Antimicrobial activity of organometallic isonicotinyl and pyrazinyl ferrocenyl-derived complexes

Article abstract of DOI:10.1039/c7dt01952a

Isonicotinyl and pyrazinyl ferrocenyl-derived complexes were prepared using various hydrazides and ferrocenyl aldehydes. Three heterobimetallic complexes were also synthesized from the Schiff base-derived isonicotinyl ferrocene complex using various platinum group metal dimers based on ruthenium, rhodium and iridium. All complexes were evaluated in vitro for antimycobacterial and antiparasitic activity. Against Mycobacterium tuberculosis H37Rv, the platinum group metal complexes showed glycerol-dependent antimycobacterial activity. The antiplasmodial activities against the NF54 chloroquine-sensitive strain of Plasmodium falciparum of some compounds were moderate, while some complexes also showed promising activity against Trichomonas vaginalis. Incorporation of the ferrocenyl-salicylaldimine moiety resulted in enhanced antimicrobial activity compared to the non-ferrocenyl compound in some cases. The bimetallic iridium-ferrocene isonicotinyl complex exhibited superior antitrichomonal activity relative to its organic counterpart, isoniazid. Furthermore, all these compounds, when screened on several normal flora bacteria of humans, showed no effect on the microbiome, emphasizing the selection of these compounds for these pathogens. The promising antimicrobial activities of the complexes thus supports incorporation of ferrocene as part of existing antimicrobial therapies in order to alter their biological activities favorably.

Full text of DOI:10.1039/c7dt01952a

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Seldon, N. Liu, D. Warner, C. Tam, L. Cheng, K. Land, P. Smith, K. Chibale and G. Smith, Dalton Trans.,
2017, DOI: 10.1039/C7DT01952A.
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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
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DOI: 10.1039/C7DT01952A
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ARTICLE
Antimicrobial activity of organometallic isonicotinyl and pyrazinyl
ferrocenyl-derived complexes
Tameryn Stringera, Ronnett Seldonb, Nicole Liuc, Digby F. Warnerd,e, Christina Tamf,
Luisa W. Chengf, Kirkwood M. Landc, Peter J. Smithg, Kelly Chibalea,b and Gregory S. Smitha,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Isonicotinyl and pyrazinyl ferrocenyl-derived complexes were prepared using various hydrazides and ferrocenyl aldehydes.
Three heterobimetallic complexes were also synthesized from the Schiff base-derived isonicotinyl ferrocene complex using
various platinum group metal dimers based on ruthenium, rhodium and iridium. All complexes were evaluated in vitro for
antimycobacterial and antiparasitic activity. Against Mycobacterium tuberculosis H37Rv, the platinum group metal
complexes showed glycerol-dependent antimycobacterial activity. The antiplasmodial activities against the NF54
chloroquine-sensitive strain of Plasmodium falciparum of some compounds were moderate, while some complexes also
showed promising activity against Trichomonas vaginalis. Incorporation of the ferrocenyl-salicylaldimine moiety resulted in
enhanced antimicrobial activity compared to the non-ferrocenyl compound in some cases. The bimetallic iridium-ferrocene
isonicotinyl complex exhibited superior antitrichomonal activity relative to its organic counterpart, isoniazid. Furthermore,
all these compounds, when screened on several normal flora bacteria of humans, showed no effect on the microbiome,
emphasizing the selection of these compounds for these pathogens. The promising antimicrobial activities of the complexes
thus supports incorporation of ferrocene as part of existing antimicrobial therapies in order to alter their biological activities
favorably.
www.rsc.org/
antiproliferative activity against various breast cancer cell lines. In
addition to this, ferrociphenols and hydroxypropyl ferrociphenols
show antiproliferative effects against triple negative breast cancer
Introduction
The development of organometallic compounds as potential
medicinal agents has been on the rise for the past decade.1-3
Although the field of bioorganometallic anticancer studies remains
one of the most prevalent research areas, recent advances in other
fields have also generated scientific interest.4 Ferrocene and
ferrocene-derived molecules have interesting biological properties.
For example, the replacement of an aromatic group in penicillin by
ferrocene resulted in a compound comparable in activity to benzyl
penicillin.5,6 Ferrocene-containing compounds also exhibit
antiproliferative activities.7-10 Hydroxyferrocifen shows promising
cells.11-14 The ferrocenyl fragment often brings about a metal-specific
mechanism of action. Since Fe(II) is redox active, the ferrocenyl
moiety is able to generate reactive oxygen species (ROS). In addition
to this, incorporation of ferrocene can alter the permeability of the
compound by altering the lipophilicity. Very recently, the
antiparasitic activity of metallocene and ferrocenyl-monepantel
derivatives were reported against Trichostrongylus colubriformis and
Dirofilaria immitis microfilariae.15,16 The versatility of the ferrocenyl
moiety in biological applications and the incorporation of ferrocene
aDepartment of Chemistry, University of Cape Town, Rondebosch, 7701, Cape Town,
South Africa
b Drug Discovery and Development Centre (H3-D), Department of Chemistry, University
of Cape Town, Rondebosch, 7701, South Africa
c Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA
d
MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of
Excellence for Biomedical TB Re-search, Department of Pathology, University of Cape
Town, Rondebosch, 7701, Cape Town, South Africa
e
Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch, 7701, Cape Town, South Africa
fFoodborne Toxins Detection and Prevention Research Unit, Agricultural Research
Service, United States Department of Agriculture, Albany, CA, 94710, USA
gDivision of Pharmacology, Department of Medicine, University of Cape Town, Medical
School, Observatory, 7925, Cape Town, South Africa
*G.S.S.: e-mail, gregory.smith@uct.ac.za; web, www.gregsmith-research.co.za; tel,
+27 21 650 5279.
Electronic Supplementary Information (ESI) available: [NMR and LC data].
This journal is © The Royal Society of Chemistry 20xx
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into organic molecules can introduce specific properties such as ROS Phase IIb clinical trials, having been reinitiated in 2V0ie1w5Airnticlae Onnelinwe
production, redox activation and lipophilicity. This supports the combination with artefenomel.31,32 Based oDnOtIh:i1s0,.1m03a9n/yC7qDuTin0o19li5n2eA-
incorporation of ferrocene for the development of new and more based ferrocenyl complexes have been evaluated extensively for
effective clinical agents as a viable approach, especially in the lesser- antiplasmodial activity and have shown promising activity.32-34 Again,
researched field of antimicrobial agents.
this supports the development of ferrocene-based complexes as
Tuberculosis (TB) is an infectious disease that has been under- potential antimicrobial agents. To the best of our knowledge,
explored in terms of potential metal-based therapies. The World ferrocene-based isoniazid/pyrazinyl complexes have not been
Health Organization (WHO) reported an estimated 10.4 million new investigated as potential antiplasmodial or antitrichomonal agents.
cases and 1.4 million deaths in 2015 worldwide.17 Moreover, the Herein, we report the synthesis, characterization and antimicrobial
increasing incidence of drug resistance has raised concerns about the activity of a small family of ferrocene-derived isoniazid and
treatment of this disease. In 2015, 480 000 cases of multidrug pyrazinamide complexes and three isonicotinyl half-sandwich
resistant (MDR)-TB were reported, which is defined as resistance to heterobimetallic complexes.
rifampicin and isoniazid.18 In order to circumvent drug resistance,
suitable therapeutic alternatives are required for treatment of this
disease. In this regard, ferrocenyl diamines (Figure 1a) were
Results and discussion
evaluated for antimycobacterial activity against the fully drug-
susceptible laboratory strain, M. tuberculosis H37Rv, and showed
potent activities in vitro.19 Based on this, ferrocenyl-containing
polyamines (Figure 1b) were prepared and evaluated for
antimycobacterial activity.20 Examples of ferrocene-conjugates of
front-line antimycobacterials have not been studied extensively.
Trichomoniasis is a parasitic disease that is caused by the parasite
Trichomonas vaginalis. The current treatment, metronidazole, has
shown decreased efficacy against some clinical isolates in
patients.21,22 As a consequence, there has been an interest in
obtaining suitable alternative therapies for this infection.22 Due to
the robust nature of ferrocene, its low toxicity and redox
properties,23,24 we have previously reported on the antitrichomonal
activity of ferrocenyl complexes (Figure 1c) that exhibit promising in
vitro activity against this protozoan.25-28
Synthesis and characterization
The preparation of the ferrocenyl complexes 1 – 6 firstly required the
synthesis of the antimycobacterial precursor compounds. Pyrazinoic
acid hydrazide (Scheme 1) was prepared from pyrazinoic acid using
published methods.35,36 Methyl pyrazinoate was prepared via an
acid-catalyzed esterification, using MeOH. The ester was then
treated with hydrazine hydrate to afford the desired hydrazide.
Ferrocenyl-containing aldehydes were used in the synthesis of 1 – 6
in order to investigate the effect of the aryl moiety on biological
activity. Ferrocenyl aldehydes (B and C, Scheme 1) were prepared
according to literature procedures.20,27 The preparation involves a
Heck cross-coupling reaction of vinylferrocene with either 4-
iodobenzaldehyde or 5-iodosalicylaldehyde giving rise to aldehydes
B and C, respectively. Pyrazinoic acid hydrazide and isoniazid were
then reacted with ferrocenecarboxaldehyde (A) and the aryl
ferrocenyl-aldehydes (B and C) via a Schiff base condensation
reaction (Scheme 2) to yield the ferrocenyl complexes (1 – 6) as
red/orange solids in low to moderate yields (32-56%). Complex 1 was
prepared as described in the literature.37 The complexes were
characterized by 1H and 13C{1H} Nuclear Magnetic Resonance (NMR)
and infrared (IR) spectroscopy, electron impact (EI) or electrospray
ionization (ESI) mass spectrometry and elemental analysis.
Fig. 1 Ferrocenyl complexes with demonstrated antimicrobial
activity.
Malaria is an infectious disease that in 2013 affected over 198
million people worldwide.29 The species of parasite responsible for
the most severe malaria infections is Plasmodium falciparum. As a
consequence of increased resistance of the parasite towards current
drug treatments, alternative therapies are required. Within the
context of malaria, the most promising ferrocene-based agent to
date is ferroquine (FQ, Figure 1d), which is active against chloroquine
(CQ) resistant strains of the causative parasite species and is now in
Scheme
1 Preparation of precursors: i) MeOH, H2SO4; ii)
NH2NH2.xH2O; iii) tri(p-tolyl)phosphine, Pd(OAc)2, Et3N, CH3CN, 24 h,
90 °C.
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Successful Schiff base condensation of 2-6 was confirmed by a singlet chiral nature of the ruthenium metal. Two doublets were observed
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for the imine proton in the region between 8.46 and 8.71 ppm in the for the protons of the isopropyl moiety. ThDeOpI:ro10to.1n0s39o/Cf 7tDheT0a1r9e5n2Ae
1H NMR spectra. Signals for the ferrocenyl Cp protons were observed moiety also appeared distinct, a consequence of the chiral metal
1
in the expected region between 4.14 and 4.77 ppm. The H NMR centre. These observations were consistent with similar chiral
spectra of complexes 2, 3, 5 and 6 showed two doublets for each ruthenium arene complexes.39-45 Since the mode of coordination was
proton of the olefinic moiety, has coupling constants ranging similar for the rhodium and iridium complexes, these complexes
between 14 and 16 Hz which suggested a trans geometry about the were also designated as chiral. Further evidence of this mode of
double bond, consistent with data obtained for similar coordination was seen in the infrared spectra. A strong absorption
structures.20,27,38 The infrared spectra of the ferrocenyl complexes band was observed at approximately 1590 cm-1 in each case. The
showed absorption bands between 1605 and 1633 cm-1 for the C=N disappearance of the C=O absorption band, suggested a change in
stretching frequencies. Absorption bands for the carbonyl stretching the tautomeric form of the ligand from the amide to the imidic form
frequencies were observed between 1660 and 1690 cm-1, confirming upon complexation, further supporting the proposed structures. The
the amido-form of complexes 2-6 in the solid state. A molecular ion ESI mass spectrum of 9 confirmed that 9 was prepared, as a base
peak was observed for each complex in the EI mass spectra peak for the protonated molecular ion was observed. EI mass
confirming the structural integrity of complexes 2 – 6.
spectrometry also confirms the integrity of the proposed structures,
owing to the observation of the molecular ion peak, in the mass
spectra of 7 and 8.
Scheme 3 Preparation of PGM complexes 7 – 9: (i) DCM, NaH, 1.5
hours, (ii) DCM, [MCl2R]2, 1.5 hours.
Antimycobacterial activity and cytotoxicity
The synthesized complexes (1 – 9) were screened for activity against
M. tuberculosis H37Rv in vitro. Ferrocenyl complexes of isoniazid and
the pyrazinoic acid hydrazide precursor were screened in order to
evaluate the effect of ferrocene on antimycobacterial activity. In
addition to this, half-sandwich (Ir, Rh and Ru) complexes of the
ferrocenyl-isoniazid conjugate (1) were prepared to determine the
effect on antimycobacterial activity by incorporating a second metal.
The data are presented in Table 1.
Scheme 2 Preparation of ferrocenyl complexes 1 – 6: Ferrocenyl
aldehyde (A, B or C: 1 eq.), EtOH, reflux, 16 hours.
All of the compounds were screened against M. tuberculosis
H37Rv::gfp in two different growth media – the glycerol-based GAST-
Fe (which does not contain bovine serum albumin) and a glucose-
based Middlebrook 7H9-ADC (which does contain BSA supplement) -
to elucidate any condition-selective antimycobacterial activity. The
majority of the compounds tested exhibited potent activity in GAST-
Fe medium, giving minimum inhibitory concentration (MIC90 and
MIC99) values lower than 1 µM on day 14. Isoniazid exhibited similar
activity profiles in both media. Complex 2 exhibited lowered activity
compared to complex 1 in both media. Isoniazid complexes 2 and 3
showed reduced activity in both growth media compared to isoniazid
itself and complex 1, perhaps as a consequence of higher lipophilicity
(Table 2). In most cases, especially for PGM complexes (7 – 9), there
appeared to be a glycerol-dependent inhibitory activity as the MICs
increased when the glucose-based 7H9 medium was used. This was
Half-sandwich complexes, specifically ruthenium compounds have
generated interest owing to their lower toxicity compared to
platinum-based drugs and their suitability as alternatives for
chemotherapy.1 The half-sandwich complexes (7-9) were prepared
by reacting ferrocenyl complex 1 with either [IrCp*Cl2]2, [RhCp*Cl2]2
or [Ru(p-cymene)Cl2]2, in the presence of NaH (Scheme 3) to yield the
complexes as orange solids in high yields (>80%). The 1H NMR spectra
of 7–9 suggests bidentate coordination of the ligand due to the
disappearance of the amido proton, which confirms its
deprotonation in the presence of the base and coordination via the
deprotonated imidic form of the ligand. In the case of complex 9,
further evidence of bidentate coordination as attested to by the
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also observed for complex 1. This profile was analogous to the Selected compounds were also screened in vitro for mammalian
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DOI: 10.1039/C7DT01952A
glycerol-dependent control, and suggested that the PGM complexes cytotoxicity against Chinese Hamster Ovarian (CHO) cells, using
and complex 1, may exert their inhibitory effects by disrupting emetine as the control. All of the complexes exhibited lower toxicity
glycerol metabolism, resulting in toxic accumulation of intermediate than the control (IC50 = 0.057 µM) at the tested concentration. This
metabolites.46-50 Selected complexes (1, 2 and 8) were tested against indicated selectivity of the compounds toward M. tuberculosis, with
the M. tuberculosis cytochrome bd oxidase deletion mutant, ΔcydKO two of the isoniazid-ferrocenyl complexes (2 and 3) exhibiting only
(Table 1), to establish whether cytochrome bc1 oxidase was a mild toxicity (IC50 > 20 µM). However, as noted above, further insight
potential cellular target. No hypersensitivity was observed towards is required into the mechanism of action of these complexes which
ΔcydKO. In addition to this, a ΔcydKO deletion mutant carrying an appear to exhibit growth medium-dependent inhibition.
Ala317Thr point mutation in qcrB51 was also screened using the
GAST/Fe assay. No significant differences in the MIC values were
observed between the two mutants (unlike the control qcrB
inhibitor), suggesting that these compounds may not target the qcrB
subunit of the respiratory bc1 complex in M. tuberculosis.
Compounds that exhibit significant differences between the MIC
values of ΔcydKO and qcrBA317T, are believed to target this
pathway.52
Table 1 Activity of complexes 1-9 against M. tuberculosis H37Rv and cytotoxicity against CHO cells
Alamar Blue
Assay: MIC99
(µM): ∆cydKO
Gaste Fe
Alamar Blue
Assay: MIC99
(µM): qcrBA317T
Gaste Fe
MTT Assay
Cytotoxicity
CHO cells:
IC50 (µM)
MIC90 (µM)
[Media:
Gaste Fe]
MIC99 (µM)
[Media:
Gaste Fe]
MIC90 (µM)
[Media: 7H9 [Media: 7H9
GLU ADC]
MIC99 (µM)
Complex
GLU ADC]
0.39
> 10
0.474
> 10
7.29
> 10
> 10
> 10
1
0.976
3.906
n.d.b
n.d.
0.976
1.953
n.d.
n.a.c
> 20
> 20
n.a.
n.a.
n.d.
n.d.
n.a.
n.d.
n.a.
n.d.
n.d.
2
41.3
> 125
120
> 125
> 125
> 125
> 125
> 125
> 10
> 125
> 125
> 125
> 125
> 125
> 10
3
45.6
4
n.d.
> 125
> 125
0.416
0.968
0.514
< 0.244
0.00811
> 125
> 125
0.6
5
n.d.
n.d.
6
n.d.
n.d.
7
8
n.d.
n.d.
1.47
0.976
n.d.
3.906
n.d.
0.751
< 0.244
0.0295
1.19
> 125
0.856
0.00459
> 125
1.26
9
isoniazid
rifampicin
GDCa
n.d.
n.d.
0.0227
> 125
0.009
n.d.
0.009
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.057 ±
0.004
emetine
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
qcrb
inhibitor
< 0.204
15.625
n.d.
aGlycerol-dependent control
bnot determined
cnot active at the highest concentration tested (100 ug mL-1)
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Table 2 Predicted logP values for 1 – 6 and isoniazid
ARTICLE
Complex 7, the iridium Cp* complex, exhibited less pVoiteewnAtrtipclaerOansliitnee
DOI: 10.1039/C7DT01952A
ClogPa
1.75
4.59
4.22
0.73
3.57
3.21
n.d.b
n.d.
growth inhibition at 80.4%, but this was higher than the rhodium and
Compound
ruthenium counterparts. Compounds 6 and 7 exhibited promising
inhibition, but were not comparable to metronidazole at the same
test concentration (data not shown - 100% inhibition). In addition,
the water-soluble isoniazid demonstrated very low inhibition along
1
2
3
4
with complexes
1 and 4. The two benzylaldimine-ferrocenyl
5
complexes (2 and 5) exhibited moderate activity, while the
pyrazinamide salicylaldimine-ferrocenyl complex 6 possessed much
greater activity than its isonicotinyl derivative (3). This further
supported the use of more lipophilic complexes as antitrichomonal
agents, an observation consistent with data obtained in previous
studies.25-28 The IC50 values of complex 6 and 7 were elucidated and
are given in Table 3. The complexes were far less active than
metronidazole at the tested concentration, but could be modified
structurally in future to afford suitable leads.
6
7
8
9
n.d.
Isoniazid
-0.69
a logP values predicted using MarvinSketch V5.9.4
according to the method described in reference 25 (values do
not take into account possible intramolecular H-bonding)
bnot determined
Table 3 IC50 values of compounds 6, 7 and metronidazole
against the G3 strain of T. vaginalis.
Compound
IC50 (µM)a
Antitrichomonal activity
6
10.41
Metronidazole has been the preferred drug used to treat
trichomoniasis, since its introduction in 1960.53 Although resistance
to metronidazole has been reported, the drug is still effective,
successfully treating more than 90% of infections. However, it does
not cure all patients, and resistance is on the rise.54 The
antitrichomonal activities of complexes (1 – 9) were evaluated
against the G3 isolate of T. vaginalis at a 50 µM drug concentration.
The results are presented in Figure 2. Given that T. vaginalis is a
mucosal pathogen that exists within a complex microbiome,
7
14.27
metronidazole
a IC50 represents the micromolar equivalents required to
inhibit parasite growth by 50%.
0.72
Antiplasmodial activity and β-Haematin inhibition
The antiplasmodial activity of compounds 1 – 9 along with isoniazid
screening of all of these compounds on several common normal flora were evaluated in vitro against the chloroquine-sensitive (CQS) P.
falciparum NF54 strain using the pLDH (Plasmodium lactate
dehydrogenase) assay. CQ was used as the reference drug in each
case and the data obtained from this study is summarized in Table 4.
bacteria found within the human microbiome at the highest
concentration did not show any effect.
100
80
Table 4 Antiplasmodial activity of compounds 1 – 9, isoniazid and CQ
against the NF54 CQS strain of P. falciparum.
Compound
IC50 ± SE (µM)
60
1
> 100
40
2
31.52 ± 6.41
1.58 ± 0.01
> 100
20
3
0
-20
4
1
1
2
3
4
5
6
7
8
9
ISO
5
10.36 ± 2.81
3.77 ± 0.86
5.00 ± 0.39
2.99 ± 0.75
7.82 ± 0.99
n.a.
6
Figure 2 Antitrichomonal activity of complexes (1 – 9) and isoniazid
against T. vaginalis.
7
8
9
All of the tested compounds, with the exception of 6 and 7, displayed
moderate to low parasite growth inhibition. The ferrocenyl-
pyrazinamide complex 6 possessed the highest growth inhibitory
activity of all of the tested compounds resulting in 88.2% inhibition.
Isoniazid
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chloroquine
0.0149 ± 0.001
enhancement in β-haematin inhibition. In fact, incorporation of the
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second PGM metal, further enhanced the abiDliOtyI:o1f0t.1h0e39c/oCm7DpTo0u1n9d52tAo
inhibit β-haematin formation. From the data, it can be proposed that
the antiplasmodial activity of some of the complexes may be related
to their ability to target haemozoin in the digestive vacuole of the
parasite.
a not active at the tested concentration
All of the tested compounds exhibited lower activity compared to
CQ, in most cases being more than 100-fold less active. Comparing
the free ferrocenyl ligands (1 – 6), the isoniazid complex 3 exhibited
the highest activity in the series as well as overall. Both complexes
derived from ferrocenecarboxaldehyde (1 and 4) showed low activity
with IC50 values greater than 100 µM. This is possibly a consequence
of the lower lipophilicity of these complexes compared to the rest of
the compounds. For the isonicotinyl and pyrazinyl moieties, the
isonicotinyl ligand 3 appeared to display enhanced activity compared
to the pyrazinyl ligand 6. Also, the addition of the salicylaldimine-
ferrocenyl moiety seemed to be beneficial for antiplasmodial
activity. This was evident in the lower activity of 2 and 5 compared
with 3 and 6, respectively. For the heterobimetallic complexes (7 –
9), the Rh-Cp* complex (8) displayed enhanced activity compared to
the ruthenium (9) and iridium (7) derivatives. Most of the complexes
exhibited higher activity compared to isoniazid at the tested
5
4
3
8
4
3
2
1
0
iso
CQ
0
1
2
3
log[concentration]
concentration, which suggested
a
positive influence on
antiplasmodial activity when ferrocene as well as a second metal was
incorporated. There did not appear to be any significant trend with
respect to antiplasmodial activity in general for complexes 1 – 6. The
weak cytotoxicity in CHO cells indicated that the activity of the
complexes may not be due to general toxicity but rather to a specific
Plasmodium falciparum-based mechanism, which was further
investigated.
Figure 3 β-Haematin inhibition studies of selected compounds (3, 4,
8, isoniazid and CQ).
Experimental
Materials. Synthetic procedures were performed under an argon
atmosphere at ambient temperatures unless otherwise stated. All
reagents were purchased from Sigma Aldrich and used as received.
Solvents were dried over Fluka Molecular Sieves with indicator.
Pyrazinoic acid hydrazide,35,36 methyl pyrazinoate,35,36 the ferrocenyl
aldehydes20,27and compound 137 were prepared according to
literature methods.
The ability of selected compounds (3, 4, 8 and isoniazid) to inhibit
β-haematin formation was examined using a detergent-mediated
assay55 to gauge some idea of a possible mechanism of action,
despite possessing relatively low antiplasmodial activity. Many
quinoline-based compounds are believed to target haemozoin
formation in the parasite and therefore it would be worthwhile to
investigate whether these isoniazid and pyrazinamide-based
complexes also act by the same mechanism. β-Haematin is a
synthetic form of haemozoin and is the target of many quinoline-
containing compounds like CQ.56,57 Haemozoin crystal growth
inhibition occurs in the digestive vacuole of the parasite. Quinolines
can bind to haematin (ferriprotoporphyrin IX), a toxic by-product of
haemoglobin degradation which prevents conversion into
haemozoin, which is less toxic. The parasite then dies owing to a
build-up of ferriprotoporphyrin IX.56,57 β-Haematin formation can be
monitored using the NP-40 detergent-mediated assay. NP-40, a
neutral detergent, is used to resemble lipids and brings about β-
haematin formation in the assay. Haemozoin formation is not
spontaneous and it is believed that haem crystallisation occurs in the
presence of neutral lipids inside of the digestive vacuole of the
parasite.58,59
Instrumentation and Methods. Nuclear magnetic resonance
(NMR) spectra were recorded on a Varian Unity XR400 spectrometer
(1H: 399.95 MHz, 13C{1H}: 100.58 MHz), Varian Mercury XR300
spectrometer (1H: 300.08 MHz, 13C{1H}: 75.46 MHz) or Bruker
Ultrashield 400 Plus spectrometer (1H: 400.20 MHz, 13C{1H}: 100.60
MHz) at 30.0°C using tetramethylsilane (TMS) as the internal
standard. Infrared (IR) absorptions were measured on a PerkinElmer
Spectrum One FT-IR spectrometer and samples were analysed using
attenuated total reflectance (ATR). EI or ESI-mass spectrometry was
used to further characterize all new compounds and determinations
were carried out using a JEOL GCmateII mass or a Waters API Quattro
instrument in the positive mode, respectively. Purity was checked
using an analytical Agilent HPLC 1260 equipped with an Agilent DAD
1260 UV/vis detector and a X Bridge C18 column (2.5 mM, 50 mm X
3 mm) or a Agilent HPLC 1220 using a Kinetex C18 column. 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-1. The gradient elution conditions were as follows: 10%
solvent B between 0 and 1 min, 10-95% solvent B between 1 and 3
min, 95% solvent B between 3 and 5 min. In some cases, isocratic
conditions were used ACN:H2O (50%). Elemental analyses were
carried out using a Fisions EA 110 elemental analyser.
The results of the β-haematin inhibition assay are shown in Figure
3. Interestingly, isoniazid did not inhibit β-haematin formation at the
highest tested concentration (500 µM). This was consistent with the
anitiplasmodial activity data as isoniazid did not exhibit any
appreciable antiplasmodial activity. Complex 8 exhibited the highest
inhibition of β-haematin formation of the tested compounds,
followed by complexes 4 and 3. It was clearly evident that
incorporating ferrocene into these systems resulted in an
6 | J. Name., 2012, 00, 1-3
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Synthesis. General procedure for compounds 2-6. To a stirred 32%). H NMR (300.08 MHz, CDCl3): (δ, ppm) 4.21 (5VHie,ws,ArCtipcl)e;O4n.l3in7e
DOI: 10.1039/C7DT01952A
solution of the appropriate hydrazide (1 eq.) in EtOH, the appropriate (2H, s, Cp); 4.55 (2H, s, Cp); 6.57 (1H, d, J = 14.40, C=CH); 6.74 (1H, d,
ferrocenyl aldehyde (1 eq.) was added. The resulting solution was J = 14.04, C=CH); 7.04 (1H, d, J = 8.52, ArH); 7.28 (1H, s, ArH); 7.45
refluxed for 24 hours, yielding (after cooling)
precipitate. The precipitate was then filtered, washed with ethanol, 9.53 (1H, s, ArH); 10.65 (1H, br s, NH); 10.93 (1H, s, OH). 13C{1H} NMR
followed by n-pentane and dried in vacuo. (100.64 MHz, CDCl3): (δ, ppm) 66.8, 69.0, 69.4, 83.9, 117.2, 117.8,
a red/orange (1H, d, J = 8.43, ArH); 8.62 (2H, br s, ArH, HC=N); 8.87 (1H, s, ArH);
Compound 2. Isonicotinylhydrazide (47.2 mg, 0.344 mmol) and 124.9, 125.4, 128.6, 129.6, 129.8, 142.5, 143.5, 144.9, 148.1, 152.3,
ferrocenylbenzaldehyde (B) (106 mg, 0.357 mmol) were reacted and 157.9, 158.4. IR (ATR): (ʋ, cm-1) 1690, 1614, 1584. Microanalysis
yielded the product as a red amorphous solid (83.4 mg, 56%). 1H NMR calculated for C24H20N4O2Fe.0.5H2O: C, 62.49; H, 4.59; found C, 62.87;
(300.08 MHz, DMSO-d6): (δ, ppm) 4.17 (5H, s, Cp); 4.36 (2H, br s, Cp); H, 4.55. EI-MS: m/z 452 ([M]+, C24H20N4O2Fe).
4.61 (2H, br s, Cp); 6.83 (1H, d, J = 16, C=CH); 7.13 (1H, d, J = 16,
General procedure for compounds 7-9. To a stirred solution of the
C=CH); 7.59 (2H, d, J = 8, ArH); 7.72 (2H, d, J = 8, ArH); 7.84 (2H, d, J = ferrocenyl isoniazid ligand (1) (2 eq.) in DCM, excess NaH (60%
5, ArH); 8.46 (1H, s, HC=N); 8.79 (2H, d, J = 5, ArH); 12.06 (1H, s, NH). dispersion in mineral oil) was added and the mixture stirred at room
13C{1H} NMR (100.64 MHz, DMSO-d6): (δ, ppm) 67.5; 69.5; 69.7; 83.3; temperature for 1.5 hours. After this time, the appropriate metal
121.9; 125.4; 126.5; 128.2; 129.4; 132.7; 140.2; 141.0; 149.3; 150.8; dimer (1 eq.) was added. The mixture was stirred for another 1.5
162.01. IR (ATR): (ʋ, cm-1) 1663; 1605; 1592. Microanalysis calculated hours at room temperature. Approximately 10 mL of MeOH was
for C25H21N3OFe.1H2O: C, 64.95; H, 5.23; found C, 65.17; H, 4.81. EI- slowly added and the mixture stirred for an additional 15 minutes.
MS: m/z 435 ([M]+, C25H21N3OFe).
After this time the mixture was filtered, the filtrate collected and the
Compound 3. Isonicotinyl hydrazide (43.4 mg, 0.316 mmol) and solvent was removed in vacuo to give a red residue. Diethyl ether was
ferrocenylsalicylaldehyde (C) (109 mg, 0.328 mmol) were reacted then added to extract the product and the solution was then filtered
and yielded the product as a red amorphous solid (48.7 mg, 34%). 1H to remove any unreacted starting materials. The filtrate was
NMR (300.08 MHz, DMSO-d6): (δ, ppm) 4.14 (5H, s, Cp); 4.30 (2H, t, J collected, the solvent removed in vacuo and the residue washed with
= 2, Cp); 4.56 (2H, t, J = 2, Cp); 6.75 (1H, d, J = 16, C=CH); 6.85 (1H, d, petroleum ether. The sample was then dried under vacuum to yield
J = 16, C=CH); 6.93 (1H, d, J = 8, ArH); 7.49 (1H, dd, J = 8, J = 2, ArH); the desired complexes as bright orange powders.
7.72 (1H, d, J = 2, ArH); 7.86 (2H, d, J = 6, ArH); 8.71 (1H, s, HC=N);
Compound 7. Ferrocenyl isoniazid ligand (1) (20.5 mg, 0.0615
8.81 (1H, d, J = 6, ArH); 11.09 (1H, s, OH); 12.28 (1H, s, NH). 13C{1H} mmol), NaH (12.0 mg, 0.500 mmol) and [Ir(Cp*)Cl2]2 (25.0 mg, 0.0314
NMR (100.64 MHz, DMSO-d6): (δ, ppm) 66.9, 69.1, 69.4, 84.1, 117.4, mmol) were reacted to yield the desired product (35.1 mg, 82%). 1H
119.4, 121.9, 125.3, 125.5, 126.5, 129.4, 129.8, 140.6, 149.0, 150.9, NMR (300.08 MHz, CDCl3): (δ, ppm) 1.52 (15H, s, Cp*); 4.32 (5H, s,
157.0, 161.9. IR (ATR): (ʋ, cm-1) 1652, 1619, 1607, 1584. Cp); 4.54 (2H, t, J = 1.83, Cp); 4.58-4.60 (1H, m, Cp); 5.90 (1H, br s,
Microanalysis calculated for: C25H21N3O2Fe.0.5H2O C, 65.23; H, 4.82; Cp); 7.98 – 8.01 (2H, m, ArH); 8.65 – 8.69 (3H, m, ArH, HC=N). 13C{1H}
found C, 65.43; H, 4.64. EI-MS: m/z 451 ([M]+, C25H21N3O2Fe).
NMR (100.64 MHz, CDCl3): (δ, ppm) 9.00; 70.12; 70.44; 71.36; 72.15;
Compound 4. Pyrazinyl hydrazide (110 mg, 0.793 mmol) and 73.06; 77.88; 85.55; 122.75; 139.17; 149.86; 158.81; 171.64. IR (ATR):
ferrocenecarboxaldehyde (A) (178 mg, 0.832 mmol) were reacted (ʋ, cm-1) 1591. Microanalysis calculated for C27H29N3OClFeIr: C,
and yielded the product as an orange amorphous solid (137 mg, 46.66; H, 4.21; found C, 46.85; H, 4.38. EI-MS: m/z 695 ([M]+,
1
52%). H NMR (300.08 MHz, CDCl3): (δ, ppm) 4.25 (5H, s, Cp); 4.48 C27H29N3OClFeIr).
(2H, s, Cp); 4.77 (2H, s, Cp); 8.32 (1H, s, ArH); 8.57 (1H, s, HC=N); 8.83
Compound 8. Ferrocenyl isoniazid ligand (1) (30.3 mg, 0.0909
(1H, s, ArH); 9.53 (1H, s, ArH); 10.48 (1H, s, NH).13C{1H} NMR (100.64 mmol), NaH (12.2 mg, 0.508 mmol) and [Rh(Cp*)Cl2]2 (28.6 mg,
MHz, CDCl3): (δ, ppm) 68.5, 69.4, 70.9, 142.4, 144.2, 144.9, 147.6, 0.0463 mmol) were reacted to yield the desired product (47.1 mg,
151.9, 158.2. IR (ATR): (ʋ, cm-1) 1684, 1611, 1580. Microanalysis 86%). 1H NMR (300.08 MHz, CDCl3): (δ, ppm) 1.53 (15H, s, Cp*); 4.32
calculated for C16H14N4OFe: C, 57.51; H, 4.22; found C, 57.16; H, 3.97. (5H, s, Cp); 4.51-4.57 (2H, m, Cp); 4.59 (1H, br s, Cp); 6.04 (1H, br s,
EI-MS: m/z 334 ([M]+, C16H14N4OFe).
Cp); 7.94 – 8.03 (2H, m, ArH); 8.60 – 8.72 (3H, m, ArH, HC=N). 13C{1H}
Compound 5. Pyrazinyl hydrazide (35.3 mg, 0.256 mmol) and NMR (100.64 MHz, CDCl3): (δ, ppm) 9.0; 69.9; 70.1; 71.3; 71.9; 72.7;
ferrocenylbenzaldehyde (B) (84.9 mg, 0.268 mmol) were reacted and 78.2; 93.9 (d, J = 73); 122.6; 140.9; 149.7; 159.3; 169.6. IR (ATR): (ʋ,
yielded the product as an orange amorphous solid (57.1 mg, 51%). 1H cm-1) 1591. Microanalysis calculated for C27H29N3OClFeRh: C, 53.54;
NMR (300.08 MHz, DMSO-d6): (δ, ppm) 4.17 (5H, s, Cp); 4.36 (2H, t, J H, 4.83; found C, 53.62; H, 4.32. EI-MS: m/z 605 ([M]+,
= 2, Cp); 4.61 (2H, t, J = 2, Cp); 6.82 (1H, d, J = 16, C=CH); 7.12 (1H, d, C27H29N3OClFeRh).
J = 16, C=CH); 7.59 (2H, d, J = 8, ArH); 7.70 (2H, d, J = 8, ArH); 8.63 Compound 9. Ferrocenyl isoniazid ligand (1) (29.3 mg, 0.0879 mmol),
(1H, s, HC=N); 8.80 (1H, m, ArH); 8.94 (1H, d, J = 2, ArH); 9.28 (1H, d, NaH (12.0 mg, 0.500 mmol) and [Ru(p-cymene)Cl2]2 (26.9 mg, 0.0440
J = 1, ArH); 12.25 (1H, s, NH). 13C{1H} NMR (100.64 MHz, DMSO-d6): mmol) were reacted to yield the desired product (43.8 mg, 83%). 1H
(δ, ppm) 67.5; 69.5; 69.7; 83.3; 125.4; 126.5; 128.2; 129.4; 132.8; NMR (300.08 MHz, CDCl3): (δ, ppm) 1.13 (3H, d, J = 4, CH3); 1.16 (3H,
140.2; 143.8; 144.6; 145.2; 148.3; 150.1; 159.9. IR (ATR): (ʋ, cm-1) d, J = 4, CH3); 2.32 (3H, s, CH3); 2.61 (1H, sept, J = 7, CH); 4.31 (5H, s,
1682, 1663, 1633, 1592. Microanalysis calculated for Cp); 4.55-4.61 (2H, m, Cp); 4.67-4.69 (1H, m, Cp); 4.85 (1H, d, J = 6, p-
C24H20N4OFe.0.3H2O: C, 65.26; H, 4.70; found C, 65.60; H, 4.60. EI-MS: cy); 5.10 (1H, d, J = 6, p-cy); 5.40-5.45 (2H, m, p-cy); 5.77 (1H, br s,
m/z 436 ([M]+, C24H20N4OFe).
Cp); 7.93 (2H, d, J = 6, ArH); 8.64 (2H, d, J = 5, ArH); 8.75 (1H, s, HC=N).
Compound 6. Pyrazinyl hydrazide (16.8 mg, 0.121 mmol) and 13C{1H} NMR (100.64 MHz, CDCl3): (δ, ppm) 18.9; 21.9; 22.6; 30.9;
ferrocenylsalicylaldehyde (C) (40.3 mg, 0.121 mmol) were reacted 69.9; 70.2; 70.9; 711.9; 72.6; 78.9; 80.9; 81.3; 82.1; 84.4; 101.2;
and yielded the product as an orange amorphous solid (17.5 mg, 101.6; 122.6; 139.8; 149.7; 160.4; 170.9. IR (ATR): (ʋ, cm-1) 1590.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Microanalysis calculated for C27H28N3OFeRuCl.0.5Et2O: C, 54.43; H, was measured on day 14 by adding 10 µL Alamar Blue (Celtic
View Article Online
5.20; found C, 54.21; H, 5.42. HR-ESI-MS: m/z 604.0394 ([M+H]+, Diagnostics) to all wells followed by incubation of the plates at 37 °C
DOI: 10.1039/C7DT01952A
[C27H28N3OFeRuCl+H]+, requires 604.0392).
for 24 hr. Wells were then viewed for a colour change (blue indicated
no growth as opposed to pink which suggested bacterial growth). The
MIC values were defined as the lowest drug concentration where a
In vitro antimycobacterial activity.
GAST/Fe MIC determinations. The broth microdilution method60 colour change was not observed.66
allows a range of concentrations to be tested using a single 96-well
In vitro antiplasmodial assay. The samples were tested in triplicate
plate to determine the minimum inhibitory concentration (MIC). A on one experiment against the chloroquine-sensitive NF54 strain and
10 mL culture of Mycobacterium tuberculosis (H37RvMa)61 was chloroquine-resistant K1 strain of P. falciparum. Continuous in vitro
grown to an OD600 of 0.6 - 0.7. The culture was then diluted 1:100 cultures of asexual erythrocyte stages of P. falciparum were
in GAST/Fe medium. In a 96-well microtitre plate, 50 μL of GAST/Fe maintained using a modified method of Trager and Jensen.67 The
medium was added to all wells in rows 2-12. The compounds were antiplasmodial activity in vitro was determined via the parasite
added to row 1 in duplicate, at a final concentration of 640 μM lactate dehydrogenase assay using a modified method described by
(stocks are made up to a concentration of 10 mM in DMSO, and Makler et al.68 The samples were prepared as a 2 mg mL-1 stock
diluted to 640 μM in GAST/Fe medium). A two-fold serial dilution was solution using DMSO and sonicated to enhance solubility. Samples
prepared by transferring 50 μL of the liquid in row 1 to row 2 and was were tested as a suspension if not completely dissolved. Stock
aspirated to mix. 50 μL of the liquid in row 2 was then transferred to solutions were stored at 20 °C. Further dilutions were prepared on
row 3 and aspirated, etc. This procedure was repeated until row 12 the day of the experiment. Chloroquine was used as the reference
was reached, from which 50 μL of the liquid was discarded so as to drug in all experiments. A full dose-response measurement was
bring the final volume in all wells to 50 μL. Finally, 50 μL of the 1:100 performed for all compounds to determine the concentration
diluted M. tuberculosis culture was added to all wells in rows 2-12. inhibiting 50% of parasite growth (IC50 value). The samples were
Cells were not added to row 1, as this served as a contamination tested at a starting concentration of 10 000 ng mL-1, which was then
control. Controls included media only, 5% DMSO, and Rifampicin. serially diluted 2-fold in complete medium to give 10 concentrations.
The microtitre plate was stored in a secondary container and The same dilution technique was used for all samples. The highest
incubated at 37 °C with a humidifier to prevent evaporation of the concentration of solvent to which the parasites were exposed to had
liquid. The lowest concentration of drug that inhibits growth of more no measurable effect on the parasite viability. The IC50 values were
than 90% of the bacterial population is considered to be the MIC90. obtained using a nonlinear dose-response curve fitting analysis via
MIC90 and MIC99 values were scored visually at 14-days post Graph Pad Prism v.5.0 software.
inoculation.
In vitro antitrichomonal assay. Cultures of T. vaginalis G3 strain
GFP assay and activity. The minimum inhibitory concentration were grown in 5 mL complete TYM Diamond's media in a 37 °C
(MIC) of compounds was determined in either 7H9 (Difco) medium incubator for 24 h. 50 mM stocks of the compounds were made by
supplemented with 0.05% Tween-80, 0.2% glycerol and dissolving in DMSO and were screened against G3 stain of T.
albumin/NaCl/ glucose (ADC) complex, and/or in GAST-Fe (glycerol– vaginalis. Cells untreated and inoculated with 5 ml DMSO are used
alanine–salts) medium pH 6.6, supplemented with 0.05% Tween- as controls. 5 mL of 50 mM stocks of the compound library were
80.62 Compounds were dissolved in DMSO to
concentration of 10 mM. MICs against H37RvMa, ΔcydKO and calculated based on counts utilizing a haemocytometer after 24 h.
ΔcydKO/A317T mutants51 were determined using the standard broth
In vitro normal flora screening assay. Non-pathogenic normal
a final stock inoculated for a final concentration of 50 mM. Results were
microdilution method described previously.63 MICs against the flora strains such as Lactobacillus reuteri (ATCC 23272, Lactobacillus
Mtb::gfp strain (H37RvMa::pMSP12GFP)64 were determined as acidophilus (ATCC 43560), and Lactobacillus rhamnosus (ATCC
described previously,65 with some modifications. Briefly, a two-fold 53103) were grown in Lactobacilli MRS at 37 °C under anaerobic
serial dilution of the compound was carried out across a 96-well plate conditions. 100 mM stock compounds as well as vehicle control
from row 2-11 (20-0.039 μM) in 7H9 or GAST-Fe with a final DMSO DMSO were diluted to 100 mM in media and incubated with empty
concentration of 5% v/v. The minimum growth control (rifampicin at BDL-sensi-discs (6 mm) for 20 min at room temperature. Discs
2xMIC) was added to row 12 and the maximum growth control (5% containing vehicle control, compounds, or various antibiotic discs
DMSO in 7H9 or GAST-Fe) was added to row 1. A Mtb::gfp stock [levofloxacin (5 µg), gentamicin (10 µg), and gentamicin (120 µg)]
culture was grown to an OD600-0.6, diluted (1:500 for 7H9 or 1:100 were placed onto the bacterial streaked agar plates and incubated
for GAST/Fe) and 50 μL added to each well. Plates were incubated at overnight at 37 °C. Vehicle, compound, or antibiotic sensitivity was
37 °C and fluorescence (excitation 485 nM; emission 520 nM) was determined via measurement of zones of inhibition around each disc
measured using a plate reader (FLUOstar OPTIMA, BMG LABTECH) at in mm.
day 14. Data were normalised to the minimum and maximum
inhibition controls to generate a dose response curve (% inhibition) cytotoxicity against a mammalian cell-line, Chinese Hamster Ovarian
from which the MIC values were calculated. (CHO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
In vitro cytotoxicity. Test samples were screened for in vitro
The microplate Alamar Blue Assay. This assay was used for MIC bromide (MTT)-assay. The MTT-assay is used as a colorimetric assay
determination of clinical ΔcydKO and qcrBA317T mutant M. for cellular growth and survival, and compares well with other
tuberculosis isolates. The broth microdilution method was adapted available assays.69,70 The tetrazolium salt MTT was used to measure
to determine the MICs of selected compounds against clinical strains all growth and chemosensitivity. The tetrazolium ring is cleaved in
of M. tuberculosis in GAST-Fe medium. The activity of the compounds active mitochondria. Thus only viable cells are able to reduce the
8 | J. Name., 2012, 00, 1-3
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water-soluble yellow colored MTT to water-insoluble purple colored the antiplasmodial activity compared to the organic analogue
View Article Online
formazan. Formazan crystals are dissolved in dimethyl sulfoxide (isoniazid). Complexes with higher lipophiliciDtyOIa: l1s0o.1s0h3o9/wC7eDnTh0a1n9c5e2dA
(DMSO).The test samples were tested in triplicate on one occasion. antiplasmodial activity. Selected compounds do exhibit the ability to
The test samples were prepared to a 20 mg mL-1 stock solution in inhibit synthetic haemozoin formation, which may be a contributing
100% DMSO. Stock solutions were stored at 20 °C. Further dilutions mechanism to the antiplasmoidal effects. The same phenomenon is
were prepared in complete medium on the day of the experiment. observed for the antitrichomonal activity. Only two compounds
Samples were tested as a suspension if not completely dissolved. exhibited parasite growth inhibition above 80%. From this, it can be
Emetine was used as the reference drug in all experiments. The initial concluded that these complexes show promising activity as potential
concentration of emetine was 100 µg mL-1, which was diluted in antimycobacterial agents. Further work to elucidate a mechanism of
complete medium with 10-fold dilutions to give 6 concentrations, the action of these complexes and the role that the ferrocenyl moiety
lowest being 0.001 µg mL-1. The same dilution technique was applied plays is thus required. In addition to this, optimization of the
to all test samples. The highest concentration of solvent to which the antimycobacterial activity in various media is also required.
cells were exposed to had no measurable effect on the cell viability.
The 50% inhibitory concentration (IC50) values were obtained from
full dose–response curves, using a non-linear dose–response curve
Acknowledgements
fitting analysis via GraphPad Prism v.5 software.
Financial support from the University of Cape Town, the
National Research Foundation (NRF; Grant Number: 90500) and
the South African Medical Research Council (MRC) is gratefully
acknowledged. Professor Timothy J. Egan (UCT) is gratefully
acknowledged for his assistance with the β-haematin inhibition
assay. CT and LWC were funded by the U. S. Department of
Agriculture, Agricultural Research Service (National Program
108, Project CRIS 5325-42000-049-00D). K.M.L. was supported
by the Department of Biological Sciences, the University of the
Pacific.
β-Haematin inhibition assay. The β-haematin assay was adapted
from the method described by Wright and co-workers.55 Compounds
were prepared as a 10 mM stock solution in 100% DMSO. Samples
were tested at a starting concentration of 500 μM and the lowest
drug concentration was 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 hemin (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 compounds
were analysed using the pyridineferrochrome method developed by
Ncokazi and Egan.71 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.
Notes
Supporting Information is available for NMR spectra and LC data
(PDF). The authors declare no competing financial interest.
References
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Conclusions
A small family of isonicotinyl and pyrazinamide-derived ferrocenyl
complexes was prepared by simple Schiff-base condensation
reactions with the aim of investigating the effect on antimicrobial
activity when incorporating ferrocene as part of these systems. Three
half-sandwich PGM complexes containing iridium, rhodium and
ruthenium were also prepared to investigate the effect on biological
activity when incorporating these metals. The compounds were
tested for antimycobacterial, antiplasmodial and antitrichomonal
activity. The compounds were found to possess relatively low
cytotoxicity against CHO cells, which suggests selectivity as
antimicrobial agents. Most of the compounds exhibited promising
antimycobacterial activity. The platinum group metal complexes
exhibit a glycerol-dependent activity which might allude to a specific
mechanism of action. In the case of antiplasmodial activity, some of
the compounds exhibit only moderate activity, lower than the
reference drug, chloroquine. However, incorporation of the
ferrocenyl moiety (specifically the salicylaldimine moiety) improves
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