Astemizole analogues with reduced hERG inhibition as potent antimalarial compounds

Article abstract of DOI:10.1016/j.bmc.2017.10.004

Astemizole is a H₁-antagonist endowed with antimalarial activity, but has hERG liabilities. Systematic structural modifications of astemizole led to the discovery of analogues that display very potent activity as inhibitors of the growth of the Plasmodium parasite, but show a decreased hERG inhibition, when compared to astemizole. These compounds can be used as starting point for the development of a new class of antimalarials.

Full text of DOI:10.1016/j.bmc.2017.10.004

Accepted Manuscript
Astemizole analogues with reduced hERG inhibition as potent antimalarial
compounds
Junjun Tian, Leen Vandermosten, Steve Peigneur, Lien Moreels, Jef Rozenski,
Jan Tytgat, Piet Herdewijn, Philippe E. Van den Steen, Steven De Jonghe
PII:
S0968-0896(17)31419-0
https://doi.org/10.1016/j.bmc.2017.10.004
BMC 14009
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
12 July 2017
26 September 2017
5 October 2017
Please cite this article as: Tian, J., Vandermosten, L., Peigneur, S., Moreels, L., Rozenski, J., Tytgat, J., Herdewijn,
P., Van den Steen, P.E., De Jonghe, S., Astemizole analogues with reduced hERG inhibition as potent antimalarial
compounds, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.10.004
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Astemizole analogues with reduced hERG inhibition as potent antimalarial compounds
Junjun Tian,a Leen Vandermosten,b Steve Peigneur,c Lien Moreels,c Jef Rozenski,a Jan
Tytgat,c Piet Herdewijn,a Philippe E. Van den Steen,b Steven De Jonghea,*
aMedicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000
Leuven, Belgium.
bImmunobiology, Rega Institute for Medical Research, Department of Microbiology and
Immunology, KU Leuven, Herestraat 49, 3000 Leuven, Belgium.
cKU Leuven, Toxicology and Pharmacology, Campus Gasthuisberg, Onderwijs en Navorsing
2, Herestraat 49, PO box 922, 3000 Leuven, Belgium.
*Corresponding author. E-mail: steven.dejonghe@kuleuven.be ; Phone : +32 16 32 26 62.
Abstract
Astemizole is a H1-antagonist endowed with antimalarial activity, but has hERG liabilities.
Systematic structural modifications of astemizole led to the discovery of analogues that
display very potent activity as inhibitors of the growth of the Plasmodium parasite, but show a
decreased hERG inhibition, when compared to astemizole. These compounds can be used as
starting point for the development of a new class of antimalarials.
Graphical abstract

1.
Introduction
Malaria is one of the most devastating diseases in developing countries.[1] According to the
world malaria report 2016 (WHO),[2] approximately 212 million cases of malaria were found
of which 428 000 deaths occurred, ~90% of these occurred in sub-Saharan Africa and 3.2
billion people residing in 97 countries were estimated to be at risk of malaria.
Malaria is caused by the parasite Plasmodium, which is transmitted by female mosquitoes of
the Anopheles genus. Human pathogenic Plasmodium species include P. falciparum, P. vivax,
P. ovale, P. malariae and P. knowlesi. P. falciparum is the most common and is highly
pathogenic, while P. vivax is also widespread. Once in the human body, the parasites infect
liver cells. Upon schizogony, the newly formed merozoites infect and multiply exponentially
in red blood cells, leading, after 7−10 days, to the onset of the typical malaria symptoms such
as fever, headache, chills, and vomiting. If untreated, imbalances between the parasite and the
immune system may lead to hyperparasitemia and organ-specific complications, resulting in
severe disease and death.[3]
The first widely used drug for the treatment of malaria infections was the natural product
quinine. Synthetic derivatives such as primaquine and chloroquine were discovered and
received marketing approval. Later on, inhibitors of dihydropteroate synthase (e.g.
sulphadoxine) and dihydrofolate reductase (e.g. pyrimethamine) were licensed for medical
use.[1a,4]
For most of the second half of the 20th century, control of acute malaria caused by all four
species of Plasmodium involved the use of chloroquine for first-line treatment and a
combination of sulphadoxine and pyrimethamine as second-line treatment. In all cases, the
emergence of Plasmodium resistant strains was observed. A major breakthrough in the
treatment of P. falciparum infections came with the discovery of artemisinin.[5] Subsequently,
derivatives with enhanced potency and improved stability or pharmacokinetics were
developed, such as dihydroartemisinin, artemether, artemotil and artesunate. Artemisinins are
fast-acting and very potent against blood-stage parasites and show activity against the early
sexual stages of the parasite, which is important to block transmission. Their major limitation
is that they have a short half-life, which is why they are partnered with longer-lasting drugs.
In order to reduce the risk of the emergence of drug resistance, combination therapy is
advised in which two drugs with a different molecular target are combined in a single tablet.
The golden standard has become fixed dose artemisinin combination therapies. Artemether–
lumefantrine (Coartem) and amodiaquine–artesunate (Coarsucam) are the most widely used,
whereas dihydroartemisinin– piperaquine (Euartesim) and artesunate–pyronaridine (Pyramax)

are the most recently approved. Chloroquine is still the recommended treatment for P. vivax
infections because resistance has not fully developed in this parasite, in contrast to P.
falciparum. In areas where chloroquine-resistant P. vivax is endemic, artemisinin combination
therapies are recommended for first-line treatment. For P. vivax infections, a 15-day course of
primaquine is required to prevent relapse and to provide a radical cure.
In search for novel drugs to treat malaria, different strategies have been followed. The
classical drug discovery approach of the past 20 years involved the target-centric approach,
Modulation of target activity provides a rational basis for the discovery of new medicines. It
offers a starting point for the identification of molecules by high-throughput screening of
compound libraries against isolated targets in biochemical assays. Well-known examples in
the malaria field are dihydroorotate dehydrogenase,[6] type II NADH dehydrogenase (NDH2)[7]
and plasmepsins.[8] More recently, there is a renewed interest in phenotypic screens for
antimalarial lead discovery. A strength of this approach is that these assays do not require
prior understanding of the molecular mechanism of action, and activity in such assays might
be translated into therapeutic impact more effectively than in target-based assays. Cellular
assays are also not biased towards particular molecular targets.[9] A third strategy for the
discovery of novel antimalarials includes the repurposing or repositioning of FDA-approved
pharmacotherapies for off-label use.[10] This is considered to be a less risky enterprise than de
novo drug discovery, because repositioning candidates have often been through several stages
of clinical development and therefore have well-known safety and pharmacokinetic profiles.
A number of repurposing whole-cell screens have been conducted in the malaria field.
Tricyclic antidepressant (promazine, nortriptyline, fluphenazine and amitriptyline) have been
associated with antiplasmodial activity displaying IC50 values in the 0.5 µM range.[11] The
NIH Chemical Genomics Center Pharmaceutical Collection which contains 2816 compounds,
registered or approved for human or animal use, has been tested against a panel of 61 P.
falciparum lines.[12] This screening campaign led to the discovery of 32 compounds with
potent in vitro activity (IC50 < 1 µM). Seven of these compounds yielded mean IC50 values
lower than 10 nM. Similarly, a library of 2687 existing drugs was screened for inhibitors of
the human malaria parasite P. falciparum, leading to the discovery of the H1-antagonist
astemizole as a potent antimalarial compound in vitro. Moreover, astemizole showed to have
activity in vivo in mouse models of malaria.[13] Later on, it was found that astemizole also
displayed activity against Plasmodium liver stages.[14]
The fact that astemizole inhibits liver-, as well as blood-stage parasites, and demonstrates in
vivo activity in a preclinical animal model of Plasmodium infection, makes it an interesting

compound for further chemistry. However, astemizole has been withdrawn from the market
because of cardiovascular adverse events, due to prolongation of the QT interval, which is
caused by inhibition of the hERG potassium ion channel.[15] Previously, a limited number of
astemizole analogues have been synthesized in order to probe into the structure-activity
relationship (SAR) studies, with respect to antimalarial activity, but hERG inhibition has not
been addressed in that study of 2013.[16]
In this publication, we report the synthesis of novel astemizole analogues and biological
antimalarial evaluation. The most promising congeners were also evaluated for hERG
inhibition.
2.
Results and discussion
2.1. Chemistry
For the synthesis of astemizole analogues, three different synthetic strategies were followed,
based upon published procedures[16,17] and with optimization of certain steps. Astemizole
analogues with structural variation at the nitrogen atom of the piperidine moiety were
synthesized as depicted in Scheme 1. Alkylation of 2-chloro-benzimidazole 1 with p-
fluorobenzyl bromide using NaH as base afforded intermediate 2. Nucleophilic aromatic
substitution reaction of 3 with ethyl 4-aminopiperidine-1-carboxylate under microwave
irradiation furnished compound 3 in excellent yield. Acidic deprotection of the carbamate
protecting group with hydrobromic acid in water under reflux afforded the desired
dihydrobromide salt of compound 4, which was used without any purification for further
reaction. This compound served as a key intermediate from which easily structural variety can
be introduced. Coupling with a series of acid chlorides in the presence of pyridine as solvent
and base at ambient temperature yielded a series of amides 5a-d (Table 1). Similar reaction
conditions were used for coupling with sulfonyl chlorides and isocyanates, yielding
sulfonamides 5e-h (Table 1) and urea 5i-p (Table 1), respectively. Initially, alkylation of the
nitrogen atom of the piperidine ring was achieved via a direct alkylation procedure using the
appropriate alkyl bromides. Later on, it was found that reductive amination reactions with
appropriate aldehydes gives also the desired compounds, which were much easier to purify
when compared to the direct alkylation procedure. Hence, this procedure was used to prepare
final compounds 5q-x (Table 2).

o
Reagents and conditions: a) NaH, DMF, 60 C, 2h, then p-fluorobenzyl bromide, overnight; b) ethyl 4-
o
aminopiperidine-1-carboxylate, NMP, microwave, 180 C, 1h; c) HBr (48 %) in water, reflux, 2 h; d) for 5a-d:
acid chloride, pyridine, rt, 18h ; for 5e-h: sulfonyl chloride, pyridine, rt, 18h ; for 5i-p: isocyanate, pyridine, rt,
18h ; for 5q-x: aldehyde, HOAc, DIPEA, EtOH, 40oC, 1 h, then NaCNBH3, 40 oC, 18 h.
Scheme 1. Synthesis of astemizole analogues with structural variation of the piperidine ring.
For alkylation of the imidazole moiety of astemizole analogues, the synthetic pathway as
shown in Scheme 2 was followed. Starting from commercially available 1-(4-
methoxyphenethyl)piperidin-4-amine 6, the isothiocyanate intermediate 7 was obtained after
reaction of 6 with carbon disulfide. The isocyanate was sufficiently stable to be isolated
through silicagel flash chromatography. Condensation of 7 with 1,2-phenylenediamine at
room temperature furnished thiourea 8 in excellent purity after crystallization. An
intramolecular cyclization with concomitant desulfurization using mercury(II) oxide furnished
benzimidazole analogue 9 as key intermediate. Alkylation in the presence of potassium
carbonate and an appropriate alkyl bromide afforded target compounds 10a-l (Table 3). Most
of these final products were obtained in pure form after silica gel based flash chromatography,
whereas other ones needed reversed-phase HPLC purification.
o
o
Reagents and conditions: a) NaOH, CS2, H2O, 0 C, 1 h, then ethyl chloroformate, 80 C, 2 h; b) 1,2-
phenylenediamine, EtOH, rt, overnight; c) yellow HgO, S, EtOH, reflux, 5 h, then HgO, S, reflux, overnight. d)
RBr, K2CO3, DMF, 70 oC overnight.

Scheme 2. Synthesis of astemizole analogues with structural variation of the imidazole
moiety.
The synthesis of an astemizole analogue with a piperazine-methylene linker is shown in
Scheme 3. Alkylation of o-phenylenediamine 11 with 4-fluorobenzylbromide under alkaline
conditions yielded 12. The formation of the imidazole moiety was achieved by refluxing 12
with chloroacetic acid in acidic conditions, affording the 2-chloromethylbenzimidazole
analogue 13.
The synthesis of the piperazine building block 16 started with the condensation of ethyl
piperazine-1-carboxylate and 1-2-(chloroethyl)-4-methoxybenzene 14. Alkaline cleavage of
the carbamate protecting group of compound 15 gave 1-(4-methoxyphenethyl)piperazine 16,
which was used subsequently as such, without any further purification, in the reaction with 13,
affording the desired molecule 17.
Reagents and conditions: a) 1-(bromomethyl)-4-fluorobenzene, K2CO3, MeOH, rt, overnight; b) 5N
HCl in water, chloroacetic acid, reflux, 4 h; c) ethyl piperazine-1-carboxylate, CH3CN-H2O, Na2CO3,
70 oC, 15 h, then aqueous NaOH; d) NaOH, EtOH-H2O, reflux, 12 h; e) Et3N, CH3CN, rt, 18 h.
Scheme 3. Synthesis of an astemizole analogue with a piperazine-methylene linker.
The synthesis of astemizole analogues with a piperazine and aminopyrrolidine linker follows
a
similar strategy (Scheme 4). Coupling of 2-chloro-1-(4-fluorobenzyl)-1H-
benzo[d]imidazole 2 with tert-butyl 3-aminopyrrolidine-1-carboxylate (as racemic mixture) or
piperazine under microwave heating led concomitantly to deprotection of the Boc group,
affording compounds 18 and 19, respectively. Reductive amination with 2-(4-
methoxyphenyl)acetaldehyde (accessible via Dess-Martin periodinane mediated oxidation of
the corresponding alcohol) under standard reactions conditions (NaCNBH3 in ethanol)
furnished final compounds 20 and 21.

Reagents and conditions: a) tert-butyl 3-aminopyrrolidine-1-carboxylate (for 18) or piperazine (for 19),
o
NMP, microwave, 180 C, 60 min; b) 2-(4-methoxyphenyl)ethan-1-ol, Dess-Martin periodinane,
CH2Cl2, reflux, 2 h; then HOAc, DIPEA, EtOH, 40 oC, 1 h, then NaCNBH3, 40 oC, 18 h.
Scheme 4. Synthesis of astemizole analogues with different linkers
2.2. Biological evaluation
2.2.1 Primary screening
All compounds were tested for their capability to inhibit the growth of the Plasmodium
falciparum 3D7a strain, using a Picogreen-based 96 well microplate assay.[18] In an initial
screening, compounds were evaluated at 4 different concentrations (10, 1, 0.1 and 0.01 µM)
and IC50 values were calculated. Astemizole was included as a reference and 100 nM
chloroquine was used as a positive control.
Three portions in astemizole were identified that were amenable for chemical modifications: a)
substitution of the piperidine moiety; b) substitutions of the imidazole moiety and c) structural
variation of the piperidine linker (Figure 1).
Figure 1. Subdivision of astemizole into three structural parts.
a) Substitution of the piperidine moiety

The nitrogen atom of the piperidine moiety offers a great chemical handle to introduce plenty
of structural variety. Amides (compounds 5a-d), sulphonamides (compounds 5e-h) and urea
(compounds 5i-p), with aliphatic, as well as aromatic side chains, were prepared. All these
analogues (Table 1) are devoid of antiparasitic activity and even at the highest tested
concentration (10 µM), they are much less active than astemizole, with an IC50 value of 3.9
µM for the most potent congener (compound 5f).
Table 1. Inhibition of P. falciparum (3D7a strain) growth by piperidine substituted astemizole
analogues (amides, sulfonamides and urea).
Average % inhibitiona of P. falciparum growth
Cmpd#
R
at
10 µM
1 µM
0.1 µM
0.01 µM
IC50 (µM) (95% CI)b
5a
5b
18 4.1
8
1.6
2.5
0
0
1.3
1.9
5
0
4.0
2.2
>10
51 4.7
6
6
10.040 (8.464 – 11.940)
5c
66 3.7
36 3.9
2.1
0
3.0
2.1
3
1.6
6.178 (5.062 – 7.538)
>10
5d
5
1
1.5
3.8
3
3
4
2
2.0
2.0
5e
5f
11 2.4
83 2.1
2.2
1
>10
6
8
3.7
3.1
2.4
4
5
1.0
3.956 (2.963 – 5.282)
5g
61 2.4
4.9
4
4.0
6.848 (5.501 – 8.523)
5h
5i
55 5.1
43 5.0
26 9.0
10 1.4
7
2
0
0.2
1.3
4.2
8
0
0
5.6
2.5
5.1
8.287 (6.431 – 10.700)
6
0
4.1
3.3
>10
>10
5j

5k
5l
35 3.3
57 2.7
66 6.0
39 4.4
25 10.9
7
5
5
4
4
0.9
1.4
3.1
3.6
5.0
3
1
2
0
2.5
3.2
2.0
1.8
0
0
1
2
0
3.5
0.9
2.1
1.9
3.0
>10
8.142 (7.003 – 9.474)
6.280 (4.946 – 7.975)
>10
5m
5n
5o
>10
>10
0
1
8.7
5.0
5p
49 9.3
99 2.5
15 5.8
91 3.8
8
0
3.0
1.5
0.162 (0.125 – 0.211)
Astemizole
37 8.1
b
aThe numbers represent the mean of at least four measurements SEM. CI; confidence
interval.
This indicates that a basic nitrogen at this position is essential for activity. It prompted us to
synthesize a series of analogues retaining a basic amine in the piperidine ring (Table 2). The
unsubstituted piperidine analogue (compound 4) is endowed with potent activity at the highest
tested concentration (10 µM), affording an IC50 value of 2.3 µM. A series of aliphatic and
cycloaliphatic substituents were introduced at that position. A methyl (compound 5q) or a
cyclopropylmethyl (compound 5s) gives complete inhibition only at the highest tested
concentration (10 µM). An isobutyl (compound 5r) or a cyclohexylmethyl (compound 5t) still
gives substantial inhibition (ca. 50%) at a concentration of 1 µM, giving rise to IC50 values of
0.8 µM. To mimic better the structure of the lead compound astemizole, a number of aromatic
residues were also incorporated. The methoxy group of astemizole is not essential for
antiplasmodial activity as its unsubstituted congener (compound 5u) is endowed with a
similar level of antiparasitic activity as astemizole. The chain length between the piperidine
nitrogen and the phenyl ring is important for activity. Shortening from a two carbon
(compound 5u) to a one carbon linker (compound 5v) led a drastic decrease in activity. On the
other hand, elongation of the linker from two (compound 5u) to three carbon atoms
(compound 5w) led to equipotent compounds. The insertion of unsaturation in the spacer
moiety (compound 5x) afforded a slightly less active compound, when compared to its fully
saturated analogue (compound 5w).

Table 2. Inhibition of P. falciparum (3D7a strain) growth by piperidine substituted astemizole
analogues (alkyl and alkylaryl substituents).
Average % inhibitiona of P. falciparum growth
at
0.1 µM
Cmpd#
R
10 µM
1 µM
0.01 µM
IC50 (µM) (95% CI)b
2.304 (1.572 – 3.384)
3.089 (2.050 – 4.662)
4
H
100 5.2
100 1.5
17 7.2
0
0
2.3
1.6
5
0
1.2
2.0
5q
2
1.5
5r
90 7.7
60 9.1
0
0
3.4
2.1
0
2.1
0.829 (0.578 – 1.191)
2.245 (1.644 – 3.074)
0.857 (0.631 – 1.155)
5s
5t
98 2.5
21 4.8
48 5.4
0
0
3.0
2.5
100 2.1
14 3.9
43 7.7
5u
5v
100 4.1
94 3.9
90 5.2
29 8.0
78 5.1
55 5.0
9
2
2
1
4.0
1.1
1.1
0.6
0.125 (0.093 – 0.168)
1.902 (1.438 – 2.524)
0.196 (0.144 – 0.269)
2
0.3
5w
5x
100 2.7
100 2.5
37 6.5
12 3.2
0.718 (0.554 – 0.926)
Astemizole
99 2.5
91 3.8
37 8.1
0
1.5
0.162 (0.125 – 0.211)
aThe numbers represent the mean of four measurements SEM. bCI; confidence interval.
b) Substitution of the imidazole nitrogen
As astemizole carries a 4-fluorobenzyl group at that position, the synthesis started with a few
very closely related analogues (Table 3), such as the benzyl (compound 10a), the 2,4-
difluorobenzyl (compound 10b) and the 4-bromobenzyl (compound 10c) derivatives. These
compounds display potent activity against P. falciparum, indicating that structural variety is
tolerated at that position. Moreover, it was discovered that the unsubstituted analogue
(compound 9) is still endowed with very potent activity. This stimulated us to study the SAR
at that position in a more systematic way. The introduction of a propyl group furnished
compound 10d that still has potent antiparasitic activity at 1 µM (80% inhibition, IC50 = 0.416
µM), whereas a methoxymethyl side chain (compound 10e) is much less active, even when

tested at 10 µM. Increasing the chain length to a hexyl (compound 10f), an octyl (compound
10g) or a decyl (compound 10h) afforded very potent compounds displaying 100% inhibition
at 1 µM and between 77-95% inhibition at 0.1 µM, yielding IC50 values between 0.029 and
0.050 µM. Branching of the aliphatic side chain by the synthesis of an isopentyl analogue
(compound 10i) and a 2-ethyl-butyl analogue (compound 10j) afforded compounds that were
clearly less potent than the long-chain alkyl analogues 10f-h. Finally, a cycloaliphatic
substituent (such as a cyclohexylmethyl moiety as in compound 10k) gives rise to another
potent compound (80% inhibition at 0.1 µM, IC50 = 0.044 µM).
Table 3. Inhibition of P. falciparum (3D7a strain) growth by imidazole substituted astemizole
analogues.
Average % inhibitiona of P. falciparum
growth at
Cmpd#
R
10 µM
1 µM
0.1 µM
0.01 µM
IC50 (µM) (95%
CI)b
10a
10b
100 3.2
80 5.8
82 6.5
14 4.8
55 7.1
0
0
2.9
1.7
0.344 (0.258 – 0.458)
0.085 (0.055 – 0.130)
100 2.0
10c
97 5.1
100 3.4
47 6.7
52 6.0
0
3.4
0.111 (0.081 – 0.151)
9
H
100 1.5 100 3.1
100 5.9 82 10.6
6
0
1.3
5.1
0.078 (0.054 – 0.112)
0.416 (0.270 – 0.640)
10d
3
0
3.5
1.4
10e
64 4.3
100 1.4 100 0.8
100 3.6 96 3.1
100 1.1 100 2.1
5
4.4
0
3.0
6.740 (5.243 – 8.672)
10f
10g
10h
10i
95 8.8
93 4.5
77 13.0
6
4
0
4.8
1.2
5.5
0.030 (0.019 – 0.046)
0.029 (0.020 – 0.041)
0.050 (0.031 – 0.081)
0.092 (0.062 – 0.136)
0.240 (0.165 – 0.349)
0.044 (0.029 – 0.066)
97 2.2
95 2.9
100 1.9
100 1.4
54 11.6
16 9.1
80 9.4
0
4.2
10j
0
1
3.9
3.7
10k
100 4.7 100 3.4

Astemizole
0.162 (0.125 – 0.211)
99 2.5
91 3.8
37 8.1
0
1.5
aThe numbers represent the mean of four measurements SEM. bCI; confidence interval.
c) Variation of the aminopiperidine linker
Three alternatives for the aminopiperidine linker of astemizole were investigated (Table 4).
The piperazine-methylene (compound 17), the aminopyrrolidine (compound 20) and the
piperazine (compound 21) linkers were all much less active than astemizole, which has an
aminopiperidine spacer.
Table 4. Inhibition of P. falciparum (3D7a strain) growth by astemizole analogues with a
modified linker moiety.
Average % inhibitiona of P. falciparum
Cmpd#
X
growth at
1 µM 0.1 µM
10 µM
0.01 µM
IC50 (µM) (95% CI)b
17
20
72 7.9
12 6.4
0
4.0
3
2.9
4.642 (3.168 – 6.770)
67 9.5
57 9.8
99 2.5
14 5.8
21 7.2
91 3.8
0
0
5.1
4.0
0
0
0
5.0
3.9
1.5
5.322 (3.376 – 8.352)
21
6.527 (3.948 – 10.650)
0.162 (0.125 – 0.211)
Astemizole
37 8.1
aThe numbers represent the mean of four measurements SEM. bCI; confidence interval.
2.2.2. Secondary screening
The most potent compounds from the initial screening were subjected to a detailed dose-
response analysis, using 8 different concentrations of the compounds ranging from 4 to 0.006
µM, allowing to determine more precise IC50 values (Table 5 and Figure 2). All compounds
were 1.5 – 6 times more potent than astemizole in inhibiting the growth of P. falciparum.
Although astemizole is endowed with strong activity against P. falciparum, its potent hERG

inhibition (IC50 = 305 nM), prevents its further development as antimalarial compound.
Therefore, compounds were also investigated for their inhibition of the hERG potassium
channel (Table 5 and Figure 3). Some of the compounds display extremely potent hERG
inhibition (e.g. 3 nM for compound 10k and 30 nM for compound 10f), and hence no
selectivity towards antiparasitic activity is observed. However, compounds 10g and 10h do
show a strongly reduced hERG inhibition, with IC50 values of 3.3 µM and 2 µM, respectively.
In combination with the potent antimalarial activities of these compounds, selectivity indexes
of 18.4 and 110 are obtained for compounds 10h and 10g, respectively. This is a major step
forward when compared to astemizole, which is endowed with a selectivity index of only 1.8.
Table 5. IC50 values for P. falciparum growth inhibition and hERG inhibition.
hERG
IC50 (nM) (95% CI)a
P. falciparum
IC50 (nM) (95% CI)a
Cmpd#
SIb
clogP
9
10b
79.7 (56.95 – 111.7)
102.3 (83.14 – 125.3)
29.0 (24.24 - ND)
163.90 (150.5 – 178.9)
178.00 (143.6 – 221.4)
29.96 (22.43 – 40.02)
3 270.00 (2 707 – 3 916)
2 069.00 (1 742 – 2 464)
211.70 (169.9 – 265.9)
3.27 (2.752 – 3.885)
2.1
1.7
4.11
5.98
6.65
7.7
10f
1.0
10g
29.6 (18.54 – 44.43)
112.7 (80.28 – 149.3)
28.0 (21.38 – 36.95)
39.4 (33.07 – 46.63)
171.1 (135.8 - 217)
110.4
18.4
7.6
10h
8.77
6.16
6.82
5.8
10i
10k
0.1
Astemizole
304.70 (207.8 - 441,8)
1.8
aCI; confidence interval. bSI; Selectivity index represents the ratio of hERG IC50/P.
falciparum IC50

−inhibition curve
Figure 2. Concentration s of astemizole and its derivatives (compounds 9,
10b, 10f, 10g, 10h, 10i, 10k) as inhibitors of P. falciparum growth.

Figure 3. Concentration−inhibition curves of astemizole and its derivatives (compounds 9,
10b, 10f, 10g, 10h, 10i, 10k) as hERG inhibitors.
3.
Conclusion
Systematic structural variation of astemizole led to the discovery of novel derivatives that are
endowed with very potent antiplasmodial activity, from which the most potent congeners
display IC50 values of 30 nM against P. falciparum. Importantly, these analogues do show a
reduced hERG inhibition, when compared to astemizole. Unfortunately, the most promising
analogue 10g (potent antiparasitic activity, while having a reduced hERG affinity) is a very
lipophilic compound (clogP = 7.7). Therefore, further work in this area should focus on
improving the drug-like properties of the compounds by reducing lipophilicity and improving
aqueous solubility. Structural variety, with respect to antimalarial activity, is tolerated at the
imidazole nitrogen of astemizole. Modifications at this position allows also to tune hERG
inhibition. Therefore, this substitution site seems to be preferred to explore further in order to
obtain novel derivatives with good antimalarial activity, reduced hERG inhibition and
improved physicochemical properties.
4.
Experimental section

4.1 Chemistry
For all reactions, analytical grade solvents were used. NMR spectra were recorded on a
Bruker Advance 300 MHz (1H NMR, 300 MHz, 13C NMR, 75 MHz) or 500 MHz (1H NMR,
1
500 MHz, 13C NMR, 125 MHz) with tetramethylsilane as internal standard for H NMR
spectra and (D6)-DMSO (39.52 ppm) or CDCl3 (77.16 ppm) or CD3OD (49.00 ppm) for 13C
NMR spectra. Abbreviations used are: s= singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad. Coupling constants are expressed in Hz. High resolution mass
spectrometry spectra were measured on a quadrupole orthogonal acceleration time-of-flight
mass spectrometer (Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3
µL/min and spectra were obtained in positive or negative ionization mode with a resolution of
15000 (FWHM) using leucine encephalin as lock mass. Pre-coated aluminum sheets (Fluka
Silica Gel/TLC-cards, 254 nm) were used for TLC. Column chromatography was performed
on ICN silica gel 63-200 60 Å. Flash column chromatography was performed on Reveleris®
X2 with column Reveleris® Silica with a flow rate of 20-30 mL/min.
Compounds 2, 3, 4, 6, 7, 8 and 9 were synthesized according to literature procedures.[16]
4.1.1. Synthesis of acyl-substituted 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine analogues
General procedure
To a solution of 4 (243 mg, 0.5 mmol) in dry pyridine (2 mL) was added an appropriate acid
chloride (0.5 mmol). The reaction mixture was stirred under a nitrogen atmosphere overnight.
Pyridine was removed under reduced pressure. Toluene (2-3 mL) was added and removed in
vacuo. This was repeated three times. The crude residue was then purified by silica gel flash
chromatography to give the desired title compounds. The following compounds were made
according to this procedure.
4.1.1.1.
(4-((1-(4-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidin-1-
1
yl)(phenyl)methanone (5a). This was obtained as a white solid (158 mg, 74%). H NMR
(300 MHz, DMSO) δ = 7.51 – 7.43 (m, 3H), 7.43 – 7.35 (m, 2H), 7.32 – 7.22 (m, 3H), 7.22 –
7.12 (m, 3H), 7.09 – 6.92 (m, 2H), 5.33 (s, 2H), 4.44 (br s, 1H), 3.61 (br s, 1H), 3.51 – 2.95
(m, 4H), 2.16 – 1.95 (m, 2H), 1.53 (br s, 2H) ppm. 13C NMR (75 MHz, DMSO) δ=169.23,
161.62 (d, J = 243.4 Hz), 152.46, 149.12, 136.34, 132.85 (d, J = 21.0 Hz), 129.62, 129.39,
129.21 (d, J = 8.3 Hz), 128.62, 126.77, 121.65, 120.05, 115.59 (d, J = 21.5 Hz), 114.23,

108.84, 59.88, 50.25, 44.12, 32.08 ppm. HRMS m/z [M + H]+ calcd for C26H26FN4O:
429.2085, found: 429.2083.
4.1.1.2.
(4-((1-(4-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidin-1-yl)(4-
1
fluorophenyl)methanone (5b). This was obtained as a white solid (122 mg, 55%). H NMR
(300 MHz, MeOD) δ = 7.56 – 7.33 (m, 3H), 7.32 – 6.94 (m, 9H), 5.40 (s, 2H), 4.63 (br s, 1H),
4.17 – 3.97 (m, 1H), 3.77 (br s, 1H), 3.23 – 2.79 (m, 2H), 2.26 – 1.94 (m, 2H), 1.70 (br s, 2H)
ppm. 13C NMR (75 MHz, MeOD) δ=171.51, 164.88 (d, J = 248.5 Hz), 163.60 (d, J = 243.8
Hz), 151.41, 149.91, 133.00 (d, J = 3.4 Hz), 132.72 (d, J = 6.0 Hz), 131.89, 130.46 (d, J = 8.6
Hz), 129.94 (d, J = 8.2 Hz), 124.70, 124.07, 116.75 (d, J = 21.9 Hz), 116.64 (d, J = 22.1 Hz),
113.69, 111.09, 52.70, 46.20, 42.47, 32.94 ppm. HRMS m/z [M + H]+ calcd for C26H25F2N4O:
447.1991, found: 447.1978.
4.1.1.3.
(4-((1-(4-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidin-1-yl)(4-
(trifluoromethyl)phenyl)methanone (5c). This was obtained as a white solid (149 mg, 60%).
1H NMR (300 MHz, MeOD) δ = 7.76 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 7.50 (dd, J
= 6.6, 1.4 Hz, 1H), 7.39 – 7.21 (m, 5H), 7.15 – 7.02 (m, 2H), 5.52 (s, 2H), 4.72 (d, J = 11.6
Hz, 1H), 4.23 – 4.05 (m, 1H), 3.72 (d, J = 11.9 Hz, 1H), 3.46 – 3.34 (m, 1H), 3.14 (t, J = 12.1
Hz, 1H), 2.19 (dd, J = 43.6, 10.6 Hz, 2H), 1.84 (br s, 2H) ppm. 13C NMR (75 MHz, MeOD) δ
= 170.81, 163.82 (d, J = 245.8 Hz), 150.30, 140.70, 132.67 (q, J = 32.2 Hz), 131.96, 131.41
(d, J = 3.2 Hz), 130.75, 130.11 (d, J = 8.3 Hz), 128.59, 126.76 (q, J = 3.5 Hz), 125.28, 125.19
(q, J = 271.6 Hz), 124.92, 116.81 (d, J = 22.0 Hz), 112.97, 111.60, 53.01, 46.49, 44.88, 32.63
ppm. HRMS m/z [M + H]+ calcd for C27H25F4N4O: 497.1959, found: 497.1964.
4.1.1.4.
(4-Chlorophenyl)(4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)amino)piperidin-1-yl)methanone (5d) was obtained as white solid (146 mg, 63%) in
1
yield. H NMR (300 MHz, MeOD) δ = 7.53 – 7.39 (m, 5H), 7.37 – 7.23 (m, 5H), 7.14 – 7.02
(m, 2H), 5.49 (s, 2H), 4.67 (br s, 1H), 4.20 – 3.96 (m, 1H), 3.78 (br s, 1H), 3.46 – 3.27 (m,
1H), 3.10 (br s, 1H), 2.18 (br s, 2H), 1.79 (br s, 2H) ppm. 13C NMR (75 MHz, MeOD) δ =
171.31, 163.86 (d, J = 246.0 Hz), 137.00, 135.31, 131.88, 131.33 (d, J = 3.1 Hz), 130.36,
130.08 (d, J = 8.3 Hz), 129.93, 129.69, 125.41, 125.08, 116.85 (d, J = 22.0 Hz), 112.87,
111.68, 53.10, 46.49, 42.40, 32.97 ppm. HRMS m/z [M + H]+ calcd for C26H25ClFN4O:
463.1695, found: 463.1686.
4.1.1.5.
(4-((1-(4-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidin-1-
yl)(morpholino)methanone (5j) was obtained as a white solid (157 mg, 72%). 1H NMR (300
MHz, MeOD) δ = 7.53 (dd, J = 6.8, 1.2 Hz, 1H), 7.39 – 7.25 (m, 5H), 7.16 – 7.06 (m, 2H),
5.51 (s, 2H), 4.04 – 3.90 (m, 1H), 3.83 (d, J = 13.5 Hz, 2H), 3.73 – 3.61 (m, 4H), 3.33 – 3.24

(m, 4H), 3.08 (t, J = 12.0 Hz, 2H), 2.14 (d, J = 10.7 Hz, 2H), 1.77 (qd, J = 12.5, 3.8 Hz, 2H)
ppm. 13C NMR (75 MHz, MeOD) δ = 165.41, 163.87 (d, J = 245.9 Hz), 150.31, 131.98,
131.43 (d, J = 3.2 Hz), 130.59, 130.01 (d, J = 8.4 Hz), 125.35, 125.00, 116.85 (d, J = 22.0 Hz),
112.89, 111.62, 67.58, 53.42, 48.56, 46.72, 46.39, 32.54 ppm. HRMS m/z [M + H]+ calcd for
C24H29FN5O2: 438.2300, found: 438.2298.
4.1.2. Synthesis of sulfonyl-substituted 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine analogues
General procedure
To a solution of 4 (243 mg, 0.5 mmol) in dry pyridine (2 mL) was added an appropriate
sulfonyl chloride (0.5 mmol). The reaction mixture was stirred under a nitrogen atmosphere
overnight. Pyridine was removed under reduced pressure. Toluene (2-3 mL) was added and
removed in vacuo. This was repeated three times. The residue was then purified by silica gel
flash column chromatographpy to give the title compounds. The following compounds were
made according to this procedure.
4.1.2.1. 1-(4-Fluorobenzyl)-N-(1-(isopropylsulfonyl)piperidin-4-yl)-1H-benzo[d]imidazol-
2-amine (5e). This was obtained as a white solid (28 mg, 13%). 1H NMR (300 MHz, MeOD)
δ = 7.47 (dd, J = 8.2, 1.4 Hz, 1H), 7.38 – 7.22 (m, 5H), 7.15 – 7.06 (m, 2H), 5.43 (s, 2H), 3.93
– 3.79 (m, 3H), 3.10 (t, J = 11.8 Hz, 2H), 2.14 (d, J = 10.1 Hz, 2H), 1.74 (ddd, J = 16.8, 11.8,
4.0 Hz, 2H), 1.33 (d, J = 6.8 Hz, 6H) ppm. 13C NMR (75 MHz, MeOD) δ = 164.00 (d, J =
246.0 Hz), 150.69, 144.46, 132.24, 131.47 (d, J = 3.0 Hz), 129.82 (d, J = 8.3 Hz), 125.40,
124.97, 116.94 (d, J = 22.1 Hz), 113.01, 111.54, 54.49, 52.85, 46.29, 33.24, 16.95 ppm.
HRMS m/z [M + H]+ calcd for C22H28FN4O2S: 431.1911, found: 431.1907.
4.1.2.2.
N-(1-((4-Chlorophenyl)sulfonyl)piperidin-4-yl)-1-(4-fluorobenzyl)-1H-
1
benzo[d]imidazol-2-amine (5f). This was obtained as a white solid (40 mg, 16%). H NMR
(300 MHz, MeOD) δ = 7.83 – 7.75 (m, 2H), 7.69 – 7.61 (m, 2H), 7.30 – 7.23 (m, 1H), 7.15 –
7.07 (m, 2H), 7.07 – 6.89 (m, 5H), 5.22 (s, 2H), 3.80 – 3.66 (m, 3H), 2.56 (td, J = 12.0, 2.5
Hz, 2H), 2.17 – 2.06 (m, 2H), 1.72 – 1.55 (m, 3H) ppm. 13C NMR (75 MHz, MeOD) δ =
161.85 (d, J = 244.5 Hz), 153.37, 141.04, 138.70, 134.74, 133.54, 131.97 (d, J = 3.1 Hz),
128.86, 128.73, 127.81 (d, J = 8.2 Hz), 120.79, 119.21, 114.70 (d, J = 21.9 Hz), 114.35,
107.45, 48.93, 44.92, 43.52, 30.88 ppm. HRMS m/z [M + H]+ calcd for C25H25ClFN4O2S:
499.1365, found: 499.1355.
4.1.2.3.
N-(1-((2,5-Dimethoxyphenyl)sulfonyl)piperidin-4-yl)-1-(4-fluorobenzyl)-1H-
benzo[d]imidazol-2-amine (5g). This was obtained as a white solid (157 mg, 60%). 1H NMR

(300 MHz, CDCl3) δ = 7.48 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 3.1 Hz, 1H), 7.17 – 6.93 (m, 9H),
5.07 (s, 2H), 4.10 (br s, 1H), 4.06 – 3.95 (m, 1H), 3.90 – 3.78 (m, 8H), 2.81 (t, J = 11.1 Hz,
2H), 2.15 (d, J = 10.3 Hz, 2H), 1.60 – 1.43 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ =
162.41 (d, J = 247.4 Hz), 153.05, 150.99, 142.05, 135.69, 134.46, 131.03 (d, J = 3.2 Hz),
128.23, 128.12, 126.85, 128.18 (d, J = 8.1 Hz), 119.95, 116.49, 116.13, 116.11 (d, J = 21.7
Hz), 113.83, 107.34, 56.60, 56.00, 49.80, 45.33, 45.00, 32.64 ppm. HRMS m/z [M + H]+
calcd for C27H30FN4O4S: 525.1966, found: 525.1962.
4.1.2.4. 1-(4-Fluorobenzyl)-N-(1-tosylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine (5h).
This was obtained as a white solid (130 mg, 52%). 1H NMR (300 MHz, CDCl3) δ = 7.57 (d, J
= 8.2 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.14 – 6.90 (m, 7H), 5.05 (s,
2H), 4.34 (d, J = 8.2 Hz, 1H), 3.95 – 3.80 (m, 1H), 3.72 (d, J = 11.8 Hz, 2H), 2.52 – 2.34 (m,
5H), 2.16 (d, J = 10.4 Hz, 2H), 1.60 (qd, J = 12.3, 4.0 Hz, 2H) ppm. 13C NMR (75 MHz,
CDCl3) δ = 162.29 (d, J = 247.1 Hz), 153.12, 143.86, 142.17, 134.49, 132.32, 131.20 (d, J =
3.2 Hz), 129.66, 128.15 (d, J = 8.2 Hz), 127.58, 121.51, 119.79, 116.40, 115.91 (d, J = 21.7
Hz), 107.35, 77.42, 77.00, 76.58, 49.44, 45.64, 44.86, 31.79, 21.50 ppm. HRMS m/z [M + H]+
calcd for C25H25ClFN4O2S: 499.1365, found: 499.1355.
4.1.3. Synthesis of carboxamide-substituted 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine analogues
General procedure
To a solution of 4 (243 mg, 0.5 mmol) in dry pyridine (2 mL) was added an appropriate
isocyanate (0.5 mmol). The reaction mixture was stirred under a nitrogen atmosphere
overnight. Pyridine was removed under reduced pressure. Toluene (2-3 mL) was added and
removed in vacuo. This was repeated three times. Finally, the crude residue was purified by
silica gel flash chromatography yielding the title compounds. The following compounds were
made according to this procedure.
4.1.3.1.
N-(tert-Butyl)-4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)amino)piperidine-1-carboxamide (5i). This was obtained as a white solid (140 mg, 66%).
1H NMR (300 MHz, MeOD) δ = 7.46 – 7.40 (m, 1H), 7.28 – 7.11 (m, 5H), 7.10 – 7.00 (m,
2H), 5.39 (s, 2H), 4.06 (d, J = 13.6 Hz, 2H), 4.00 – 3.85 (m, 1H), 2.92 (t, J = 12.0 Hz, 2H),
2.07 (d, J = 10.3 Hz, 2H), 1.72 – 1.52 (m, 2H), 1.33 (s, 9H) ppm. 13C NMR (75 MHz, MeOD)
δ = 163.73 (d, J = 245.5 Hz), 159.36, 151.86, 134.48, 133.02, 132.12 (d, J = 3.2 Hz), 129.84
(d, J = 8.3 Hz), 124.41, 123.65, 116.69 (d, J = 22.0 Hz), 113.85, 110.79, 52.95, 51.68, 46.01,

44.25, 32.78, 29.73 ppm. HRMS m/z [M + H]+ calcd for C24H31FN5O: 424.2507, found:
424.2503.
4.1.3.2. 4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)-N-phenylpiperidine-1-
1
carboxamide (5k). This was obtained as a white solid (122 mg, 55%). H NMR (300 MHz,
DMSO) δ = 8.92 (d, J = 7.9 Hz, 1H), 8.63 (s, 1H), 7.54 – 7.42 (m, 4H), 7.38 (dd, J = 8.7, 5.4
Hz, 2H), 7.33 – 7.16 (m, 6H), 6.93 (t, J = 7.3 Hz, 1H), 5.51 (s, 2H), 4.24 (d, J = 13.6 Hz, 2H),
4.05 – 3.89 (m, 1H), 2.93 (t, J = 12.1 Hz, 2H), 2.08 (d, J = 12.9 Hz, 2H), 1.73 – 1.50 (m, 2H)
ppm. 13C NMR (75 MHz, DMSO) δ = 161.89 (d, J = 244.2 Hz), 155.12, 148.92, 140.69,
131.03 (d, J = 3.1 Hz), 130.51, 129.52 (d, J = 8.3 Hz), 129.22, 128.42, 123.95, 123.52, 121.88,
119.78, 115.83 (d, J = 21.6 Hz), 111.79, 110.71, 51.70, 44.86, 43.18, 31.34 ppm. HRMS m/z
[M + H]+ calcd for C26H27FN5O: 444.2194, found: 444.2196.
4.1.3.3.
N-(2,4-Dimethoxyphenyl)-4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)amino)piperidine-1-carboxamide (5l). This was obtained as a white solid (121 mg, 48%).
1H NMR (300 MHz, MeOD) δ = 7.39 – 7.30 (m, 2H), 7.19 – 7.10 (m, 2H), 7.09 – 6.90 (m,
5H), 6.51 (d, J = 2.6 Hz, 1H), 6.41 (dd, J = 8.7, 2.6 Hz, 1H), 5.24 (s, 2H), 4.12 (d, J = 13.4 Hz,
2H), 3.99 (br s, 1H), 3.77 (s, 3H), 3.70 (s, 3H), 3.05 (t, J = 11.8 Hz, 2H), 2.08 (d, J = 10.1 Hz,
2H), 1.69 – 1.52 (m, 2H) ppm. 13C NMR (75 MHz, MeOD) δ = 163.36 (d, J = 244.5 Hz),
158.95, 158.14, 154.75, 154.30, 141.99, 135.01, 133.54 (d, J = 3.0 Hz), 129.76 (d, J = 8.2 Hz),
126.41, 122.54, 122.08, 121.04, 116.29 (d, J = 21.8 Hz), 115.76, 109.30, 105.18, 99.66, 56.32,
55.98, 51.60, 45.40, 44.41, 32.99 ppm. HRMS m/z [M + H]+ calcd for C28H31FN5O3:
504.2405, found: 504.2409.
4.1.3.4.
N-(2,3-Dihydro-1H-inden-5-yl)-4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)amino)piperidine-1-carboxamide (5m). This was obtained as a white solid (147 mg,
61%). 1H NMR (300 MHz, MeOD) δ = 7.33 (d, J = 7.6 Hz, 1H), 7.24 – 7.13 (m, 3H), 7.11 –
6.93 (m, 7H), 5.29 (s, 2H), 4.18 (d, J = 13.2 Hz, 2H), 4.00 (br s, J = 10.9 Hz, 1H), 3.06 (t, J =
12.0 Hz, 2H), 2.81 (q, J = 7.7 Hz, 4H), 2.18 – 1.92 (m, 4H), 1.70 – 1.53 (m, 2H) ppm. 13C
NMR (75 MHz, MeOD) δ = 163.49 (d, J = 244.7 Hz), 158.13, 154.81, 145.52, 141.93, 140.06,
138.83, 135.05, 133.57 (d, J = 3.2 Hz), 129.75 (d, J = 8.2 Hz), 124.92, 122.60, 121.11, 120.94,
119.01, 116.35 (d, J = 21.8 Hz), 115.74, 109.33, 51.74, 45.46, 44.50, 33.82, 33.17, 33.09,
26.67 ppm. HRMS m/z [M + H]+ calcd for C29H31FN5O: 484.2507, found: 484.2509.
4.1.3.5.
N-(2,4-Difluorophenyl)-4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-
yl)amino)piperidine-1-carboxamide (5n). This was obtained as a white solid (129 mg, 54%).
1H NMR (300 MHz, MeOD) δ = 7.53 – 7.47 (m, 1H), 7.45 – 7.35 (m, 1H), 7.35 – 7.22 (m,
5H), 7.15 – 7.07 (m, 2H), 7.02 – 6.86 (m, 2H), 5.46 (s, 2H), 4.28 (d, J = 13.7 Hz, 2H), 4.09 –

3.90 (m, 2H), 3.14 (t, J = 12.1 Hz, 2H), 2.17 (d, J = 10.1 Hz, 2H), 1.74 (qd, J = 12.5, 3.9 Hz,
13
2H) ppm. C NMR (75 MHz, MeOD) δ = 163.88 (d, J = 245.7 Hz), 163.05 (d, J = 11.5 Hz),
159.73 (dd, J = 11.2 Hz), 157.87, 156.36 (d, J = 12.4 Hz), 151.00, 132.45, 132.23, 131.71 (d,
J = 3.2 Hz), 129.92 (d, J = 8.3 Hz), 129.31 (dd, J = 9.6, 2.6 Hz), 125.03, 124.51, 116.83 (d, J
= 22.0 Hz), 113.33, 111.80 (dd, J = 22.3, 3.7 Hz), 111.31, 104.86 (dd, J = 26.5, 24.8 Hz),
53.11, 46.24, 44.44, 32.70 ppm. HRMS m/z [M + H]+ calcd for C26H25F3N5O: 480.2006,
found: 480.1985.
4.1.3.6. N-Benzyl-4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)piperidine-1-
carboxamide (5o). This was obtained as white solid (169 mg, 74%). 1H NMR (300 MHz,
MeOD) δ = 7.53 – 7.45 (m, 1H), 7.37 – 7.22 (m, 9H), 7.20 – 7.05 (m, 3H), 5.46 (s, 2H), 4.35
(s, 2H), 4.18 (d, J = 13.7 Hz, 2H), 3.02 (t, J = 12.0 Hz, 2H), 2.10 (d, J = 10.2 Hz, 2H), 1.66
(qd, J = 12.4, 3.9 Hz, 2H) ppm. 13C NMR (75 MHz, MeOD) δ = 163.90 (d, J = 245.9 Hz),
159.77, 150.22, 141.52, 131.93, 131.37 (d, J = 3.1 Hz), 130.35, 129.97 (d, J = 8.3 Hz), 129.32,
128.15, 127.79, 125.43, 125.10, 116.87 (d, J = 22.0 Hz), 112.83, 111.66, 53.49, 46.38, 45.24,
44.07, 32.62 ppm. HRMS m/z [M + H]+ calcd for C27H29FN5O: 458.2350, found: 458.2354.
4.1.3.7. 4-((1-(4-Fluorobenzyl)-1H-benzo[d]imidazol-2-yl)amino)-N-phenethylpiperidine-
1-carboxamide (5p). This was obtained as a white solid (193 mg, 82%). 1H NMR (300 MHz,
MeOD) δ = 7.48 (d, J = 7.3 Hz, 1H), 7.32 – 7.13 (m, 10H), 7.10 – 7.01 (m, 2H), 5.45 (s, 2H),
4.12 (d, J = 13.3 Hz, 2H), 4.02 – 3.88 (m, 1H), 3.42 – 3.28 (m, 2H), 2.98 (t, J = 12.1 Hz, 2H),
2.85 – 2.68 (m, 2H), 2.08 (d, J = 11.5 Hz, 2H), 1.73 – 1.54 (m, 2H) ppm. 13C NMR (75 MHz,
MeOD) δ = 163.77 (d, J = 245.7 Hz), 159.68, 150.80, 140.83, 132.32, 132.01, 131.65 (d, J =
3.2 Hz), 129.92 (d, J = 8.3 Hz), 129.79, 129.35, 127.12, 124.97, 124.46, 116.77 (d, J = 22.0
Hz), 113.24, 111.29, 53.23, 46.24, 43.95, 43.55, 37.45, 32.62 ppm. HRMS m/z [M + H]+
calcd for C28H31FN5O: 472.2507, found: 472.2519.
4.1.5. Synthesis of alkyl and alkylaryl substituted 1-(4-fluorobenzyl)-N-(piperidin-4-yl)-
1H-benzo[d]imidazol-2-amine analogues
General procedure
To a solution of 4 (121 mg, 0.25 mmol) and an appropriate aldehyde (0.30 mmol) in EtOH (5
mL), was added diisopropylethylamine (83 µL, 0.5 mmol) and acetic acid (14 µL, 0.75 mmol).
o
The mixture was stirred at 40 C for 1 hour. Then, sodium cyanoborohydride (19 mg, 0.3
mmol) was added and the mixture was stirred at 40 oC overnight. The solvents were removed
under reduced pressure. The residue was diluted with a saturated NaHCO3 solution and
extracted with EtOAc. The combined organic layers were dried over Na2SO4 and the solvent

was removed under reduced pressure. The crude residue was purified by silica gel flash
chromatography furnishing the title compounds. The following compounds were made
according to this procedure.
4.1.5.1.
1-(4-Fluorobenzyl)-N-(1-methylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(5q). This was obtained as a white solid (59 mg, 70%). 1H NMR (300 MHz, CDCl3) δ = 7.51
(dt, J = 7.8, 0.8 Hz, 1H), 7.17 – 7.07 (m, 3H), 7.07 – 6.96 (m, 4H), 5.04 (s, 2H), 3.99 – 3.81
(m, 2H), 2.65 (d, J = 10.3 Hz, 2H), 2.24 (s, 3H), 2.21 – 2.01 (m, 4H), 1.50 – 1.33 (m, 2H)
ppm. 13C NMR (75 MHz, CDCl3) δ = 162.55 (d, J = 247.5 Hz), 153.40, 142.44, 134.59,
131.22 (d, J = 3.3 Hz), 128.34 (d, J = 8.2 Hz), 121.63, 119.86, 116.65, 116.27 (d, J = 21.7 Hz),
107.21, 54.28, 49.31, 46.24, 45.11, 32.73 ppm. HRMS m/z [M + H]+ calcd for C20H24FN4:
339.1979, found: 339.1980.
4.1.5.2.
1-(4-Fluorobenzyl)-N-(1-isobutylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(5r) was obtained as a white solid (64 mg, 73%). 1H NMR (300 MHz, CDCl3) δ = 7.52 (d, J =
7.8 Hz, 1H), 7.17 – 7.09 (m, 3H), 7.08 – 6.98 (m, 4H), 5.04 (s, 2H), 4.02 – 3.85 (m, 1H), 3.78
(d, J = 8.0 Hz, 1H), 2.64 (d, J = 11.1 Hz, 2H), 2.19 – 1.98 (m, 6H), 1.82 – 1.64 (m, 1H), 1.50
– 1.33 (m, 2H), 0.87 (d, J = 6.6 Hz, 6H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.62 (d, J =
247.3 Hz), 153.50, 142.59, 134.69, 131.30 (d, J = 3.2 Hz), 128.36 (d, J = 8.2 Hz), 121.65,
119.84, 116.75, 116.35 (d, J = 21.7 Hz), 107.16, 67.07, 52.55, 49.94, 45.18, 32.84, 25.83,
21.09 ppm. HRMS m/z [M + H]+ calcd for C23H30FN4: 381.2449, found: 381.2440.
4.1.5.3.
N-(1-(Cyclopropylmethyl)piperidin-4-yl)-1-(4-fluorobenzyl)-1H-
1
benzo[d]imidazol-2-amine (5s). This was obtained as a white solid (61 mg, 65%). H NMR
(300 MHz, CDCl3) δ = 7.49 (d, J = 7.8 Hz, 1H), 7.17 – 7.07 (m, 3H), 7.07 – 6.95 (m, 4H),
5.06 (s, 2H), 4.27 (br s, 1H), 3.97 (br s, 1H), 2.95 (d, J = 10.2 Hz, 2H), 2.33 – 2.18 (m, 4H),
2.11 (d, J = 12.9 Hz, 2H), 1.65 – 1.47 (m, 2H), 0.91 – 0.78 (m, 1H), 0.58 – 0.47 (m, 2H), 0.10
(q, J = 4.7 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.45 (d, J = 247.2 Hz), 153.43,
142.37, 134.55, 131.29 (d, J = 3.2 Hz), 128.32 (d, J = 8.2 Hz), 121.57, 119.80, 116.50, 116.15
(d, J = 21.7 Hz), 107.25, 63.29, 51.94, 49.55, 45.03, 32.04, 8.01, 4.08 ppm. HRMS m/z [M +
H]+ calcd for C23H28FN4: 379.2292, found: 379.2282.
4.1.5.4. N-(1-(Cyclohexylmethyl)piperidin-4-yl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazol-
1
2-amine (5t). This was obtained as a white solid (69 mg, 66%). H NMR (300 MHz, CDCl3)
δ = 7.51 (dt, J = 7.8, 0.9 Hz, 1H), 7.17 – 7.05 (m, 3H), 7.06 – 6.95 (m, 4H), 5.01 (s, 2H), 4.01
– 3.80 (m, 2H), 2.62 (d, J = 11.3 Hz, 2H), 2.18 – 1.94 (m, 6H), 1.84 – 1.57 (m, 5H), 1.52 –
1.31 (m, 3H), 1.32 – 1.07 (m, 3H), 0.97 – 0.71 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ =
162.50 (d, J = 247.3 Hz), 153.46, 142.53, 134.61, 131.25 (d, J = 3.2 Hz), 128.27 (d, J = 8.2

Hz), 121.55, 119.74, 116.62, 116.23 (d, J = 21.8 Hz), 107.11, 65.79, 52.64, 49.89, 45.05,
35.42, 32.75, 32.03, 26.86, 26.21 ppm. HRMS m/z [M + H]+ calcd for C26H34FN4: 421.2762,
found: 421.2754.
4.1.5.5. 1-(4-Fluorobenzyl)-N-(1-phenethylpiperidin-4-yl)-1H-benzo[d]imidazol-2-amine
(5u). This was obtained as a white solid (74 mg, 30%). 1H NMR (300 MHz, CDCl3) δ = 7.50
(d, J = 7.8 Hz, 1H), 7.26 (dd, 2H), 7.22 – 7.14 (m, 3H), 7.14 – 7.05 (m, 3H), 7.05 – 6.95 (m,
4H), 5.04 (s, 2H), 4.19 – 4.06 (m, 1H), 3.98 (br s, 1H), 2.88 – 2.72 (m, 4H), 2.63 – 2.52 (m,
2H), 2.24 (t, J = 10.4 Hz, 2H), 2.11 (d, J = 12.6 Hz, 2H), 1.57 – 1.38 (m, 2H) ppm. 13C NMR
(75 MHz, CDCl3) δ = 162.28 (d, J = 247.3 Hz), 153.22, 142.23, 139.91, 134.39, 131.09 (d, J
= 3.2 Hz), 128.52, 128.28, 128.19, 128.13 (d, J = 8.2 Hz), 121.40, 119.63, 116.37, 115.99(d, J
= 21.7 Hz), 107.05, 60.21, 51.95, 49.51, 44.86, 33.49, 32.31 ppm. HRMS m/z [M + H]+ calcd
for C27H30FN4: 429.2449, found: 429.2440.
4.1.5.6. N-(1-Benzylpiperidin-4-yl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-amine (5v).
This was obtained as a white solid (87 mg, 84%). 1H NMR (300 MHz, CDCl3) δ = 7.47 (dd, J
= 7.7 Hz, 1H), 7.35 – 7.24 (m, 5H), 7.16 – 6.95 (m, 7H), 5.07 (s, 2H), 4.04 – 3.90 (m, 1H),
3.53 (s, 2H), 2.77 (d, J = 11.4 Hz, 2H), 2.27 (t, J = 10.4 Hz, 2H), 2.12 – 2.01 (m, 2H), 1.54 (td,
J = 13.8, 3.6 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.51 (d, J = 247.2 Hz), 153.11,
141.54, 136.48, 134.35, 131.13 (d, J = 3.3 Hz), 129.53, 128.51, 128.40 (d, J = 8.2 Hz), 127.72,
121.82, 120.18, 116.26, 116.21 (d, J = 21.7 Hz), 107.52, 62.70, 51.83, 49.54, 45.13, 31.88
ppm. HRMS m/z [M + H]+ calcd for C26H28FN4: 415.2292, found: 415.2299.
4.1.5.7. 1-(4-Fluorobenzyl)-N-(1-(3-phenylpropyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-
amine (5w). This was obtained as a white solid (85 mg, 77%). 1H NMR (300 MHz, CDCl3) δ
= 7.51 (d, J = 7.8 Hz, 1H), 7.31 – 7.23 (m, 2H), 7.22 – 7.07 (m, 6H), 7.06 – 6.95 (m, 4H),
5.04 (s, 2H), 4.05 – 3.89 (m, 2H), 2.80 (d, J = 10.8 Hz, 2H), 2.61 (t, J = 7.7 Hz, 2H), 2.47 –
2.32 (m, 2H), 2.19 (t, J = 11.5 Hz, 2H), 2.09 (d, J = 13.6 Hz, 2H), 1.89 – 1.74 (m, 2H), 1.57 –
13
1.39 (m, 2H) ppm. C NMR (75 MHz, CDCl3) δ = 162.56 (d, J = 247.4 Hz), 153.39, 142.41,
141.94, 134.61, 131.26 (d, J = 3.2 Hz), 128.46, 128.35 (d, J = 8.2 Hz), 125.96, 121.67, 119.90,
116.66, 116.28 (d, J = 21.7 Hz), 107.25, 57.89, 52.07, 49.65, 45.12, 33.77, 32.34, 28.48 ppm.
HRMS m/z [M + H]+ calcd for C28H32FN4: 443.2605, found: 443.2604.
4.1.5.8. N-(1-Cinnamylpiperidin-4-yl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-amine
(5x). This was obtained as white solid (92 mg, 84%). 1H NMR (300 MHz, CDCl3) δ = 7.49 (d,
J = 7.8 Hz, 1H), 7.40 – 7.18 (m, 5H), 7.17 – 7.05 (m, 3H), 7.05 – 6.93 (m, 4H), 6.50 (d, J =
15.8 Hz, 1H), 6.29 – 6.15 (m, 1H), 5.04 (s, 2H), 4.32 – 4.04 (m, 1H), 4.04 – 3.89 (m, 1H),
3.13 (d, J = 6.8 Hz, 2H), 2.89 – 2.73 (m, 2H), 2.21 (t, J = 11.2 Hz, 2H), 2.09 (d, J = 10.5 Hz,

2H), 1.57 – 1.39 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.46 (d, J = 247.4 Hz),
153.32, 142.21, 136.73, 134.50, 133.72, 131.21 (d, J = 3.2 Hz), 128.62, 128.32 (d, J = 8.2 Hz),
127.69, 126.40, 125.74, 121.62, 119.88, 116.48, 116.17 (d, J = 21.7 Hz), 107.28, 60.82, 52.01,
49.59, 45.03, 32.29 ppm. HRMS m/z [M + H]+ calcd for C28H30FN4: 441.2449, found:
441.2439.
4.1.6.
Synthesis
of
1-substituted-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-
benzo[d]imidazol-2-amine
General procedure
A mixture of 9 (140 mg, 0.4 mmol), an appropriate alkyl bromide (0.4 mmol), anhydrous
K2CO3 (56 mg, 0.4 mmol) in DMF (2 mL) was heated at 70 oC overnight. The cooled reaction
mixture was partitioned between water and EtOAc. The aqueous phase was further extracted
with EtOAc. The combined organic phases were washed with water and brine and dried over
anhydrous Na2SO4. The solvents were removed under reduced pressure. The crude residue
was purified by silica gel flash column chromatography affording the title compounds. Some
of these analogues were further purified by HPLC. The following compounds were made
according to this procedure.
4.1.6.1.
1-Benzyl-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-
amine (10a). This was obtained as a white solid (62 mg, 35%). 1H NMR (300 MHz, CDCl3) δ
= 7.51 (d, J = 7.7 Hz, 1H), 7.35 – 7.28 (m, 3H), 7.18 – 7.03 (m, 7H), 6.87 – 6.79 (m, 2H),
5.13 (s, 2H), 4.54 – 4.18 (m, 1H), 4.14 – 3.97 (m, 1H), 3.78 (s, 3H), 3.04 (d, J = 11.0 Hz, 2H),
2.88 – 2.78 (m, 2H), 2.77 – 2.67 (m, 2H), 2.46 (t, J = 10.5 Hz, 2H), 2.15 (d, J = 9.9 Hz, 2H),
1.77 – 1.59 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 158.25, 153.13, 141.62, 135.28,
134.48, 130.70, 129.59, 129.19, 128.21, 126.56, 121.59, 119.98, 116.28, 114.02, 107.42,
59.87, 55.25, 51.83, 48.92, 45.74, 31.62, 31.24 ppm. HRMS m/z [M + H]+ calcd for
C28H33N4O: 441.2649, found: 441.2638.
4.1.6.2.
1-(2,4-Difluorobenzyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine (10b). This was further purified by HPLC and obtained as a white
1
solid (52 mg, 22%). H NMR (300 MHz, CDCl3) δ = 9.06 (s, 1H), 7.56 (d, J = 7.2 Hz, 1H),
7.32 – 7.16 (m, 2H), 7.15 – 7.01 (m, 4H), 6.92 – 6.67 (m, 4H), 5.33 (s, 2H), 4.60 (br s, 1H),
3.79 (s, 3H), 3.59 (d, J = 12.1 Hz, 2H), 3.31 (br s, 2H), 3.17 (br s, 2H), 2.98 – 2.89 (m, 2H),
2.33 (s, 4H) ppm. 13C NMR (75 MHz, CDCl3) δ = 164.93 (d, J = 12.0 Hz), 161.92 (dd, J =
48.9, 12.4 Hz), 159.18, 149.14, 130.20, 129.99 (dd, J = 9.7, 5.1 Hz), 129.79, 129.71, 127.18,
124.79, 124.07, 118.45, 116.64 (dd, J = 14.8, 3.8 Hz), 114.69, 113.38, 112.19 (dd, J = 21.5,

3.4 Hz), 109.71, 104.76 (t, J = 25.3 Hz), 58.77, 55.44, 52.00, 49.02, 40.58, 29.81, 29.01 ppm.
HRMS m/z [M + H]+ calcd for C28H31F2N4O: 477.2460, found: 477.2459.
4.1.6.3.
1-(4-Bromobenzyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine (10c). This was further purified by HPLC and obtained as a white
solid 125 mg, 31%). 1H NMR (300 MHz, CDCl3) δ = 9.15 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 7.9
Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.31 – 7.14 (m, 1H), 7.11 – 6.95 (m, 5H), 6.85 (d, J = 8.6
Hz, 2H), 5.27 (s, 2H), 4.63 (br s, 1H), 3.79 (s, 3H), 3.54 (d, J = 10.2 Hz, 2H), 3.30 (br s, 2H),
3.17 – 3.07 (m, 2H), 2.98 – 2.88 (m, 2H), 2.29 (s, 4H) ppm. 13C NMR (75 MHz, CDCl3) δ =
159.18, 149.26, 132.50, 132.43, 130.50, 130.03, 129.73, 128.51, 127.22, 124.72, 124.00,
122.76, 114.72, 113.59, 109.68, 58.75, 55.47, 52.03, 48.87, 45.70, 29.80, 29.01 ppm. HRMS
m/z [M + H]+ calcd for C28H32BrN4O: 519.1754, found: 519.1747.
4.1.6.4.
N-(1-(4-Methoxyphenethyl)piperidin-4-yl)-1-propyl-1H-benzo[d]imidazol-2-
amine (10d). This was obtained as a white solid (85 mg, 54%). 1H NMR (300 MHz, CDCl3) δ
= 7.47 (d, J = 7.5 Hz, 1H), 7.17 – 7.01 (m, 5H), 6.87 – 6.80 (m, 2H), 5.49 (br s, 1H), 4.12 –
3.97 (m, 1H), 3.82 (t, J = 7.1 Hz, 2H), 3.78 (s, 3H), 3.04 (d, J = 11.8 Hz, 2H), 2.82 – 2.72 (m,
2H), 2.68 – 2.58 (m, 2H), 2.32 (t, J = 11.7 Hz, 2H), 2.22 (d, J = 12.4 Hz, 2H), 1.79 (dt, J =
14.6, 7.3 Hz, 2H), 1.71 – 1.59 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H) ppm. 13C NMR (75 MHz,
CDCl3) δ = 158.13, 153.21, 142.22, 134.48, 131.95, 129.69, 121.20, 119.53, 116.30, 113.99,
107.40, 60.43, 55.35, 52.34, 49.80, 43.83, 32.54, 32.48, 22.33, 11.61 ppm. HRMS m/z [M +
H]+ calcd for C24H33N4O: 393.2649, found: 393.2641.
4.1.6.5.
1-(Methoxymethyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine (10e). This was obtained as a slight yellow solid (121 mg, 77%).
1H NMR (300 MHz, CDCl3) δ = 7.48 (dd, J = 6.6, 2.1 Hz, 1H), 7.18 – 6.99 (m, 5H), 6.88 –
6.79 (m, 2H), 5.26 (d, J = 12.5 Hz, 2H), 4.06 – 3.90 (m, 1H), 3.77 (s, 3H), 3.32 (s, 3H), 2.99
(d, J = 11.8 Hz, 2H), 2.76 (dd, J = 10.4, 5.3 Hz, 2H), 2.65 – 2.56 (m, 2H), 2.39 – 2.14 (m, 4H),
1.76 – 1.55 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 158.05, 154.04, 142.46, 134.34,
132.13, 129.64, 121.79, 119.87, 116.58, 113.92, 106.85, 73.89, 60.55, 56.16, 55.30, 52.26,
49.53, 32.63, 32.40 ppm. HRMS m/z [M + H]+ calcd for C23H31N4O2: 395.2441, found:
395.2434.
4.1.6.6.
1-Hexyl-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-
amine (10f). This was obtained as a white solid (113 mg, 65%). 1H NMR (300 MHz, CDCl3)
δ = 7.52 – 7.44 (m, 1H), 7.17 – 7.00 (m, 5H), 6.88 – 6.78 (m, 2H), 4.22 – 3.94 (m, 2H), 3.92 –
3.72 (m, 5H), 3.00 (d, J = 11.6 Hz, 2H), 2.82 – 2.71 (m, 2H), 2.67 – 2.53 (m, 2H), 2.41 – 2.13
(m, 4H), 1.80 – 1.55 (m, 4H), 1.44 – 1.20 (m, 6H), 0.87 (t, J = 6.9 Hz, 3H) ppm. 13C NMR

(75 MHz, CDCl3) δ = 158.08, 153.14, 142.27, 134.40, 132.15, 129.66, 121.16, 119.50, 116.30,
113.94, 107.33, 60.59, 55.32, 52.36, 49.87, 42.28, 32.75, 32.69, 31.50, 28.95, 26.75, 22.56,
14.04 ppm. HRMS m/z [M + H]+ calcd for C27H39N4O: 435.3118, found: 435.3109.
4.1.6.7. N-(1-(4-Methoxyphenethyl)piperidin-4-yl)-1-octyl-1H-benzo[d]imidazol-2-amine
1
(10g). This was obtained as a white solid (133 mg, 72%). H NMR (300 MHz, MeOD) δ =
7.30 (dd, J = 6.8, 1.8 Hz, 1H), 7.15 – 6.94 (m, 5H), 6.87 – 6.78 (m, 2H), 3.97 (t, J = 7.1 Hz,
2H), 3.88 – 3.77 (m, 1H), 3.74 (s, 3H), 3.13 (d, J = 11.5 Hz, 2H), 2.83 – 2.72 (m, 2H), 2.71 –
2.57 (m, 2H), 2.37 (t, J = 11.1 Hz, 2H), 2.12 (d, J = 11.9 Hz, 2H), 1.80 – 1.58 (m, 4H), 1.26
(d, J = 11.2 Hz, 10H), 0.87 (t, J = 6.7 Hz, 3H) ppm. 13C NMR (75 MHz, MeOD) δ = 159.70,
154.98, 142.62, 135.33, 132.61, 130.60, 122.10, 120.65, 115.87, 114.99, 108.80, 61.37, 55.64,
53.61, 51.04, 42.68, 32.95, 32.85, 32.58, 30.39, 30.28, 29.68, 27.71, 23.64, 14.42 ppm.
HRMS m/z [M + H]+ calcd for C29H42N4O: 463.3431, found: 463.3429.
4.1.6.8. 1-Decyl-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-amine
1
(10h). This was obtained as a white solid (122 mg, 62%). H NMR (300 MHz, CDCl3) δ =
7.48 (dt, J = 7.7, 1.0 Hz, 0H), 7.16 – 7.00 (m, 5H), 6.87 – 6.79 (m, 2H), 4.12 – 3.94 (m, 2H),
3.83 (t, J = 7.2 Hz, 2H), 3.78 (s, 3H), 2.97 (d, J = 11.9 Hz, 2H), 2.82 – 2.71 (m, 2H), 2.65 –
2.54 (m, 2H), 2.36 – 2.17 (m, 4H), 1.78 – 1.53 (m, 4H), 1.40 – 1.20 (m, 15H), 0.88 (t, J = 6.7
Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3) δ = 158.07, 153.16, 142.39, 134.45, 132.34, 129.67,
121.16, 119.48, 116.39, 113.94, 107.32, 60.74, 55.34, 52.43, 49.93, 42.31, 32.96, 32.89, 31.92,
29.57, 29.53, 29.38, 29.33, 29.03, 27.13, 22.73, 14.18 ppm. HRMS m/z [M + H]+ calcd for
C31H47N4O: 491.3744, found: 491.3736.
4.1.6.9. 1-Isopentyl-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-
amine (10i). This was obtained as a white solid (79 mg, 47%). 1H NMR (300 MHz, MeOD) δ
= 7.28 (dd, J = 6.6, 2.2 Hz, 1H), 7.16 – 6.92 (m, 5H), 6.87 – 6.72 (m, 2H), 4.05 – 3.89 (m,
2H), 3.85 – 3.62 (m, 4H), 3.09 (d, J = 11.7 Hz, 2H), 2.82 – 2.65 (m, 2H), 2.65 – 2.47 (m, 2H),
2.28 (t, J = 12.0 Hz, 2H), 2.11 (d, J = 12.0 Hz, 2H), 1.77 – 1.44 (m, 5H), 0.97 (d, J = 6.2 Hz,
6H) ppm. 13C NMR (75 MHz, MeOD) δ = 159.67, 155.11, 142.95, 135.28, 133.20, 130.57,
122.05, 120.57, 115.98, 115.00, 108.68, 61.74, 55.69, 53.76, 51.34, 41.25, 38.33, 33.32, 32.92,
26.97, 22.95 ppm. HRMS m/z [M + H]+ calcd for C26H36N4O: 421.2962, found: 421.2963.
4.1.6.10.
1-(2-Ethylbutyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine (10j). This was obtained as a white solid (94 mg, 54%). 1H NMR
(300 MHz, MeOD) δ = 7.31 (dd, J = 6.9, 1.9 Hz, 1H), 7.18 – 6.94 (m, 5H), 6.89 – 6.76 (m,
2H), 3.96 – 3.79 (m, 3H), 3.77 (s, 3H), 3.17 (d, J = 11.9 Hz, 2H), 2.86 – 2.75 (m, 2H), 2.74 –
2.63 (m, 2H), 2.41 (t, J = 11.8 Hz, 2H), 2.14 (d, J = 11.9 Hz, 2H), 1.94 – 1.83 (m, 1H), 1.81 –

1.64 (m, 2H), 1.43 – 1.23 (m, 4H), 0.91 (t, J = 7.4 Hz, 6H) ppm. 13C NMR (75 MHz, MeOD)
δ = 159.74, 155.44, 142.62, 135.51, 132.59, 130.60, 122.13, 120.63, 115.95, 115.02, 109.21,
61.39, 55.66, 53.60, 51.07, 46.61, 41.17, 32.96, 32.55, 24.31, 11.15 ppm. HRMS m/z [M +
H]+ calcd for C27H38N4O: 435.3118, found: 435.3113.
4.1.6.11.
1-(Cyclohexylmethyl)-N-(1-(4-methoxyphenethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-amine (10k). This was obtained as a white solid (71 mg, 40%). 1H NMR
(300 MHz, CDCl3) δ = 7.47 (d, J = 7.6 Hz, 1H), 7.16 – 7.01 (m, 5H), 6.87 – 6.79 (m, 2H),
5.17 (br s, 1H), 4.14 – 3.98 (m, 1H), 3.78 (s, 3H), 3.66 (d, J = 7.1 Hz, 2H), 3.04 (d, J = 11.8
Hz, 2H), 2.82 – 2.74 (m, 2H), 2.69 – 2.60 (m, 2H), 2.33 (t, J = 11.7 Hz, 2H), 2.23 (d, J = 12.3
Hz, 2H), 1.85 – 1.78 (m, 1H), 1.71 (d, J = 10.6 Hz, 7H), 1.17 (d, J = 8.0 Hz, 2H), 1.09 – 0.97
(m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 158.14, 153.45, 142.17, 134.89, 131.94, 129.68,
121.17, 119.47, 116.30, 113.99, 107.72, 60.49, 55.35, 52.36, 49.74, 48.81, 38.09, 32.55, 31.27,
26.20, 25.75 ppm. HRMS m/z [M + H]+ calcd for C28H39N4O: 447.3118, found: 447.3114.
4.1.7. 1-(4-Fluorobenzyl)-N-(pyrrolidin-3-yl)-1H-benzo[d]imidazol-2-amine (18).
A
mixture of 2-chloro-1-(4-fluorobenzyl)-1H-benzo[d]imidazole (2) (261 mg, 1 mmol) and tert-
butyl 3-aminopyrrolidine-1-carboxylate (370 µL, 2 mmol) in NMP (2 mL) was heated at 180
oC under microwave for 60 minutes. After cooling down to room temperature, the mixture
was treated with a saturated NaHCO3 solution and extracted with EtOAc. The combined
organic phases was washed with water (2 times), dried over Na2SO4, and evaporated in vacuo.
The crude residue was purified by silica gel flash column chromatography to afford the title
compound 18 (168 mg, 54%). 1H NMR (300 MHz, CDCl3) δ = 7.53 (d, J = 7.8 Hz, 1H), 7.18
– 7.05 (m, 3H), 7.05 – 6.91 (m, 4H), 5.20 (s, 2H), 3.77 – 3.60 (m, 3H), 3.54 (ddd, J = 9.7, 7.9,
6.2 Hz, 1H), 3.26 (dd, J = 9.4, 4.1 Hz, 1H), 2.25 (br s, 2H), 2.18 – 2.06 (m, 1H), 1.79 – 1.65
(m, 1H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.11 (d, J = 246.1 Hz), 156.76, 142.16,
135.93, 132.52 (d, J = 3.1 Hz), 127.43 (d, J = 8.0 Hz), 121.98, 120.25, 116.76, 115.87 (d, J =
21.6 Hz), 108.28, 58.73, 51.21, 48.95, 47.08, 34.70 ppm. HRMS m/z [M + H]+ calcd for
C18H20FN4: 311.1666, found: 311.1662.
4.1.8. 2-(4-Methoxyphenyl)acetaldehyde. 2-(4-Methoxyphenyl)ethan-1-ol (300 µL, 2 mmol)
was dissolved in CH2Cl2 (30 mL). Dess-Matin periodinane (975 mg, 2.25 mmol) was added
and the resulting reaction mixture was refluxed for 2 hours. The solvents were removed under
reduced pressure, and subjected to flash column chromatography to give the product 2-(4-
methoxyphenyl)acetaldehyde (250 mg, 85%). 1H NMR (300 MHz, CDCl3) δ = 9.69 (t, J = 2.4

Hz, 1H), 7.13 – 7.09 (m, 2H), 6.92 – 6.87 (m, 2H), 3.78 (s, 3H), 3.60 (d, J = 2.4 Hz, 2H) ppm.
13C NMR (75 MHz, CDCl3) δ = 199.51, 158.82, 130.53, 123.61, 114.29, 55.09, 49.50 ppm.
4.1.9.
1-(4-Fluorobenzyl)-N-(1-(4-methoxyphenethyl)pyrrolidin-3-yl)-1H-
benzo[d]imidazol-2-amine (20). To a solution of 1-(4-fluorobenzyl)-N-(pyrrolidin-3-yl)-1H-
benzo[d]imidazol-2-amine (78 mg, 0.25 mmol) and 2-(4-methoxyphenyl)acetaldehyde (50 mg,
0.30 mmol) in EtOH (5 mL), was added diisopropylethylamine (83 µL, 0.50 mmol) and acetic
o
acid (14 µL, 0.75 mmol). The mixture was stirred at 40 C for 1 hour. Then, sodium
o
cyanoborohydride (19 mg, 0.30 mmol) was added and the mixture was stirred at 40 C
overnight. The solvents were removed under reduced pressure and the residue was diluted
with a saturated NaHCO3 solution, extracted with EtOAc and dried over Na2SO4. The solvent
was removed under reduced pressure. The crude residue was purified by silica gel flash
1
column chromatography to give the title compound as slight yellow solid (96 mg, 86%). H
NMR (300 MHz, MeOD) δ = 7.38 (d, J = 7.8 Hz, 1H), 7.16 – 6.96 (m, 9H), 6.86 – 6.78 (m,
2H), 5.33 (s, 2H), 3.72 (s, 3H), 3.70 – 3.59 (m, 2H), 3.58 – 3.50 (m, 1H), 3.48 – 3.34 (m, 2H),
2.87 – 2.66 (m, 4H), 2.24 – 2.10 (m, 1H), 1.88 – 1.74 (m, 1H) ppm. 13C NMR (75 MHz,
MeOD) δ = 163.54 (d, J = 244.7 Hz), 159.76, 157.35, 142.56, 136.96, 134.47 (d, J = 3.2 Hz),
132.44, 130.59, 128.89 (d, J = 8.2 Hz), 123.13, 121.66, 116.64 (d, J = 21.4 Hz), 115.02,
109.79, 58.43, 56.10, 55.65, 50.57, 49.83, 47.83, 35.59, 31.89 ppm. HRMS m/z [M + H]+
calcd for C27H30FN4O: 445.2398, found: 445.2403.
4.1.10. N-1-(4-Fluorobenzyl)benzene-1,2-diamine (12). o-Phenylenediamine (11) (2.7 g, 25
mmol) was dissolved in MeOH (15 mL). 1-(bromomethyl)-4-fluorobenzene (1.0 g, 5.3 mmol)
and K2CO3 (2.0 g, 14.5 mmol) were added. The mixture was allowed to stir at ambient
temperature overnight. The solvent was removed under reduced pressure. The crude residue
was purified by silica gel flash column chromatography to give the title compound as a
viscous liquid (0.7 g, 61%). 1H NMR (300 MHz, CDCl3) δ = 7.40 – 7.30 (m, 2H), 7.08 – 6.97
(m, 2H), 6.85 – 6.60 (m, 4H), 4.27 (s, 2H), 3.34 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3) δ =
162.21 (d, J = 245.1 Hz), 137.62, 135.22 (d, J = 2.9 Hz), 134.34, 129.41 (d, J = 8.0 Hz),
120.89, 119.16, 115.53 (d, J = 21.3 Hz), 115.39, 112.22, 48.06 ppm.
4.1.11.
2-(Chloromethyl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazole
(13).
N-1-(4-
fluorobenzyl)benzene-1,2-diamine (12) (0.7 g, 3.2 mmol) was dissolved in 5 N HCl (25 mL),
then the colour changed to light red. Chloroacetic acid (0.39 g, 4.1 mmol) was added and the

mixture was stirred at reflux for 4 hour. The reaction was cooled to room temperature,
basified by the addition of a saturated NaHCO3 solution and extracted with EtOAc. The
combined organic layers were washed with brine, dried over NaSO4, and concentrated in
vacuo. The crude residue was purified by silica gel flash column chromatography to give the
title product 13 as a white solid (0.66 g, 74%). 1H NMR (300 MHz, CDCl3) δ = 7.78 (dd, J =
6.1, 2.8 Hz, 1H), 7.33 – 7.15 (m, 3H), 7.11 – 7.04 (m, 2H), 6.97 (t, J = 8.6 Hz, 2H), 5.42 (s,
2H), 4.75 (s, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ = 162.40 (d, J = 247.2 Hz), 149.00,
142.10, 135.59, 131.14 (d, J = 3.3 Hz), 128.19 (d, J = 8.2 Hz), 123.98, 122.92, 120.38, 116.01
(d, J = 21.8 Hz), 110.11, 46.86, 36.92 ppm.
4.1.12. Ethyl 4-(4-methoxyphenethyl)piperazine-1-carboxylate (15). 1-(2-Chloroethyl)-4-
methoxybenzene (14) (1.13 mL, 7.5 mmol) and ethyl piperazine-1-carboxylate (0.36 mL, 2.5
mmol) were added to a mixture of CH3CN-H2O (25 mL: 15 mL). Then Na2CO3 (1.33 g, 12.5
o
mmol) was added and the resulting reaction mixture was refluxed at 70 C for 15 hours. The
solvents were removed under reduced pressure. An aqueous NaOH solution was added to
adjust the pH to 14. The aqueous phase was extracted with CH2Cl2, and the combined organic
phase was dried over Na2SO4, concentrated, and subjected for flash column chromatography
to afford the product (15) (0.38 g, 52%) as colorless oil. 1H NMR (300 MHz, CDCl3) δ = 7.16
– 7.06 (m, 2H), 6.90 – 6.77 (m, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 3.57 – 3.43 (m,
4H), 2.78 – 2.68 (m, 2H), 2.64 – 2.53 (m, 2H), 2.53 – 2.42 (m, 4H), 1.26 (t, J = 7.1 Hz, 3H)
ppm. 13C NMR (75 MHz, CDCl3) δ = 158.05, 155.54, 132.14, 129.61, 113.90, 61.35, 60.72,
55.27, 52.95, 43.70, 32.62, 14.72 ppm.
4.1.13. 1-(4-Methoxyphenethyl)piperazine (16). Ethyl 4-(4-methoxyphenethyl)piperazine-1-
carboxylate (15) (585 mg, 2 mmol) was added to a solution of NaOH (1 g) in H2O (5 mL) and
EtOH (5 mL). The resulting reaction mixture was refluxed for 12 hours. Then, the mixture
was cooled down to room temperature and extracted with EtOAc. The combined organic
layers were dried over Na2SO4, evaporated in vacuo, affording the title compound 16. The
crude residue was used as such without further purification.
4.1.14.
1-(4-Fluorobenzyl)-2-((4-(4-methoxyphenethyl)piperazin-1-yl)methyl)-1H-
benzo[d]imidazole (17). Crude ethyl 4-(4-methoxyphenethyl)piperazine-1-carboxylate 16
(220 mg, 1 mmol) and 2-(chloromethyl)-1-(4-fluorobenzyl)-1H-benzo[d]imidazole 13 (275
mg, 1 mmol) were dissolved in dry CH3CN (4 mL) and Et3N (1 mL) was added. The mixture
was stirred for 18 h at room temperature. Then, an aqueous 1 N NaOH (10 mL) solution was