A Diverse Range of Hemozoin Inhibiting Scaffolds Act on Plasmodium falciparum as Heme Complexes

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A Diverse Range of Hemozoin Inhibiting Scaﬀolds Act on

Plasmodium falciparum as Heme Complexes

Roxanne Openshaw, Keletso Maepa, Stefan J. Benjamin, Lauren Wainwright, Jill M. Combrinck,

Roger Hunter, and Timothy J. Egan *

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ABSTRACT: A diverse series of hemozoin-inhibiting quinolines, benza-

mides, triarylimidazoles, quinazolines, benzimidazoles, benzoxazoles, and

benzothiazoles have been found to lead to exchangeable heme levels in

cultured Plasmodium falciparum (NF54) that ranged over an order of

magnitude at the IC₅₀. Surprisingly, less active compounds often exhibited

higher levels of exchangeable heme than more active ones. Quantities of

intracellular inhibitor measured using the inoculum eﬀect exhibited a linear

correlation with exchangeable heme, suggesting formation of heme−inhibitor

complexes in the parasite. In an eﬀort to conﬁrm this, the presence of a Br

atom in one of the benzimidazole derivatives was exploited to image its

distribution in the parasite using electron spectroscopic imaging of Br, an

element not naturally abundant in cells. This showed that the compound

colocalized with iron, consistent with its presence as a heme complex. Direct

evidence for this complex was then obtained using confocal Raman

microscopy. Exchangeable heme and inhibitor were found to increase with decreased rate of killing, suggesting that slow-acting

compounds have more time to build up exchangeable heme complexes. Lastly, some but not all compounds evidently cause pro-

oxidant eﬀects because their activity could be attenuated with N-acetylcysteine and potentiated with t-butyl hydroperoxide.

Collectively, these ﬁndings suggest that hemozoin inhibitors act as complexes with free heme, each with its own unique activity.

KEYWORDS: hemozoin, antimalarial, electron spectroscopic imaging, Raman microscopy, ROS

nism, we have investigated a diverse range of hemozoin

inhibiting scaﬀolds (Figure 1).

emozoin microcrystals are a striking feature in the

H_{digestive vacuole (DV) of malaria parasites. They are}

formed following digestion of host hemoglobin, sequestering

≈95% of the released heme in a nontoxic insoluble form.¹

Inhibition of hemozoin formation has been implicated in the

mechanism of action of chloroquine as well as amodiaquine

and piperaquine, currently used in artemisinin combination

therapy (ACT).²⁻⁶Studies have shown that chloroquine

decreases formation of hemozoin, increases exchangeable

heme, disrupts the hemozoin crystal lattice, and redistributes

heme into the parasite cytoplasm. The dose-dependent

increase in exchangeable heme coincides with decreased

parasite survival.⁷Similar behavior has been shown with

other clinical and experimental hemozoin inhibiting com-

pounds.^6,8−15

Here, we show that the amounts of intracellular exchange-

able heme and intracellular test compound coincide. Using

electron spectroscopic imaging, we demonstrate that a

derivative bearing a Br atom (compound 13) colocalizes

with iron, suggesting the presence of a heme complex in the

DV, a ﬁnding strongly supported by confocal Raman

microscopy. Higher levels of exchangeable heme are associated

with a decreased rate of killing, while heme-related pro-oxidant

activity is involved in the antiparasitic activity of some but not

all compounds. Based on these ﬁndings, we propose a

comprehensive model for the antimalarial mechanism of

action of hemozoin inhibitors.

It has been hypothesized that inhibition of hemozoin

formation causes a buildup of heme, killing the parasite via pro-

oxidant activity, membrane disruption, or osmotic pres-

sure.¹⁶⁻¹⁹Surprisingly, levels of exchangeable heme (all

heme other than undigested hemoglobin and hemozoin) at

the IC₅₀values of a range of hemozoin inhibitors are

remarkably variable.^6,8−15To better understand this mecha-

Received: September 27, 2020

Published: January 12, 2021

https://dx.doi.org/10.1021/acsinfecdis.0c00680

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Figure 1. Compounds investigated in this study fall into seven scaﬀolds: quinolines (chloroquine 1 and amodiaquine 2), benzamides (3−5),

triarylimidazoles (6−9), quinazolines (10−12), benzimidazoles (13−16), benzothiazole (17), and benzoxazole (18).

with an appropriate amine to produce a 2-chloroquinazoline

derivative followed by a second S_NAr reaction to yield the ﬁnal

products. The benzazoles were also prepared in three steps,

this time starting with an S_NAc reaction between 2-amino-

phenol or 2-aminothiophenol and 3-aminobenzoic acid to

produce an azole intermediate, which was then reacted with an

acyl halide to aﬀord the product (Scheme 1b). The purity of all

ﬁnal products was conﬁrmed to be >95% by HPLC.

RESULTS

■

Chloroquine and amodiaquine are commercially available,

while the syntheses of compounds 3−9 and 13−16 have been

previously reported.⁹⁻¹¹The quinazolines 10−12 and

benzazoles 17 and 18 have not previously been reported.

Compounds 3−18 are all based on scaﬀolds previously

discovered in a high throughput screening campaign for β-

hematin inhibitors.²⁰The quinazolines were synthesized in

three steps from benzoylene urea (Scheme 1a). This involved

reﬂuxing with a large excess of POCl₃to produce 2,4-

dichloroquinazoline, followed by a regioselective S_NAr reaction

The simplest explanation for the activity of hemozoin

inhibitors is that they cause a buildup of free heme, which

directly kills the parasite. Exchangeable heme levels should

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Scheme 1. Synthesis of Quinazolines (a) and Benzoxazoles and Benzothiazoles (b)

then be the same at the parasite growth IC₅₀of each

compound. While the IC₅₀values of diﬀerent compounds

could vary because of the diﬀerent extents of accumulation in

the DV through pH trapping and diﬀerent hemozoin inhibiting

strengths, the toxicity of heme to the parasite should remain

constant. In previous studies, we have shown that the level of

exchangeable heme at the IC₅₀varies over several fold,^6,8−14

making this hypothesis untenable. It is tempting to suggest that

these compounds act on a target other than heme, with

adventitious inhibition of hemozoin formation causing a

buildup of exchangeable heme with no role in parasite killing.

This argument is also untenable, however, because no match

would then be expected between the dose-dependent increase

in exchangeable heme and decrease in parasite survival for a

range of compounds. For example, a compound that inhibited

hemozoin formation more strongly than its drug target would

build up exchangeable heme levels at doses below the IC₅₀,

while one that inhibited the drug target more strongly than

hemozoin formation would only exhibit rising exchangeable

heme levels at doses well above the IC₅₀. This is not what we

have previously observed.⁸⁻¹⁰Rather, the decrease in survival

is closely linked to the increase in exchangeable heme. This

seems to leave one alternative; that inhibition of hemozoin

formation causes a buildup of exchangeable heme in the form

of a unique complex, with each complex having its own speciﬁc

level of activity. The implication is that heme complexes with

drugs or test compounds should be observable in the parasite

with variable levels of heme at the IC₅₀of each complex.

The β-hematin and parasite growth inhibition IC₅₀values for

all compounds are presented in Table S1. We have reported

most of these previously, but quinazolines 10−12, benzothia-

zole 17, and benzoxazole 18 are new compounds. While we

have reported levels of exchangeable heme upon treatment

with test compounds previously for compounds 3−9 and

13,⁹⁻¹¹we have now also measured these for the remaining ten

compounds. There is a wide range of exchangeable heme levels

at the respective IC₅₀of each compound with no deﬁnite trend

relating exchangeable heme and parasite IC₅₀. A counter-

intuitive tendency for higher exchangeable heme levels to be

associated with higher IC₅₀is evident, especially if chloroquine,

amodiaquine, and compounds 6 and 15 are disregarded

(Figure 2a). Not only do these results conﬁrm recent

observations of variable levels of exchangeable heme at the

IC₅₀, but they also illustrate a tendency for higher levels to be

associated with weaker activity. This surprising result further

invalidates the hypothesis that exchangeable heme alone is

responsible for parasite death.

Next, we measured amounts of selected active compounds

accumulated in the parasite to establish whether they

stoichiometrically correlate with levels of exchangeable heme

as might be expected if they act as complexes. To this end, we

measured the accumulation of 11 of the active compounds in

the parasite by using the inoculum eﬀect (Figure 2b and Table

S2). These consisted of all compounds with parasite growth

IC₅₀values ≤2 μM. A direct 1:1 correlation was found between

amounts of exchangeable heme and active compound in NF54

parasites (Figure 2c), with a linear correlation exhibiting a

slope of 1 and intercept of 0. Runs tests of the data showed no

deviation from linearity.

The statistical correlation between the amount of test

compound accumulated in the parasite and exchangeable heme

over seven scaﬀolds with a wide range of inhibitory activities

suggests they are present as complexes. Several compounds

(10, 13, 14, 16, and 17) contain either F or Br atoms that do

not occur naturally at high concentrations in cells and can be

exploited as labels for electron spectroscopic imaging (ESI)

based on electron energy loss spectroscopy (EELS). The EELS

K edge of F is close to the L₂and L₃edges of Fe making it

unsuitable for elemental mapping in this Fe rich system,²¹

ruling out mapping of compound 17. Compound 10 was

unsuitable because exchangeable heme at the IC₅₀represents

only a very small fraction of the total heme in the parasite. This

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exchangeable heme, making up ≈25% of total heme iron at

the IC₅₀. In untreated trophozoites, the distribution of Fe was

readily observed using ESI and was almost exclusively conﬁned

to hemozoin (Figure 3a and b). At the Br edge, no signal was

observed (Figure 3c), conﬁrming no signiﬁcant overlap in the

EELS of Fe and Br and no measurable Br in the untreated cell.

When parasites were treated with 13, ESI images of the

cryoﬁxed parasitized red blood cells at the Br edge readily

permitted the distribution of the compound to be visualized

(Figure 3f). Comparison with the Fe distribution (Figure 3d)

showed almost perfect colocalization of Fe and high

concentration regions of Br (Figure 3e). This region of

colocalization coincided with hemozoin and in view of the lack

of any other strong Fe signals, also the region occupied by

most of the exchangeable heme. This colocalization supports

the hypothesis of a complex between exchangeable heme and

compound 13 and likely association of the compound with

hemozoin as well.

A confocal Raman microscope with a 532 nm laser was used

to further investigate parasites treated with 13. Figure 4a shows

average Raman spectra obtained from various parts of an

infected RBC treated with compound 13 that were obtained

using the built-in true component analysis tool in Witec Suite

FIVE software from large area scan spectra obtained separately

from 10−15 cells. Extracted spectra were averaged and

produced distinct spectra of oxyhemoglobin, deoxyhemoglo-

bin, and hemozoin following demixing. An additional spectrum

obtained near the hemozoin and not seen in untreated

parasites was ﬂagged as a possible complex between heme and

compound 13. This spectrum diﬀered from other heme

species, including hematin and hemin and from compound 13

alone, but resembled a synthetically prepared complex of

hematin and compound 13 (Figure 4b). This visual similarity

was conﬁrmed using principal components analysis (PCA).

For this purpose, second derivatives of Raman spectra

normalized to the strongest peak were used, and only second

derivative peaks lying outside 95th percentile of the mean were

considered. For each representative part of the infected RBC,

150 such spectra were recorded from multiple cells, and the

dimensionality of these data was reduced to two using PCA

which clustered similar spectra (Figure 5a and b). As a

Figure 2. Comparison of levels of exchangeable heme, parasite growth

inhibition, and compound accumulation in NF54 P. falciparum. (a)

Exchangeable heme levels at the IC₅₀(black bars, left axis) and the

corresponding IC₅₀values of each compound (gray bars, right axis)

showing no direct correlation. Compounds are arranged in order of

increasing IC₅₀. (b) Inoculum eﬀect for compound 13, r²= 0.95. (c)

A plot of exchangeable heme versus amount of intracellular test

compound at the IC₅₀. Slope 1.0 0.2, intercept 0.05 0.04, r²=

0.70, P = 0.0013, P-value (runs test) = 0.61.

left compounds 13, 14, and 16. Compound 13 was ﬁnally

chosen for further investigation because it was the most active

against the parasite and produced fairly high levels of

Figure 3. Distribition of Fe and Br in untreated P. falciparum infected red blood cells and cells treated with compound 13. (a) Electron

spectroscopic imaging using EELS showing distribution of Fe (green) and (c) Br in an untreated parasite with (b) TEM image. (d−f) A parasite

treated with 13 is shown with the (d) Fe map, (f) Br map (yellow), and (e) manual overlay.

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Figure 4. Raman spectra obtained from infected RBCs treated with 13 and compared with pure hemin, compound 13, and a complex of 13 with

hemin in 40% aqueous DMSO in the presence of a 9-fold excess of compound 13. (a) Typical sampling sections where individual species were

dominant are illustrated in the inset by 1 μm²boxes which are ≈5× the lateral resolution of the instrument. Spectra represent (i) oxyhemoglobin,

(ii) deoxyhemoglobin, (iii) hemozoin, and (iv) putative heme-13 complex. (b) Raman spectra of hemin (top), 13 (middle), 10:1 mixture of hemin

and compound 13 in 40% DMSO (bottom in orange), and spectrum iv from (a) added to the spectrum of compound 13 at a ratio of 9:1 (bottom

in black). The vertical dotted line marks 1080 cm⁻¹, where a unique peak occurs in the complex. Peak positions in the bottom spectra are very

similar. Diﬀerences in relative intensities may reﬂect the fact that the synthetic complex was recorded in a precipitate obtained from an aqueous

DMSO solution, whereas the parasite species represents a solid formed in the cell which may exhibit diﬀerent heme−heme interactions.

statistical measure of separation of these clusters, we computed

the centroid of each cluster and projected the data onto a line

coincident with the vector joining the cluster centroids and

applied a Welch’s t-test. This showed that the spectrum of the

putative heme-13 complex was statistically distinct from all

other heme species (oxy- and deoxyhemoglobin, hemozoin,

hemin, hematin and β-hematin) as well as from the spectrum

of pure compound 13 (P < 0.0001) but was statistically

indistinguishable from the synthetically prepared complex (P =

0.8043, Table S3). A unique peak was observed in the

synthetic complex at 1080 cm⁻¹as well as in the putative

complex in the cell. This likely involves a shift in the

δ_as(=CH₂) vinyl mode in the porphyrin which occurs at 1090

cm⁻¹in hemozoin and oxyhemoglobin and at 1099 cm⁻¹in

deoxyhemoglobin. This peak was subsequently used for Raman

imaging of the complex in the cell.

Raman imaging was used to establish the distribution of the

complex in the cell (Figure 5c). A spectral unmixing protocol

like that described above used to collect single scan spectra was

applied, but the single scan spectra previously identiﬁed were

used as reference spectra. Use of a ﬁlter viewer tool then

improved the quality of the images by reducing background

noise. This procedure produced an image at 1080 cm⁻¹using

the spectrum of the complex as basis spectrum that was totally

absent in the untreated sample and in images based on any of

the other component spectra. This image could thus be

ascribed to the complex alone and was in close agreement with

the evidence from the EELS data, showing that the complex

colocalized with hemozoin (see also Figures S1 and S2).

The evidence for heme−inhibitor complexes in the parasite

presented above supports the hypothesis that such complexes

are responsible for parasite killing. One explanation for the

variability in exchangeable heme relates to speed of killing. A

heme−inhibitor complex that kills the parasite slowly or later

in the lifecycle would permit more hemoglobin digestion,

resulting in more heme available to form a complex with the

inhibitor than if it kills early in the lifecycle. To explore this, we

inoculated ring-stage parasitized RBCs with chloroquine,

amodiaquine, and compounds 3, 6, 13, and 18, with the test

compound washed out after 4, 8, 12, 24, or 48 h. Parasite

growth was measured at 72 h postinoculation using ﬂow

cytometry with SYBR Green I staining (Figure S3). It was

evident that some compounds kill parasites much earlier in the

lifecycle than others (Figure 6a), with an overall increase in

exchangeable heme as the rate of killing decreases as measured

from the ratio of IC₅₀with 24 h washout to that without

washout, both measured at 72 h (Figure 6b).

Finally, we investigated the basis of inhibitor toxicity to the

parasite of several inhibitors. It has long been known that

hemin is toxic to parasites,¹⁶probably arising from pro-oxidant

eﬀects of the Fe center of hemin.¹⁷To investigate this, we ﬁrst

incubated parasitized RBCs with various doses of hemin,

determining an IC₅₀of 31 7 μM (Figure 6c). The reductant

and glutathione precursor N-acetylcysteine (NAC) at 10 mM,

a concentration that did not interfere with the assay, protected

the parasites from hemin up to 100 μM, the highest

concentration that could be tested. This is consistent with a

pro-oxidant eﬀect of hemin. Interestingly, zinc(II) proto-

porphyrin IX (Zn(II)PPIX) also inhibited parasite growth, but

consistent with the redox inactivity of Zn(II) was unaﬀected by

10 mM NAC, showing that this metalloporphyrin acted by a

diﬀerent mechanism (Figure 6c). The IC₅₀of Zn(II)PPIX was

slightly lower than that of hemin (24

4 μM), and we

speculate that this may involve deactivation of crucial

hemoproteins in the parasite by replacement of heme in

their active sites with catalytically inactive Zn(II)PPIX.

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Figure 5. PCA analysis of Raman spectra and Raman imaging of P. falciparum infected RBCs treated with compound 13. (a) PCA scores plot of a

solution of hemin with excess compound 13 (orange crosses) and spectra obtained from the parasite (purple triangles) used to constitute the

average shown in Figure 5a spectrum iv (left panel) and scatter plots along the line coinciding with the vector joining the centroids of the two series

in the PCA plot (right panel). (b) PCA scores plot for compound 13 alone (blue diamonds) and spectra obtained from the parasite (purple

triangles, left panel) and scatter plots along the line coinciding with the vector joining the centroids of the two series in the PCA plot (right panel).

(c) Raman imaging of a parasitized RBC obtained using unmixed spectra based on the average spectra as shown in Figure 5a as components with

application of a ﬁlter tool: oxyhemoglobin (yellow), deoxyhemoglobin (orange), hemozoin (red), and heme-13 complex (green) imaged at 1090,

1642, 754, and 1080 cm⁻¹, respectively (left and central panels), and an overlay of images (right panel).

Chloroquine is the most studied hemozoin inhibitor, so we

used it as a benchmark for studying oxidative eﬀects.

Consistent with a pro-oxidant eﬀect of chloroquine, the

presence of 10 mM NAC caused a signiﬁcant increase in its

IC₅₀. This increase was dependent on the concentration of

NAC up to 10 mM, but above this concentration, leveled oﬀ,

probably because higher concentrations of NAC started to

interfere with the parasite survival assay (Figure 6d). In the

case of mammalian (CHO) cells, where chloroquine is far less

toxic, NAC had no eﬀect at 10 mM (Figure 6e). This shows

that chloroquine alone has no evident pro-oxidant eﬀects and

that it is the presence of heme that is responsible for the eﬀect

in the parasite. To further, explore whether chloroquine has an

oxidative mechanism of action in the parasite, we introduced t-

butyl hydroperoxide (Luperox) at a dose found to have no

eﬀect on parasite growth (50 μM). This signiﬁcantly

potentiated the activity of chloroquine. This pro-oxidant eﬀect

of Luperox could be completely abrogated by 10 mM NAC

(Figure 6f). These ﬁndings are consistent with the hypothesis

that chloroquine kills the parasite through the pro-oxidant

eﬀects of heme.

also found not to inhibit parasite hemozoin formation but

displayed moderate growth inhibition activity. For the ﬁrst two

combinations, the isobolograms were strongly antagonistic,

consistent with compounds 4 and 13 competing with

chloroquine for intracellular heme, while for the last

combination, the relationship was merely additive, consistent

with independent targets (Figure 6g). Finally, we investigated

the eﬀects of NAC and Luperox on compounds 4, 5, and 13

(Figure 6h). As expected, the parasite was protected from

compound 4 by NAC, while Luperox potentiated its eﬀect.

There was no eﬀect of either NAC or Luperox on compound

5, consistent with a mechanism not involving hemozoin

inhibition. Unexpectedly, compound 13 was also unaﬀected by

either NAC or Luperox despite the antagonistic relationship

with chloroquine and the strong evidence of complex

formation with heme presented earlier. This shows that not

all heme−inhibitor complexes act in the same way. Because

heme-13 apparently does not kill the parasite via pro-oxidant

eﬀects, it must kill it in some other way, possibly involving

inhibition of a protein target in the DV.

DISCUSSION

For comparison with this benchmark compound, we

constructed isobolograms of chloroquine in combination

with two hemozoin inhibitors (4 and 13) and a close analogue

of 4 (compound 5) that is not a β-hematin inhibitor and was

■

From the data collated in Figure 2a, it is evident that the

amount of exchangeable heme at the parasite growth inhibition

IC₅₀values across the diverse scaﬀolds investigated in this

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Figure 6. Speed and basis of killing of selected compounds. (a) IC₅₀values for test compounds washed out at various times (IC₅₀^t) relative to that

with no washout (IC₅₀^72h). Where no bar is shown, there was no inhibition of parasite growth after that length of exposure. (b) Trend of increasing

exchangeable heme levels as a function of prolonged duration of killing. (c) Eﬀects of externally introduced Fe(III)PPIX and Zn(II)PPIX on

parasite survival with and without NAC. (d) Eﬀects of varying concentration of NAC on the relative IC₅₀of chloroquine (1). (e) Lack of eﬀect of

NAC on toxicity of chloroquine (1) in CHO cells. (f) Protective eﬀect of NAC and potentiating eﬀect of Luperox on parasites in the presence of

chloroquine (1). NAC was found to abrogate the eﬀect of Luperox. (g) Isobolograms showing strong antagonism between compound 4 and

chloroquine (1) and compound 13 and chloroquine (1) but an additive eﬀect between the nonhemozoin inhibitor 5 and chloroquine (1). Dotted

line represents one standard deviation about the line. (h) Protective eﬀect of NAC on the eﬀects of compound 4 and potentiating eﬀect of Luperox,

but lack of eﬀect of either NAC or Luperox on the activities of compounds 5 and 13. Statistical signiﬁcance, calculated using a two-tailed t-test

(error bars showing 95% CI), is expressed using asterisks. *P < 0.05, **P < 0.01, ***P < 0.001.

study is not constant. Rather, it covers an order of magnitude

range from 0.05 to 0.52 fmol/cell (Table S2). This implies that

free heme on its own cannot be responsible for inhibition of

parasite growth in all cases, invalidating the supposition that all

hemozoin-inhibiting antimalarials kill the parasite merely by

causing a buildup of free heme alone.

The linear relationship between the amount of exchangeable

heme and amount of accumulated active compound at the

parasite inhibition IC₅₀shown in Figure 2c strongly suggests

that the exchangeable heme is present as a complex with the

inhibitor in each case. There has been only one previous report

of direct spectroscopic evidence for a complex between

chloroquine and heme in malaria parasites based on photo-

acoustic spectroscopy.²²While Raman microscopy has

provided evidence of eﬀects on the spectra of parasitized

RBCs and the heme species therein,²³changes in heme spectra

are quite subtle, and direct evidence for a complex has proven

elusive. It is clear from Figure 2a and c that the amount of

complex present in the case of chloroquine is low (Table S2),

making its detection in the presence of a large hemozoin

background extremely challenging. An advantage of using a

wide range of scaﬀolds was the presence of compounds that

produce substantial quantities of heme complex and which

contain substituents that could be exploited for imaging.

Taking advantage of the Br atom in compound 13, it was

possible to demonstrate colocalization of this compound with

Fe, strongly indicating the presence of a complex with heme.

That this is in fact a complex between compound 13 and heme

was demonstrated by Raman spectroscopy. This observation

supports the contention that the direct linear relationship

between exchangeable heme and inhibitor amount in the

parasite is a result of complex formation. It is therefore likely

that hemozoin inhibitors either act as complexes or that their

activity is strongly inﬂuenced by complex formation.

Interestingly, a very recent soft X-ray ﬂuorescence imaging

study of P. falciparum treated with bromoquine, an analogue of

chloroquine with a 7-Br, rather than 7-Cl substituent has

shown that this compound also colocalizes with hemozoin, but

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is found in the DV membrane as well, which the authors

interpreted as evidence of its presence as a heme−bromoquine

complex.²⁴

resulting in increased free Fe(III) heme (H) in the DV; and

(iii) precipitation of free Fe(III) heme, which is poorly soluble

at DV pH. It has been reported that the solubility of hemin at

pH 5 is 0.1 μM,²⁷with the result that the overwhelming

majority of the observed free heme in the DV is in the solid

state. (iv) The compound forms a complex with free heme

(HD) in solution. (v) The complex is also likely to be relatively

insoluble in this environment. The observed exchangeable

heme is a combination of mainly precipitated Fe(III) heme

and the complex, itself probably largely solid. (vi) The

dissolved complex may inhibit enzymes or transporters in

the DV or (vii) it may diﬀuse or be transported out of the DV

into the cytoplasm where the concentration of compound D is

much lower, driving the equilibrium toward dissociation. (viii)

Finally, soluble free heme in the cytoplasm may generate ROS,

killing the parasite.

The level of exchangeable heme attained with diﬀerent

compounds can evidently be traced to the speed of killing, with

slow killing compounds leading to high levels of exchangeable

heme and fast killing compounds resulting in only low levels of

exchangeable heme. A possible explanation for this is that slow

acting complexes allow the parasite to grow further into the

trophozoite stage relative to fast acting compounds, with

consequent increased digestion of hemoglobin, resulting in

more heme being available to bind to the test compound. This

would account for the trend seen in Figure 6b.

Several authors have suggested that the inhibition of

hemozoin formation by chloroquine and other quinolines

results in increased reactive oxygen species (ROS) owing to

the pro-oxidant eﬀect of free heme.^17,25,26This hypothesis is

consistent with the observation that the activities of

chloroquine and compound 4 are signiﬁcantly reduced with

the use of NAC which is expected to raise the concentration of

cytosolic glutathione protecting the cell from ROS, while also

being able to act directly as an antioxidant. Further support for

the ROS hypothesis is the fact that the activities of these

compounds were enhanced by the pro-oxidant Luperox. A

notable point, however, is that not all hemozoin inhibitors

seem to act in this way, because neither NAC nor Luperox had

any eﬀect on the activity of compound 13. Thus, the killing

mechanism of these compounds is not always the same.

Interestingly, despite the diﬀerence in parasite killing

mechanism between chloroquine and compound 13, they

nonetheless have an antagonistic relationship as revealed in the

isobologram study. This is to be expected if the ﬁrst step in the

antiparasitic process is inhibition of hemozoin formation in

both cases.

In terms of this hypothesis, if the compound delivers heme

to the cytoplasm, increased glutathione produced by the

introduction of NAC would protect the parasite from the

eﬀects of ROS, while the pro-oxidant Luperox would

exacerbate them. On the other hand, if the complex inhibits

a target in the DV, neither NAC nor Luperox would have any

eﬀect on its activity. This would explain the diﬀerential eﬀects

of NAC and Luperox on chloroquine and compound 4 on the

one hand versus compound 13 on the other. In the case of a

compound that delivers free heme to the cytoplasm, the

solubility of the complex would determine the rate of delivery.

For a very insoluble complex, a relatively long time would be

needed for heme to reach toxic levels, and consequently,

hemoglobin digestion would continue for a longer time, with

consequent buildup of a larger quantity of exchangeable heme

in the form of precipitated complex. Conversely, for a more

soluble complex, delivery of heme to the cytoplasm would be

much faster, with consequent faster killing of the parasite and

less time to build-up the solid complex in the DV. Diﬀerential

rates of transport of complexes out of the DV by transporters

would have a similar eﬀect. In the case of a complex that

inhibits a target in the DV, the time to appearance of the target

would determine the speed of killing. Once again, if the target

were expressed early, the killing speed would be fast, with little

time to build-up the precipitated complex and vice versa in the

case of a target that appears late. This can explain the

relationship between speed of killing and levels of exchange-

able heme. Key factors governing the concentrations of Fe(III)

heme complexes are likely to be the solubility products (K_sp) of

Fe(III) heme and its complex as well as the equilibrium

constant for formation of the complex (K). The availability of

free Fe(III) heme for complexation is determined by inhibition

of hemozoin formation. This is dependent on the strength of

inhibition of hemozoin formation and degree of accumulation

of the inhibitor in the DV by pH trapping. This accumulation

will also inﬂuence the position of the equilibrium formation of

the Fe(III) heme complex. Inhibition of hemozoin formation is

the key initiating step in the mechanism. Thus, even when

compounds have diﬀerent subsequent eﬀects, they will show

an antagonistic relationship as seen for chloroquine and

compound 13. This complicated process, involving an

interplay of multiple factors, would explain why no simple

relationship between antiparasitic activity and β-hematin

inhibition activity or levels of exchangeable heme has been

found.

Taking account of all the above ﬁndings, we propose a

hypothesis that can integrate these observations. This is

presented in summary in Figure 7. It involves: (i)

accumulation of the charged protonated form of the

compound (D) in the DV via pH trapping; (ii) inhibition of

hemozoin (HZ) formation by this protonated compound,

Figure 7. Model of the factors involved in the activity of hemozoin

inhibitors. Protonation states and charges are omitted for the sake of

simplicity. Refer to the main text for the numbered steps.

Abbreviations: D, drug; H, heme; HZ, hemozoin.

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derivatives (1.1−1.7 equiv) with dichloroquinazoline (19, 1.0

equiv) and Et₃N (1.5−2 equiv) in THF (10−15 mL) for 3−12

h at 60 °C.

TLC was used to monitor the reaction progress in

combination with UV (254 nm) absorption and staining

using iodine, ninhydrin or anisaldehyde.

Upon reaction completion, the reaction vessel was allowed

to cool to room temperature before the reaction contents were

diluted in EtOAc and DCM (50:50), after which the contents

were neutralized with a saturated solution of Na₂CO₃and left

to stir for several minutes. The aqueous and organic layers

were separated and the former washed multiple times with

DCM, after which the DCM fractions were pooled and

concentrated under high vacuum. Chromatography of the

residue followed by recrystallization from boiling MeOH or

DCM, produced solid products that were characterized and

evaluated for analytical purity.

METHODS

■

Synthesis. All chemicals and solvents were bought from

Sigma-Aldrich or Fluka and unless otherwise speciﬁed were

used as received. Tetrahydrofuran (THF) and dichloro-

methane (DCM) were freshly distilled under N₂and dried

over Na wire with benzophenone and P₄O₁₀, respectively.

Reactions were monitored by TLC using precoated silica-gel

60 F254 (0.2 mm) mounted on aluminum-backed plates,

commercially available from Merck. Compounds were

detected using a UV light with an absorption of 254 nm and

stained for visible detection using either iodine ﬁlings,

ninhydrin, or anisaldehyde. Puriﬁcation procedures involved

the use of ﬂash column chromatography and single or double

solvent recrystallization techniques using a Biotage Isolera 4

EXP and analytical reagent grade solvents, respectively.

Characterization procedures comprised: the determination of

the melting points of the compounds using a Reichert-Jung

Thermovar hot stage microscope; IR spectra recorded on a

N-(4-Bromophenyl)-2-chloroquinazolin-4-amine (21). 4-

Bromoaniline (0.50 g, 2.9 mmol, 1.4 equiv) and Et₃N (0.51

mL, 3.5 mmol, 1.7 equiv) were added to a suspension of 19

(0.40 g, 2.0 mmol) for the synthesis of 21. Compound 21 was

puriﬁed by recrystallization from boiling MeOH and obtained

as a white solid (0.51 g, 75%), mp 167−168 °C, R_f= 0.3

Bruker Tensor 27 FT-IR spectrometer; and NMR spectra

recorded on Bruker Ultrashield 400 Plus (for ¹H,

C

,

13

stan

HSQC, and COSY). The NMR spec solvents used were

deuterated acetone ((CD₃)₂CO), deuterated dimethyl sulf-

oxide ((CD₃)₂SO), and deuterated chloroform (CDCl₃)

with reference peaks occurring at 2.05, 2.50, and 7.26 ppm in

1

(EtOAc/hexane, 30:70). H NMR (400 MHz, DMSO-d₆) δ_H

1

the H NMR and 29.84, 39.52, and 77.16 ppm in the ¹³C

10.21 (1H, s), 8.55 (1H, ddd, J 8.4, 1.2, 0.5 Hz), 7.89 (1H, td,

J 8.4, 1.2 Hz), 7.82−7.76 (2H, m), 7.72 (1H, ddd, J 8.4, 1.2,

NMR spectra, respectively. All chemical shifts are stated in

ppm and coupling constants in Hz; HPLC was conducted

using an Agilent 1220 LC system V; HRMS (ESI) was

conducted at the University of Stellenbosch, Central Analytical

facilities (CAF) using a Waters Synapt G2 instrument.

0.5 Hz), 7.65 (1H, td, J 8.4, 1.2 Hz), 7.63−7.59 (2H, m); ¹³

C

NMR (101 MHz, DMSO-d₆) δ_C159.2 (C-q), 155.9 (C-q),

150.8 (C-q), 137.6 (C-q), 134.1, 131.4, 126.9, 126.6, 124.6,

123.4, 116.5 (C-q), 113.7 (C-q); HRMS (ESI) m/z: Found

333.9713. Calculated 333.9747, C₁₄H₁₀BrClN₃[M + H]⁺;

HPLC purity: 99%.

2,4-Dichloroquinazoline (19). Et₃N (1.4 mL, 10 mmol)

was added to a suspension of benzoylene urea 20 (0.82 g, 5.1

mmol) in POCl₃(9.2 mL, 51 mmol) under inert conditions

and set to reﬂux at 106 °C for approximately 18 h. The

reaction was monitored using TLC (MeOH/DCM, 1:99, R_f=

0.85) until the presence of the starting material diminished,

indicating the completion of the reaction. After completion,

the reaction mixture was diluted with DCM (80 mL) and

poured over crushed ice, after which the slurry was neutralized

using a saturated solution of Na₂CO₃(approximately 30 mL)

and allowed to stir for 1 h. The aqueous layer was extracted

several times with DCM, after which all the DCM fractions

were pooled, dried over sodium sulfate (Na₂SO₄) and the

organic fraction concentrated under vacuum. The crude

material was puriﬁed using ﬂash column chromatography

(MeOH/DCM 1:99, R_f= 0.85), followed by recrystallization

from boiling MeOH to obtain compound 19 as a white solid

2-Chloro-N-(furan-2-ylmethyl)quinazolin-4-amine (22).

Furfurylamine (0.16 mL, 1.8 mmol, 1.1 equiv) and Et₃N

(0.36 mL, 2.5 mmol, 1.5 equiv) were added to a suspension of

19 (0.34 g, 1.7 mmol). Compound 21 was puriﬁed by

recrystallization from boiling MeOH and obtained as a white

solid (0.33 g, 50%), mp 158−159 °C, R_f= 0.2 (EtOAc/hexane,

20:80). ¹H NMR (400 MHz, DMSO-d₆) δ_H9.18 (1H, t, J 5.5

Hz), 8.30 (1H, ddd, J 8.4, 1.2, 0.5 Hz), 7.81 (1H, td, J 8.4, 1.2

Hz), 7.63 (1H, ddd, J 8.4, 1.2, 0.5 Hz), 7.60 (1H, dd, J 1.8, 0.9

Hz), 7.54 (1H, td, J 8.4, 1.2 Hz), 6.42 (1H, dd, J 3.2, 1.8 Hz),

6.37 (1H, dd, J 3.2, 0.9 Hz), 4.74 (2H, d, J 5.5 Hz); ¹³C NMR

(101 MHz, DMSO-d₆) δ_C160.9 (C-q), 156.7 (C-q), 151.2 (C-

q), 150.3 (C-q), 142.2, 133.7, 126.6, 126.2, 123.1, 113.4 (C-q),

110.5, 107.7, 37.3; HRMS (ESI) m/z: Found 260.0538.

Calculated 260.0591, C₁₃H₁₁ClN₃O [M + H]⁺; HPLC purity:

99%.

(0.87 g, 87%), mp 117−118 °C (Lit:²⁸118−120 °C). H

1

2-Chloro-N-phenylquinazolin-4-amine (23). Aniline (0.26

mL, 2.8 mmol, 1.1 equiv) and Et₃N (0.53 mL, 3.8 mmol, 1.5

equiv) were added to a suspension of 19 (0.50 g, 2.5 mmol).

Compound 23 was puriﬁed by recrystallization from boiling

DCM and obtained as a white solid (0.57 g, 89%), mp 187−

188 °C, R_f= 0.3 (EtOAc/hexane, 30:70). ¹H NMR (400 MHz,

DMSO-d₆) δ_H10.16 (1H, s), 8.57 (1H, ddd, J 8.4, 1.2, 0.5

Hz), 7.87 (1H, td, J 8.4, 1.2 Hz), 7.82−7.75 (2H, m), 7.71

(1H, ddd, J 8.4, 1.2, 0.5 Hz), 7.64 (1H, td, J 8.4, 1.2 Hz),

7.48−7.39 (2H, m), 7.22−7.18 (1H, m); ¹³C NMR (101

MHz, DMSO-d₆) δ_C159.4 (C-q), 156.2 (C-q), 150.8 (C-q),

138.2 (C-q), 134.0, 128.6, 126.8, 126.5, 124.7, 123.4, 122.9,

113.7 (C-q); HRMS (ESI) m/z: Found 256.0633. Calculated

256.0642, C₁₄H₁₁ClN₃[M + H]⁺; HPLC purity: 100%.

NMR (400 MHz, Acetone-d₆) δ_H8.33 (1H, ddd, J 8.4, 1.4, 0.7

Hz), 8.16 (1H, td, J 8.4, 1.4 Hz), 8.01 (1H, ddd, J 8.4, 1.4, 0.7

Hz), 7.90 (1H, td, J 8.4, 1.4 Hz); ¹³C NMR (101 MHz,

Acetone-d₆) δ_C164.5 (C-q), 155.5 (C-q), 153.4 (C-q), 137.5,

130.7, 128.7, 126.8, 123.2 (C-q); HRMS (ESI) m/z: Found

198.9822. Calculated 198.9830, C₈H₅Cl₂N₂[M + H]⁺; HPLC

purity: 99%.

General Procedure for the Synthesis of 2-Chloro-

quinazolin-4-amine Intermediates (21−23). The follow-

ing general procedure for the synthesis of intermediates (21−

23) was followed unless otherwise stated in the speciﬁc

intermediate experimental details.

The 2-chloro-quinazolin-4-amine intermediates were synthe-

sized on a 0.50−1.0 g scale by reacting the preferred amine

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General Procedure for the Synthesis of 2,4-Diamino-

quinazoline (10−12). The quinazoline-2,4-diamines were

synthesized on a 0.05−0.30 g scale by reacting the required 2-

chloro-quinazolin-4-amine intermediate (1 equiv) with the

desired amine compound (1−2 equiv) in a sealed tube

overnight at a temperature of 150 °C in isopropanol (2−5

mL). TLC was used to monitor the reactions in combination

with UV (254 nm) absorption and staining using iodine,

ninhydrin or anisaldehyde.

Upon reaction completion, the reaction contents were

allowed to cool to ambient temperature before being diluted

with EtOAc, after which they were neutralized with a solution

of NaOH (1 M, 3−4 mL), which was allowed to stir for several

minutes. The aqueous and organic layers were separated and

the former washed multiple times with EtOAc, after which the

EtOAc fractions were pooled and concentrated under high

vacuum. Chromatography of the residue, followed by

recrystallization from boiling MeOH or DCM, produced

solid products.

121.5, 120.1, 110.9 (C-q), 49.2, 32.8, 25.4, 25.0; HRMS (ESI)

m/z: Found 319.1917. Calculated 319.1923, C₂₀H₂₃N₄[M +

H]⁺; HPLC purity: 99%.

3-(Benzo[d]thiazol-2-yl)aniline (24). 3-Aminobenzoic

acid (2.500 g, 18.637 mmol) was added to 2-aminothiophenol

(2 mL, 18.637 mmol) and polyphosphoric acid (PPA) (25.00

g, 10 equiv). The mixture was continually stirred while heated

at 180 °C until completion (3 h) as conﬁrmed by TLC (5:95

MeOH:DCM, product R_f= 0.67). After completion, the

reaction mixture was cooled and poured into ice cold Na₂CO₃

solution (400 mL, 10%) while stirring in order to neutralize

residual PPA. The resulting mixture was ﬁltered and washed

with cold water (5 mL). The ﬁltrate was retained and dried

under vacuum at 60 °C to produce crude 24. The product was

recrystallized in boiling MeOH and water to aﬀord pure 24

(3.623 g, 16.028 mmol, 86%) as a green solid, mp 138−139

°C. IR (ATR) ν/cm⁻¹: 3440, 3323 (NH str.), 3062 (Ar−H

str.), 1627 (C = N str.), 1604 (NH bend), 761 (m. disub.

1

benzene); H NMR (400 MHz, DMSO-d₆) δ_H8.02 (d, J 7.9

N2, N4-Bis(4-bromophenyl)quinazoline-2,4-diamine (10).

4-Bromoaniline (0.17 g, 1.0 mmol, 1.3 equiv) was reacted with

compound 21 (0.25 g, 0.75 mmol) to produce 10, which was

puriﬁed by recrystallization from boiling MeOH and obtained

as a light-brown solid (0.29 g, 82%), mp 183−184 °C, R_f= 0.6

Hz, 1H), 8.02 (d, J 8.1 Hz, 1H), 7.52 (t, J 7.2 Hz, 1H), 7.43 (t,

J 8.0 Hz 1H), 7.38 (m, 1H), 7.19 (m, 2H), 6.77 (d, J 6.6 Hz,

1H), 5.44 (br. s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ_C

168.3 (Cq), 153.7 (Cq), 149.4 (Cq), 134.4 (Cq), 133.6 (Cq),

129.9, 126.6, 125.4, 122.5, 117.0, 114.9, 112.1. EI-MS m/z:

Found 226. Calculated 226, C₁₃H₁₀N₂S [M]⁺. HPLC purity:

100%.

1

(EtOAc/hexane, 50:50). H NMR (400 MHz, DMSO-d₆) δ_H

9.69 (1H, s), 9.31 (1H, s), 8.38 (1H, dd, J 7.5, 1.2 Hz), 7.92−

7.88 (4H, m), 7.69 (1H, td, J 7.5, 1.2 Hz), 7.57 (2H, d, J 8.9

Hz), 7.52 (1H, dd, J 7.5, 1.2 Hz), 7.41 (2H, d, J 8.9 Hz), 7.31

(1H, td, J 7.5, 1.2 Hz); ¹³C NMR (101 MHz, DMSO-d₆) δ_C

158.2 (C-q), 155.9 (C-q), 151.4 (C-q), 140.5 (C-q), 138.8 (C-

q), 133.1, 131.2, 130.9, 125.7, 124.4, 123.1, 122.2, 120.7, 115.2

(C-q), 112.1 (C-q), 111.8 (C-q); HRMS (ESI) m/z: Found

468.9663. Calculated 468.9663, C₂₀H₁₅Br₂N₄[M + H]⁺;

HPLC purity: 98%.

3-(Benzo[d]oxazol-2-yl)aniline (25). 3-Aminobenzoic acid

(2.500 g, 18.229 mmol) was added to 2-aminophenol (1.989g,

18.229 mmol) and PPA (25 g, 10 equiv). The mixture was

continually stirred while heated at 180 °C until completion (3

h) as conﬁrmed by TLC (5:95 MeOH:DCM, product R_f=

0.63). After completion, the reaction mixture was cooled and

poured into ice cold Na₂CO₃solution (400 mL, 10%) while

stirring in order to neutralize residual PPA. The resulting

mixture was ﬁltered and washed with cold water (5 mL). The

ﬁltrate was retained and dried under vacuum at 60 °C to

produce crude 25. The product was recrystallized in boiling

MeOH and water to aﬀord pure 25 (3.411 g, 16.225 mmol,

89%) as a red solid. mp 179−180 °C (Lit.:²⁹178 °C). IR

(ATR) ν/cm⁻¹: 3458, 3305 (NH str.), 3091 (Ar−H str.), 1625

N4-(Furan-2-ylmethyl)-N2-phenylquinazoline-2,4-dia-

mine (11). Phenylamine (0.11 mL, 1.2 mmol, 1.2 equiv) was

reacted with compound 22 (0.26 g, 1.0 mmol). Compound 11

was puriﬁed by recrystallization from boiling MeOH and

obtained as a white solid (0.11 g, 36%) mp 103−104 °C, R_f=

1

0.4 (EtOAc/hexane, 30:70). H NMR (400 MHz, DMSO-d₆)

1

δ_H9.10 (1H, s), 8.64 (1H, t, J 5.6 Hz), 8.12 (1H, dd, J 8.4, 1.0

Hz), 7.86 (2H, dd, J 8.6, 1.0 Hz), 7.62 (1H, td, J 8.4, 1.0 Hz),

7.59 (1H, dd, J 1.8, 0.8 Hz), 7.43 (1H, dd, J 8.4, 1.0 Hz),

7.29−7.23 (2H, m), 7.19 (1H, td, J 8.4, 1.0 Hz), 6.95−6.86

(1H, m), 6.40 (1H, dd, J 3.2, 1.8 Hz), 6.36 (1H, dd, J 3.2, 0.8

Hz), 4.78 (2H, d, J 5.6 Hz); ¹³C NMR (101 MHz, DMSO-d₆)

δ_C159.8 (C-q), 156.4 (C-q), 152.3 (C-q), 150.4 (C-q), 141.9,

141.0 (C-q), 132.8, 128.2, 124.8, 122.8, 121.6, 120.8, 118.9,

111.5 (C-q), 110.4, 107.1, 37.2; HRMS (ESI) m/z: Found

317.1393. Calculated 317.1402, C₁₉H₁₇N₄O [M + H]⁺; HPLC

purity: 97%.

(C = N str.), 1550 (NH bend), 746 (m. disub. benzene); H

NMR (400 MHz, DMSO-d₆) δ_H7.76 (m, 2H), 7.46 (t, J 1.8

Hz, 1H), 7.40 (m, 2H), 7.35 (m, 1H), 7.23 (t, J 7.8 Hz, 1H),

6.81 (ddd, J 8.0, 2.3, 1.0, 1H), 5.45 (br. S, 2H); ¹³C NMR

(101 MHz, DMSO-d₆) δ_C163.1 (Cq), 150.2 (Cq), 149.5

(Cq), 141.7 (Cq), 129.8, 127.0 (Cq), 125.3, 124.8, 119.7,

117.4, 114.7, 112.2, 110.8. EI-MS m/z: Found 210. Calculated

210, C₁₃H₁₀N₂O [M]^+.HPLC purity: 96%.

Acid Chloride Formation and Amide Bond Forma-

tion. To 5-(triﬂuoromethyl)pyridine-3-carboxylic acid (2

mmol) was added thionyl chloride (SOCl₂) (8 equiv), and

the reaction mixture heated under reﬂux at 80 °C until

completion. TLC was performed to monitor the reaction using

10:90 MeOH/DCM and stained with I₂. After completion

residual thionyl chloride was removed under pressure and the

product dried under vacuum.

The acid chloride was dissolved in THF and added to the

required intermediate (24 or 25) (1 mmol) dissolved in

pyridine (2 mL) under an inert atmosphere until completion.

Reactions were maintained at −40 °C using an acetonitrile/

liquid N₂bath. Reaction progress was monitored using TLC

5:95 MeOH/DCM and stained using ninhydrin. The starting

material (amine) appeared dark pink and the product (amide)

Synthesis of Benzazole Aniline Intermediates (24 and

25). Cyclohexylamine (0.11 mL, 0.89 mmol, 1.2 equiv) was

reacted with compound 23 (0.20 g, 0.78 mmol) to form 12,

which was puriﬁed by ﬂash chromatography, R_f= 0.3 (EtOAc/

Hexane, 30:70) as a white solid (0.18 g, 72%), mp 133−134

1

°C. H NMR (400 MHz, DMSO-d₆) δ_H9.35 (1H, s), 8.28

(1H, dd, J 8.0, 1.2 Hz), 7.96 (2H, d, J 7.9 Hz), 7.54 (1H, td, J

8.0, 1.2 Hz), 7.35 (2H, t, J 7.9 Hz), 7.30 (1H, dd, J 8.0, 1.2

Hz), 7.15−7.03 (2H, m), 6.57 (1H, s), 3.79 (1H, s), 1.94−

1.29 (8H, m), 1.61−1.58 (1H, m), 1.21−1.17 (1H, m); ¹³C

NMR (101 MHz, DMSO-d₆) δ_C158.1 (C-q), 158.0 (C-q),

152.4 (C-q), 139.8 (C-q), 132.5, 128.2, 124.6, 123.0, 122.8,

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stained light pink. Once complete the reaction was quenched

with MeOH and dried under vacuum to aﬀord the appropriate

amide. Following puriﬁcation by column chromatography

(MeOH: DCM, from 2:98 to 20:80) the product was

recrystallized in boiling MeOH.

inoculum sizes (IS) ranging from 1 to 5 and fractional volumes

(V_f) of parasitized erythrocytes from 0.0001 to 0.001. The

relationship was extrapolated via linear regression to give an

absolute IC₅₀(IC₅₀at inﬁnite parasite dilution) from the y-

intercept in Graph Pad Prism v7.0. Experimental CAR values

were calculated from a linear plot of IC₅₀values plotted as a

function of their corresponding V_fin accordance with eq 1.

N-(3-(Benzo[d]oxazol-2-yl)phenyl)-5-(triﬂuoromethyl)-

nicotinamide (17). The product of the reaction between 24

(0.226 g, 1 mmol) and the acid chloride (2 mmol) dissolved in

THF (2 mL) was puriﬁed by column chromatography (5:95

MeOH: DCM, product R_f= 0.56) and the appropriate

fractions recrystallized to aﬀord pure 17 (0.295 g, 0.739 mmol,

74%) as a brown solid. mp 212−213 °C. IR (ATR) ν/cm⁻¹

3302 (NH str.), 3053 (Ar−H str.), 1650 (C = O str.), 1656 (C

= N str.), 1139 (C−F str.), 710 (m. disub. benzene); ¹H NMR

(400 MHz, DMSO-d₆) δ_H10.84 (s, 1H), 9.43 (d, J 2.0 Hz,

1H), 9.20 (m, 1H), 8.75 (m, 1H), 8.62 (t, J 1.8 Hz, 1H), 8.16

(d, J 7.3 Hz, 1H), 8.08 (d, J 7.5 Hz, 1H), 8.03 (ddd, J 8.2 Hz,

2.1 Hz, 0.9 Hz, 1H), 7.86 (dt, J 8.1 Hz, 1.2 Hz, 1H), 7.60 (m,

1H), 7.56 (m, 1H), 7.48 (m, 1H); ¹³C NMR (400 MHz,

DMSO-d₆) δ_C166.9 (Cq), 162.7, 153.4 (Cq), 152.7, 148.6

(Cq), 139.4 (Cq), 134.4 (Cq),133.3, 132.6 (Cq), 132.5 (Cq),

130.3 (Cq), 129.9, 126.6, 125.6, 125.0, 122.9, 122.8, 122.8,

122.3, 118.6. EI-MS m/z: Found 399. Calculated 399 [M]⁺

C₂₀H₁₂F₃N₃OS. HPLC purity: 99%.

IC₅₀(obs) = {IC₅₀(absolute) × CAR} × V

f

+ IC₅₀(absolute)

(1)

hematocrit × % parasitemia

where V =

and hematocrit × %para-

f

10 000

sitemia is the IS.

The total amount of intracellular test compound (ITC) was

then calculated using eq 2:

(CAR × V × IC₅₀(4))

cell

ITC (fmol) =

(2)

1 000 000

Where V_cellis the average volume of an infected RBC in fL

(84.3

5.7 fL)³⁷and IC₅₀(4) the 50% inhibitory

concentration in μM at IS = 4, the standard parasite culturing

conditions used for cellular heme measurements.

Electron Microscopy. TEM and EELS experiments were

carried out as described previously^1,7except for the use of

polysaccharide gel encapsulation and high pressure freezing. A

4% agarose gel solution was prepared using a modiﬁed

microwave method previously described.³⁸The ﬁxed PRBCs

were washed twice with 1 mL PBS before the samples were

suspended in an equal volume of the 4% agarose slurry. The

suspension was allowed to solidify before adding 50 μL of PBS

to avoid sample dehydration.

Agarose-ﬁxed PRBC samples were high pressure frozen and

embedded using a modiﬁed method previously described by

Yamada et al.³⁹⁻⁴¹Brieﬂy, the agarose-ﬁxed PRBC samples

were cut into thin sections (80−100 nm) using a diamond

knife and sandwiched between two single-holed copper grids

under a light microscope. Samples were rapidly frozen with

liquid nitrogen at a pressure of 2100 bar to a temperature of

−81 °C within 2 min using a Leica EM High Pressure Freezer

100. High pressure frozen samples were transferred to a Leica

EM AFS embedding system after removing the copper grids

from the Leica sample carrier. Samples were embedded at −80

°C in acetone for 24 h. After 24 h, acetone was replaced with

OsO₄to maintain lipids and proteins within the sample for

another 24 h. The samples were left in the Leica EM AFS UC7

embedding system in low viscosity Spurr’s resin for 5−7 days

while the temperature was gradually increased to RT. Samples

were removed and imaged using TEM with EELS.

N-(3-(Benzo[d]oxazol-2-yl)phenyl)-5-(triﬂuoromethyl)-

nicotinamide (18). The product of the reaction between 25

(0.210 g, 1 mmol) and the acid chloride (2 mmol) dissolved in

THF (6 mL) and DMF (0.1 mL) was puriﬁed by column

chromatography (5:95 MeOH:DCM, product R_f= 0.67). The

appropriate fractions were recrystallized in boiling MeOH to

aﬀord pure 18 (0.198 g, 0.518 mmol, 52%) as a cream solid,

mp 229−230 °C. IR (ATR) ν/cm⁻¹3259 (NH str.), 3051

(Ar−H str.), 1649 (C = O str.), 1635 (C = N str.), 1128 (C−F

1

str.), 736 (m. disub. benzene); H NMR (400 MHz, DMSO-

d₆) δ_H10.83 (s, 1H), 9.41 (d, J 1.6 Hz, 1H), 9.18 (d, J 0.9 Hz,

1H), 8.73 (d, J 1.6 Hz, 2H), 8.02 (m, 1H), 7.96 (m, 1H), 7.80

(m, 2H), 7.62 (t, J 8.0 Hz, 1H), 7.42 (m, 2H); ¹³C NMR (400

MHz, DMSO-d₆) δ_C163.3, 162.5 (Cq), 153.3, 150.7 (Cq),

149.2 (Cq), 142.0 (Cq), 139.9 (Cq), 133.2, 130.8 (Cq), 130.4,

127.4 (Cq), 126.1, 125.4, 124.7, 123.9, 123.4, 120.4, 119.4,

111.4. EI-MS m/z: Found 383. Calculated 383 [M]⁺

C₂₀H₁₂F₃N₃O₂. HPLC purity: 97%.

β-Hematin Inhibition, Parasite Culturing, and Cellular

Heme Measurements. The detergent mediated assay for β-

hematin inhibition was based on that developed by Carter et

al.³⁰modiﬁed to use pyridine to detect unconverted hematin as

previously described,³¹with modiﬁcations reported in detail

elsewhere.³²

Parasites were cultured according to the method of Trager

and Jensen with minor modiﬁcations.³³Parasite growth

inhibition experiments were carried out using previously

reported methods. For most experiments the parasite lactate

dehydrogenase assay was used to measure parasite growth,³⁴

but for the experiments investigating speed of killing and the

eﬀects of NAC and Luperox a SYBR Green I assay was

used.^35,36

Raman Microscopy. Slides for confocal Raman micros-

copy were prepared as follows: a drop of clear nail polish was

smeared evenly onto a clean glass microscope slide to create a

thin ﬁlm. Precut aluminum foil (1.5 × 3.5 cm) strips were

carefully placed on the ﬁlm using thin tipped tweezers. The

aluminum foil strips were then carefully pressed, using nitrile

gloves, to create a smooth surface.

Measurement of cellular hemoglobin, exchangeable heme

and hemozoin was performed as previously described in

detail.⁸

Cellular Accumulation Ratios. The inoculum eﬀect

analysis was carried out using a method similar to that

previously reported.⁴IC₅₀values measured using a modiﬁed

version of the pLDH assay were determined in triplicate at

Trophozoite cultures were mixed and decanted into four 15

mL centrifuge tubes (250 μL). The decanted trophozoite

cultures were left to stand for 10 min at rt before mixing and

gently smearing 2 μL onto an aluminum foil covered glass

microscope slide. Glutaraldehyde ﬁxation was avoided, because

this is known to oxidize hemoglobin to methemoglobin. The

sample slide was left to air-dry at rt for 15 min before analysis.

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Samples for Raman imaging of the synthetically prepared

material were prepared as follows: Solid aggregates of β-

hematin crystals were gently broken up in a glass beaker before

placing on an aluminum foil covered microscope glass slide for

analysis; a 2 μL drop of 1 mM 13 in DMSO was dried onto an

aluminum foil covered microscope glass slide using an

incandescent lamp at RT before further analysis; a 2 μL

drop of 1 mM solutions of hematin or hemin were dried onto

separate aluminum foil covered microscope glass slides using

an incandescent lamp at RT before further analysis; a 1:10 (v/

v) mixture of hemin and 13 was prepared by titrating 5 mL of a

1 mM solution of hemin into 50 mL solution of 1 mM of

compound 13 in a 100 mL glass beaker. DMSO was added to a

ﬁnal volume of 100 mL. The container was covered with

paraﬁlm and the solution was gently heated in a water bath (60

°C) for 30 min and cooled to RT. Roughly 2 μL of the mixture

was dried, using an incandescent lamp, onto an aluminum foil

covered glass microscope slide for further analysis.

Raman spectra were collected using a confocal Raman

microspectrometer alpha300 R (WITec GmbH, Germany)

equipped with a CCD camera cooled to −60 °C and three air-

calibrated objective lenses (10×, 50×, and 100× ). All spectra

were collected using the 100× objective lens. This Raman

microspectrometer uses a green Nd:YAG laser with excitation

wavelength of 532 nm and a laser spot of 1−2 μm with a

resolution of 1−2 cm⁻¹that was set at a default grating of

2050. The Raman microspectrometer was controlled using

WITec Control FIVE v5.0.0 software. Spectra were recorded in

the iron porphyrin dominated spectral region of 1700−500

cm⁻¹.

Single scan Raman spectra were recorded on uninfected

RBCs, untreated and treated infected RBCs and synthetic

samples. For cells, an average of 30 single scan Raman spectra

from selected regions were collected from each of 10 randomly

selected cells. For synthetic complexes, the same number of

single scan Raman spectra were obtained from 5 randomly

selected regions of the slide. Before performing scans, the

oscillator was adjusted and calibrated to the aluminum foil

covered microscope glass slide to correct for any background

signals. This was also done to reduce the comic ray eﬀect

which can cause false-positive peaks. All experiments were

carried out in triplicate, and each single scan Raman spectrum

was collected using a total of 100 accumulations at an

integration time of 0.05 s. The spectra obtained from cells,

including RBCs, were collected with an optimal laser power of

0.1 mW to maximize the signal-to-noise ratio, while avoiding

hemolysis and photodamage of cells. Furthermore, cells were

allowed to rest for intervals of 10 min before each scan.

Synthetic complexes were analyzed using a laser power of 1

mW.

integration times and laser power were set as low as possible to

obtain acceptable signal-to-noise ratios, namely 0.05 s and 0.1

mW. For synthetic materials an integration time of 0.1 s at

laser power 1 mW was used. These spectra were corrected for

cosmic rays, smoothed, averaged and background subtracted

using the same parameters described above for single scan

Raman spectra.

For PCA analysis, spectra were normalized by subtracting

the minimum relative intensity count (c_min) from each data

point (c_i), divided by the relative intensity of the most intense

peak (c_max). Next, the normalized spectra were converted to

their corresponding second derivative spectra, thus deﬁning

peak positions based on their curvature rather than intensity.

The data points ( a_i) at each peak position of the second

derivative spectra were ﬁltered based on whether they were

above or below the 95th percentile (positive + p_0.05or negative

− p_0.05) to eliminate noise.

All resulting replicate peak positions for pairs of heme

components analyzed (e.g., oxyhemoglobin versus hemozoin)

were transferred to Molegro Data Modeler Executive software.

This software was used to generate the eigenvectors (PC1 and

PC2) and corresponding eigenvalues.

A simpliﬁed approach was taken to test the statistical

signiﬁcance of separation of clusters in each PCA plot for pairs

of heme components. In brief, the eigenvalues for PC1 and

PC2 for a given pair of Fe(III)PPIX species were used to

calculate the centroids μ^a_xand μ_y^a(a = I or II) given by eqs 3

and 4:

n_a

i=1

∑

x_i^a

μ^a=

x

n

(3)

a

n_a

∑

_i=1y^a

i

μ^a=

y

n

(4)

a

where n_ais the number of points in cluster a. The points were

then shifted so that one of the clusters had a centroid at (0,0)

by applying eqs 5 and 6.

x_i^a′= x_i^a− μ^I

(5)

x

y^a′= y^a− μ^I

y

(6)

i

The new position of centroid II in polar coordinates is then

given by eqs 7 and 8

μ^II′= R^IIcosθ

(7)

(8)

x

μ^II′= R^IIsinθ

y

where R^II= (μ^II′)²+ (μ^II′)²

All single scan spectra were further processed using WITec

Project FIVE v5.0.0.40 software. This involved cosmic ray

correction (ﬁlter size 4; dynamic factor 50), smoothing using a

Savitzky−Golay algorithm (9 smoothing points; derivative 0),

averaging (spectral 9; spatial 0), and subtraction of background

(shape size 100; noise factor 4).

Large-scale image Raman spectra were also recorded for

cells. Every large-scale image Raman spectrum has a

corresponding image where each pixel corresponds to a single

spectrum. An average of 15 lines per image and 15 points per

line for each large-scale image Raman spectrum were recorded

for an average collection time of about 35 min. Because there is

a risk of photodamage to cells with this laser exposure time, the

x

y

(9)

II′

i

y

μ

j

z

j

^yz

z

and θ = sin⁻¹

z

j

z

II

j

z

R

(10)

k

{

Finally, rotation of the axes through angle θ resulted in a new

set of axes in which both centroids lay on the new x-axis with

no separation of the clusters along the y-direction and

maximum separation along x. The positions of points along

the new x-axis were given by eq 11

x_i^a″= x_i^a′cosθ + y^a′sinθ

(11)

i

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The statistical signiﬁcance of the separations of the means μ^I_x′′

and μ^I_x^I″ were evaluated using a Welch’s t-test which allows for

unequal variances in the data.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00680.

■

sı

Speed of Killing Measurements. For speed of killing

measurements, an early ring-stage sorbitol synchronized

parasite culture at 2% parasitemia and 2% hematocrit was

prepared in complete medium, and 200 μL was added to each

well of a 96-well ﬂat-bottom plate. For each compound, 5

concentrations were prepared in complete medium from the

original 2 mg/mL stock. Twenty microliters of these individual

concentrations were added to the plate such that the ﬁnal

volume in each well was 220 μL and the ﬁnal concentration in

each well corresponded to a multiple of the IC₅₀of each

compound (0.5× , 1×, 1.5×, 2×, and 3× ). Each assay plate

had at least eight drug-free control wells. The assay plates were

incubated at 37 °C in a gassing chamber with 3% O₂, 4% CO₂,

and N₂. Compound wash-out steps were performed by

washing oﬀ the compound containing medium and replacing

it with new complete medium at the time points shown in

Figure S3. The assay plates were then left to incubate for 72 h.

After 72 h, the plates were removed from the gassing chamber,

the medium aspirated and the pellet washed with 0.22 μm

ﬁltered phosphate buﬀered saline (1 × PBS). Following this,

100 μL of freshly prepared SYBR green I in 0.22 μm ﬁltered

PBS (1 μL SYBR green I: 9 mL PBS) was added to each well

of the 96-well plate. This was left to incubate for at least 30

min in the dark at RT. After incubation the plates were

resuspended and read on a BD AccuriTM C6 Plus ﬂow

cytometer with C-sampler software. For each sample measure-

ment 20 000 events were collected. The acquired data was

analyzed using FlowJo software version 10.5.3. A gating

method was employed. The uninfected and infected red blood

cells in each sample were characterized using forward (FSC)

and side scatter (SSC), as well as the ﬂuorescence intensity

(FL1) parameter to determine SYBR green I positive cells.

Isobolograms. Isobolograms were constructed according

to the method of Fivelman et al.⁴²For each isobologram, two

compounds were tested alone and in ﬁxed concentration ratios

of 1:4, 2:3, 3:2, and 4:1. All combinations were prepared on

the day of the experiment, and IC₅₀values were measured in

triplicate using a modiﬁed version of the pLDH assay. Five

IC₅₀values were determined for each compound for each

isobologram. The IC₅₀values were used to determine the

fractional inhibitory concentration (FIC) for each compound,

for each combination according to eqs 12 and 13:

IC₅₀values for inhibition of β-hematin formation and

parasite growth in the chloroquine-sensitive NF54 strain

of P. falciparum; amounts of cellular exchangeable heme,

cellular accumulation ratios, and amounts of accumu-

lated inhibitor per cell for selected compounds studied;

statistical analyses of overlap between Raman spectra of

various heme compounds, both in the cell and

synthetically prepared; Raman microscopy images of

untreated infected red blood cells; additional Raman

microscopy images of infected red blood cells treated

with compound 13; speed of killing experiment strategy

(PDF)

AUTHOR INFORMATION

Corresponding Author

■

Timothy J. Egan − Department of Chemistry and Institute of

Infectious Disease and Molecular Medicine, University of

Cape Town, Cape Town 7701, South Africa; orcid.org/

0000-0001-7720-8473; Email: timothy.egan@uct.ac.za

Authors

Roxanne Openshaw − Department of Chemistry, University of

Cape Town, Cape Town 7701, South Africa

Keletso Maepa − Division of Clinical Pharmacology,

Department of Medicine, University of Cape Town, Cape

Town 7925, South Africa

Stefan J. Benjamin − Department of Chemistry, University of

Cape Town, Cape Town 7701, South Africa

Lauren Wainwright − Department of Chemistry, University of

Cape Town, Cape Town 7701, South Africa

Jill M. Combrinck − Division of Clinical Pharmacology,

Department of Medicine, University of Cape Town, Cape

Town 7925, South Africa

Roger Hunter − Department of Chemistry, University of Cape

Town, Cape Town 7701, South Africa; orcid.org/0000-

0001-8775-083X

Complete contact information is available at:

https://pubs.acs.org/10.1021/acsinfecdis.0c00680

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

IC₅₀(A + B)

FIC A =

We thank Miranda Waldron for assistance with Raman

microscopy and Mohamed Jaﬀer for assistance with TEM.

R.O., K.M., and S.J.B. thank the National Research Foundation

of South Africa, and J.M.C. thanks CIDRI Africa for ﬁnancial

support. The research reported in this publication was

supported by the United States National Institute of Allergy

and Infectious Diseases of the National Institutes of Health

under Award R01AI110329 and for the speed of killing studies

R01AI143521. The content is solely the responsibility of the

authors and does not necessarily represent the oﬃcial views of

the National Institutes of Health.

IC₅₀(A)

(12)

where IC₅₀(A + B) = 50% inhibitory concentration of

compound A and B at a ﬁxed concentration ratio and IC₅₀A =

50% inhibitory concentration of compound A alone

IC₅₀(B + A)

FIC B =

IC₅₀(B)

(13)

where IC₅₀(B + A) = 50% inhibitory concentration of

compound B and A at a ﬁxed concentration ratio and IC₅₀B =

50% inhibitory concentration of compound B alone. An

isobologram was constructed for each combination by plotting

FIC A as a function of FIC B.

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A Diverse Range of Hemozoin Inhibiting Scaffolds Act on Plasmodium falciparum as Heme Complexes

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