Alkoxide coordination of iron(III) protoporphyrin IX by antimalarial quinoline methanols: A key interaction observed in the solid-state and solution

Article abstract of DOI:10.1039/c5dt02671g

The quinoline methanol antimalarial drug mefloquine is a structural analogue of the Cinchona alkaloids, quinine and quinidine. We have elucidated the single crystal X-ray diffraction structure of the complexes formed between racemic erythro mefloquine and ferriprotoporphyrin IX (Fe(iii)PPIX) and show that alkoxide coordination is a key interaction in the solid-state. Mass spectrometry confirms the existence of coordination complexes of quinine, quinidine and mefloquine to Fe(iii)PPIX in acetonitrile. The length of the iron(iii)-O bond in the quinine and quinidine complexes as determined by Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy unequivocally confirms that coordination of the quinoline methanol compounds to Fe(iii)PPIX occurs in non-aqueous aprotic solution via their benzylic alkoxide functional group. UV-visible spectrophotometric titrations of the low-spin bis-pyridyl-Fe(iii)PPIX complex with each of the quinoline methanol compounds results in the displacement of a single pyridine molecule and subsequent formation of a six-coordinate pyridine-Fe(iii)PPIX-drug complex. We propose that formation of the drug-Fe(iii)PPIX coordination complexes is favoured in a non-aqueous environment, such as that found in lipid bodies or membranes in the malaria parasite, and that their existence may contribute to the mechanism of haemozoin inhibition or other toxicity effects that lead ultimately to parasite death. In either case, coordination is a key interaction to be considered in the design of novel antimalarial drug candidates.

Full text of DOI:10.1039/c5dt02671g

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Alkoxide coordination of iron(III) protoporphyrin IX
by antimalarial quinoline methanols: a key
Cite this: DOI: 10.1039/c5dt02671g
interaction observed in the solid-state and solution†
Johandie Gildenhuys,a Chandre J. Sammy,a Ronel Müller,a Victor A. Streltsov,b
Tanya le Roex,a David Kuter*a and Katherine A. de Villiers*a
The quinoline methanol antimalarial drug mefloquine is a structural analogue of the Cinchona alkaloids,
quinine and quinidine. We have elucidated the single crystal X-ray diffraction structure of the complexes
formed between racemic erythro mefloquine and ferriprotoporphyrin IX (Fe(III)PPIX) and show that alkox-
ide coordination is a key interaction in the solid-state. Mass spectrometry confirms the existence of
coordination complexes of quinine, quinidine and mefloquine to Fe(III)PPIX in acetonitrile. The length of
the iron(III)–O bond in the quinine and quinidine complexes as determined by Extended X-ray Absorption
Fine Structure (EXAFS) spectroscopy unequivocally confirms that coordination of the quinoline methanol
compounds to Fe(III)PPIX occurs in non-aqueous aprotic solution via their benzylic alkoxide functional
group. UV-visible spectrophotometric titrations of the low-spin bis-pyridyl-Fe(III)PPIX complex with each of
the quinoline methanol compounds results in the displacement of a single pyridine molecule and sub-
sequent formation of a six-coordinate pyridine–Fe(III)PPIX-drug complex. We propose that formation of the
drug–Fe(III)PPIX coordination complexes is favoured in a non-aqueous environment, such as that found in
lipid bodies or membranes in the malaria parasite, and that their existence may contribute to the mechan-
ism of haemozoin inhibition or other toxicity effects that lead ultimately to parasite death. In either case,
coordination is a key interaction to be considered in the design of novel antimalarial drug candidates.
Received 14th July 2015,
Accepted 25th August 2015
DOI: 10.1039/c5dt02671g
www.rsc.org/dalton
synthesis of libraries of derivatives of a particular scaffold.
This may be informed by activity data collected for earlier gen-
Introduction
According to the World Health Organization (WHO), almost erations of derivatives, however is frequently guided by avail-
half of the world’s population remains at risk of contracting ability of starting materials and the particular methodological
malaria,1 a parasitic disease caused by Plasmodium species. expertise within a research group. The alternative approach
While prevention and control measures have curbed the relies on understanding both the drug target and, in the case
spread of the disease to the point that the WHO has observed of malaria chemotherapy, the antimalarial pharmacophore. In
a decrease in mortality rates in recent years, resistance to both this regard, limited definitive structural data detailing antima-
historically-relevant quinoline treatments and the newer larial drug interactions with proposed targets has to date pre-
artemisinin-based combination therapies remains a motivat- cluded such rational drug design.
ing factor to develop novel antimalarial chemotherapies.1
Haemozoin formation is a biocrystallisation process
Efforts to do so typically fall into two categories, the first being exploited by the malaria parasite P. falciparum to rid itself of
toxic ferriprotoporphyrin IX (Fe(III)PPIX) released during hae-
moglobin digestion.2 Fe(III)PPIX is widely considered to be the
aStellenbosch University, Department of Chemistry and Polymer Science, Private Bag
target of the clinically-relevant 4-aminoquinoline and quino-
X1, Matieland, 7602 Stellenbosch, South Africa. E-mail: kdev@sun.ac.za;
line methanol antimalarial drugs,3–8 however the mechanism
Fax: +27-21-808-3360; Tel: +27-21-808-2741
bManufacturing Flagship, Commonwealth Scientific and Industrial Research
of quinoline antimalarial drug action remains unclear.
Hypotheses in the literature include complexation between the
drug and the haemozoin precursor (namely Fe(III)PPIX) in
solution,5,9–12 direct interaction between the drug and haemo-
zoin,13,14 or between drug–Fe(III)PPIX complexes and haemo-
zoin.15,16 We recently reported a kinetic model describing the
inhibition of β-haematin (synthetic haemozoin) formation,
Organization, Parkville, Victoria 3050, Australia
†Electronic supplementary information (ESI) available: SCD data tables and
additional figures, as well as MS, EXAFS and UV-visible spectroscopic data tables
and spectra are presented. The SCD structural data for MQ-Fe(III)PPIX and QD-Fe(III)-
PPIX determined in the current work have been deposited with the Cambridge
Crystallographic Data Centre. CCDC 1057567 and 1057568. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c5dt02671g
This journal is © The Royal Society of Chemistry 2015
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which incorporates aspects of these different hypotheses.17 At the absolute stereochemistry of C8 and C9 in QD afforded the
low drug concentration, adsorption of a monolayer of drug to required geometry within its coordination complex with Fe(III)
the surface of the β-haematin crystal is presumed to slow its PPIX more readily than QN.
rate of further growth, while at higher concentrations of drug,
In the current work, we report the first SCD structure of
the irreversible precipitation of Fe(III)PPIX as its drug complex racemic MQ complexed with Fe(III)PPIX, and a second SCD
was proposed as a competing process. Insight into the specific structure of QD-Fe(III)PPIX, both as acetonitrile solvates. These
interactions between drug and target for either of these pro- structures are complementary to that of QN-Fe(III)PPIX
cesses is limited. By means of a computational docking study, reported previously,24 which is also a solvate of acetonitrile.
Leiserowitz and co-workers suggested that adsorption of qui- We show that the geometries within the two MQ-Fe(III)PPIX
noline antimalarial drugs is most likely to occur on the fastest- complexes are analogous to those observed in QN- and
growing crystal face of β-haematin, and they propose several QD-Fe(III)PPIX, and suggest that this may account for the
intermolecular interactions between surface Fe(III)PPIX mole- observed difference in activity between the (+) and (−) erythro
cules and quinoline antimalarial drugs (including chloroquine MQ isomers (see Fig. 1b).28 Furthermore, we present Electro-
(CQ), amodiaquine (AQ), quinine (QN) and mefloquine (MQ)) spray Ionization Mass Spectrometry (ESI-MS), Extended X-ray
that appear favourable.13 The literature concerning the struc- Absorption Fine Structure (EXAFS) and UV-visible spectro-
ture and stoichiometry of Fe(III)PPIX-drug complexes in solu- scopic evidence that strongly supports the existence of these
tion is extensive and has been recently reviewed.18,19 In the alkoxide-bound coordination complexes in non-aqueous solu-
case of the quinoline methanol compounds specifically, tion. We propose that the interactions reported in this struc-
debate has centred around the structure of their coordination tural study may be of relevance in the lipid environments of
complexes with urohaemin20 and Fe(III)PPIX.9,21–23 Recently, the malaria parasite’s digestive vacuole where haemozoin
we determined the first single crystal X-ray diffraction (SCD) formation,29–34 and most likely drug action as a direct conse-
structures of the coordination complexes formed between quence,35 have recently been considered to take place.
Fe(III)PPIX and two active antimalarial compounds, the Cinch-
ona alkaloids QN and quinidine (QD, see Fig. 1a).24 A three-
point binding mode, which had been predicted using compu-
Experimental
tation,25 was confirmed. The SCD structures provide indisputa-
Materials and chemicals
ble evidence that coordination takes place via the
All chemicals and organic solvents used in this study were pur-
chased from Sigma-Aldrich. Antimalarial drugs were pur-
chased as salts, namely QN hemisulfate monohydrate, QD
sulfate dihydrate, MQ hydrochloride and CQ diphosphate.
Haemin (Cl–Fe(III)PPIX) and haematin (H2O–Fe(III)PPIX) were
both of porcine origin. Free base forms of QN and QD were
prepared by precipitating the respective salt from aqueous
solution using concentrated NaOH, followed by freeze drying.
Free base MQ was obtained by precipitating its HCl salt from
methanol using concentrated NaOH, followed by centrifu-
gation and drying over phosphorous pentoxide. Dimethyl sulf-
oxide (DMSO) and acetonitrile were reagent grade or better
and were used without further purification.
deprotonated benzylic alcohol (alkoxide) functional group of
both QN and QD, at least in the solid-state. As expected,
π-stacking between the porphyrin and quinoline aryl systems
of Fe(III)PPIX and the alkaloids respectively, is also observed.
Finally,
a charge-assisted intramolecular hydrogen bond
formed between the protonated tertiary amine of the quinucli-
dine moiety of the drug molecules and the deprotonated pro-
pionate side chain of Fe(III)PPIX is
a feature in both
coordination complexes. Interestingly, QD is known to be
more active than QN.26,27 The computational investigation
resolved that the strain energy within these complexes follow-
ing formation of the intramolecular hydrogen bond was less in
the case of QD compared to QN.25 The authors proposed that
Single crystal X-ray diffraction
MQ-Fe(III)PPIX complexes. Crystals of MQ-Fe(III)PPIX were
obtained from an acetonitrile solution of Fe(III)PPIX, MQ
hydrochloride and CQ free base. MQ hydrochloride (5.0 mg,
12.05 μmol) and CQ free base (5.0 mg, 15.63 μmol) were com-
pletely dissolved in 5.0 mL acetonitrile with rapid stirring.
Haemin (4.0 mg, 6.14 μmol) was then added and the resultant
solution stirred at 50 °C for 20 min, after which it was filtered
through a polytetrafluoroethylene (PTFE) filter disc (0.45 μm
pore size) to remove undissolved porphyrin. The glass vial con-
taining the filtrate was then capped and placed in a water bath
set at 37 °C for a further 24 hours. Small plate-shaped crystals
were observed on the bottom of the vial following this period
of incubation. A single crystal of dimensions 0.20 × 0.10 ×
0.05 mm was selected for X-ray diffraction. The crystal data
Fig. 1 (a) The molecular structures of quinine (QN: 8S,9R) and quini-
dine (QD: 8R,9S), two naturally-occurring Cinchona alkaloids. (b) The
molecular structures of the erythro isomers of mefloquine (MQ). These
are designated (−)-MQ and (+)-MQ, and have 11R,12S and 11S,12R
configurations, respectively.43
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and structural refinement parameters are summarised in (v0.8.061), and later fitted to
Table S1.†
a
proposed model using
ARTEMIS (v0.8.014).37–39 Within this suite of programs, the
QD-Fe(III)PPIX complex. QD sulfate dihydrate (5.0 mg, AUTOBK algorithm was applied for background removal and
12.77 μmol) and CQ free base (5.0 mg, 15.63 μmol) were dis- normalization.40 In the case of QD-Fe(III)PPIX, both the solid-
solved in 5.0 mL acetonitrile, after which haematin (4.0 mg, state and frozen solution EXAFS spectra could be simul-
6.31 μmol) was added. The resultant solution was stirred at taneously refined, owing to the similarity between the two data
100 °C for five minutes, after which it was filtered through a sets. For QN- and MQ-Fe(III)PPIX on the other hand, the EXAFS
PTFE filter disc (0.45 μm pore size) into a clean glass vial data sets of the solid-state and frozen solution samples were
before further incubation (four days) in a water bath set at refined independently. A previously-built model of the por-
37 °C. Small triangle-shaped crystals were observed on the phyrin core of Fe(III)PPIX41 served as the basis for the struc-
bottom of the vial and a single crystal of dimensions 0.39 × tural fitting in each case. The SCD structures reported
0.24 × 0.12 mm was selected for X-ray diffraction. The crystal previously24 and in the current work were used to identify non-
data and structural refinement parameters are summarised in hydrogen atoms (within 5.0 Å of the absorbing Fe atom)
Table S2.†
belonging to either the coordinated quinoline methanol drug
(designated as axial atoms) or the neighbouring π-stacked
complex (designated as atoms outside of the complex). Of the
Mass spectroscopy
Samples of the mother liquors from the crystallisation experi- axial atoms, the alkoxide oxygen atom, the benzylic carbon
ments were freshly prepared. Measurements were performed atom and the quinoline and quinuclidinium/piperidinium
using a Waters Synapt G2 mass spectrometer equipped with carbon atoms immediately adjacent to this were explicitly
an electrospray ionization (ESI) source in positive ion mode. refined. All other C and N atoms (both axial and outside of the
Aliquots were delivered via direct injection.
complex) were refined as one of five arbitrary atom types (Cp1,
Cp2, Cp3, Cp4 or Cn1), and assigned initial Fe–X distances in
the range 3.5 to 5.0 Å. For each Fe(III)PPIX-drug complex,
X-ray absorption studies
EXAFS spectra were recorded of the single crystals of the fitting of experimental data to the model required inclusion of
QD-Fe(III)PPIX and QN-Fe(III)PPIX complexes previously- different numbers of each atom type (see Results and discus-
reported,24 as well as those of the MQ-Fe(III)PPIX complex sion for further details). The initial distances, together with
reported in the current work. Solutions that contained higher the associated Debye–Waller factors, were allowed to refine
concentrations of both Fe(III)PPIX and drugs compared to the during the fitting process.
crystallisation experiments, but with both individual com-
ponents present in their original ratios, were also investigated.
The target concentration of Fe(III)PPIX (∼10 mM) represents an
8 to 11 fold increase in concentration relative to that used in
the crystallisation experiments. In practice however, owing to
the poor solubility of haemin in acetonitrile, this could not be
easily attained, especially for QN- and MQ-Fe(III)PPIX. The raw
EXAFS scans (χ(k)) were collected at the X-ray absorption spec-
troscopy (XAS) beamline (1.9 T Wiggler) at the Australian Syn-
chrotron in Melbourne. Crystals were placed between two
Kapton tapes with as much of the mother liquor removed as
possible and mounted on a Teflon cell. All liquid samples were
prepared immediately prior to the EXAFS experiments being
conducted, and solutions were filtered through a PTFE filter
disk (0.45 μm pore size) to remove undissolved haemin and to
obtain homogeneous samples for analysis. These were then
injected into Teflon cells and flash frozen in liquid nitrogen. A
cryogenic temperature (18 K) was maintained for the duration
of the measurements to limit thermal disorder and avoid
sample degradation. For each sample, at least three iron
Kα-edge (E0 = 7112 eV) scans were recorded in fluorescence
mode up to k = 12 Å−1 using a 100-element Ge detector (cooled
UV-visible spectrophotometric titrations
Fe(III)PPIX-drug and -pyridine complexes. A 1.23 mM stock
solution of haematin was prepared in DMSO, owing to its poor
solubility in acetonitrile. A Fe(III)PPIX working solution was
prepared by adding 20 μL of the stock solution to 2.48 mL of
acetonitrile in a quartz cuvette (1 cm path length). To deter-
mine the association between drugs and Fe(III)PPIX, aliquots
of free base QD, QN or MQ solutions (2 mM in acetonitrile)
were added to the working solution and the resulting spectra
recorded between 300 and 800 nm after each addition. Note
that the free bases were selected in preference of the salt/CQ
free base combination used in the crystallisation and MS
experiments in order to simplify the system. Data were cor-
rected for dilution and analysed using the HypSpec software
package42 according to eqn (1) in order to obtain the equili-
brium constant β1 and predicted spectra of Fe(III)PPIX (M) and
its drug complex (MD).
β1
M þ D Ð MD
ð1Þ
The association of pyridine with Fe(III)PPIX was measured
by liquid N2). Data collections were made from several by adding aliquots of pyridine to a fresh sample of the same
locations within the samples to determine the extent of Fe(III)PPIX working solution described above and recording the
sample homogeneity and to limit sample damage due to con- spectrum after each addition. A total volume of 290 μL pyri-
tinuous exposure to the X-ray beam.
dine (approximately 10% (v/v)) was added. Data were corrected
Replicate data sets for each sample were merged using the for dilution and analysed in HypSpec according to the equili-
XFIT AVERAGE program.36 Data were processed using ATHENA bria described by eqn (2) and (3) in order to obtain equili-
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brium constants β2 and β3. To minimise variables in the refine- formed over the μ-propionato dimer. Interestingly, there is no
ment process, the spectrum of Fe(III)PPIX (M) was constrained. evidence of CQ in either of the structures.
Spectra of the monopyridyl- and bis-pyridyl-Fe(III)PPIX com-
The MQ salt used in this study was obtained as a racemate
plexes (MPy and M(Py)2, respectively) were allowed to freely of erythro enantiomers. The absolute configurations of these
refine.
(−) and (+) isomers have been determined as (11R,12S) and
(11S,12R), respectively.43 It is not unexpected therefore, that
the X-ray diffraction structure of MQ-Fe(III)PPIX includes two
unique complexes in the asymmetric unit (Fig. S1†). These are
designated as I and II for clarity (Fig. 2), and include the (−)
and (+) isomers of MQ, respectively.
β2
M þ Py Ð MPy
ð2Þ
ð3Þ
β3
M þ 2Py Ð MðPyÞ2
Competition titrations. Working solutions of the bis-
pyridyl-Fe(III)PPIX complex were prepared by mixing 20 μL of a
haematin stock solution (1.23 mM in DMSO), 400 μL pyridine
and 2.08 mL acetonitrile in a quartz cuvette (1 cm path
length). The concentration of pyridine in this solution was
16% (v/v). Free base drug solutions were prepared by dissol-
ving QD (7 mg, 2 mM), QN (13 mg, 4 mM) or MQ (35 mg,
10 mM) in an acetonitrile solution containing 16% (v/v) pyri-
dine, which was necessary in order to prevent a change in
solvent composition upon their titration into the bis-pyridyl-
Fe(III)PPIX working solution. Aliquots of drug solutions were
added to the working solution and the spectra recorded follow-
ing each addition. Data were corrected for dilution and ana-
lysed in HypSpec.42 The equilibria described by eqn (1)–(3), as
well as that described by eqn (4), were used in the refinements.
To reduce variable parameters and aid fitting, the values of β1,
β2 and β3, as well as the spectra of Fe(III)PPIX (M), the drug–
Fe(III)PPIX complex (MD), the monopyridyl–Fe(III)PPIX complex
(MPy) and the bis-pyridyl-Fe(III)PPIX species (M(Py)2), were con-
strained to those obtained in the non-competitive titrations
described above. The spectrum of the six-coordinate pyridine–
Fe(III)PPIX-drug complex (PyMD) and the value of β4 were
allowed to freely refine.
As observed previously for QN-Fe(III)PPIX and QD-Fe(III)
PPIX,24 coordination of MQ to the metal centre of Fe(III)PPIX
occurs via the deprotonated benzylic alcohol functional group.
The Fe–O bond lengths in I and II are 1.899(2) and 1.892(2) Å
respectively, which are comparable to other alkoxide-bound
species.24,44–47 π-Stacking is also a feature within and between
MQ-Fe(III)PPIX complexes. This is best appreciated when
viewing the unit cell down the crystallographic b-axis as shown
in Fig. S2.† π-Stacking between the unligated faces of Fe(III)
PPIX molecules in adjacent complexes is observed, resulting in
dimers consisting of either two molecules of I or two mole-
cules of II. The distance between the mean porphyrin planes is
3.81 and 3.70 Å in dimers of I and II respectively, with the
Fe(III) centres offset relative to one another. Dimers of I align
to form rows parallel to the b-axis, as do dimers of II. These
rows pack together in an alternating fashion to form sheets in
the bc plane, which stack together to form the three dimen-
sional structure (Fig. S3†). In addition to ligand-metal coordi-
nation and π-stacking, intramolecular hydrogen bonding is
also a prominent feature within each complex. The first of
three such hydrogen bonds exists between the propionic acid
and propionate side chains of Fe(III)PPIX (labelled (i) in Fig. 2).
β4
M þ Py þ D Ð PyMD
ð4Þ
Results and discussion
Solid-state structures of quinoline methanol complexes of
Fe(III)PPIX
We have shown previously that QN and QD form coordination
complexes with Fe(III)PPIX in the solid-state via their benzylic
alcohol functional groups.24 Somewhat surprisingly, the μ-pro-
pionato dimer of Fe(III)PPIX that constitutes haemozoin and Fig. 2 The asymmetric unit of the MQ-Fe(III)PPIX structure includes two
unique complexes, I and II, which have been reoriented relative to one
another for clarity such that their enantiomeric relationship is evident.
Complex I includes the (−) erythro isomer of MQ, while complex II
includes the (+) erythro isomer. The absolute configurations of the two
β-haematin was recently crystallised from a DMSO solution in
the presence of the antimalarial CQ (as its free base).17 It was
therefore of interest to determine whether, in the presence of
CQ free base, the formation of the coordination complexes of
stereochemical centres are (C11R,C12S) in I and (C11S,C12R) in II. Posi-
the quinoline methanol antimalarial drugs (Fig. 1) with Fe(III)- tion 11 corresponds to C46 and C97 in the SCD structures of I and II,
respectively, while position 12 corresponds to C47 and C98 in the SCD
structures of I and II. Selected atom labels have been included for later
reference. Intramolecular hydrogen bonds are shown as red dashed
lines and labelled as (i), (ii), and (iii). Solvent molecules and non-relevant
PPIX would be favoured over the formation of the μ-propionato
dimer. Single crystals were obtained from acetonitrile solu-
tions of Fe(III)PPIX, which contained CQ free base and either
QD or MQ salts. In both cases, the coordination complex,
H atoms have been removed for clarity. Atom colour labelling: grey – C,
namely QD-Fe(III)PPIX or MQ-Fe(III)PPIX, was preferentially cyan – Fe, dark blue – N, light blue – F, red – O and white – H.
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The second and third (labelled (ii) and (iii), respectively, in
Fig. 2) occur in turn between each of these groups and the
diprotic piperidinium nitrogen atom of MQ. The salt bridge
formed in the third instance is reminiscent of that originally
predicted25 and later observed24 in the QN-Fe(III)PPIX and
QD-Fe(III)PPIX complexes. This network of intramolecular
hydrogen bonds is shown in the ESI (Fig. S4†), and the hydro-
gen bond geometries are reported in Table S3.† An interesting
observation is the absence of intermolecular hydrogen bonds
between the propionate and propionic acid side chains of
neighbouring complexes as were observed in the QN-Fe(III)
PPIX and QD-Fe(III)PPIX complexes. Rather, disordered solvent
molecules (acetonitrile and water, since no precautions were
taken to ensure that the acetonitrile was thoroughly dry before
use) occupy spaces between adjacent complexes.
An earlier computational study examined the influence of
the charge-assisted intramolecular hydrogen bond (salt bridge)
on the resultant geometries of the QN- and QD-Fe(III)PPIX com-
plexes.25 The investigation focused on the energies of each
complex as two dihedral angles, designated φ and ψ (where φ
refers to the axial Fe–O bond dihedral and ψ refers to the di-
hedral about the C8–C9 bond in the coordinated drug), were
varied relative to one another. Of the two complexes, QD-Fe(III)
PPIX was found to incur the lowest strain energy. It was argued
that the absolute configuration (at C8 and C9) of QD, the more
active isomer,27 confers a greater degree of geometrical pre-
organization, which in turn facilitates the formation of the salt
bridge between the drug and the porphyrin. Parallels can be
drawn between the erythro isomers of MQ and the Cinchona
alkaloids QN and QD. Importantly, the two chiral centres in
QN and QD that are considered relevant for their activity (C8
and C9)48 are preserved in MQ (C11 and C12). Furthermore, it
has been shown that the (+) erythro isomer of MQ is almost
twice as active as the (−) erythro isomer.28 With the present elu-
cidation of the SCD structures of MQ-Fe(III)PPIX, it was possi-
ble to compare the geometries of I and II to their analogous
alkaloid complex and explore whether a similar trend exists
between their structures and the reported activities of the
unbound MQ stereoisomers. It was not possible during the
current study to extend the original Hyperchem calculations25
to examine the strain energy surfaces for each of the hydrogen-
bonded MQ-Fe(III)PPIX complexes. Rather, a qualitative analy-
sis was carried out. The values of the dihedral angles φ and ψ
in I and II were compared to those in QN-Fe(III)PPIX and
QD-Fe(III)PPIX, respectively (Table 1). In general, close agree-
ment is observed. An overlay of the observed MQ dihedral
angles with the strain energy surfaces previously computed for
the QN- and QD-Fe(III)PPIX complexes provides visual appreci-
ation (Fig. S5†). It would appear that the complex induced by
the less active (−)-MQ isomer (I) adopts a conformation that is
Table 1 Dihedral angles in MQ-Fe(III)PPIX complexes (I and II) com-
pared to previously-reported Cinchona alkaloid complexes of Fe(III)-
PPIX24
I
QN-Fe(III)PPIX
II
QD-Fe(III)PPIX
φa
ψb
141.4(2)
169.5(2)
155.7(4)
158.3(6)
312.9(2)
192.6(2)
300.3(3)
208.7(3)
a I: N2–Fe1–O5–C46; II: N8–Fe2–O10–C97 (see Fig. 2). b I: C37–C46–
C47–N6; II: C88–C97–C98–N12 (see Fig. 2).
complexes is between 2 and 3 kcal mol−1. This energy barrier
is relatively small, and given that the crystallisation procedure
involved heating the mother liquor at 50 °C for 20 minutes,
and 37 °C for a further 24 hours, both complexes would be
accessible in solution, though in differing proportions. The
fact that I and II appear in the crystal structure in a 1 : 1 ratio
suggests that additional factors (e.g. crystallisation energy,
entropy) may have strongly influenced the composition of the
crystal, however such an investigation is not within the scope
of the current work.
In addition to the SCD structure of MQ-Fe(III)PPIX pres-
ented above, a new structure of QD-Fe(III)PPIX has been
obtained under the same conditions. The molecular structure
of this complex is in good agreement with that of the species
recovered from a 3 : 7 (v/v) DMSO-methanol solution.24 The
asymmetric unit of the acetonitrile solvate includes two
unique complexes (labelled III and IV), and the three-point
binding mode between the drug and Fe(III)PPIX that is dis-
cussed above for MQ is observed in both (Fig. S6†). The com-
plexes π-stack in the solid-state via their unligated porphyrin
faces, and the iron centres are laterally-shifted relative to one
another. Close packing is assisted by a series of intermolecular
hydrogen bonds between the porphyrin propionate and pro-
pionic acid groups, as well as between the suspected propionic
acid group and the quinoline nitrogen atom of adjacent com-
plexes of IV (Table S4†). As before, the presence of a charge-
assisted intramolecular hydrogen bond between the proto-
nated quinuclidine nitrogen atom of QD and the porphyrin
propionate side chain reduces conformational freedom within
each coordination complex. Notably, the crystallisation
medium does not appear to strongly influence the magnitudes
of the φ and ψ dihedral angles. These differ by no more than
six degrees compared to those measured in the complex recov-
ered from DMSO-methanol (Table 1 and Table S5†).24
Coordination of quinoline methanol antimalarials to
Fe(III)PPIX in solution
higher in strain energy than the complex induced by the more While the SCD structures conclusively demonstrate coordi-
active (+)-MQ isomer (II). This is consistent with what was nation of the benzylic alcohol (as an alkoxide) moiety of quino-
observed for the QN- and QD-Fe(III)PPIX complexes, and line methanol antimalarial drugs to the iron(III) centre of Fe(III)
suggests that the (+) erythro isomer of MQ may be structurally PPIX in the solid-state, evidence for this binding mode in solu-
preorganised in a similar manner as QD. The φ/ψ strain energy tion is more contested.9,21–23 Since single crystals of QN-, QD-
analysis suggests that the difference in energy between the two and MQ-Fe(III)PPIX were recovered from acetonitrile, a solvent
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routinely used in mass spectrometry, this provided a serendipi- atoms arising from either the axially-bound quinoline metha-
tous opportunity to investigate whether these coordination nol drug or the porphyrin of a neighbouring π-stacked
complexes persist in the mother liquors. As expected, peaks complex. In the case of QD-Fe(III)PPIX, there was a high degree
arising from the individual components that comprised the of congruency between the experimental spectra obtained for
samples, namely haemin, MQ, QN, QD and CQ, were observed the crystals and mother liquor. To increase the number of
(Table S6†). More importantly, peaks corresponding to the independent points, the model was refined to both data sets
coordination complexes of all species were also detected, con- simultaneously. On the other hand, differences in the spectra
firming the existence of these complexes in non-aqueous solu- of QN-Fe(III)PPIX in the solid-state and in solution necessitated
tion. The MQ-Fe(III)PPIX complex is observed at m/z 994.2946 their independent refinement. The EXAFS spectrum for
(Fig. S7†), which corresponds to [M + H]+ (calculated m/z = MQ-Fe(III)PPIX in solution was noisy, most likely owing to a
994.2940). M is considered to be the species observed by SCD, lower concentration of Fe(III)PPIX solubilised in acetonitrile in
i.e. the neutral coordination complex formed between the zwit- the presence of MQ, and a reliable fit could not be achieved.
terionic drug and a Fe(III)PPIX molecule with one ionised pro- Fitting was possible however, for the solid-state spectrum. In
pionate group. Subsequent protonation (via the medium) addition to those atoms refined explicitly, 17, 18 and 25
could occur at this latter site, or alternatively at the quinoline additional atoms were required to yield acceptable fits of the
nitrogen atom of the drug when feasible. Similarly, the QN- experimental spectra of QD-, QN- and MQ-Fe(III)PPIX, respect-
and QD-Fe(III)PPIX complexes are observed at m/z values of ively (see Table 2 for refinement statistics). Best fits of the
940.3632 (Fig. S8†) and 940.3629 (Fig. S9†), respectively, which experimental spectra are presented in R-space (Fig. 3) and k-
are in very good agreement with the calculated monoisotopic space (Fig. S10†), while fitted distances of all atoms relative to
mass of the [M + H]+ species (m/z = 940.3611). Previously, the absorbing iron(III) centre are given in Table S7.† The com-
Alumasa et al. reported similar MS data for a QN-Fe(III)PPIX parative distances determined by SCD of all explicitly-refined
adduct precipitated from basic aqueous solution and redis- atoms relative to the absorbing iron(III) centre are given in
solved in acetonitrile for analysis.49 Taking this and other Table S8,† while tentative assignments of the additional C and
spectroscopic data into account, the authors proposed that the N atoms for each complex are presented in Table S9.† To aid
complex present in aqueous solution involves non-covalent appreciation of the goodness of fit of each EXAFS structure
coordination of QN free base to the unligated iron(III) centre of (for solid-state samples), an overlay of all atoms at their
a fully ionized Fe(III)PPIX molecule via the benzylic alcohol refined distances is shown relative to the respective SCD struc-
group of the drug. The structure is notably different to the ture in Fig. S11.†
coordination complex observed by SCD, in which coordination
is facilitated by the deprotonated alkoxide of the QN QD- and QN-Fe(III)PPIX, respectively, in the solid-state struc-
zwitterion.24
tures determined by EXAFS (Table 3). These values compare
The Fe–O bond length refined to 1.86(2) and 1.86(3) Å for
MS alone is not able to distinguish between the two above- favourably to the corresponding bond lengths observed in the
mentioned structural interpretations, and consequently we SCD structures (1.862(2) and 1.866(4) Å, respectively).24 While
undertook an EXAFS spectroscopic investigation to probe the a less favourable agreement is observed for MQ-Fe(III)PPIX, the
local environment around the iron(III) centre. This technique fitted EXAFS Fe–O distance (1.85(2) Å) is within three standard
has been successfully applied in various investigations of deviations of the SCD equivalent (1.899(2) Å and 1.892(2) Å for
Fe(III)PPIX50–53 and haemozoin (β-haematin),54 as well as syn- I and II respectively). These observations confirm the coordi-
thetic analogues of the latter.55–58 Knowing that the Fe–O
bond length of a bound alkoxide (1.816(2)–1.866(4) Å)24,44–47 is
considerably shorter than that of a bound alcohol (2.112(8)–
2.160(8) Å),59,60 we were particularly interested to apply EXAFS
spectroscopy to study the length of Fe–O bonds in the com-
plexes present in non-aqueous solution, with the aim of differ-
entiating between the structure implicated by SCD (alkoxide-
bound) and that proposed by Roepe and co-workers (alcohol-
bound).49 EXAFS spectra were therefore recorded of the frozen
solutions of each complex. To facilitate analysis, we further
recorded spectra of the corresponding solid-state samples.
Atoms comprising the porphyrin core (Cα, Cβ, Cmeso and N),
selected non-hydrogen axial atoms from the Cinchona alka-
loids (C4′, C8, C9 and O(H) in Fig. 1a) or MQ (C4, C11, C12
and O(H) in Fig. 1b), and the iron(III) atom of a neighbouring
Table 2 Refinement statistics and associated errors (in parentheses)
determined from EXAFS fitting of the solid-state and frozen solution
spectra of quinoline methanol–Fe(III)PPIX complexes
QN-Fe(III)PPIX
QD-Fe(III)PPIXa Solid-state Solution MQ-Fe(III)PPIXd
nind
nvar
R-factor
45.03
19
20.03
14
0.022
23.11
15
0.044
25.00
16
0.021
0.033b
0.029c
Reduced χ2 1.38
0.91
0.81(9)
0.1(2.0)
1.62
1.63
2
S0
0.89(9)
1.00(10) 0.65(6)
2.1(1.6) 0.7(1.5)
ΔE0 (eV)
−0.3(1.4)b
1.3(1.3)c
π-stacked complex were identified, and their distances from a EXAFS data collected from crystalline and solution samples were
fitted simultaneously. b Parameters pertain to the solid-state sample.
the absorbing iron(III) centre refined explicitly. As outlined in
the experimental methods, arbitrary C and N atoms at longer
c Parameters pertain to the frozen solution sample. d Only data
obtained from the crystalline sample of MQ-Fe(III)PPIX were
distances (3.5–5.0 Å) were used as descriptors for additional successfully fit.
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Fig. 3 Experimental and fitted Fourier transformed (R-space) EXAFS spectra. (a) The best fit of the model (grey line) to the experimental spectra of
QD-Fe(III)PPIX in the solid-state (black solid line) and frozen solution (black dashed line). Spectra were fitted over the range 2.0 ≤ k ≤ 10.0 Å−1 and
0.0 ≤ R ≤ 5.0 Å. (b) The best fit of the model (grey line) to the experimental spectrum of a crystalline sample of MQ-Fe(III)PPIX (black solid line).
Spectra were fitted over the range 2.0 ≤ k ≤ 10.0 Å−1 and 0.0 ≤ R ≤ 5.0 Å. (c) The best fit of the model (grey line) to the experimental spectrum of a
crystalline sample of QN-Fe(III)PPIX (black solid line). Spectra were fitted over the range 2.0 ≤ k ≤ 10.0 Å−1 and 1.0 ≤ R ≤ 5.0 Å. (d) The best fit of the
model (grey line) to the experimental spectrum of the frozen solution of QN-Fe(III)PPIX (black dashed line). Spectra were fitted over the range 2.0 ≤
k ≤ 9.4 Å−1 and 0.0 ≤ R ≤ 5.0 Å. All spectra (a to d) were fitted with k3-weighting in R-space at the final step.
Previously, changes in the UV-visible spectrum of Fe(III)
PPIX in acetonitrile following titration with QN24 were shown
to be consistent with those arising from a QN-Fe(III)PPIX
coordination complex prepared in benzene.9 Coordination in
the latter case was presumed to occur via the quinuclidine N
atom. Based on SCD evidence however, the solution structure
was reinterpreted, and coordination of QN to Fe(III)PPIX was
proposed to occur in the same manner via the alkoxide.24
Indeed, in the current study, EXAFS spectroscopy has con-
firmed this to be the case. In order to obtain a quantitative
measure of the coordinating ability of each drug to Fe(III)PPIX
Table 3 Comparison of Fe–O bond lengths (Å) determined by SCD and
EXAFS spectroscopy for quinoline methanol–Fe(III)PPIX complexes
QN-Fe(III)PPIX
MQ-Fe(III)PPIX
II
QD-Fe(III)PPIX Solid-state Solution
I
Fe–OSCD
Fe–OEXAFS 1.86(2)
1.862(2)
1.866(4)
1.86(3) 1.85(3)
1.899(2) 1.892(2)
1.85(2)
nation of the alkoxide species in all cases in the solid-state, in acetonitrile, association constants for complexation (β1)
and demonstrate the sensitivity of the EXAFS technique to were determined from spectrophotometric titrations (Table 4).
provide reliable structural information regarding the first The trend in binding strength reported for the three quinoline
coordination shell of Fe(III)PPIX. For the mother liquors of QN- methanol antimalarial drugs to Fe(III)PPIX in 40% (v/v)
and QD-Fe(III)PPIX, Fe–O bond lengths from fitted EXAFS aqueous DMSO5,26 is also observed in acetonitrile, with QD
spectra confirm the presence of a coordinated alkoxide displaying the strongest interaction and MQ the weakest.
species, with values in very good agreement with the corres- However, the absolute values of the association constants are
ponding SCD structures (Table 3). The data is particularly reso- between five and twenty-four fold larger in acetonitrile. To
lute in the case of QD-Fe(III)PPIX, so much so that the solution further probe the relative stability of the coordination inter-
EXAFS spectrum could be simultaneously refined together action observed in solution between the three drugs and Fe(III)-
with the solid-state spectrum. Despite a lower precision in PPIX, we considered the effect of a competing ligand, namely
the fit for QN-Fe(III)PPIX in solution, the Fe–O bond length pyridine. This compound readily coordinates to free Fe(III)PPIX
(1.85(3) Å) is within one standard deviation of the value in solution to form a peach-coloured low-spin bis-pyridyl-Fe(III)
resolved by SCD.
PPIX complex.61,62 The latter species has a characteristic
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above. The spectrum of MPy was predicted following analysis
of the titration data for Fe(III)PPIX and pyridine. These four
spectra together with their respective association constants (β1,
β2 and β3) were constrained during the refinement of the data
obtained from titration of M(Py)2 with drug. This left only two
unknown parameters, namely the spectrum of PyMD and the
association constant β4 (Table 4), which were optimized to
reproduce the experimental data. The predicted UV-visible
spectrum of PyMD is unique, and can thus be distinguished
from all other Fe(III)PPIX species that may be present in the
experiment (Fig. 4). In the Soret band region of PyMD, there is
generally a decrease in the intensity of the peak relative to the
bis-pyridyl-Fe(III)PPIX complex. The shoulder feature at shorter
wavelength is somewhat more pronounced than in the case of
M(Py)2, however is significantly less intense compared to MPy
(see Fig. 4a). Compared to the spectra of the three MD species,
the Soret band of PyMD in each case is redshifted by approxi-
mately 10 nm and displays a somewhat less intense shoulder
feature at shorter wavelength. In the longer wavelength region
(450–700 nm), the characteristic charge-transfer band at
600 nm in the spectra of MD is retained in the spectra of
PyMD, however is consistently observed at a reduced intensity.
Furthermore, the spectroscopic envelope that is characteristic
of M(Py)2 is also observed in each case of PyMD, although this
varies with intensity for the three different drugs. This feature
is most prominent in the spectrum of PyMD arising from the
MQ species, and least evident in the spectrum arising from
the QD species.
Table 4 Association constants determined from spectrophotometric
titration data using HypSpec analysis for Fe(III)PPIX and quinoline anti-
malarial drugs in acetonitrile
QD
QN
MQ
log β1
5.7 0.1
5.48 0.02
(4.10 0.02)b
7.0 0.2
4.2 0.2
1.6 0.2
4.78 0.08
(3.90 0.08)b
6.61 0.05
3.78 0.05
1.83 0.09
(5.02 0.03)a
7.32 0.06
4.49 0.07
1.6 0.1
log β4
log K1
log K2
Values in brackets determined in 40% (v/v) aqueous DMSO. a Ref. 26.
b Ref. 5.
UV-visible spectrum identified by a sharp Soret peak at
405 nm and a distinctive spectroscopic envelope between 510
and 580 nm. The stepwise coordination of pyridine to Fe(III)-
PPIX in acetonitrile was monitored spectrophotometrically
(Fig. S12,† a and b), and the resultant equilibrium constants
for the processes described by eqn (2) and (3) were determined
as 1.76
0.07 (log β2) and 2.83
0.03 (log β3), respectively.
These represent
a
10–17 fold enhancement over values
obtained in aqueous DMSO.63 Having confirmed that pyridine
indeed behaves as a coordinating ligand in acetonitrile, its
ability to displace each quinoline methanol drug from the
respective drug–Fe(III)PPIX complex was probed through com-
petition titrations (Fig. S12, c–h†). These were conducted in
two different ways. In the first case, increasing concentrations
of pyridine were added to acetonitrile solutions of preformed
drug–Fe(III)PPIX complexes. The alternative approach involved
the titration of drug into an acetonitrile solution containing
the preformed bis-pyridyl-Fe(III)PPIX complex. Both methods
were expected to yield the same overall results, however differ-
ences in the fitted equilibrium constants were observed. These
were attributed to the significant changes in solvent compo-
sition arising from the addition of relatively large volumes of
neat pyridine required in the first method. Since the solvent
composition of the titrant (drug solution) used in the second
approach was the same as that of the working solution (see
Experimental section for details), the results obtained are con-
sidered more reliable, and are therefore presented here. For
comparative purposes however, the results obtained using the
first approach are presented in the ESI (Table S10†).
The displacement of a single pyridine ligand from M(Py)2
by the quinoline methanol drug to form the observed PyMD
species is given by eqn (5a), and the respective step-wise equili-
brium constant by eqn (5b) (see the ESI† for the derivation).
The association of pyridine with the five-coordinate drug–
Fe(III)PPIX complex (MD) to yield PyMD is described by
eqn (6a), and the equilibrium constant K2 is obtained from
eqn (6b) (see the ESI† for the derivation).
K1
MðPyÞ2 þ D Ð PyMD þ Py
ð5aÞ
ð5bÞ
log K1 ¼ log β4 ꢀ log β3
K2
MD þ Py Ð PyMD
ð6aÞ
ð6bÞ
log K2 ¼ log β4 ꢀ log β1
The spectrophotometric titration data of the bis-pyridyl-
Fe(III)PPIX complex, M(Py)2, with each of the quinoline metha-
nol compounds could not be successfully fit using the specia-
tion equilibria described by eqn (1)–(3) only. Rather,
incorporation of an additional equilibrium process involving
the formation of a unique six-coordinate Fe(III)PPIX species
ligated by the drug and a single pyridine molecule (PyMD) was
essential (see eqn (4)). In the fitting procedure, spectra of all
Fe(III)PPIX species were summed together in varying ratios
(that were governed by β values) to reproduce the experimental
data at each wavelength. The spectrum of M, as well as those
of MD and M(Py)2, were obtained from initial and endpoints,
respectively, of the spectrophotometric titrations discussed
The initial intention behind the spectrophotometric titra-
tions was to probe the ability of pyridine to act as a competing
ligand for the coordination site occupied by the alkoxide drug
moiety in each MD complex. It is evident from the spectro-
scopic data and extracted equilibrium constants however, that
this does not occur in the acetonitrile system. The large posi-
tive values determined for log K1 (Table 4) instead support the
formation of a six-coordinate PyMD species over either MPy or
M(Py)2. The values follow the trend observed for log β1, which
describe the formation of the three MD complexes, however
they are approximately one order of magnitude weaker. The
difference can be attributed to the necessary dissociation of
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ordinated quinoline methanol antimalarial drugs, at least in
acetonitrile. This is not unexpected given a previous report,
which showed that in the absence of proton competition (such
as in acetonitrile), coordination to Fe(III)PPIX is dependent on
the pKa of the ligand.63 The pKa of pyridine (5.23 63) is lower
than that of an alkoxide (predicted value for QN and QD =
13.89 64), and hence coordination is expected to be stronger in
the case of the latter. Indeed, a recent computational study
has shown the alkoxide-iron(III) porphine interaction to be par-
ticularly favourable compared to coordination by neutral
ligands.65 These current findings may have important impli-
cations for detergent- and lipid-based pyridine hemochrome
assays that rely on the bis-coordination of free Fe(III)PPIX by
pyridine to quantitate and evaluate the ability of compounds
to inhibit the formation of β-haematin.17,62,66 We have shown
in non-aqueous solution, at least for quinoline methanol anti-
malarial drugs, that the major Fe(III)PPIX species that exists in
the presence of pyridine is not the anticipated bis-pyridyl
complex, but rather the six-coordinate pyridine–Fe(III)PPIX-
drug complex. The latter species has a unique UV-visible spec-
trum, which bears some resemblance to that of the low-spin
bis-pyridyl–Fe(III)PPIX complex in the longer wavelength
region. At first glance, this may be mistaken as an indication
of the presence of the bis-pyridyl-Fe(III)PPIX species. If this
were the case, the reduced intensity of the Soret band would
be suggestive of a lower concentration of free Fe(III)PPIX. This
in turn would result in a prospective inhibitor displaying a
lower than actual activity. Follow-up studies using techniques
such as electron paramagnetic resonance and nuclear mag-
netic resonance spectroscopy would most certainly be necess-
ary in order to confirm the existence and structure of the six-
coordinate PyMD species, however are beyond the scope of the
current investigation.67,68
We have suggested previously, that owing to its absolute
stereochemistry at C8 and C9, QD may be structurally pre-
organised compared to QN and therefore possess an inherent
predisposition to form a complex with Fe(III)PPIX.25 The inter-
action is primarily facilitated by coordination, which we have
shown occurs in both the solid-state and non-aqueous solution
via the alkoxide zwitterionic form of the quinoline methanol
antimalarial drug. An early study by Behere and Goff demon-
strated that an aliphatic amine is able to promote the for-
mation of an alkoxide–iron(III) porphyrin complex in aprotic
solution by enabling proton transfer from an alcohol.21 Given
however, that MQ hydrochloride and QD sulfate dihydrate
were used in the current study, the aliphatic amine would not
Fig. 4 UV-visible spectra of Fe(III)PPIX species (10 μM) in acetonitrile
solution following spectrophotometric titration. (a) The initial spectrum
of Fe(III)PPIX (black solid line) and predicted spectra of mono- and bis-
pyridyl-Fe(III)PPIX complexes (grey solid and grey dashed lines, respect-
ively). (b–d) Spectra of five-coordinate drug–Fe(III)PPIX complexes (black
solid lines) and predicted spectra of six-coordinate pyridine–Fe(III)PPIX–
drug complexes (grey solid lines). In each case, the longer wavelength
region has been expanded, and the spectrum of the bis-pyridyl–Fe(III)-
PPIX species included (black dashed line) for comparison. The drugs
present in each case are quinidine (b), quinine (c) and mefloquine (d).
one pyridine molecule from M(Py)2 before the drug is able to
coordinate. The PyMD species presumably contains a single have been able to facilitate the proton transfer necessary for
pyridine molecule bound to the unligated face of the five- alkoxide formation in either case. While the quinoline nitro-
coordinate drug–Fe(III)PPIX complexes. Indeed, the strength of
gen atom may have served as a basic site in the case of QD,
this ligation by pyridine (described by eqn (6a)) is the same at the this would not have been a viable base in the case of MQ
95% confidence level as that found for the association of one owing to the steric hinderance of the –CF3 substituents on the
pyridine molecule to Fe(III)PPIX in acetonitrile (log β2 reported
above). The moderate values of log K2 for this process (Table 4) mental solutions to possess the free aliphatic amine functional
quinoline ring of MQ. The only component in both experi-
are essentially the same irrespective of the drug present.
group necessary to facilitate alkoxide formation would have
been CQ free base. Almost three decades ago, Blauer reported
The UV-visible spectroscopic data above conclusively
demonstrate that pyridine is unable to displace the co- reversible changes with pH in the circular dichroism spectra of
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Fe(III)PPIX in the presence of QD in aqueous solution. He
attributed the changes to an ionisable functional group that
Acknowledgements
would be of uttermost importance in the formation of the opti- This material is based upon work supported in part by the
cally-active complexes, and considered the tertiary amine of National Research Foundation (NRF) of South Africa under the
the quinuclidine ring the most likely candidate.23 Following Thuthuka Programme (KdV, Grant no. 76320 and TlR, Grant
coordination, structural preorganisation almost certainly pro- no. 69103). Beamtime at the Australian Synchrotron was made
motes the subsequent formation of the intramolecular charge- possible following a successful application by VS (Ref AS131/
assisted hydrogen bond. As observed for each complex in the XAS/5839). Travel by JG to and from the Australian Synchrotron
solid-state, the tertiary amine of the quinuclidine ring (QD and to participate in the EXAFS experiments was made possible by
QN) and the secondary amine of the piperidine ring (MQ) are a travel grant under the NRF Research Equipment Programme
directly involved in this important interaction. Unfortunately, (Grant no. 87336). We gratefully acknowledge Professor
it has not been possible to confirm the presence of this hydro- Timothy Egan at the University of Cape Town for use of
gen bond in the solution-state coordination complexes investi- HypSpec software. Any opinions, findings, or conclusions
gated in the current study.
expressed in this material are those of the authors and do not
necessarily reflect the views of the NRF.
Conclusions
Notes and references
The data reported herein provide insight into the speciation
and drug–Fe(III)PPIX interactions that may be relevant in non-
aqueous environments within the digestive vacuole of the
malaria parasite, such as lipid bodies or membranes. The
determination of the structure of MQ-Fe(III)PPIX supports the
three point binding model that is evident in the solid-state
structures of the Cinchona alkaloid complexes of Fe(III)PPIX,
and we have been able to reconcile the differences in activity
of the erythro isomers of MQ as a result. Previously, the differ-
ence in activity of (+) and (−) erythro MQ has been attributed
to the orientation of the benzylic alcohol and terminal amine
functional groups and their subsequent hydrogen bonding
capacity relative to cellular constituents.43 The current study
implicates Fe(III)PPIX as a feasible drug target, and infers that
differences in activity are directly related to the ability of a
compound to complex free Fe(III)PPIX. We have provided con-
clusive evidence that the latter process is facilitated by coordi-
nation of the quinoline methanol benzylic alcohol (alkoxide)
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