Bioisosteric ferrocenyl aminoquinoline-benzimidazole hybrids: Antimicrobial evaluation and mechanistic insights

Article abstract of DOI:10.1016/j.ejmech.2019.06.069

Phenyl- and bioisosteric ferrocenyl-derived aminoquinoline-benzimidazole hybrid compounds were synthesised and evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive NF54 and multi-drug resistant K1 strains of the human mal

Full text of DOI:10.1016/j.ejmech.2019.06.069

Accepted Manuscript
Bioisosteric ferrocenyl aminoquinoline-benzimidazole hybrids: Antimicrobial
evaluation and mechanistic insights
N. Baartzes, T. Stringer, R. Seldon, D.F. Warner, D. Taylor, S. Wittlin, K. Chibale,
G.S. Smith
PII:
S0223-5234(19)30596-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.069
EJMECH 11472
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 April 2019
Revised Date: 8 June 2019
Accepted Date: 25 June 2019
Please cite this article as: N. Baartzes, T. Stringer, R. Seldon, D.F. Warner, D. Taylor, S. Wittlin, K.
Chibale, G.S. Smith, Bioisosteric ferrocenyl aminoquinoline-benzimidazole hybrids: Antimicrobial
evaluation and mechanistic insights, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.069.
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Bioisosteric ferrocenyl aminoquinoline-benzimidazole hybrids:
antimicrobial evaluation and mechanistic insights
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N. Baartzes,a T. Stringer,a R. Seldon,b D. F. Warner,c,d D. Taylore , S. Wittlin,f,g
K. Chibalea,h and G. S. Smitha,*
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a Department of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
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Drug Discovery and Development Centre (H3D), Institute of Infectious Disease and Molecular
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Medicine, University of Cape Town, Rondebosch 7701, Cape Town, South Africa.
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SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for
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Biomedical TB Research, Department of Clinical Laboratory Sciences, University of Cape Town,
Rondebosch 7701, Cape Town, South Africa.
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d Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town, Rondebosch
7701, South Africa.
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H3D, Division of Clinical Pharmacology, Department of Medicine, University of Cape Town,
Observatory 7925, Cape Town, South Africa.
f Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland.
g University of Basel, 4003 Basel, Switzerland.
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South African Medical Research Council, Drug Discovery and Development Research Unit,
Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of
Cape Town, Rondebosch 7701, South Africa.
* Corresponding author. Tel.: +27-21-650 5279
E-mail address: gregory.smith@uct.ac.za
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Keywords:
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Bioorganometallic chemistry
Aminoquinolines
Benzimidazoles
Hybrids
Ferrocene
Antiplasmodial
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Abstract
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Phenyl- and bioisosteric ferrocenyl-derived aminoquinoline-benzimidazole hybrid compounds were
synthesised and evaluated for their in vitro antiplasmodial activity against the chloroquine-sensitive
NF54 and multi-drug resistant K1 strains of the human malaria parasite, Plasmodium falciparum. All
compounds were active against the two strains, generally showing enhanced activity in the K1 strain,
with resistance indices less than 1. Cytotoxicity studies using Chinese hamster ovarian cells revealed
that the hybrids were relatively non-cytotoxic and demonstrated selective killing of the parasite.
Based on favourable in vitro antiplasmodial and cytotoxicity data, the most active phenyl (4c) and
ferrocenyl (5b) hybrids were tested in vivo against the rodent Plasmodium berghei mouse model. Both
compounds caused a reduction in parasitemia relative to the control, with 5c displaying superior
activity (92 % reduction in parasitemia at 4×50 mg/kg oral doses). The most active phenyl and
ferrocenyl derivatives showed inhibition of β-haematin formation in a NP-40 detergent-mediated
assay, indicating a possible contributing mechanism of antiplasmodial action. The most active
ferrocenyl hybrid did not display appreciable reactive oxygen species (ROS) generation in a ROS-
induced DNA cleavage gel electrophoresis study. The compounds were also screened for their in vitro
activity against Mycobacterium tuberculosis. The hybrids containing a more hydrophobic substituent
had enhanced activity (< 32.7 µM) compared to those with a less hydrophobic substituent (> 62.5
µM).
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1. Introduction
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Infectious microbial diseases such as malaria and tuberculosis are a major global health burden,
particularly affecting the developing world. The World Health Organisation (WHO) estimates that 2
million deaths were caused by these diseases worldwide in 2017 [1, 2]. Malaria is a parasitic disease
caused by protists in the Plasmodium genus and current treatment utilises artemisinin-based
combination therapy (ACT) [3]. ACT involves the use of artemisinin or a derivative together with
another antimalarial of known efficacy and a different mechanism of action [4]. On the other hand,
tuberculosis (TB) is a bacterial disease caused by Mycobacterium tuberculosis, and is typically treated
using a six-month four-drug regimen, in drug-susceptible (DS-TB) cases [2]. The drugs involved are
isoniazid, rifampicin, pyrazinamide and ethambutol, all of which have different mechanisms of action,
and inhibit the growth of M. tuberculosis [2, 5]. However, resistance to current antimalarial and anti-
TB treatment has arisen, and thus the search for new therapeutic strategies to combat rising
antimicrobial resistance is required.
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An emerging strategy showing promise in tackling rising resistance is the use of hybrid therapeutic
agents, combining two or more pharmacophores into a single compound. The hybrid agent may have
multiple modes of action [6, 7] and enhanced uptake, resulting in decreased emergence of resistance
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and improved efficacy, compared to the individual parent drugs [8, 9]. There are numerous examples
of hybrids applied in malaria, incorporating almost all classes of antimalarials [9-12]. With regard to
ferrocenyl derivatisation, ferroquine, a ferrocenyl-aminoquinoline antimalarial currently in Phase IIb
combination studies, represents a promising endeavour [13]. Notably, Robert et al. reported on the
potent antiplasmodial activity of a series of trioxaferroquines, organometallic hybrid complexes
incorporating a 1,2,4-trioxane group covalently linked to ferroquine [14]. There thus remains great
scope for hybrid antimicrobial agents and their organometallic derivatives.
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Within the context of targeting resistant strains of parasites, work within our research group has
focussed on the preparation of metal complexes of heterocycles such as quinolines [15-18]. Many of
these examples exhibited potent inhibition of the P. falciparum parasite, particularly resistant strains
[15-18]. However, the possibility of cross-resistance, similar to that of chloroquine, remains a
problem [15-18]. In addition, organometallic ferrocenyl-benzothiazoles [19] and PGM-containing
benzimidazoles [20] have been explored, which also displayed promising antiplasmodial activity. This
study explores the combination of two heterocycles, a quinoline and benzimidazole ring, and the
effect on antimicrobial activity. Herein, we report the synthesis and characterisation of a series of new
aminoquinoline-benzimidazole hybrids and their ferrocenyl analogues. We evaluated their in vitro
antiplasmodial activity against sensitive and resistant strains of P. falciparum, selected compounds
were tested for in vivo activity against a rodent P. berghei mouse model of malaria, and explored their
possible mechanism of action with respect to the haemoglobin degradation pathway. We also report
their potential in vitro antimycobacterial activity.
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2. Results and discussion
2.1. Synthesis and characterisation
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The nitro-containing precursors 2a – 2e were synthesised via nucleophilic aromatic substitution
reactions of compound 1 with various substituted nitrobenzene derivatives in N,N-dimethylformamide
(Scheme 1). Compounds 2a – 2e were reacted with zinc powder, in the presence of ammonium
chloride and under anhydrous conditions, to reduce the aryl nitro group to the primary amines of 3a –
3e group. Condensation and subsequent cyclisation of the 1,2-diamines with either benzaldehyde or
ferrocenecarboxaldehyde, respectively, in the presence of a catalytic amount of trifluoroacetic acid
(TFA) and anhydrous magnesium sulphate, yielded the phenyl (4a – 4e) and ferrocenyl (5a – 5e)
hybrids. The overall synthetic route is shown in Scheme 1. In this study the 5-position of the
benzimidazole moiety was varied with different substituents chosen from the Craig Plot, which is
often used in rational drug design. Specific substituents (-CH3, -CF3, -SO2CH3 and -CN) were chosen
in order to probe the effect of the varying hydrophobic and electron withdrawing/donating properties
on the biological activity.
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Scheme 1 Synthesis of phenyl- and ferrocenyl-derived aminoquinoline benzimidazole hybrids.
Reagents and conditions: i) 1,3-diaminopropane / 140 °C / 17 h; ii) nitrobenzene derivative / DMF/ rt
/ 24 h; iii) Zn / NH4Cl / MeOH / rt / 24 h; iv) benzaldehyde / TFA / MgSO4 / EtOH / 80 °C / 24 h; v)
ferrocenecarboxaldehyde / TFA / MgSO4 / EtOH / 80 °C / 48 h.
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Successful synthesis of the nitro-containing precursors was confirmed in the H NMR spectra by the
presence of aromatic proton signals for the newly incorporated phenyl moiety, four signals for
compound 2a and three signals for compounds 2b – 2e. The absence of the two primary amine proton
signals of 1 and appearance of a triplet for the proton of the newly-formed secondary amine, provides
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further evidence. In addition, the IR spectra of compounds 2a – 2e display two ν(N-O) absorption
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bands around 1500 cm-1, confirming the presence of the nitro group. The H NMR spectra of 1,2-
diamines 3a – 3e remain largely the same as their nitro precursors, however, the aromatic proton
signals of the phenyl ring display slight upfield shifts as a result of the shielding effect of the electron-
donating nature of the new amine group (Ar-NH2). Successful reduction of the nitro group is shown
by the presence of broad peak in the range 4.5 – 5 ppm, corresponding to the new primary amine
group. This is also evident in the IR spectra of compounds 3a – 3e, as the absorption bands for the
nitro group are no longer present. Cyclisation of the 1,2-diamine group with the aldehyde to form
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benzimidazole moiety is evidenced in the H NMR spectra of compounds 4a – 4e and 5a – 5e. In
these spectra, the only amine proton signal (triplet) present corresponds to the secondary amine of the
quinoline moiety. The primary amine proton signals of the phenyl ring are no longer present,
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suggesting that this nitrogen now forms part of the heterocycle. In the H NMR spectra for
compounds 5a – 5e, the ferrocenyl entity reveals a singlet for the protons of the unsubstituted
cyclopentadienyl (Cp) ring and two triplets for the four protons of the substituted Cp ring.
Significantly, the IR spectra of these hybrids do not display two separate absorptions bands for the
two C=N moieties (quinoline and benzimidazole) in their structures. A ν(C=N) absorption band
corresponding to both moieties is present between 1609 and 1611 cm-1.
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2.2. Molecular structures of 4b and 5c
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Crystals of compounds 4b and 5c suitable for single-crystal X-ray diffraction were obtained and
analysed. Crystals of compound 4b were obtained by crystallisation in DMSO-d6, forming colourless
block crystals in a monoclinic crystal system with the P21/c space group. Crystals of compound 5c
were obtained by slow evaporation in dichloromethane, forming red block crystals in a triclinic crystal
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system with P1 space group. The molecular structures of 4b and 5c are shown in Figure 1 and Figure
2. Selected bond lengths and bond angles are reported in Table 1. The C(benzimidazole)-
C(phenyl/ferrocenyl) bonds of 4b and 5c are similar in length, as well as the C(benzimidazole)-
C(CH3/CF3) bonds. The C-F bonds of compound 5c vary in length (1.330, 1.314 and 1.341 Å), in the
range corresponding with lengths expected for this strong polar covalent bond. It is worth noting that
in both 4b and 5c, the benzimidazole and its 2-substituted ring (phenyl for 4b and substituted Cp for
5c) are not co-planar. The torsion angle between the two ring systems is 45.2° in 4b and a
significantly lower value of 17.5° in 5c.
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Figure 1 Molecular structures of compounds 4b at 50 % probability ellipsoids (CCDC 1907165).
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Figure 2 Molecular structures of compounds 5c at 50 % probability ellipsoids (CCDC 1907167).
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Table 1 Selected bond lengths and torsion angles
4b
5c
Bond lengths (Å)
C(13) – C(20)
1.471(2)
–
C(1) – C(6)
–
1.456(3)
–
C(16) – C(26)
1.508(3)
C(9) – C(13)
–
–
–
–
1.498(4)
1.330(3)
1.341(3)
1.314(4)
C(13) – F(1)
C(13) – F(2)
C(13) – F(3)
Torsion angles (°)
N(3) – C(13) – C(20) – C(25)
C(2) – C(1) – C(6) – N(2)
45.2(2)
–
–
17.5(4)
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2.3. In vitro antiplasmodial evaluation, in vivo antimalarial efficacy and cytotoxicity
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The phenyl- and ferrocenyl-derived aminoquinoline-benzimidazole hybrids 4a – 4e and 5a – 5e were
evaluated for their in vitro antiplasmodial activity using the Plasmodium lactate dehydrogenase
(pLDH) assay. All compounds were screened against the chloroquine-sensitive NF54 and multi-drug
resistant K1 strains of Plasmodium falciparum. Chloroquine and artesunate were used as control
drugs against these parasite strains. The IC50 values, resistance index and LogP values are listed in
Table 2.
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Table 2 In vitro antiplasmodial data and resistance indices of compounds 4a – 4e and 5a – 5e.
IC50 (µM) ± SE
Compound
R1
R2
H
aLogP
5.60
6.12
6.48
4.44
NF54
K1
bRI
0.0362
0.583
0.350
3.28
5.55 ± 0.0627
0.784 ± 0.0163
0.431 ± 0.0115
0.559 ± 0.0105
0.201 ± 0.00268
0.457 ± 0.0945
0.151 ± 0.00351
1.83 ± 0.0797
4a
4b
4c
4d
CH3
CF3
SO2CH3
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CN
5.46
0.975 ± 0.0174
2.88 ± 0.0479
0.329 ± 0.005
1.22 ± 0.0996
0.848 ± 0.0069
0.909 ± 0.0194
0.0102 ± 0.0023
0.0098 ± 0.0011
0.179 ± 0.00365
0.339 ± 0.0113
0.283 ± 0.00376
0.658 ± 0.0392
0.579 ± 0.00770
0.565 ± 0.00751
0.205 ± 0.00709
cND
0.183
0.118
0.860
0.538
0.683
0.622
20.2
4e
H
CH3
CF3
SO2CH3
CN
-
-
5a
-
5b
-
-
5c
5d
-
5e
Chloroquine
Artesunate
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-
3.93
3.10
-
-
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aLogP values calculated using MarvinSketch V17.3.13
bRI = IC50(K1) / IC50(NF54)
cND = not determined
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All of the phenyl- and ferrocenyl-derived hybrids displayed good antiplasmodial activity, with most
IC50 values in the submicromolar range. In some cases, introduction of the organometallic ferrocenyl
moiety resulted in enhanced antiplasmodial activity, however, this trend was not consistent across the
series and the different strains. This is contrary to what was expected. With regards to ferrocenyl
derivatisation, literature reports comparable in vitro activity of chloroquine and ferroquine in the CQS
strain, while in the CQR strain ferroquine proved twenty-fold more active than chloroquine [21]. In
the chloroquine-sensitive NF54 strain, incorporation of the substituent (R2) at the 6-position of the
benzimidazole significantly enhanced the activity. This derivatisation resulted in mostly
submicromolar IC50 values across the 2-phenyl and 2-ferrocenyl series, compared to the unsubstituted
analogues (R2 = H). As a result of their mostly submicromolar IC50 values, all hybrids were evaluated
against the multidrug-resistant K1 parasite strain. In general, most compounds were more active in the
resistant strain, with IC50 values comparable or lower than that of CQ (0.205 µM). Compounds 4c and
4e were more potent than CQ in the resistant strain with IC50 values of 0.151 µM and 0.179 µM,
respectively. All hybrids had resistance indices (RIs) less than 1, lower than that of CQ (20.2),
indicating that they are potentially not susceptible to cross-resistance, and suggesting potential greater
applicability against resistant strains of P. falciparum. The most active compound in the chloroquine-
sensitive strain was the 2-ferrocenyl-derived 5b, while in the resistant strain the 2-phenyl-derived 4c
was most active. Interestingly, both active analogues have a more hydrophobic substituent at the 6-
position of the benzimidazole. However, there was no correlation between lipophilicity, determined
using calculated LogP values, and the antiplasmodial activity (Table 2).
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The hybrids were also screened for cytotoxicity against a non-tumorigenic Chinese hamster ovarian
(CHO) cell line, using emetine as the control drug. Compounds 4a and 4c – 4e displayed low toxicity
against the CHO cells, while 4b and 5a – 5e displayed no cytotoxicity at the highest concentration
tested. Calculated selectivity index (SI) values, shown in Table 3, were greater than 7 and 100 for the
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sensitive and resistant parasite strains, respectively. This indicates that these compounds display
selectivity towards the Plasmodium parasites, and suggests that their activity may attributed to
mechanisms other than general cytotoxicity.
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Table 3 Cytotoxicity data and selectivity indices
Compound
IC50 (µM) CHO
42.01
> 50
aSI1
bSI2
7.57
209
4a
4b
-
-
16.16
40.93
38.87
> 50
37.5
107
4c
73.2
22.4
4d
39.9
217
4e
-
-
-
-
-
-
-
-
-
-
-
-
5a
> 50
5b
> 50
5c
> 50
5d
> 50
5e
Emetine
0.0162
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aSI1 = IC50(CHO) / IC50(NF54); bSI2 = IC50(CHO) / IC50(K1)
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The most active phenyl and ferrocenyl aminoquinoline-benzimidazole hybrids (4c and 5b,
respectively) were evaluated for the in vivo antimalarial efficacy in Plasmodium berghei-infected
mice (Table 4). The mice were dosed orally at 4 × 50 mg/kg and reduction in parasitemia relative to
the control determined. None of the dosed mice were cured at this concentration and thus lower
concentrations were not administered. Dosing with the ferrocenyl hybrid resulted in significantly
enhanced inhibition (92 %) of parasitemia compared to the phenyl hybrid (58 %). Despite this vast
difference in parasite inhibition, mice administered with either compound had the same mean survival
time of seven days. This may suggest that compound 4c has some immune modulating properties.
Conversely, it may also suggest that the pharmacokinetics profile of compound 5b is not optimal, with
solubility-limited adsorption a likely contributing factor.
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Table 4 In vitro and in vivo data for compounds 4c and 5b.
Compound
R1
R2
Pf(NF54) Pf(K1)
Oral dose
(mg/kg)
% reduction in
parasitemia (MSD)a
Cured/infected
IC50
IC50 (µM)
(µM)
CF3
0.431
0.329
0.151
0.283
4 x 50
4 x 50
58 (7)
92 (7)
0/3
0/3
4c
CH3
5b
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a MSD = mean survival days
2.4. β-haematin inhibition activity
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Chloroquine, a quinoline-containing antimalarial drug, is known to target the parasite-induced host
haemoglobin degradation pathway by preventing the parasite from converting the toxic haem to the
non-toxic haemozoin [22-24]. This results in the build-up of the toxic haem, which in turn kills the
parasite [22-24]. In order to elucidate whether these aminoquinoline-benzimidazole compounds also
inhibit haemozoin formation as their mechanism of antiplasmodial action, the most active phenyl- and
ferrocenyl-derived hybrid (4c and 5b) were evaluated for their ability to inhibit β-haematin (synthetic
haemozoin) formation. The inhibition properties of these aminoquinoline-benzimidazole hybrids were
investigated using the NP-40 detergent-mediated assay developed by Sandlin et al [25].
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The dose-response curves of 4c and 5b, obtained from the NP-40 detergent-mediated assay, are
shown in Fig. 2, with chloroquine (CQ) as a control. Both of the tested hybrids displayed potent β-
haematin inhibition activity, exhibiting IC50 values of about 16 µM, at least four times more potent
than CQ (74 µM) This result suggests that this series of hybrids may act by inhibition of haemozoin
formation. However, this result obtained in the cell-free assay would require validation in the parasite.
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3
2
1
0
CQ
4c
5b
0.0
0.5
1.0
1.5
2.0
2.5
log [compound]
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Figure 3 Dose-response curves of compounds 4c, 5c and chloroquine (CQ).
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2.5. Reactive oxygen species (ROS) studies
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The presence of a ferrocenyl group in hybrids 5a – 5e brings a potential additional mechanism of
antiplasmodial action into consideration. Ferroquine, the ferrocene-containing analogue of
antimalarial drug chloroquine, is proposed to have a multifaceted mode of action, including the ability
to generate reactive oxygen species (ROS) [26-29]. This is ascribed to the ability of the redox-active
Fe2+ centre to oxidise to Fe3+ (ferrocene to ferrocenium), via a Fenton-like process, generating the
ROS and ultimately causing parasite damage [26-29]. Thus, a preliminary study into whether these
hybrids are able to generate ROS was performed using the most active ferrocenyl hybrid 5b. These
experiments work on the principle that radicals are generated by reaction of the compound of interest
with hydrogen peroxide and ascorbic acid. Cleavage of one or both strands of circular plasmid DNA
(pUC57), which is used as the reporter molecule, is a result of and indicates the formation of ROS.
Results of the dose-dependent DNA-cleavage of pUC57 measured by agarose gel electrophoresis is
shown in Fig. 4 below.
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Figure 4 Agarose gel electrophoresis displaying DNA cleavage.
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The gel displayed a slight increase in the amount of linear DNA on the addition of the hybrid 5b. The
small degree of DNA cleavage may be due to solubility issues of the compound in the mixtures over a
period of time, as there was evidence of precipitation in wells at the 50 and 100 µM concentrations.
Comparing lane 6 versus lane 7, it can be noted that hydrogen peroxide was not completely necessary
for DNA cleavage, while ascorbic acid was required. This assay suggests that the generation of
reactive oxygen species is not a valid mechanism of activity for these hybrid compounds.
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2.6. In vitro antimycobacterial screening
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Within the context of repurposing known drugs and scaffolds [30], all hybrid compounds were also
screened for their in vitro antimycobacterial activity against the Mycobacterium tuberculosis H37Rv.
Rifampicin was used as a control, and the 7-day MIC90 values are summarised in Table 5.
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Table 5 Antimycobacterial activity of compounds 4a – 4e and 5a – 5e.
Compound
Visual MIC90
(µM) [media:
Alamar blue assay:
Visual MIC90
(µM) [media:
Alamar blue assay:
Calculated MIC90 (µM)
Calculated MIC90 (µM)
7H9 GLU ADC] [media: 7H9 GLU ADC] 7H9 GLU CAS]
[media: 7H9 GLU CAS]
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62.5
38.8
>62.5
89.1
62.5
31.2
15.6
50.6
32.7
15.8
4a
4b
4c
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>125
>125
62.5
>62.5
>62.5
60.1
125
62.5
31.2
15.6
15.6
125
>125
>125
31.3
4d
4e
5a
31.2
29.7
28.7
5b
62.5
64.2
10.8
5c
5d
>125
>125
0.018
>125
>125
0.0156
>125
>62.5
0.0217
31.2
0.018
5e
Rifampicin
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Compounds were screened in two different growth media, glucose-based Middlebrook 7H9-ADC
(albumin-dextrose complex) and 7H9-CAS (casitone). Selected hybrids were moderately active
against M. tuberculosis, generally exhibiting better activity in the 7H9-CAS medium. Considering
the calculated MIC90 values determined in 7H9-CAS media, compounds 4a – 4c and 5a – 5c showed
moderate antimycobacterial activity (10.8 – 50.6 µM), while 4d – 4e and 5d – 5e were inactive at the
tested concentration. The more active hybrids (b and c) have the more hydrophobic substituents (CH3
and CF3), while the inactive hybrids (d and e) have the less hydrophobic substituents (SO2CH3 and
CN). It is worth noting that there is a correlation between the MIC90 values of a – c and their
calculated LogP values (see Table 3), indicating that antimycobacterial activity increases with
increasing lipophilicity. Recent literature has shown a positive correlation between antimycobacterial
activity and lipophilicity [31, 32]. Some studies have also indicated that selected lipophilic drugs had
favourable activity against dormant and replicating M. tuberculosis compared to selected hydrophilic
drugs [33]. Additionally, for the active hybrids (4a – 4c and 5a – 5c), the introduction of the
organometallic ferrocenyl moiety (5a – 5c) resulted in an overall enhancement in the activity
compared to the phenyl-derived hybrids (4a – 4c). The MIC90 values of the hybrids were significantly
less active than the clinically-available drug, rifampicin (0.0217 µM).
269
3. Conclusions
270
271
272
273
274
275
276
277
278
279
A series of phenyl-derived aminoquinoline-benzimidazole hybrid compounds and their bioisosteric
ferrocenyl analogues were synthesised and evaluated against the chloroquine-sensitive NF54 and
multi-drug resistant K1 strains of Plasmodium falciparum, displaying good in vitro activity. Results of
the screening revealed enhanced activity in the resistant strain compared to the sensitive strain, with
resistance indices less than 1 in most cases. All hybrids proved to be non-toxic in the non-tumorigenic
Chinese hamster ovarian (CHO) cell line with selectivity indices indicating selectivity of the hybrids
towards the Plasmodium parasite. In light of their promising activity and favourable cytotoxicity,
phenyl-derived 4c and ferrocenyl-derived 5b were assessed for in vivo efficacy in Plasmodium
berghei-infected mice. Treatment with either compound saw a decrease in parasitemia, with 5b
demonstrating superior activity. With regards to a possible mechanism of action, selected derivatives
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showed inhibition of synthetic haemozoin formation, indicating that for this series of hybrids, the
haemoglobin degradation pathway is a possible target. A preliminary study of the reactive oxygen
species (ROS)-generating ability of ferrocenyl-derived 5b revealed very low ROS generation. This
suggests that ROS-induced parasite damage is likely not a contributing mode of action of the
ferrocenyl hybrids. In a screening against the H37Rv strain of Mycobacterium tuberculosis, the
hybrids with higher lipophilicity showed enhanced activity. Furthermore, incorporation of the
ferrocenyl moiety resulted in improved activity compared to the phenyl analogues. It can be
concluded that these hybrids show great promise as potential antimicrobial agents. Further work to
elucidate additional contributing mechanisms of action is ongoing.
289
290
291
4. Experimental
4.1. Materials and methods
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
Infrared (IR) absorptions were measured on Perkin-Elmer Spectrum 100 FT-IR Spectrometer using
Attenuated Total Reflectance (ATR). Nuclear Magnetic Resonance (NMR) Spectra were recorded on
13
a Varian Unity XR400 MHz (1H at 399.95 MHz, C at 100.58 MHz), Varian Mercury XR300 MHz
(1H at 300.08 MHz, 13C at 75.46 MHz) or a Bruker Biospin GmbH (1H at 400.22 MHz, 13C at 100.65
MHz) spectrometer at 30 °C. Chemical shifts are reported using tetramethylsilane (TMS) as the
internal standard. Mass spectrometry determinations were carried out using Electron Impact (EI) on a
JEOL GC Matell instrument or Electrospray Ionisation (ESI) on a Waters API Quattro Micro triple
quadrupole mass spectrometer with data recorded using both the positive. Melting points were
determined using a Büchi Melting Point Apparatus B-540. Purity was determined using analytical
Agilent HPLC 1260 equipped with an Agilent DAD 1260 UV/vis detector and a X Bridge C18
column (2.5 µM, 50 mm × 3 mm). The compounds were eluted using a mixture of solvent A (10 mM
NH4OAc/H2O) and solvent B (10 mM NH4OAc/MeOH) at a flow rate of 0.9 mL/min (4a – 4e) or
0.5 mL/min (5a – 5e). The gradient elution conditions for 4a – 4e were as follows: 15 % solvent B
between 0 and 0.5 min, 15–100 % solvent B between 0.5 and 1.2 min, 100 % solvent B between 1.2
and 4.5 min. The gradient elution conditions for 5a – 5e were as follows: 10 % solvent B between 0
and 2 min, 10–90 % solvent B between 2 and 8 min, 90 % solvent B between 8 and 9 min. All
compounds were confirmed to have >95 % purity. Single‒crystal X‒ray diffraction data were
collected on a Bruker KAPPA APEX II DUO diffractometer using graphite-monochromated Mo‒Kα
radiation (χ = 0.71073 Å). All gel electrophoresis experiments were carried out using hand-cast 1.25
% agarose gels dissolved in 1x TAE buffer (Sigma-Aldrich, 10x, diluted with Type 1 ultrapure water,
40 mM Tris-acetate and 1 mM EDTA, pH 8.3) in a Mini-Sub-Cell GT® Agarose Gel Electrophoresis
System (Bio-Rad). Compound 1 was prepared using a modified literature method [34].
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4.2. General procedure for synthesis of nitro-containing aminoquinoline precursors (2a – 2e).
316
317
318
319
320
321
322
323
324
A nitrobenzene derivative (1.5 eq.) was added to a stirring solution of 1 (1 eq.) in anhydrous DMF (5
ml), and the reaction mixture was stirred at room temperature under nitrogen for 24 hours. Thereafter,
1 M NaOH (50 ml) was added to basify the mixture, which was then extracted with DCM or EtOAc
(3 × 50 ml). The organic fractions were collected and washed with brine (3 × 50 ml). The organic
layer was collected, dried over anhydrous Na2SO4 and filtered by gravity. The solvent of the filtrate
was removed to afford the crude product as a bright yellow or orange solid. The crude product was
purified by column chromatography (silica gel) using DCM:MeOH (19:1). Subsequent trituration in
diethyl ether afforded the desired products 2a – 2e as bright yellow or orange powders, which were
collected by suction filtration.
325
4.2.1. N1-(7-chloroquinolin-4-yl)-N3-(2-nitrophenyl)-propane-1,3-diamine (2a).
326
327
328
329
330
331
332
333
334
335
1-Fluoro-2-nitrobenzene (1.02 ml, 9.67 mmol), 1 (1.52 g, 6.45 mmol). Product: Yellow powder.
1
Yield: 1.20 g, 52.2 %. Rf(DCM:MeOH, 19:1): 0.25. M.p. 181.9 – 183.6 °C. H NMR (400 MHz,
3
3
DMSO-d6): δ (ppm) = 8.39 (d, JHH = 5.5 Hz, 1H, ArH), 8.25 (d, JHH = 9.1 Hz, 1H, ArH), 8.20 (t,
3
4
3
3JHH = 5.7 Hz, 1H, NH), 8.06 (dd, JHH = 8.6, JHH = 1.6 Hz, 1H, ArH), 7.79 (d, JHH = 2.3 Hz, 1H,
3
3
ArH), 7.50 (m, 1H, ArH), 7.45 (dd, JHH = 9.1, 4JHH = 2.3 Hz, 1H, ArH), 7.35 (t, JHH = 5.4 Hz, 1H,
3
4
3
NH), 7.08 (dd, JHH = 8.7, JHH = 0.9 Hz, 1H, ArH), 6.68 (m, 1H, ArH), 6.49 (d, JHH = 5.5 Hz, 1H,
3
3
3
ArH), 3.51 (q, JHH = 6.6 Hz, 2H, -CH2), 3.40 (q, J
= 6.6 Hz, 2H, -CH2), 2.03 (p, JHH = 6.8 Hz,
HH
2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4, 150.5, 149.6, 145.6, 137.0, 133.9,
131.6, 128.0, 126.8, 124.5, 118.0, 115.6, 114.9, 99.2, 40.5, 40.4, 27.5. IR (ATR, cm-1) ν = 3377 (N-
H), 3306 (N-H), 1621 (C=N), 1568 (C=C), 1530 (N-O), 1508 (N-O). EI-MS: m/z 356 (100 %, [M]+).
336
4.2.2. N1-(7-chloroquinolin-4-yl)-N3-(4-methyl-2-nitrophenyl)propane-1,3-diamine (2b).
337
338
339
340
341
342
343
344
345
346
4-Fluoro-3-nitrotoluene (0.992 g, 6.40 mmol), 1 (1.00 g, 4.24 mmol). Product: Orange powder. Yield:
1
0.688 g, 43.7 %. Rf(DCM:MeOH, 19:1): 0.23. M.p. 198.1 – 199.9 °C. H NMR (400 MHz, DMSO-
d6): δ (ppm) = 8.39 (d, 3JHH = 5.5 Hz, 1H, ArH), 8.26 (d, 3JHH = 9.0 Hz, 1H, ArH), 8.12 (t, 3JHH = 5.7
3
Hz, 1H, NH), 7.87 (m, 1H, ArH), 7.79 (d, 4JHH = 2.3 Hz, 1H, ArH), 7.45 (dd, JHH = 9.0, 2.3 Hz, 1H,
ArH), 7.38 – 7.32 (overlapping m, 2H, NH/ArH), 7.01 (d, 3JHH = 9.0 Hz, 1H, ArH), 6.49 (d, 3JHH = 5.5
3
3
Hz, 1H, ArH), 3.49 (q, JHH = 6.6 Hz, 2H, -CH2), 3.39 (q, JHH = 6.6 Hz, 3H, -CH2), 2.22 (s, 3H, -
3
CH3), 2.01 (p, JHH = 6.8 Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4,
150.5, 149.6, 144.0, 138.5, 133.9, 131.1, 128.0, 125.7, 124.7, 124.5, 118.0, 115.0, 99.2, 40.5, 40.4,
27.6, 19.9. IR (ATR, cm-1) ν = 3417 (N-H), 3361 (N-H), 1586 (N-H), 1609 (C=N), 1570 (C=C), 1520
(N-O). EI-MS: m/z 370 (100 %, [M]+).
347
348
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4.2.3. N1-(7-chloroquinolin-4-yl)-N3-(2-nitro-4-(trifluoromethyl)phenyl)propane-1,3-diamine (2c).
350
351
352
353
354
355
356
357
358
359
4-Chloro-3-nitrobenzotrifluoride (01.43 ml, 9.58 mmol), 1 (1.51 g, 6.41 mmol). Product: Yellow
1
powder. Yield: 1.54 g, 56.6 %. Rf(DCM:MeOH, 19:1): 0.21. M.p. 210.4 – 212.8 °C. H NMR (400
3
MHz, DMSO-d6): δ (ppm) = 8.56 (t, 3JHH = 5.8 Hz, 1H, NH), 8.39 (d, JHH = 5.5 Hz, 1H, ArH), 8.30
(dd, 3JHH = 2.3, 4JHH = 0.9 Hz, 1H, ArH), 8.24 (d, 3JHH = 9.1 Hz, 1H, ArH), 7.79 (d, 4JHH = 2.3 Hz, 1H,
3
3
ArH), 7.75 (dd, JHH = 9.2, 4JHH = 2.3 Hz, 1H, ArH), 7.45 (dd, JHH = 9.0, 4JHH = 2.3 Hz, 1H, ArH),
3
3
7.34 (t, 3JHH = 5.4 Hz, 1H, NH), 7.28 (d, JHH = 9.2 Hz, 1H, ArH), 6.50 (d, JHH = 5.5 Hz, 1H, ArH),
3
3
3
3.58 (q, JHH = 6.6 Hz, 2H, -CH2), 3.40 (q, JHH = 6.6 Hz, 2H, -CH2), 2.02 (p, JHH = 6.7 Hz, 2H, -
CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4, 150.5, 149.6, 147.2, 133.9, 132.3,
130.7, 128.0, 124.5, 124.5, 118.0, 116.4, 99.2, 40.8, 40.3, 27.3. IR (ATR, cm-1) ν = 3361 (N-H), 1609
(C=N), 1568 (C=C), 1536 (N-O), 1324 (C-F). EI-MS: m/z 424 (100 %, [M]+).
360
4.2.4. N1-(7-chloroquinolin-4-yl)-N3-(4-(methylsulfonyl)-2-nitrophenyl)propane-1,3-diamine (2d).
361
362
363
364
365
366
367
368
369
370
371
1-Fluoro-4-methanesulfonyl-2-nitrobenzene (0.848 g, 3.87 mmol), 1 (0.625 g, 2.65 mmol). Product:
1
Yellow powder. Yield: 0.516 g, 46.0 %. Rf(DCM:MeOH, 19:1): 0.17. M.p. 202.0 – 204.4 °C. H
4
NMR (400 MHz, DMSO-d6): δ (ppm) = 8.69 (t, 3JHH = 5.9 Hz, 1H, NH), 8.50 (d, JHH = 2.3 Hz, 1H,
ArH), 8.40 (d, 3JHH = 5.4 Hz, 1H, ArH), 8.24 (d, 3JHH = 9.1 Hz, 1H, ArH), 7.89 (dd, 3JHH = 9.1, 4JHH
=
4
3
2.2 Hz, 1H, ArH), 7.79 (d, JHH = 2.2 Hz, 1H, ArH), 7.46 (dd, JHH = 9.0, 4JHH = 2.3 Hz, 1H, ArH),
3
3
7.34 (t, 3JHH = 5.4 Hz, 1H, NH), 7.30 (d, JHH = 9.3 Hz, 1H, ArH), 6.51 (d, JHH = 5.5 Hz, 1H, ArH),
3.61 (q, 3JHH = 6.5 Hz, 2H, -CH2), 3.39 (q, 3JHH = 6.5 Hz, 2H, -CH2), 3.20 (s, 3H, -CH3) 2.03 (p, 3JHH
= 6.7 Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4, 150.5, 149.5, 147.8,
133.9, 130.5, 128.0, 127.5, 126.7, 124.6, 124.5, 118.0, 116.1, 99.2, 65.4, 44.3, 27.2, 15.6. IR (ATR,
cm-1) ν = 3544 (N-H), 3381 (N-H), 1617 (C=N), 1582 (C=C), 1570 (N-O), 1520 (N-O), 1368 (S=O),
1136 (S=O). EI-MS: m/z 436 (100 %, [M]+).
372
4.2.5. 4-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)-3-nitrobenzonitrile (2e).
373
374
375
376
377
378
379
380
381
382
4-Chloro-3-nitrobenzonitrile (0.587 g, 3.22 mmol), 1 (0.513 g, 2.18 mmol). Product: Yellow powder.
1
Yield: 0.415 g, 51.8 %. Rf(DCM:MeOH, 19:1): 0.19. M.p. 205.7 – 207.9 °C. H NMR (400 MHz,
DMSO-d6): δ (ppm) = 8.66 (t, 3JHH = 5.8 Hz, 1H, NH), 8.50 (d, 4JHH = 2.0 Hz, 1H, ArH), 8.39 (d, 3JHH
3
= 5.5 Hz, 1H, ArH), 8.23 (d, JHH = 9.1 Hz, 1H, ArH), 7.80 – 7.77 (overlapping m, 2H, ArH), 7.45
(dd, 3JHH = 9.1, 4JHH = 2.3 Hz, 1H, ArH), 7.33 (t, 3JHH = 5.4 Hz, 1H, NH), 7.23 (d, 3JHH = 9.2 Hz, 1H,
ArH), 6.50 (d, 3JHH = 5.5 Hz, 1H, ArH), 3.58 (q, 3JHH = 6.6 Hz, 2H, -CH2), 3.39 (q, 3JHH = 6.6 Hz, 2H,
3
-CH2), 2.01 (p, JHH = 6.7 Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4,
150.4, 149.5, 147.3, 138.1, 133.9, 132.4, 131.3, 128.0, 124.5, 124.5, 118.7, 118.0, 116.2, 99.2, 96.8,
40.8, 40.3, 27.2. IR (ATR, cm-1) ν = 3369 (N-H), 3306 (N-H), 2222 (C≡N), 1621 (C=N), 1578 (C=C),
1562 (N-O), 1522 (N-O). EI-MS: m/z 381 (100 %, [M]+).
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383
4.3. General procedure for synthesis of 1,2-diamine aminoquinoline precursors (3a – 3e).
384
385
386
387
388
389
390
391
392
393
Ammonium chloride (10 eq.) was added to a stirring mixture of the appropriate nitro-containing
aminoquinoline precursor (2a – 2e) (1 eq.) in anhydrous methanol (100 ml) and the yellow mixture
was stirred under nitrogen for 15 min. Zinc powder (20 eq.) was then added, and the resulting grey
mixture was stirred vigorously at room temperature under nitrogen for 24 hours. Thereafter, 1 M
NaOH (20 ml) was added to basify the mixture, which was then filtered through Celite and washed
with excess MeOH. The MeOH was removed under reduced pressure, and the aqueous mixture
extracted using DCM (3 × 50 ml). The organic fractions were collected and washed with brine (2 × 50
ml), followed by water (1 × 50 ml) The organic layer was collected, dried over anhydrous Na2SO4 and
filtered by gravity. The solvent of the filtrate was removed to afford the desired products 3a – 3e as
purple, pink or brown powders.
394
4.3.1. N1-(3-((7-chloroquinolin-4-yl)amino)propyl)benzene-1,2-diamine (3a).
395
396
397
398
399
400
401
402
403
Ammonium chloride (0.757 g, 14.2 mmol), 2a (0.503 g, 1.41 mmol), zinc powder (1.87 g, 28.6
mmol). Product: Brown powder. Yield: 0.440 g, 95.5 %. M.p. 169.1 – 170.8 °C. 1H NMR (400 MHz,
3
3
DMSO-d6): δ (ppm) = 8.40 (d, JHH = 5.4 Hz, 1H, ArH), 8.30 (d, JHH = 9.0 Hz, 1H, ArH), 7.80 (d,
3
4
3
4JHH = 2.2 Hz, 1H, ArH), 7.44 (dd, JHH = 9.0, JHH = 2.3 Hz, 1H, ArH), 7.33 (t, JHH = 5.3 Hz, 1H,
NH), 6.57 (dd, 3JHH = 7.3, 4JHH = 1.5 Hz, 1H, ArH), 6.53-6.40 (m, 4H, ArH), 4.45 (m, 2H, NH2), 3.42
3
3
13
(q, JHH = 6.8 Hz, 3H, -CH2), 3.18 (m, 2H, -CH2), 2.01 (p, JHH = 6.8 Hz, 2H, -CH2). C{1H} NMR
(101 MHz, DMSO-d6): δ (ppm) = 152.4, 150.6, 149.6, 136.4, 135.8, 133.8, 128.0, 124.6, 124.5, 118.1,
118.0, 117.4, 114.6, 110.5, 99.2, 41.7, 41.1, 28.0. IR (ATR, cm-1) ν = 3413 (N-H), 3349 (N-H), 1611
(C=N), 1580 (C=C). EI-MS: m/z 326 (100 %, [M]+).
404
4.3.2. N1-(3-((7-chloroquinolin-4-yl)amino)propyl)-4-methylbenzene-1,2-diamine (3b).
405
406
407
408
409
410
411
412
413
414
Ammonium chloride (0.953 g, 17.8 mmol), 2b (0.655 g, 1.75 mmol), zinc powder (2.29 g, 35.0
mmol). Product: Purple powder. Yield: 0.589 g, 97.8 %. M.p. 134.7 – 138.7 °C. 1H NMR (400 MHz,
3
3
DMSO-d6): δ (ppm) = 8.39 (d, JHH = 5.4 Hz, 1H, ArH), 8.29 (d, JHH = 9.0 Hz, 1H, ArH), 7.79 (d,
3
4
3
4JHH = 2.3 Hz, 1H, ArH), 7.44 (dd, JHH = 9.0, JHH = 2.3 Hz, 1H, ArH), 7.34 (t, JHH = 5.2 Hz, 1H,
NH), 6.50 (d, 3JHH = 5.5 Hz, 1H, ArH), 6.40 (d, 4JHH = 1.7 Hz, 1H, ArH), 6.36 (d, 3JHH = 7.9 Hz, 1H,
ArH), 6.29 (dd, 3JHH = 7.9, 4JHH = 1.7 Hz, 1H, ArH), 4.44 (br s, 2H, NH2), 4.25 (m, 1H, NH), 3.40 (q,
3
3
3JHH = 6.8 Hz, 2H, -CH2), 3.13 (q, JHH = 6.8 Hz, 2H, -CH2), 2.09 (s, 3H, -CH3), 1.98 (p, JHH = 6.8
Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4, 150.6, 149.6, 136.0, 134.1,
133.8, 128.0, 125.9, 124.6, 124.5, 118.3, 118.0, 115.6, 110.9, 99.2, 42.1, 41.1, 28.0, 20.9. IR (ATR,
cm-1) ν = 3349 (N-H), 1607 (C=N), 1578 (C=C). EI-MS: m/z 340 (100 %, [M]+).
415
416
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4.3.3. N1-(3-((7-chloroquinolin-4-yl)amino)propyl)-4-(trifluoromethyl)benzene-1,2-diamine (3c).
418
419
420
421
422
423
424
425
426
427
Ammonium chloride (0.953 g, 17.8 mmol), 2c (0.755 g, 1.78 mmol), zinc powder (2.42 g, 37.0
mmol). Product: Purple powder. Yield: 0.674 g, 96.1 %. M.p. 174.9 – 176.0 °C. 1H NMR (400 MHz,
3
3
DMSO-d6): δ (ppm) = 8.39 (d, JHH = 5.4 Hz, 1H, ArH), 8.29 (d, JHH = 9.1 Hz, 1H, ArH), 7.79 (d,
3
4
3
4JHH = 2.3 Hz, 1H, ArH), 7.45 (dd, JHH = 9.0, JHH = 2.3 Hz, 1H, ArH), 7.33 (t, JHH = 5.3 Hz, 1H,
NH), 6.82 (d, 4JHH = 2.1 Hz, 1H, ArH), 6.79 (d, 3JHH = 8.7 Hz, 1H, ArH), 6.51 (m, 2H, ArH), 5.10 (t,
3JHH = 5.3 Hz, 1H, NH), 4.94 (s, 2H, NH2), 3.41 (q, 3JHH = 6.8 Hz, 2H, -CH2), 3.25 (q, 3JHH = 6.8 Hz,
2H, -CH2), 2.01 (p, 3JHH = 6.8 Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.4,
150.6, 149.6, 139.3, 135.6, 133.9, 128.0, 124.6, 124.5, 118.0, 115.0, 114.9, 109.8, 109.8, 108.7, 99.2,
41.3, 40.9, 27.6. IR (ATR, cm-1) ν = 3377 (N-H), 1611 (C=N), 1581 (C=C), 1328 (C-F). EI-MS: m/z
394 (100 %, [M]+).
428
4.3.4. N1-(3-((7-chloroquinolin-4-yl)amino)propyl)-4-(methylsulfonyl)benzene-1,2-diamine (3d).
429
430
431
432
433
434
435
436
437
438
Ammonium chloride (0.242 g, 4.52 mmol), 2d (0.192 g, 0.454 mmol), zinc powder (0.592 g, 9.05
mmol). Product: Brown powder. Yield: 0.148 g, 80.5 %. M.p. 227.2 – 229.5 °C. 1H NMR (400 MHz,
3
3
DMSO-d6): δ (ppm) = 8.40 (d, JHH = 5.4 Hz, 1H, ArH), 8.29 (d, JHH = 9.1 Hz, 1H, ArH), 7.79 (d,
3
4
3
4JHH = 2.2 Hz, 1H, ArH), 7.44 (dd, JHH = 9.1, JHH = 2.2 Hz, 1H, ArH), 7.33 (t, JHH = 5.1 Hz, 1H,
NH), 7.02 (m, 2H, ArH), 6.55 (m, 1H, ArH), 6.51 (d, 3JHH = 5.5 Hz, 1H, ArH), 5.38 (t, 3JHH = 5.1 Hz,
1H, NH), 5.02 (br s, 2H, NH2), 3.41 (m, 2H, -CH2), 3.33 (s, 3H, -CH3), 3.31 – 3.24 (m, 2H, -CH2),
2.02 (p, 3JHH = 6.8 Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO): δ (ppm) = 152.4, 150.6, 149.6,
140.5, 135.4, 133.9, 128.0, 127.5, 124.6, 124.5, 118.0 (2C), 111.4, 108.3, 99.3, 45.1, 41.2, 40.8, 27.6.
IR (ATR, cm-1) ν = 3401 (N-H), 3345 (N-H), 1611 (C=N), 1580 (C=C), 1370 (S=O), 1124 (S=O). EI-
MS: m/z 405 (100 %, [M+H]+).
439
4.3.5. 3-Amino-4-((3-((7-chloroquinolin-4-yl)amino)propyl)amino)benzonitrile (3e).
440
441
442
443
444
445
446
447
448
449
Ammonium chloride (0.592 g, 11.1 mmol), 2e (0.406 g, 1.10 mmol), zinc powder (1.45 g, 22.2
1
mmol). Product: Pink powder. Yield: 0.294 g, 75.7 %. M.p. 250.1 – 253.4 °C. H NMR (300 MHz,
3
3
DMSO-d6): δ (ppm) = 8.39 (d, JHH = 5.4 Hz, 1H, ArH), 8.29 (d, JHH = 9.1 Hz, 1H, ArH), 7.79 (d,
3
4
3
4JHH = 2.2 Hz, 2H, ArH), 7.45 (dd, JHH = 9.1, JHH = 2.2 Hz, 1H, ArH), 7.33 (t, JHH = 5.2 Hz, 2H,
3
4
3
4
NH), 7.02 (dd, JHH = 8.2, JHH = 2.0 Hz, 1H, ArH), 6.91 (dd, JHH = 8.2, JHH = 2.0 Hz, 1H, ArH),
6.79 (d, 4JHH = 2.0 Hz, 1H, ArH), 6.50 (d, 3JHH = 5.4 Hz, 1H, ArH), 5.42 (t, 3JHH = 5.1 Hz, 2H, -CH2),
3
3
4.97 (s, 2H, NH), 3.41 (m, 2H, Hl), 3.26 (q, JHH = 6.8 Hz, 3H, -CH2), 2.00 (p, JHH = 6.8 Hz, 2H, -
CH2). 13C{1H} NMR (101 MHz, DMSO) δ 152.4, 150.6, 149.6, 140.5, 135.7, 133.8, 128.0, 124.6,
124.5, 123.3, 121.5, 118.0, 115.4, 109.2, 99.3, 97.1, 41.0, 40.8, 27.6. IR (ATR, cm-1) ν = 3417 (N-H),
3393 (N-H), 2202 (C≡N), 1609 (C=N), 1580 (C=C). EI-MS: m/z 352 (100 %, [M+H]+).
450
18

ACCEPTED MANUSCRIPT
451
452
4.4. General procedure for synthesis of phenyl-benzimidazoles (4a – 4e).
453
454
455
456
457
458
459
460
461
462
463
A 1,2-diamine precursor (3a – 3e) (1 eq.), benzaldehyde (1.5 eq.) and TFA (0.1 eq.) were dissolved in
ethanol (25 ml), anhydrous MgSO4 (5 eq.) was added and the brown reaction mixture was refluxed for
24 hours. Thereafter, the resulting brown mixture was filtered by gravity and the solvent of the filtrate
was removed to give a brown residue. To this crude residue, 1 M NaOH (50 ml) was added and the
mixture was extracted using DCM (3 × 50 ml). The organic fractions were collected and washed with
brine (2 × 50 ml), followed by water (1 × 30 ml). The organic layer was collected, dried over
anhydrous Na2SO4 and filtered by gravity. The solvent of the filtrate was removed to afford the crude
product as a dark orange residue. The crude product was purified by column chromatography using
EtOAc (100 %). Subsequent precipitation from DCM/pet. ether (40 – 60 °C) afforded the desired
products 4a – 4e as white, pale-yellow or beige powders, which were collected by suction filtration
and dried in vacuo.
464
4.4.1. 7-Chloro-N-(3-(2-phenyl-1H-benzo[d]imidazole-1-yl)propyl)quinolin-4-amine (4a).
465
466
467
468
469
470
471
472
473
474
475
Compound 3a (0.502 g, 1.54 mmol), benzaldehyde (0.235 ml, 2.31 mmol), TFA (11.7 µl, 0.153
mmol) and anhydrous MgSO4 (0.927 g, 7.70 mmol). Product: Pale-yellow powder. Yield: 0.407 g,
64.2 %. Rf(EtOAc, 100%): 0.15. M.p. 180.9 – 182.5 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) =
8.35 (d, 3JHH = 5.4 Hz, 1H, ArH), 8.13 (d, 3JHH = 9.1, 1H, ArH), 7.80 (d, 4JHH = 2.2 Hz, 1H, ArH), 7.75
– 7.61 (m, 4H, ArH), 7.44 (dd, 3JHH = 9.1, 4JHH = 2.2 Hz, 1H, ArH), 7.40 – 7.24 (m, 5H, ArH), 7.21 (t,
3JHH = 4.8 Hz, 1H, NH) 6.30 (d, 3JHH = 5.5 Hz, 1H, ArH), 4.47 (t, 3JHH = 7.4 Hz, 2H, -CH2), 3.19 (m,
3
2H, -CH2), 2.13 (p, JHH = 6.8 Hz, 2H, -CH2).13C{1H} NMR (101 MHz, DMSO) δ 153.4, 152.3,
150.3, 149.5, 143.1, 136.1, 133.9, 130.8, 129.9, 129.4 (2C), 129.0 (2C), 127.9, 124.5 (2C), 122.9,
122.5, 119.7, 118.0, 111.3, 99.1, 42.5, 39.8, 28.4. IR (ATR, cm-1) ν = 3433 (N-H), 1611 (C=N), 1578
(C=C). ESI-MS: m/z 413.1534 (100 %, [M+H]+), calculated 413.1528. HPLC purity > 99 %
(tR = 2.30 min).
476
4.4.2. 7-Chloro-N-(3-(5-(methyl)-2-phenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-amine (4b).
477
478
479
480
481
482
483
484
Compound 3b (0.405 g, 1.19 mmol), benzaldehyde (0.181 ml, 1.78 mmol), TFA (8.98 µl, 0.119
mmol) and anhydrous MgSO4 (0.716 g, 5.95 mmol). Product: Pale-yellow powder. Yield: 0.307 g,
1
60.5 %. Rf(EtOAc, 100%): 0.14. M.p. 248.2 – 250.2 °C. H NMR (300 MHz, DMSO-d6): δ (ppm) =
8.35 (d, 3JHH = 5.4 Hz, 1H, ArH), 8.13 (d, 3JHH = 9.1 Hz, 1H, ArH), 7.80 (d, 4JHH = 2.2 Hz, 1H, ArH),
7.75 – 7.60 (m, 2H, ArH), 7.57 (d, 3JHH = 8.3 Hz, 1H, ArH), 7.48 (m, 1H, ArH), 7.43 (dd, 3JHH = 9.1,
4JHH = 2.2 Hz, 1H, ArH), 7.39 – 7.26 (overlapping m, 3H, ArH), 7.21 (t, 3JHH = 5.2 Hz, 1H, NH), 7.10
(d, 3JHH = 8.2 Hz, 1H, ArH), 6.29 (d, 3JHH = 5.4 Hz, 1H, ArH), 4.44 (t, 3JHH = 7.3 Hz, 1H, -CH2), 3.18
3
(q, JHH = 6.3 Hz, 2H, -CH2), 2.43 (s, 3H, -CH3) 2.11 (m, 2H, -CH2). 13C{1H} NMR (101 MHz,
19

ACCEPTED MANUSCRIPT
485
486
487
488
DMSO) δ 153.3, 152.3, 150.3, 149.5, 143.5, 134.3, 133.9, 131.5, 130.9, 129.8, 129.4 (2C), 128.9
(2C), 127.9, 124.5, 124.5, 124.3, 119.4, 118.0, 110.9, 99.1, 42.5, 39.9, 28.4, 21.6. IR (ATR, cm-1) ν =
3250 (N-H), 1609 (C=N), 1578 (C=C). ESI-MS: m/z 214.0882 (100 %, [M+2H]2+), calculated
214.0878; 427.1690 (30 %, [M+H]+), calculated 427.1684. HPLC purity > 99 % (tR = 2.37 min).
489
490
4.4.3. 7-Chloro-N-(3-(5-(trifluoromethyl)-2-phenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-
amine (4c).
491
492
493
494
495
496
497
498
499
500
501
502
Compound 3c (0.250 g, 0.633 mmol), benzaldehyde (0.0965 ml, 0.950 mmol), TFA (4.84 µl, 0.0633
mmol) and anhydrous MgSO4 (0.381 g, 3.17 mmol). Product: White powder. Yield: 0.248 g, 81.4 %.
Rf(EtOAc, 100%): 0.26. M.p. 197.8 – 199.6 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 8.35 (d,
3JHH = 5.4 Hz, 1H, ArH), 8.10 (d, 3JHH = 9.1 Hz, 1H, ArH), 8.06 (m, 1H, ArH) 7.97 (d, 3JHH = 8.5 Hz,
3
1H, ArH), 7.80 (d, 4JHH = 2.2 Hz, 1H, ArH), 7.74 – 7.67 (overlapping m, 2H, ArH), 7.61 (dd, JHH
=
8.6, 4JHH = 1.1 Hz, 1H, ArH), 7.43 (dd, 3JHH = 9.0, 4JHH = 2.2 Hz, 1H, ArH), 7.40 – 7.28 (overlapping
m, 3H, ArH), 7.19 (t, 3JHH = 5.1 Hz, 1H, NH), 6.30 (d, 3JHH = 5.4 Hz, 1H, ArH), 4.54 (t, 3JHH = 7.4 Hz,
1H, -CH2), 3.20 (q, 3JHH = 6.3 Hz, 2H, -CH2), 2.13 (p, 3JHH = 6.8 Hz, 1H, -CH2). 13C{1H} NMR (101
MHz, DMSO-d6): δ (ppm) = 155.8, 152.3, 150.3 (2C), 149.5, 142.5, 138.4, 133.9 (2C), 130.4, 130.1,
129.5 (2C), 129.1 (2C), 127.9, 124.5, 124.5, 119.5, 118.0, 117.1, 112.5, 99.2, 42.9, 39.7, 28.2. IR
(ATR, cm-1) ν = 3278 (N-H), 1611 (C=N), 1578 (C=C), 1326 (C-F). ESI-MS: m/z 481.1410 (100 %,
[M+H]+), calculated 481.1401. HPLC purity 99 % (tR = 2.44 min).
503
504
4.4.4. 7-Chloro-N-(3-(5-(methylsulfonyl)-2-phenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-
amine (4d).
505
506
507
508
509
510
511
512
513
514
515
516
517
Compound 3d (0.0600 g, 0.148 mmol), benzaldehyde (0.0226 g, 0.222 mmol), TFA (1.13 µl, 0.0148
mmol) and anhydrous MgSO4 (0.0891 g, 0.740 mmol). Product: Beige powder. Yield: 0.0453 g, 62.3
%. Rf(EtOAc, 100%): 0.06. M.p. 94.1 – 96.5 °C. 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 8.35 (d,
3JHH = 5.4 Hz, 1H, ArH), 8.24 (d, 4JHH = 1.6 Hz, 1H, ArH), 8.11 (d, 3JHH = 9.1 Hz, 1H, ArH), 8.00 (d,
3
4
4
3JHH = 8.6 Hz, 1H, ArH), 7.83 (dd, JHH = 8.6, JHH = 1.6 Hz, 1H, ArH), 7.80 (d, JHH = 2.2 Hz, 1H,
3
4
ArH), 7.74 – 7.67 (m, 2H, ArH), 7.44 (dd, JHH = 9.1, JHH = 2.2 Hz, 1H, ArH), 7.41 – 7.29
(overlapping m, 3H, ArH), 7.19 (t, 3JHH = 4.9 Hz, 1H, NH), 6.30 (d, 3JHH = 5.5 Hz, 1H, ArH), 4.55 (t,
3JHH = 7.3 Hz, 1H, -CH2), 3.24 (s, 3H, -SO2CH3), 3.19 (overlapping m, 2H, -CH2), 2.13 (p, 3JHH = 6.8
Hz, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 156.3, 152.3, 150.3, 149.5, 142.4,
139.2, 135.2, 133.9, 130.5, 129.9, 129.5 (2C), 129.1 (2C), 127.9, 124.5, 124.5, 121.4, 119.3, 118.0,
112.4, 99.2, 44.7, 42.9, 39.8, 28.2. IR (ATR, cm-1) ν = 3397 (N-H), 1609 (C=N), 1578 (C=C), 1368
(S=O), 1136 (S=O). ESI-MS: m/z 246.0693 (100 %, [M+2H]2+), calculated 246.0688; 491.1313 (80
%, [M+H]+), calculated 491.1303. HPLC purity > 99 % (tR = 2.20 min).
518
519
4.4.5. 1-(3-((7-Chloroquinolin-4-yl)amino)propyl)-2-phenyl-1H-benzo[d]imidazole-5-carbonitrile
(4e).
20

ACCEPTED MANUSCRIPT
520
521
522
523
524
525
526
527
528
529
530
531
Compound 3e (0.141 g, 0.401 mmol), benzaldehyde (0.0607 ml, 0.597 mmol), TFA (3.04 µl, 0.0397
mmol) and anhydrous MgSO4 (0.244 g, 1.99 mmol). Product: Beige powder. Yield: 0.115 g, 65.5 %.
1
Rf(EtOAc, 100%): 0.12. M.p. 228.8 – 230.5 °C. H NMR (300 MHz, DMSO-d6): δ (ppm) = 8.35 (d,
3JHH = 5.5 Hz, 1H, ArH), 8.25 (d, 4JHH = 1.0 Hz, 1H, ArH), 8.10 (d, 3JHH = 9.1 Hz, 1H, ArH), 7.97 (d,
4
3JHH = 8.4 Hz, 1H, ArH), 7.80 (d, JHH = 2.2 Hz, 1H, ArH), 7.72 – 7.69 (overlapping m, 3H, ArH),
3
4
3
7.44 (dd, JHH = 9.1, JHH = 2.2 Hz, 1H, ArH), 7.40 – 7.27 (overlapping m, 3H, ArH), 7.21 (t, JHH
=
5.1 Hz, 1H, NH), 6.30 (d, 3JHH = 5.5 Hz, 1H, ArH), 4.53 (t, 3JHH = 7.3 Hz, 2H, -CH2), 3.19 (m, 2H, -
CH2), 2.13 (m, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 156.1, 152.3, 150.2,
149.5, 142.6, 139.1, 133.9, 130.5, 129.8, 129.5 (2C), 129.1 (2C), 128.0, 126.3, 124.6, 124.5, 124.5,
120.3, 118.0, 113.0, 104.8, 99.2, 42.9, 39.7, 28.2. IR (ATR, cm-1) ν = 3175 (N-H), 1611 (C=N), 1580
(C=C), 2214 (C≡N). ESI-MS: m/z 219.5773 (100 %, [M+2H]2+), calculated 219.5776; 438.1470 (90
%, [M+H]+), calculated 438.1480. HPLC purity > 99 % (tR = 2.30 min).
532
4.5. General procedure for synthesis of ferrocenyl-benzimidazoles (5a – 5e).
533
534
535
536
537
538
539
540
541
542
A 1,2-diamine precursor (3a – 3e) (1 eq.), ferrocenecarboxaldehyde (1.5 eq.) and TFA (0.1 eq.) were
dissolved in EtOH (25 ml), anhydrous MgSO4 (5 eq.) was added and the dark red reaction mixture
was refluxed for 48 hours. Thereafter, the resulting dark red mixture was filtered by gravity and the
solvent of the filtrate was removed to give a red residue. To this crude residue, 1 M NaOH (50 ml)
was added and the mixture was extracted using DCM (3 × 50 ml). The organic fractions were
collected and washed with brine (2 × 50 ml), followed by water (1 × 30 ml). The organic layer was
collected, dried over anhydrous Na2SO4 and filtered by gravity. The solvent of the filtrate was
removed to afford the crude product as a red residue. The crude product was purified by column
chromatography using EtOAc (100 %). The desired products 5a – 5e were isolated as orange powders
and dried in vacuo.
543
4.5.1. 7-Chloro-N-(3-(2-ferrocenyl-1H-benzo[d]imidazole-1-yl)propyl)quinolin-4-amine (5a).
544
545
546
547
548
549
550
551
552
553
Compound 3a (0.102 g, 0.312 mmol), ferrocenecarboxaldehyde (0.100 g, 0.467 mmol), TFA (2.50 µl,
0.0327 mmol) and anhydrous MgSO4 (0.184 g, 1.53 mmol). Product: Orange powder. Yield: 0.0742
1
g, 45.6 %. Rf(EtOAc, 100%): 0.26. M.p. 227.0 – 228.3 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
3
3
4
= 8.44 (d, JHH = 5.5 Hz, 1H, ArH), 8.37 (d, JHH = 9.1 Hz, 1H, ArH), 7.87 (d, JHH = 2.2 Hz, 1H,
4
ArH), 7.69 – 7.57 (overlapping m, 3H, NH/ArH), 7.56 (d, JHH = 2.1 Hz, 1H, ArH), 7.28 – 7.10
3
3
(overlapping m, 2H, ArH), 6.63 (d, JHH = 5.5 Hz, 1H, ArH), 4.75 (t, JHH = 1.8 Hz,, 2H, substituted
3
CpH), 4.62 (m, 2H, -CH2), 4.14 (t, JHH = 1.8 Hz, 2H, substituted CpH), 3.99 (s, 5H, unsubstituted
CpH), 3.60 (q, 3JHH = 5.7 Hz, 2H, -CH2), 2.23 (m, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ
(ppm) = 152.5, 152.5, 150.4, 149.8, 143.2, 136.5, 134.0, 128.1, 124.7, 124.6, 122.1 (2C), 118.7,
118.1, 110.3, 99.5, 74.1, 70.1 (2C), 69.7 (5C), 69.1 (2C), 42.3, 39.5, 29.1. IR (ATR, cm-1) ν = 3437
21

ACCEPTED MANUSCRIPT
554
555
(N-H), 1609 (C=N), 1574 (C=C). ESI-MS: m/z 521.1207 (100 %, [M+H]+), calculated 521.1190.
HPLC purity > 99 % (tR = 6.75 min).
556
557
4.5.2. 7-Chloro-N-(3-(5-(methyl)-2-ferrocenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-amine
(5b).
558
559
560
561
562
563
564
565
566
567
568
569
570
Compound 3b (0.0813 g, 0.239 mmol), ferrocenecarboxaldehyde (0.0774 g, 0.362 mmol), TFA (2.30
µl, 0.0301 mmol) and anhydrous MgSO4 (0.146 g, 1.21 mmol). Product: Orange powder. Yield:
0.0718 g, 56.3 %. Rf(EtOAc, 100%): 0.29. M.p. 114.2 – 115.9 °C. 1H NMR (400 MHz, DMSO-d6): δ
3
3
4
(ppm) = 8.44 (d, JHH = 5.5 Hz, 1H, ArH), 8.37 (d, JHH = 9.1 Hz, 1H, ArH), 7.87 (d, JHH = 2.3 Hz,
1H, ArH), 7.65 (t, 3JHH = 5.5 Hz, 1H, NH), 7.57 (dd, 3JHH = 9.1, 4JHH = 2.3 Hz, 1H, ArH), 7.49 (d, 3JHH
= 8.3 Hz, 1H, ArH), 7.36 (m, 1H, ArH), 7.03 (dd, 3JHH = 8.3, 4JHH = 1.2 Hz, 1H, ArH), 6.62 (d, 3JHH
=
5.5 Hz, 1H, ArH), 4.74 (t, 3JHH = 1.9 Hz, 2H, substituted CpH), 4.58 (m, 2H, -CH2), 4.13 (t, 3JHH = 1.9
Hz, 2H, substituted CpH), 3.98 (s, 5H, unsubstituted CpH) 3.58 (q, 3JHH = 6.0 Hz, 2H, -CH2), 2.41 (s,
3H, -CH3), 2.21 (m, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 152.5, 152.3, 150.4,
149.8, 143.6, 134.7, 134.0, 131.1, 128.1, 124.7, 124.6, 123.4, 118.5, 118.1, 109.8, 99.5, 74.3, 70.0
(2C), 69.7 (5C), 69.0 (2C), 42.3, 39.7, 29.1, 21.7. IR (ATR, cm-1) ν = 3254 (N-H), 1609 (C=N), 1580
(C=C). ESI-MS: m/z 268.0718 (100 %, [M+2H]2+), calculated 267.0710; 535.1367 (23 %, [M+H]+),
calculated 535.1346. HPLC purity 99 % (tR = 6.98 min).
571
572
4.5.3. 7-Chloro-N-(3-(5-(trifluoromethyl)-2-ferrocenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-
amine (5c).
573
574
575
576
577
578
579
580
581
582
583
584
Compound 3c (0.140 g, 0.355 mmol), ferrocenecarboxaldehyde (0.117 g, 0.547 mmol), TFA (3.43 µl,
0.0355 mmol) and anhydrous MgSO4 (0.214 g, 1.78 mmol). Product: Orange powder. Yield: 0.107 g,
1
51.2 %. Rf(EtOAc, 100%): 0.39. M.p. 184.2 – 185.7 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) =
3
3
8.44 (d, JHH = 5.4 Hz, 1H, ArH), 8.36 (d, JHH = 9.0 Hz, 1H, ArH), 7.92 (m, 1H, ArH), 7.90 – 7.85
3
3
4
(overlapping m, 2H, ArH), 7.67 (t, JHH = 5.2 Hz, 1H, NH), 7.58 (dd, JHH = 9.0, JHH = 2.0 Hz, 1H,
3
3
ArH), 7.54 (d, JHH = 8.7 Hz, 1H, ArH), 6.64 (d, JHH = 5.3 Hz, 1H, ArH), 4.79 (m, 2H, substituted
CpH), 4.67 (m, 2H, -CH2), 4.16 (m, 2H, substituted CpH), 4.00 (s, 5H, unsubstituted CpH), 3.61 (m,
2H, -CH2), 2.24 (m, 2H, -CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 155.5, 152.5, 150.4,
149.8, 142.7, 138.9, 134.0, 128.1, 124.7, 124.5, 118.7, 118.1, 115.9, 115.8, 111.3, 99.5, 73.0, 70.5
(2C), 69.9 (5C), 69.3 (2C), 42.7, 39.6, 29.0. IR (ATR, cm-1) ν = 3266 (N-H), 1611 (C=N), 1582
(C=C), 1328 (C-F). ESI-MS: m/z 295.0572 (100 %, [M+2H]2+), calculated 295.0568; 589.1068 (32 %,
[M+H]+), calculated 589.1064. HPLC purity > 99 % (tR = 8.73 min).
585
586
4.5.4. 7-Chloro-N-(3-(5-(methylsulfonyl)-2-ferrocenyl-1H-benzo[d]imidazol-1-yl)propyl)quinolin-4-
amine (5d).
22

ACCEPTED MANUSCRIPT
587
588
589
590
591
592
593
594
595
596
597
598
Compound 3d (0.159 g, 0.393 mmol), ferrocenecarboxaldehyde (0.130 g, 0.607 mmol), TFA (3.01 µl,
0.0393 mmol) and anhydrous MgSO4 (0.239 g, 1.99 mmol). Product: Orange powder. Yield: 0.128 g,
1
54.4 %. Rf(EtOAc, 100%): 0.10. M.p. 164.0 – 166.0 °C. H NMR (300 MHz, DMSO-d6): δ (ppm) =
8.45 (d, 3JHH = 5.5 Hz, 1H, ArH), 8.37 (d, 3JHH = 9.0 Hz, 1H, ArH), 8.10 (d, 4JHH = 1.3 Hz, 1H, ArH),
3
4
3
4
7.92 (d, JHH = 8.5 Hz, 1H, ArH), 7.87 (d, JHH = 2.1 Hz, 1H, ArH), 7.76 (dd, JHH = 8.5, JHH = 1.5
Hz, 1H, ArH), 7.68 (t, 3JHH = 5.4 Hz, 1H, NH), 7.58 (dd, 3JHH = 9.0, 4JHH = 2.1 Hz, 1H, ArH), 6.65 (d,
3JHH = 5.5 Hz, 1H, ArH), 4.80 (m, 2H, substituted CpH), 4.68 (m, 2H, -CH2), 4.18 (m, 2H, substituted
CpH), 4.00 (s, 5H, unsubstituted CpH), 3.62 (m, 2H, -CH2), 3.22 (s, 3H, -SO2CH3), 2.24 (m, 2H, -
CH2). 13C{1H} NMR (101 MHz, DMSO-d6): δ (ppm) = 156.2, 152.5, 150.4, 142.6, 139.7, 134.9,
134.0, 128.2, 124.7 (2C), 124.6, 120.6, 118.0, 111.2, 72.8, 70.6 (2C), 69.9 (5C), 69.4 (2C), 44.7, 42.8,
39.6, 29.0. IR (ATR, cm-1) ν = 3242 (N-H), 1609 (C=N), 1580 (C=C), 1366 (S=O), 1136 (S=O). ESI-
MS: m/z 599.0969 (100 %, [M+H]+), calculated 599.0965. HPLC purity 97 % (tR = 7.55 min).
599
600
4.5.5. 1-(3-((7-Chloroquinolin-4-yl)amino)propyl)-2-ferrocenyl-1H-benzo[d]imidazole-5-
carbonitrile (5e).
601
602
603
604
605
606
607
608
609
610
611
612
Compound 3e (0.141 g, 0.398 mmol), ferrocenecarboxaldehyde (0.131 g, 0.614 mmol), TFA (3.04 µl,
0.0398 mmol) and anhydrous MgSO4 (0.240 g, 1.99 mmol). Product: Orange powder. Yield: 0.0954
1
g, 43.9 %. Rf(EtOAc, 100%): 0.21. M.p. 222.9 – 224.4 °C. H NMR (300 MHz, DMSO-d6): δ (ppm)
= 8.44 (d, 3JHH = 5.4 Hz, 1H, ArH), 8.37 (d, 3JHH = 9.1 Hz, 1H, ArH), 8.09 (m, 1H, ArH), 7.92 – 7.83
(overlapping m, 2H, ArH), 7.71 (t, 3JHH = 5.2 Hz, 1H, NH), 7.67 – 7.52 (m, 2H, ArH), 6.64 (d, 3JHH
=
5.4 Hz, 1H, ArH), 4.77 (m, 2H, substituted CpH), 4.66 (m, 2H, -CH2), 4.15 (m, 2H, substituted CpH),
13
4.00 (s, 5H, unsubstituted CpH), 3.61 (m, 2H, -CH2), 2.21 (m, 2H, -CH2). C{1H} NMR (101 MHz,
DMSO-d6): δ (ppm) = 156.0, 152.5, 150.4, 149.8, 142.7, 139.6, 134.0, 128.1, 125.6, 124.7, 124.5,
123.2, 120.5, 118.1, 111.9, 104.3, 99.5, 72.7, 70.7 (2C), 69.9 (5C), 69.4 (2C), 42.7, 39.5, 29.0. IR
(ATR, cm-1) ν = 3242 (N-H), 1609 (C=N), 1580 (C=C), 2222 (C≡N). ESI-MS: m/z 273.5657 (100 %,
[M+2H]2+), calculated 273.5608; 546.1166 (47 %, [M+H]+), calculated 546.1142. HPLC purity 97 %
(tR = 8.02 min).
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4.6. X-ray structure determination.
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Single-crystal X-ray diffraction data were collected on a Bruker KAPPA APEX II DUO
diffractometer using graphite-monochromated Mo-Kα radiation (χ = 0.71073 Å). Data collection was
carried out at 173(2) K. Temperature was controlled by an Oxford Cryostream cooling system
(Oxford Cryostat). Cell refinement and data reduction were performed using the program SAINT
[35]. The data were scaled, and absorption correction performed using SADABS [36]. The structures
were solved by direct methods using SHELXS-97 [36] and refined by full-matrix least-squares
methods based on F2 using SHELXL-2014 [36] and using the graphics interface program X-Seed [37,
38]. The programs X-Seed and POV-Ray [39] were used to prepare molecular graphic images.
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For 4b, the amino hydrogen H2 was located in the difference electron density map and refined
independently. The structure of 4b was refined to R factor of 0.0381. For 5c, there is solvent
accessible void of 100.6 Å3 per unit cell volume of 1384.0 Å3 (~7.3 %). Attempts to model as discrete
atomic sites failed, probably due to severe disorder. PLATON SQUEEZE [40] was employed to
calculate the contribution to the diffraction from the missing solvent molecules and it produced a set
of partial-solvent-free diffraction intensities. This set of intensities was used for final refinements.
SQUEEZE estimated a total count of 41 electrons per unit cell contributed by the missing solvents. It
corresponds to one dichloromethane molecule per unit cell. This was included in the formula and
subsequent calculation of molecular weight, density etc. The structure of 5c was refined to R factor of
0.0438.
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4.7. Plasmodium falciparum assay.
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The samples were tested in triplicate on one experiment against the chloroquine-sensitive NF54 strain
and chloroquine-resistant K1 strain of P. falciparum. Continuous in vitro cultures of asexual
erythrocyte stages of P. falciparum were maintained using a modified method of Trager and Jensen
[41]. The antiplasmodial activity in vitro was determined via the parasite lactate dehydrogenase assay
using a modified method described by Makler et al [42]. The samples were prepared as a 20 mg/ml
stock solution using DMSO and sonicated to enhance solubility. Samples were tested as a suspension
if not completely dissolved. Stock solutions were stored at -20 °C. Further dilutions were prepared on
the day of the experiment. Chloroquine was used as the reference drug in all experiments. A full dose-
response measurement was performed for all compounds to determine the concentration inhibiting 50
% of parasite growth (IC50 value). The samples were tested at a starting concentration of 100 µg/ml,
which was then serially diluted 2-fold in complete medium to give 10 concentrations; with the lowest
concentration being 0.2 µg/ml. The same dilution technique was used for all samples. CQ was tested
at a starting concentration of 1000 ng/cm3 against the CQS strain. The highest concentration of
solvent to which the parasites were exposed to had no measurable effect on the parasite viability. The
IC50 values were obtained using a non-linear dose-response curve fitting analysis via Graph Pad Prism
v.4.0 software.
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4.8. In vitro cytotoxicity assay.
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In vitro cytotoxicity of potent target compounds against the Chinese Hamster Ovarian (CHO) cells
was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay,
which determines cellular growth and survival. For each test compound, a stock solution (2 mg/mL)
was prepared in 10% DMSO, and then diluted with the assay medium to yield a starting concentration
of a 100 µg/mL. This was then serially 10-fold diluted to give six assay concentrations ranging from
100–0.001 µg/mL. Emetine was subjected to similar dilutions and used as the positive assay control.
In all cases, triplicate samples were prepared. Negative control was the highest concentration of
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DMSO used, which had to be demonstrated that it had no measurable effect on the viability and
survival of CHO cells. A solution of MTT was then added after 44 hours of exposure of the cells to
the test compound. This was followed by a further 4 hours of incubation at 37 ºC, after which the
supernatant was separated from the cells by suction. Crystals of the reduced dye were then dissolved
by addition of DMSO to each well. The amount of formazan in each well was determined by
measuring the absorbance at 540 nm, which was then used to prepare dose-response curves. A non-
linear dose-response curve analysis, using GraphPad Prism software, generated the 50% inhibitory
concentration (IC50) values.
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4.9. In vivo antimalarial efficacy studies.
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In vivo efficacy against P. berghei was performed using mice (n = 3) which were infected with a
GFP-transfected P. berghei ANKA strain (donated by A. P. Waters and C. J. Janse, Glasgow and
Leiden Universities respectively). Parasitemia was determined using standard flow cytometry
techniques. The detection limit was 1 parasite per 1000 erythrocytes (that is, 0.1%). Activity was
calculated as the difference between the mean % parasitemia for the control and treated groups
expressed as a % relative to the control group. Compounds were dissolved or suspended in 90/10
Tween80/ethanol (v/v), diluted 10 times with water and administered orally as four consecutive daily
doses (4, 24, 48 and 72 h after infection). Animals were considered cured if there were no detectable
parasites on day 30 post-infection as determined by light microscopy.
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4.10. β-Haematin inhibition assay.
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The β-haematin formation assay was adapted from the method described by Wright and co-workers
[25]. Test compounds were prepared as a 10 mM stock solution in 100% DMSO. Test samples were
tested at a starting concentration of 500µM and the lowest drug concentration being 5 µM. The stock
solution was serially diluted to give 12 concentrations in a 96 well flat-bottom assay plate. NP-40
detergent was then added to mediate the formation of β-haematin (30.55 µM, final concentration). A
25 mM stock solution of haematin was prepared by dissolving haemin (16.3 mg) in dimethyl
sulfoxide (1 ml). A 177.76 µl aliquot of haematin stock was suspended in 20 ml of a 2 M acetate
buffer, pH 4.7. The haematin suspension was then added to the plate to give a final haematin
concentration of 100 µM. The plate was then incubated for 16 hours at 37 °C. The assay was analysed
using the pyridineferrochrome method developed by Ncokazi and Egan [43]. 32 µl of a solution of
50% pyridine, 20% acetone, 20% water, and 10 % 2M HEPES buffer (pH 7.4) was added to each
well. To this, 60 µl acetone was then added to each well and mixed. The absorbance of the resulting
complex was measured at 405 nm on a SpectraMax 340PC plate reader. The IC50 values were
obtained using a non-linear dose-response curve fitting analysis via Graph Pad Prism v.5.0 software.
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4.11. DNA cleavage assay
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Compound-DNA interaction assays (EMSA) were performed in tubes containing 25 ng of pUC57
plasmid DNA (ThermoFisher), an appropriate amount of compound dissolved in DMSO (to a final
concentration of 10% DMSO) in 1x TAE buffer (Sigma-Aldrich, pH 8.3) for a total volume of 10 µL.
The samples were incubated at 37 °C for 30 minutes. 4 uL of loading dye (Sigma-Aldrich, 0.25%
bromophenol blue, 0.25% xylene cyanol, and 40% sucrose) was added to each sample and 6 µL
aliquots were then loaded onto the gels and electrophoresed at a constant 75 V for 90 minutes in 1x
TAE buffer. The DNA was stained with an Ethidium Bromide solution (0.5 mg/L in Type 1 ultrapure
water) for 30 minutes, washed in Type 1 ultrapure water for 20 minutes and visualized using a G:Box
Chemi XRQ gel doc system (Syngene) with midwave transillumination and a UV filter (GeneSys
1.4.6.0).
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4.12. Mycobacterium tuberculosis assay.
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The minimum inhibitory concentration (MIC) was determined using the standard broth micro dilution
method [44], where a 10 mL culture of Mycobacterium tuberculosis H37Rv [45], was grown to an
absorbance (OD600) of 0.6 – 0.7. The media used were Middlebrook 7H9 media (Difco)
supplemented with 0.2 % Glucose, Middlebrook albumin-dextrose- catalase (ADC) enrichment
(Difco) and 0.05 % Tween [46], as well as Middlebrook 7H9 supplemented with 0.03 % casitone, 0.4
% glucose, and 0.05 % tyloxapol [47]. Cultures grown in these media are diluted 1:500, prior to
inoculation of the MIC assay. The compounds to be tested were reconstituted to a concentration of 10
mM in DMSO. Duplicate two-fold serial dilutions of the test compound are prepared across a 96-well
micro titre plate, in a volume of 50 µL, after which, 50 µL of the diluted M. tuberculosis cultures is
added to each well in the plate (including control wells). The final volume per well is 100 µL. The
plate layout is a modification of the method previously described [48]. Controls used are a minimum
growth control (Rifampicin at 2 × MIC), and a maximum growth control (5% DMSO). The micro titre
plates are sealed in a secondary container and incubated at 37 °C with 5% CO2 and humidification.
Alamar Blue reagent is added to each well of the assay plate, 24 hours prior to the assay end data,
after which the assay is re-incubated for 24 hours. Relative fluorescence (excitation 485 nM; emission
520 nM) is measured using a plate reader (FLUOstar OPTIMA, BMG LABTECH), at day 7 and day
14. The raw fluorescence data was archived and analysed using the CDD Vault from Collaborative
Drug Discovery, in which, data was normalised to the minimum and maximum inhibition controls to
generate a dose response curve (% inhibition), using the Levenberg-Marquardt damped least squares
method, from which the MIC90 was calculated [49]. The lowest concentration of drug that inhibited
growth of more than 90 % of the mycobacterial population was considered to be the MIC90.
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Conflicts of interest
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There are no conflicts to declare.
Acknowledgements
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Financial support from the University of Cape Town and the National Research Foundation (NRF;
UID:111707) of South Africa is gratefully acknowledged. The University of Cape Town, South
African Medical Research Council, and South African Research Chairs Initiative of the Department of
Science and Technology, administered through the South African National Research Foundation are
gratefully acknowledged for support (K.C). We gratefully acknowledge Professor Orde Munro and Dr
Cathryn Slabber for their assistance with ROS studies.
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