the molecule was
replaced with a methyl group (5d; 155 µM).
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Table 1: Antiplasmodium activity and cytotoxicity of AST and the synthesized analogues.
5
6
7
8
9
N
X
N
R1
R2
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
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Antiplasmodium
Cytotoxicity
Activity IC50
(µM)a,b
CHOd IC50
(µM)
Compound
AST
R1
X
R2
PfNF54 PfK1 R.I.c
S.I.e
HN
N
OMe
OH
0.086
0.37
4.3
29.6
344
F
F
F
F
HN
HN
N
N
DM-AST
0.040
1.1
0.03
0.92
-
0.8
0.8
-
23.0
--
575
--
Nor-AST
----
OMe
OMe
OMe
N
N
3
1.9
--
--
HN
HN
N
N
5c
5d
H
0.047
0.35
1.7
2.2
36.2
6.3
40.4
--
860
--
H3C
HN
N
OMe
7
0.068
0.31
4.6
43.8
644
F
HN
HN
N
N
OMe
OMe
8
9
0.075
0.078
0.34
0.36
4.5
4.6
40.4
21.2
539
272
F
NC
HN
HN
HN
N
N
N
OMe
OMe
OMe
10
11
12
0.055
0.033
1.5
0.33
0.18
-
6.0
5.5
-
39.7
43.6
--
722
1321
--
CN
CN
HO
O
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Antiplasmodium
Activity IC50
(µM)a,b
Cytotoxicity
CHOd IC50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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Compound
13
R1
X
R2
PfNF54 PfK1 R.I.c
(µM)
S.I.e
H2N
HN
HN
HN
N
OMe
OMe
OMe
0.41
0.23
0.12
2.2
1.3
5.4
5.7
3.6
--
--
O
O
N
N
14
15
--
--
--
--
S
O
0.43
O
N
HN
HN
N
N
OMe
OH
16
17
1.7
--
--
--
--
0.028
0.041
1.5
38.5
1376
NC
HN
N
18
19
0.089
0.077
0.43
0.32
4.8
4.2
41.0
43.1
461
560
F
OMe
HN
HN
N
N
F
F
MeO
20
0.086
0.004
0.37
0.14
4.3
32.4
--
377
--
Chloroquine
35.0
Artesunate
Emetine
0.003
0.001
0.3
--
--
0.95
aAll the in vitro antiplasmodium testing was performed in Modified [3H]-hypoxanthine incorporation
assay at Swiss TPH except compounds 16 and 17 which were evaluated in parasite lactate dehydrogenase
assay at UCT; b Readout is mean from two independent experiments (n = 2) for multidrug resistant
(PfK1) and CQ-sensitive (PfNF54) strains of P. falciparum; cRI
=
Resistance
d
e
Index (PfK1-IC50/PfNF54-IC50); CHO = Chinese hamster ovarian cells; SI = Selectivity Index
(CHO IC50/PfNF54-IC50).
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Gametocyte and Liver Stage Activity: While the ability of AST to inhibit proliferation of P.
berghei liver stage parasites has been documented,10 there are no reports on the gametocytocidal
potential of this class of compounds. We therefore tested AST, DM-AST and nine other AST
analogues (3, 5c, 10, 11, 14, 15 and 18-20) against late (LG; > 95% stage IV/V) gametocytes.
Only two analogues showed activity, with 5c (IC50 = 1.9 µM) being the most potent against late
stage gametocytes compared to AST (IC50 = 3.4 μM) and 18 (IC50 = 4.1 μM) (Table 2). None of
the compounds showed improved activity against early stage gametocytes (> 90% stage II/III)
compared to AST (data not shown). To the best of our knowledge, this data represents the first
evidence of activity of AST and its derivatives against gametocytes.
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11
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15
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45
46
47
48
49
50
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Table 2: Gametocyte and liver stage activity of AST and analogues:
N
X
N
R1
R2
IC50 (µM)
bLiver
Cell
aLate
gametocyt
e
Blood
stage
confluency
(% of
R1
X
R2
stage
Compound
control)
stage
>73%
>91%
>91%
0.66 ±
0.79
HN
HN
N
N
OMe
AST
3.4
NSc
NSc
0.086
0.04
1.9
F
0.14 ±
0.031
0.21 ±
0.12
OH
DM-AST
F
F
OMe
OMe
N
N
3
NSd
NSd
HN
HN
N
N
5c
H
1.9
4.1
NSd
0.047
0.089
18
NSd
8.4
F
Primaquine
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MB
0.14
0.00
Chloroquine
6
4
7
8
9
b
aLate gametocyte stage: Data was obtained in a single experiment (n = 1) as a technical triplicate; Liver
stage: Data represents the mean ± SD of n = 3 independent experiments performed in triplicate obtained for
inhibition of the infection of human hepatoma cells (Huh7) by P berghei; NSc: Not selected for IC50
determination as they showed < 50% inhibition in an initial dual-point screen at 1 and 5 µM; NSd: Not
selected for IC50 determination due to < 50% reduction in liver stage parasite load at ≤ 1.0 µM
concentration; MB = methylene blue.
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The potential for inhibition of liver stage infection was probed using a model of P. berghei-
infection of the Huh7 human hepatoma cell line.13 Preliminary screening for liver stage activity of
AST, DM-AST, Nor-AST, 3, 5c, 7-15 and 17-20 at four different concentrations (10 μM, 1 μM,
0.1 μM and 0.01 μM) showed that only DM-AST and analogue 3 exhibited > 50% reduction of
parasite load at ≤ 1 μM with IC50 values of 0.14 μM and 0.21 μM, respectively (Table 2). In
parallel, Huh7 cell confluency was assessed in the presence of each compound concentration, as a
measurement of cytotoxicity (Table 2). The activity of AST in our analysis was, however, lower
than reported by Derbyshire et al (AST: IC50 = 0.114 µM),10 which might result from the fact that
different cell lines were employed in two protocols (HepG2 versus HuH7) or from distinct varying
cell densities in each assay.10
Mechanistic Studies: In humans, AST competitively binds to histamine H1 receptor sites.14
Although Chong and colleagues implicated the inhibition of heme crystallization within the
parasite in AST’s activity, the drug’s exact MoA in Plasmodium is yet to be established.5
Employing a pyridine-based, detergent-mediated in vitro model that partially recapitulates the
micro-environment within the DV and quantifies the inhibition of formation of synthetic Hz, β-
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haematin (βH),15 we assessed the ability of AST derivatives to block βH formation as surrogate
for the inhibition of heme detoxification by the parasite. Invoking a discriminatory IC50 of 100 µM
to identify active inhibitors, four of the twenty evaluated compounds blocked βH formation, albeit
less potently than the standard H formation inhibitors amodiaquine (IC50: 11 µM) and CQ (IC50:
23 µM) (Supplementary Information Table S1). Although inhibition was not directly linked to
any specific structural modification of AST, all the active derivatives comprised analogues bearing
nitrile or carboxylic acid substitution of the fluorine at the C4 position on the LHS phenyl ring.
Seeking possible structure-activity relationships, we observed a strong and significant positive
correlation (R2 = 0.6759, p < 0.0001) between βH inhibition and activity against PfNF54
(Supplementary Information Figure S1), As a caveat, we reiterate that this assay represents an
in vitro set-up with the attendant limitations of mimicking the physiological complexities of
intracellular drug activity, which include membrane permeation and vacuolar accumulation. These
data support inhibition of the heme detoxification pathway as a possible contributing mechanism
of the antiplasmodium action of the AST derivatives analysed in the present study.
10
11
12
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15
16
17
18
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To establish whether or not this series actually inhibits intracellular Hz formation in P. falciparum,
DM-AST and compound 10 (with βH inhibitory profiles) were progressed into a cellular heme
fractionation assay designed to delineate a dose-dependent effect of compounds on the various
heme species (Hb, free heme and Hz) in the parasite.16 Exposure of synchronized ring stage
PfNF54 parasites to each of the two compounds for 32 h led to statistically significant
concentration-dependent increases in the proportions of free heme and a corresponding decrease
in Hz, similarly to what is observed for CQ (Figure 3). Indeed, this concentration-dependent
profile persisted for both derivatives when the analysis was focused on the amount of heme
(expressed as mass of heme Fe) measured per cell. Thus, these results, corroborate the findings of
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Chong et al.,5 as they strongly suggest that inhibition of Hz formation is a likely, although not
necessarily the only, potential MoA of these two AST analogues. Indeed, given the liver stage
activity of DM-AST, there must be other targets, suggesting a possible pleiotropic mechanism, at
least for this compound.
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11
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Figure 3: Dose-dependent effect of DM-AST, 10 and CQ on Hb (left panels), ‘free’ heme
(middle panels), and Hz (right) panels in PfNF54. Significant increases in Hb and heme and
decreases in Hz relative to the control were calculated using a two-tailed t-test and are denoted in
asterisk: (∗) P < 0.05; (∗∗) P < 0.01; (∗∗∗) P < 0.001.
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CONCLUSION:
5
6
7
8
As follow up on earlier work on the antimalarial activity of AST, we have presented an
investigation on the derivatization and evaluation of the cytotoxicity, activity against different
stages of the parasite life cycle, and potential MoA of AST and seventeen analogues. Structural
modifications of the AST pharmacophore showed that replacement of the 4-fluoro group on the
LHS with a nitrile group was particularly favoured for blood stage activity, especially when
accompanied by a hydroxyl substitution for the methoxy group on the RHS of the molecule.
Appreciable activity against late stage gametocytes and liver stage parasites was also observed,
thus introducing the possibility for optimization towards dual- or triple-action antimalarial agents.
To this end, 5c represents a promising starting point due to its good asexual blood and moderate
sexual gametocyte stage activities while further optimization of 3 should be aimed at improving
its asexual blood stage activity to match its good liver stage activity. In addition, our data confirm
previous findings of the likely contribution of inhibition of heme detoxification as a contributing
mechanism to the antiplasmodium effect of AST. Finally, the high selectivity observed for these
molecules highlights their cellular safety, at least in lower mammals. Further optimization and
evaluation of additional AST derivatives is thus warranted.
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10
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17
18
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EXPERIMENTAL:
All commercially available chemicals were purchased from Sigma-Aldrich or Combi-Blocks,
South Africa. All solvents were dried by appropriate techniques. Unless otherwise stated, all
1
solvents used were anhydrous. H-NMR and 13C-NMR spectra were recorded on Bruker
13
13
spectrometer at 400 MHz (1H 400.2 MHz; C 100.6 MHz) or Bruker-600 (1H 600.3 MHz; C
150.9 MHz). Melting points were determined on a Lasec automatic melting point machine.
Analytical thin-layer chromatography (TLC) was performed on aluminium-backed silica-gel 60
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F254 (70-230 mesh) plates with detection and visualization done using (a) UV lights (254/366 nm),
(b) iodine vapor and (c) ninhydrin spray reagent. Column chromatography was performed with
Merck silica-gel 60 (70-230 mesh). Chemical shifts (δ) are given in ppm downfield from TMS as
the internal standard. Coupling constants were recorded in Hertz (Hz). Purity was determined by
Agilent 1260 Infinity binary pump, Agilent 1260 Infinity diode array detector (DAD), Agilent
1290 Infinity column compartment, Agilent 1260 Infinity standard autosampler, and Agilent 6120
quadrupole (single) mass spectrometer, equipped with APCI and ESI multimode ionization source,
and all compounds tested for biological activity were confirmed to have ≥ 95% purity. The HPLC
method used is described in material and method section. Any data not shown below is supplied
in the Supporting files.
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11
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1-(4-fluorobenzyl)-2-(piperazin-1-yl)-1H-benzo[d]imidazole (2)
A solution 1b (0.2 grams, 0.071 mmol) and piperazine (0.66 grams, 0.71 mmol) in t-BuOH was
treated with triethyl amine (0.77 grams, 1.1 mL, 0.71 mmol). The resulting reaction mixture was
stirred at 120 C in a closed vessel for 48 h. After the completion of reaction, reaction was
quenched with aqueous saturated solution of NaHCO3 and extracted with ethyl acetate (4 × 10
mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to
afford crude product. The recrystallization of crude product from a mixture of pentane and DCM
1
furnished pure compound 2 (0.2 grams, 87% yield). m.p. 107.9-108.9 C; H-NMR (400 Hz,
CD3OD @ 50 C): δppm 7.53 (d, J = 7.6 Hz, 1H,), 7.25-7.10 (m, H-1, 5H), 7.05 (t, J = 8.4 Hz, 2H),
5.32 (s, 2H), 3.27 (t, J = 5.2 Hz, 4H), 3.04 (t, J = 4.8 Hz, 4H); 13C-NMR (101 Hz, MeOD @ 50
C) δppm 162.29 (d, J = 244.9 Hz, C-F), 157.7, 140.7, 134.7, 132.41 (d, J = 3.0 Hz), 128.10 (d, J =
8.1 Hz, 2C), 122.0, 121.7, 117.1, 115.23 (d, J = 21.9 Hz, 2C), 109.6, 50.4 (2C), 46.4 and 44.4 (2C);
LC-MS (APCI/ESI)+: m/z [M+H]+ = 311.2 (calculated for C18H19FN4, 310.16).
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1-(4-fluorobenzyl)-2-(4-(4-methoxyphenethyl)piperazin-1-yl)-1H-benzo[d]imidazole (3)
A solution of 2 (0.15 grams, 0.48 mmol), 1-(2-bromoethyl)-4-methoxybenzene (0.21 grams, 0.151
mL, 0.57 mmol) and triethyl amine (0.12 grams, 0.17 mL, 0.12 mmol) in toluene was refluxed
(110 C) for 48 h. After the completion of reaction, concentrated in vacuo and residue was
dissolved in ethyl acetate (20 mL). This mixture was washed with sat. NaHCO3 (2 × 15 mL), dried
over sodium sulfate and concentrated in vacuo. Purification of residue by biotage flash
chromatography at 5% MeOH: DCM and recrystallization from a mixture of pentane and DCM
provided 3 as a white solid (0.12 grams, 50%); 1H-NMR (400 Hz, CD3OD @ 50 C): δppm 7.53 (d,
J = 8.0 Hz, 1H), 7.23-7.12 (m, 6H), 7.13-7.09 (m, 1H), 7.08-7.03 (m, 2H), 6.84 (d, J = 8.8 Hz,
2H), 5.31 (s, 2H), 3.77 (s, 3H), 3.35-329 (m, 4H), 2.81-2.75 (m, 2H), 2.69 (m, 4H), 2.67-2.61 (m,
2H); 13C-NMR (101 Hz, MeOD @ 50 C) δppm 162.31 (d, J = 244.9 Hz, C-F), 158.3, 157.8, 140.8,
134.8, 132..46 (d, J = 2.7 Hz), 132.0, 129.2 (2C), 128.08 (d, J = 8.2 Hz, 2C), 121.9, 121.6, 117.0,
115.23 (d, J = 21.9, 2C), 113.6 (2C), 109.5. 60.2, 54.4, 52.4 (2C), 50.1 (2C), 46.5 and 31.8; LC-
MS (APCI/ESI)+, found m/z = 445.2 [M+H]+ (calculated for C27H29FN4O, 444.23); Purity = 99.9%
(tr = 4.035 min).
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41
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48
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Synthesis of 4-N-Boc-((1H-benzo[d]imidazol-2-yl) amino) piperidine (5a). A mixture of 2-
chlorobenzimidazole (1a) (2.0 grams, 13.2 mmol) and 4-amino-N-Boc-piperidine (3.15 grams,
15.7 mmol) in TEA (5.50 mL, 39.4 mmol) where stirred under a nitrogen atmosphere at 150 C
for 2 h. After the completion of reaction, dissolved in DCM (25 mL) followed by washing with
NaHCO3 (2 × 20 mL), brine (2 × 20 mL), dried over anhydrous Na2SO4 and concentrated in vacuo.
Residue was washed with EtOAc to obtained 5a as a white solid (2.49 grams, 60%). 1H-NMR (300
MHz, DMSO-d6) δ 10.59 (s, 1H), 7.12 (t, J = 6.9 Hz, 2H), 6.86 (dt, J = 12.4 and 7.2 Hz, 2H), 6.50
(d, J = 8.0 Hz, 1H), 3.90 (d, J = 13.3 Hz, 2H), 3.83 – 3.68 (m, 1H), 2.87 (d, J = 13.5 Hz, 2H), 1.93
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(dd, J = 12.6 and 3.7 Hz, 2H), 1.41 (s, 9H), 1.39 – 1.27 (m, 2H). LC-MS (APCI/ESI)+, found m/z
5
6
= 317.1 [M+H]+ (cal. for C17H24N4O2, 316.19).
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Ethyl 4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidine-1-carboxylate (5b)
A neat mixture of 1b (1.5 grams, 5.75 mmol) and ethyl 4-aminopiperidine-1-carboxylate (2.0
grams, 11.62 mmol) was heated to 170 C and stirred for 4 h. After completion of the reaction, it
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was cooled to ambient temperature (23 C), dissolved in DCM (50 mL), washed with saturated
NaHCO3 (2 × 20 mL), dried over sodium sulfate and concentrated in vacuo. Purification by column
chromatography using 0.2-1% MeOH: DCM afforded 5b as a white solid (2.3 grams, 98% yield);
m.p. 175.8-178.7 °C; 1H-NMR (400 Hz, CDCl3): δppm 7.55 (d, J = 8.0 Hz, 1H), 7.22-7.11 (m, H-
1, 3H), 7.10-7.00 (m, 4H), 5.14 (s, 2H), 4.22-3.90 (m, 3H), 4.11 (q, J = 8.0 Hz, 2H), 4.24 (q, J =
8.0 Hz, 2H), 2.10 (m, 2H), 1.33 (m, 2H) and 1.25 (t, J = 8.0 Hz, 3H); LC-MS (APCI/ESI)+, found
m/z = 397.2 [M+H]+ (calculated for C22H25FN4O2, 396.20).
N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-amine (5c)
A neat mixture of 1a (0.1 grams, 0.657 mmol) and 1-(4-methoxyphenethyl)piperidin-4-amine
(0.31 grams, 1.323 mmol) was heated to 170 C and stirred for 4 h. After completion of the
reaction, the reaction mixture was cooled down to ambient temperature (23 C), dissolved in DCM
(20 mL), washed with sat. NaHCO3 (2 × 10 mL), dried over sodium sulfate and concentrated in
vacuo. Purification by column chromatography using 0-1% MeOH: DCM afforded 5c as a white
solid (0.11 grams, 48% yield). 1H-NMR (Methanol-d4, 400 MHz) δ 7.24-7.17 (m, 2H), 7.13 (d, J
= 8.8 Hz, 2H), 700-6.94 (m, 2H), 6.85 (d, J = 8.4 Hz, 2H), 3.76 (s, 3H), 3.72-3.62 (m, 1H), 3.10-
2.96 (m, 2H), 2.82-2.71 (m, 2H), 2.64-2.54 (m, 2H), 2.29 (t, J = 9.2 Hz, 2H), 2.15-2.10 (m, 2H),
1.71-1.54 (m, 2H); 13C-NMR (101 MHz, MeOD) δ 158.7, 154.3, 137.6, 131.7, 129.2 (2C), 119.9,
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found m/z = 351.2 [M+H]+ (calculated for C21H26N4O, 350.2), Purity = 97.1% (tr = 0.197 min).
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4-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzonitrile (5d)
A solution of 1a (0.3 grams, 1.967 mmol) and methyl iodide (1.5 grams, 10.709 mmol) in
DMF (3 mL) was charged with potassium carbonate (2.95 grams, 19.67 mmol). The resulting
reaction mixture was stirred at room temperature (23 C) for 30 mins. After the completion of
reaction, quenched with water (10 mL), extracted with ethyl acetate (5 × 10 mL), dried over sodium
sulphate and concentrated to obtain the crude product (0.225 grams, 68% yield), which was washed
with n-pentane and used in next step without further purification.
A neat mixture of methylated product from previous step (0.075 grams, 0.450 mmol) and 1-(4-
methoxyphenethyl)piperidin-4-amine (0.2 grams, 0.90 mmol) was heated to 170 C and stirred for
8 h. After the completion of the reaction, it was cooled to ambient temperature (23 C), dissolved
in DCM (20 mL), washed with sat. NaHCO3 (2 × 10 mL), dried over sodium sulfate and
concentrated in vacuo. Purification by column chromatography using 0-1% MeOH: DCM afforded
5d as a yellow solid (0.055 grams, 30% yield). 1H-NMR (Methanol-d4, 400 MHz) δ 7.33-7.27 (m,
1H), 7.21-7.11 (m, J = 8.4 Hz, 3H), 7.10-7.0 (m, 2H), 6.87 (d, J = 8.0 Hz, 2H), 3.88-3.80 (m, 1H),
3.76 (s, 3H), 3.55 (s, 3H), 3.21-3.11 (m, 2H), 2.87-2.76 (m, 2H), 2.74-2.65 (m, 2H), 2.39 (t, J =
9.2 Hz, 2H), 2.21-2.12 (m, 2H), 1.80-1.66 (m, 2H); 13C-NMR (101 MHz, MeOD) δ 158.4, 154.2,
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141.2, 134.6, 131.4, 129.2 (2C), 120.7, 119.3, 114.4, 113.6 (2C), 107.0, 60.1, 54.3, 52.3 (2C), 49.7,
31.7, 31.2 (2C) and 27.2; LC-MS (APCI/ESI)+, found m/z = 365.2 [M+H]+ (calculated for
C22H28N4O, 364.2), Purity = 95.5% (tr = 0.200 min).
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4-((2-(piperidin-4-ylamino)-1H-benzo[d]imidazol-1-yl)methyl)benzonitrile (6a).
A mixture of 5a (2.20 grams, 6.96 mmol), 4-(bromomethyl) benzonitrile (1.64 grams, 8.4 mmol) and
K2CO3 (2.40 grams, 17.4 mmol) in DMF (9 mL) was stirred under a nitrogen atmosphere at 70 oC
for 3 h. After the completion of the reaction, it was cooled to ambient temperature (23 C) and
diluted with EtOAc (50 mL). The resulting mixture was washed with water (3 × 30 mL), 5% LiCl
(20 mL × 2) and brine (20 mL × 2), dried over sodium sulfate and concentrated in vacuo. The
residue was washed with diethyl ether and used in next step without further purification.
A suspension of N-Boc protected compound from the previous step in 4N HCl in dioxane (1 mL
per 0.100 grams) was stirred at ambient temperature (20 C) for 2 h. After the completion of
reaction, dioxane was removed under vacuum and residue was taken in EtOAc (25 mL) and
neutralized with 15% NaOH pH > 8) while stirring in an ice bath. The organic phase was dried
over anhydrous Na2SO4 and evaporated under vacuum to afford 6a (2.50 grams, 5.80 mmol, 1 eq)
as a pale yellow solid (1.54 grams, 80% yield). 1H-NMR (300 MHz, Methanol-d4) δ 7.70 – 7.64
(m, 2H), 7.32 (dt, J = 7.8, 0.9 Hz, 1H), 7.28 – 7.22 (m, 2H), 7.05 (ddd, J = 7.9, 6.9, 1.7 Hz, 1H),
6.99 (ddd, J = 7.9, 1.7, 0.7 Hz, 1H), 6.94 (ddd, J = 7.9, 6.9, 1.1 Hz, 1H), 5.37 (s, 2H), 3.89 (tt, J =
11.1, 4.1 Hz, 1H), 3.07 (dt, J = 12.9, 3.5 Hz, 2H), 2.74 (td, J = 12.5, 2.6 Hz, 2H), 2.06 (dd, J =
12.7, 3.7 Hz, 2H), 1.46 (qd, J = 11.9, 4.1 Hz, 2H). 13C-NMR (101 MHz, Methanol-d4) δ 153.95,
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142.34, 141.70, 133.89, 132.25, 127.13, 121.29, 119.54, 118.05, 114.85, 111.02, 107.51, 50.16,
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44.66, 44.23, 32.45. LC-MS (APCI/ESI): found m/z = 331.9 [M+H] + (cal. for C20H21N5, 331.18).
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1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-benzo[d]imidazol-2-amine (Nor-AST)
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A solution of 5b (2.3 grams, 5.80 mmol) in HBr (48%, 17 mL) was refluxed (120 C) for 3 h. After
completion of the reaction, it was cooled to 0 C and treated with 2.5 M aqueous NaOH in dropwise
manner to neutralized excess of HBr (pH = 12-14). The extraction of organic compounds was done
with chloroform (5 × 20 mL). The combined organic layers were dried over sodium sulfate and
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concentrated in vacuo to obtain Nor-AST as an orange solid (1.89 grams, 85% yield); H-NMR
(400 Hz, CDCl3): δppm 7.52 (d, J = 8.0 Hz, 1H), 7.19-7.08 (m, 3H), 7.07-6.98 (m, 4H), 5.07 (s,
2H), 3.04 (dt, J = 12.4 and 4.8 Hz, 2H), 2.75 (td, J = 12.0 and 2.8 Hz, 2H), 2.41 (bs, 1H), 2.10 (m,
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2H) and 1.32 (m, 2H); C-NMR (101 Hz, CDCl3): δppm 162.44 (d, J = 247.3 Hz, C-F), 153.3,
142.4, 134.5, 131.19 (d, J = 3.3 Hz), 128.22 (d, J = 8.3 Hz, 2C), 121.5, 119.8, 116.6, 116.13 (d, J
= 21.8 Hz, 2C), 107.2, 50.2, 45.2 (2C), 45.0 and 33.8 (2C); LC-MS (APCI/ESI)+, found m/z =
325.2 [M+H]+ (calculated for C19H21FN4, 324.18), Purity = 95.2% (tr = 2.819 min).
General procedure 1 for the synthesis of AST and compounds 7-16: A solution of 2-chloro-
1H-benzo[d]imidazole (1 equivalent) in acetone was charged with K2CO3 (2.5 equivalent) and
respective benzyl bromide (1.5 equivalent). The resulting reaction mixture was stirred at room
temperature (23 C) for 4-6 hours. After completion of the reaction, it was filtered and
concentrated. The crude product was washed with n-pentane to obtain 4 and used in next step
without further purification.
A mixture of respective benzylated benzimidazole intermediate 4 (1 equivalents) and 1-(4-
methoxyphenethyl)piperidin-4-amine (2 equivalents) was heated to 170 C and stirred for 6-12 h.
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After the completion of reaction, cooled to ambient temperature (23 C) and resulting solid was
dissolved in DCM, washed with saturated solution of sodium bicarbonate (twice), water (twice)
and brine. The organic phase was dried over sodium sulphate and concentrated in vacuo to obtain
crude product, which was purified by column chromatography using 1-10% MeOH: DCM to
afford pure compounds.
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1-(4-fluorobenzyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(AST)
White solid (72% yield); 1H-NMR (400 Hz, CD3OD): δppm 7.32 (d, J = 8.0 Hz, 1H), 7.19-7.10 (m,
4H), 7.08-7.00 (m, 4H), 6.95 (td, J = 8.0 and 0.8 Hz, 1H) 6.84 (dd, J = 8.8 and 2.4 Hz, 2H), 5.25
(s, 2H), 3.84 (tt, J = 12.0 and 4.0 Hz, 1H), 3.76 (s, 3H), 3.01 (m, 2H), 2.76 (m, 2), 2.58 (m, 2H),
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2.27 (m, 2H), 2.09 (m, 2H), 1.69-1.53 (m, 2H); C-NMR (101 Hz, CD3OD): δ 162.19 (d, J =
242.4 Hz), 158.2, 154.0, 141.7, 134.0, 132.4 (d, J = 2.02 Hz) , 131.8, 129.2 (2C), 128.23 (d, J =
8.1 Hz; 2C), 121.1, 119.4, 115.06 (d, J = 21.8 Hz, 2C), 114.7, 113.6 (2C), 107.7, 60.4, 54.3, 52.2,
49.9, 43.9, 32.0 and 31.5; LC-MS (APCI/ESI)+, found m/z = 459.2 [M+H]+ (calculated for
C28H31FN4O, 458.25); Purity = 95.7%, (tr = 3.525 min).
1-(3-fluorobenzyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(7)
White solid (70%);1H-NMR (Methanol-d4, 400 MHz) δ 7.37-7.28 (m, 2H), 7.18-7.12 (d, J = 8.8
Hz, 2H), 7.09-7.01 (m, 2H), 7.0-6.91 (m, 2H), 6.98-6.80 (m, 2H), 5.31 (s, 2H), 3.86 (tt, J = 12.0
and 4.0 Hz, 1H), 3.77 (s, 3H), 3.10-2.95 (m, 2H), 2.83-2.70 (m, 2H), 2.66-2.52 (m, 2H), 2.29 (td,
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J = 9.2 and 2.0 Hz, 2H), 2.15-2.05 (m, 2H), 1.72-1.59 (m, 2H); C-NMR (101 MHz, MeOD) δ
163.09 (d, J = 245.3 Hz, C-F), 158.2, 154.0, 141.6, 139.45 (d, J = 6.98 Hz), 134.0, 131.8, 130.25
(d, J = 8.27 Hz), 129.2 (2C), 122.0, 121.1, 119.5, 114.7, 113.89 (d, J =21.3 Hz), 113.6 (2C), 113.07
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(d, J = 22.6 Hz), 107.6, 60.4, 54.3, 52.3 (2C), 49.9, 44.0, 31.9, 31.3 (2C); LC-MS (APCI/ESI)+,
found m/z = 459.1 [M+H]+ (calculated for C28H31FN4O, 458.25); Purity = 97.8%, (tr = 3.989 min).
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1-(2-fluorobenzyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(8)
White solid (64%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.35-7.32 (m, 1H), 7.31-7.36 (m, 1H,),
7.18-7.11 (m, 3H), 7.09-7.00 (m, 3H), 6.95 (ddd, J = 8.4, 7.2 and 1.2 Hz), 6.89-6.81 (m, 3H), 5.34
(s, 2H), 3.86 (tt, J = 12.0 and 4.0 Hz, 1H), 3.78 (s, 3H), 3.09-3.0 (m, 2H), 2.83-2.71 (m, 2H), 2.64-
2.54 (m, 2H), 2.30 (td, J = 12 and 3.6 Hz, 2H), 2.16-2.05 (m, 2H), 1.69-1.53 (m, 2H); 13C-NMR
(101 MHz, MeOD) δ 160.56 (d, J = 245.2 Hz, C-F), 158.2, 154.1, 141.7, 133.9, 131.9, 129.2 (2C),
128.8, 127.9 (d, J = 3.7 Hz), 124.2, 123.34 (d, J = 14.7 Hz), 121.1, 119.4, 115.03 (d, J = 21.2 Hz),
114.9, 114.7, 113.6 (2C), 107.5, 60.4, 54.3, 52.2 (2C), 50.0, 39.0, 31.9 and 31.2 (2C); LC-MS
(APCI/ESI)+, found m/z = 459.1 [M+H]+ (calculated for C28H31FN4O, 458.25); Purity = 99.2%,
(tr = 3.886 min).
4-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzonitrile (9)
Pink solid (40%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.69 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0
Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 7.05 - 7.0(m,
1H), 7.0 - 6.95 (m, 1H), 6.86 (d, J = 8.4 Hz, 2H), 5.40 (s, 2H), 3.94-3.83 (m, 1H), 3.77 (s, 3H),
3.22-3.07 (m, 2H), 2.88-2.77 (m, 2H), 2.77-2.66 (m, 2H), 2.45 (t, J = 12.4 Hz, 2H), 2.21-2.10 (m,
2H), 1.78-1.61 (m, 2H); 13C-NMR (101 MHz, MeOD) δ 158.4, 153.9, 142.3, 141.6, 132.3 (2C),
131.1, 129.2 (3C), 127.1 (2C), 121.4, 119.7, 118.0, 114.9, 113.7 (2C), 111.0, 107.6, 59.9, 54.3,
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52.0 (2C), 49.7, 44.3, 31.4, and 31.0 (2C); LC-MS (APCI/ESI)+, found m/z = 466.2. [M+H]+
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(calculated for C29H31N5O, 465.25); Purity = 99.9%, (tr = 1.974 min).
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3-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzonitrile (10)
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White solid (15%); H-NMR (Methanol-d4, 400 MHz) δ 7.68-7.67 (m, 1H), 7.55-7.45 (m, 2H),
7.43-7.33 (m, 2H), 7.14 (d, J = 7.8 Hz, 2H), 7.11-7.01 (m, 2H), 7.03-6.95 (m, 1H), 6.85 (d, J = 8.0
Hz, 2H), 5.35 (s, 2H), 3.98-3.83 (m, 1H), 3.77 (s, 3H), 3.20-3.07 (m, 2H), 2.87-2.77 (m, 2H), 2.76-
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2.66 (m, 2H), 2.43 (t, J = 12.0 Hz, 2H), 2.21-2.09 (m, 2H), 1.80-1.60 (m, 2H); C-NMR (101
MHz, MeOD) δ 158.4, 154.0, 141.7, 138.4, 133.9, 131.6, 131.0 (2C), 129.9, 129.6, 129.2 (2C),
127.4, 119.7, 118.0, 115.0, 113.8 (2C), 112.6, 107.6, 59.9, 54.3, 52.1 (2C), 49.8, 44.0, 31.7, and
31.2 (2C); LC-MS (APCI/ESI)+, found m/z = 466.2. [M+H]+ (calculated for C29H31N5O, 465.25);
Purity = 98.9%, (tr = 1.977 min).
2-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzonitrile (11)
White solid (30%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.80 (dd, J = 6.8 and 0.96 Hz, 1H), 7.52
(td, J = 7.60 and 1.2 Hz, 1H), 7.44 (td, J = 7.60 and 0.8 Hz, 1H), 7.36 (d, J = 7.60 Hz, 1H), 7.14
(d, J = 8.4 Hz, 2H), 7.08 (ddd, J = 8.4, 6.0 and 2.4 Hz, 1H), 6.98-6.90 (m, 2H), 6.88-6.77 (m, J =
8.4 Hz, 3H), 5.52 (s, 2H), 3.95-3.82 (m, 1H), 3.77 (s, 3H), 3.14-3.02 (m, 2H), 2.81-2.74 (m, 2H),
2.68-2.59 (m, 2H), 2.34 (t, J = 12.0 Hz, 2H), 2.19-2.09 (m, 2H), 1.75-1.61 (m, 2H); 13C-NMR (101
MHz, MeOD) δ 158.3, 154.1, 141.7, 141.0, 133.9, 131.6, 131.1, 131.5, 129.2 (2C), 127.9, 126.2,
121.4, 119.6, 116.2, 114.9, 113.6 (2C), 110.7, 107.5, 60.2, 54.3, 52.2 (2C), 49.9, 43.3, 31.8, and
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Purity = 99.01% (tr = 2.027 min).
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4-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzoic acid (12)
White solid (15%); 1H-NMR (DMSO-d6, 400 MHz @ 80 ˚C) δ 10.74 (s, 1H), 8.04 (s, 1H), 7.82
(d, J = 8.2Hz), 7.38 (d, J = 8.3 Hz), 7.16 (d, J = 8.6 Hz, 2H), 7.03-6.91 (m, 4H), 6.87 (d, J = 8.6
Hz, 2H), 5.04 (s, 2H), 3.97-3.82 (m, 1H), 3.06-2.92 (m, 2H; overlapping with DMSO water peak),
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2.89-2.72 (m, 6H), 1.96-1.86 (m, 2H), 1.81-1.65 (m, 2H); C-NMR (101 MHz, DMSO-d6) C
NMR (101 MHz, DMSO) δ 166.1, 158.3, 154.8, 140.7, 134.3, 130.5, 130.3, 130.1 (2C), 128.8,
128.1 (2C), 127.6 (2C), 121.6, 121.0, 114.3 (2C), 109.4, 108.5, 55.5 (2C), 51.9 (2C), 50.5, 49.9,
49.6 and 43.33 (2C); LC-MS (APCI/ESI)+, found m/z = 485.2 [M+H]+ (calculated for C29H32N4O3,
458.25); Purity = 97.0%, (tr = 3.989 min).
4-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)benzamide (13)
White solid (29%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.82 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.8
Hz, 1H), 7.20 (d, J = 6.8 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 7.11-7.01(m, 2H), 6.99-6.91 (m, 1H),
6.85 (d, J = 8.4 Hz, 2H), 5.37 (s, 2H), 3.90-3.80 (m, 1H), 3.77 (s, 3H), 3.08-2.94 (m, 2H), 2.81-
2.71 (m, 2H), 2.63-2.53 (m, 2H), 2.28 (t, J = 12.0 Hz, 2H), 2.14-2.04 (m, 2H), 1.72-1.56 (m, 2H);
13C-NMR (101 MHz, MeOD) δ 170.4, 158.2, 154.1, 141.7, 140.6, 134.0, 132.9, 131.8, 129.2 (2C),
127.7 (2C), 126.3 (2C), 121.1, 119.4, 114.7, 113.6 (2C), 107.6, 60.4, 54.2, 52.2 (2C), 49.9, 44.3,
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31.9, and 31.4 (2C); LC-MS (APCI/ESI)+, found m/z = 483.9 [M+H]+ (calculated for C29H33N5O2,
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483.9); Purity = 95.81% (tr = 2.264 min).
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N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1-(4-(methylsulfonyl)benzyl)-1H-
benzo[d]imidazol-2-amine (14)
White solid (38%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.88 (d, J = 8.4 Hz, 2H), 7.42-7.31 (m,
3H), 7.13 (d, J = 8.6 Hz, 2H), 7.07 (td, J = 6.9 and 1.2 Hz, 1H), 7.03-6.99 (m, 1H), 6.99-6.91 (m,
1H), 6.85 (d, J = 8.4 Hz, 2H), 5.37 (s, 2H), 3.90-3.80 (m, 1H), 3.77 (s, 3H), 3.06 (s, 3H), 3.04-2.94
(m, 2H), 2.81-2.71 (m, 2H), 2.63-2.53 (m, 2H), 2.28 (t, J = 12.0 Hz, 2H), 2.14-2.04 (m, 2H), 1.72-
1.56 (m, 2H); 13C-NMR (101 MHz, MeOD) δ 13C-NMR (101 MHz, MeOD) δ 158.3, 154.1, 143.0,
141.8, 140.1, 134.0, 132.0, 129.2 (2C), 127.4, 127.3 (2C), 121.3, 119.6, 115.0, 113.7 (2C), 107.6,
102.1, 60.1, 54.4, 52.1 (2C), 50.0, 44.3, 43.0, 31.9, 31.4 (2C); LC-MS (APCI/ESI)+, found m/z =
519.2 [M+H]+ (calculated for C29H34N4O3, 518.68); Purity = 98.49% (tr = 1.991 min).
1-(4-((2-((1-(4-methoxyphenethyl)piperidin-4-yl)amino)-1H-benzo[d]imidazol-1-
yl)methyl)phenyl)ethan-1-one (15)
White solid (30%); 1H-NMR (Methanol-d4, 400 MHz) δ 7.92 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0
Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 7.07(td, J = 8 and 1.2 Hz, 1H), 7.03-
6.99 (m, 1H), 6.99-6.92 (m, 1H), 6.84 (d, J = 8.4 Hz, 2H), 5.36 (s, 2H), 3.92-3.82 (m, 1H), 3.76
(s, 3H), 3.15-2.99 (m, 2H), 2.81-2.71 (2H, m), 2.68-2.58 (m, 2H), 2.37 (t, J = 12.0 Hz, 2H), 2.19-
2.07 (m, 2H), 1.77-1.60 (m, 2H); 13C-NMR (101 MHz, MeOD) δ 198.5, 158.3, 154.0, 142.1, 141.6,
136.2, 134.0, 131.4, 129.2 (2C), 128.5 (2C), 126.5 (2C), 121.2, 119.5, 114.8, 113.6 (2C), 107.7,
29
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