Polyhydroxybenzoic acid derivatives as potential new antimalarial agents

Article abstract of DOI:10.1002/ardp.202100190

With more than 200 million cases and 400,000 related deaths, malaria remains one of the deadliest infectious diseases of 2021. Unfortunately, despite the availability of efficient treatments, we have observed an increase in people infected with malaria since 2015 (from 211 million in 2015 to 229 million in 2019). This trend could partially be due to the development of resistance to all the current drugs. Therefore, there is an urgent need for new alternatives. We have, thus, selected common natural scaffolds, polyhydroxybenzoic acids, and synthesized a library of derivatives to better understand the structure–activity relationships explaining their antiplasmodial effect. Only gallic acid derivatives showed a noticeable potential for further developments. Indeed, they showed a selective inhibitory effect on Plasmodium (IC₅₀ ~20 μM, SI > 5) often associated with interesting water solubility. Moreover, this has confirmed the critical importance of free phenolic functions (pyrogallol moiety) for the antimalarial effect. Methyl 4-benzoxy-3,5-dihydroxybenzoate (39) has, for the first time, been recognized as a potential lead for future research because of its marked inhibitory activity against Plasmodium falciparum and its significant hydrosolubility (3.72 mM).

Full text of DOI:10.1002/ardp.202100190

Received: 17 May 2021
Revised: 27 June 2021
Accepted: 1 July 2021
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DOI: 10.1002/ardp.202100190
F U L L P A P E R
Polyhydroxybenzoic acid derivatives as potential new
antimalarial agents
Gilles Degotte1,2
| Bernard Pirotte1
| Michel Frédérich2
| Pierre Francotte1
1Department of Pharmacy, Laboratory of
Medicinal Chemistry, CIRM, University of
Liège, Liège, Belgium
Abstract
With more than 200 million cases and 400,000 related deaths, malaria remains one of
the deadliest infectious diseases of 2021. Unfortunately, despite the availability of
efficient treatments, we have observed an increase in people infected with malaria
since 2015 (from 211 million in 2015 to 229 million in 2019). This trend could partially
be due to the development of resistance to all the current drugs. Therefore, there is an
urgent need for new alternatives. We have, thus, selected common natural scaffolds,
polyhydroxybenzoic acids, and synthesized a library of derivatives to better under-
stand the structure–activity relationships explaining their antiplasmodial effect. Only
gallic acid derivatives showed a noticeable potential for further developments. Indeed,
they showed a selective inhibitory effect on Plasmodium (IC50 ~20 µM, SI > 5) often
associated with interesting water solubility. Moreover, this has confirmed the critical
importance of free phenolic functions (pyrogallol moiety) for the antimalarial effect.
Methyl 4‐benzoxy‐3,5‐dihydroxybenzoate (39) has, for the first time, been recognized
as a potential lead for future research because of its marked inhibitory activity against
Plasmodium falciparum and its significant hydrosolubility (3.72 mM).
2Department of Pharmacy, Laboratory of
Pharmacognosy, CIRM, University of Liège,
Liège, Belgium
Correspondence
Pierre Francotte, Department of Pharmacy,
CIRM, University of Liège, Quartier Hôpital,
B36 Tower 4, +5, Ave. Hippocrate 15,
4000 Liège, Belgium.
Email: pierre.francotte@uliege.be
Funding information
Fonds pour la Formation à la Recherche dans
l'Industrie et dans l'Agriculture,
Grant/Award Number: FC23283; Fondation
Léon Frédéricq
K E Y W O R D S
gallic acid, hydroxybenzoic acid, malaria, medicinal chemistry, Plasmodium
(Pf), P. vivax, P. ovale, P. malariae, and P. knowlesi.[4] Among them, Pf is
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1
INTRODUCTION
more problematic because it is responsible for more than 95% of
Among all the public health problems, the response to infectious
diseases remains a major challenge, as demonstrated by the recent
outbreak of SARS‐Cov‐2. This could be explained by a lack of in-
novations or satisfactory treatments and the quick development of
resistance. Moreover, infections are a high burden for low‐ and
middle‐income countries with millions of cases annually and costing
billions of dollars.[1,2]
cases worldwide and because of its higher mortality.[5]
This parasitosis results in more than 200 million cases and
400,000 related deaths annually, mainly of children under five.[5]
Unfortunately, after more than a decade of constant decreases, the
last few years have been characterized by an increase in patients with
malaria (211 million in 2015 to 229 million in 2019). This trend could
be partially explained by the development of resistances to all the
recommended active principles, for example, in the Greater Mekong
subregion and even in Africa (Rwanda).[6–8] However, it could also
be a result of the development of resistances for the Plasmodium
vectors to the common insecticides (mainly pyrethroids).[5] A vaccine
(RTS,S/AS01 or Mosquirix®), developed by GSK and the Walter Reed
Army Institute of Research, is now in pilot trials in some African
countries, and the only vaccine that significantly reduces the
Therefore, malaria, one of the deadliest infections, is a cause of
concern even in 2021. It is commonly characterized by cyclic fevers,
headaches, and chills, but could lead to more severe symptoms:
cerebral malaria, severe anemia, and finally death, if untreated.[3] This
pathology is caused by a multistage parasite belonging to Plasmodium
spp., and transmitted to humans through mosquitoes. Five species
are currently reported as human pathogens: Plasmodium falciparum
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incidence of malaria (30% reduction). Currently, it cannot be used
extensively.[5,9]
Moreover, it has excellent hydrosolubility (>10 mg/ml), even in acidic
conditions, and has already been studied for some ADME (absorp-
tion, distribution, metabolism, and excretion) properties (tmax = 60 min;
Cmax = 0.71 µM).[15–17] This phenolic compound also showed some
pharmacological activities, including antitrypanosome, antibacterial,
and antiplasmodial, without high toxicity in various cell lines, or during
in vivo studies (ED50 > 80 mg/kg in mice) (Table 1).[18–28] On the
contrary, some derivatives from this scaffold have already been stu-
died for anti‐Plasmodium purposes and have shown interesting in-
hibitory effects (IC50 ~10 µM).[20,29]
Consequently, in terms of the available drugs, the first to be
reported was quinine (1, Figure 1), an alkaloid from Cinchona sp. bark.
It has been followed by the development of other 4‐aminoquinoline
derivatives including chloroquine, hydroxychloroquine, and amodia-
quine (2–4, Figure 1).[10–12] After that, different classes of molecules,
mostly inspired by natural compounds, were developed, including
sulfadoxine, pyrimethamine, and lumefantrine (5–7, Figure 1).[12]
However, the last major discovery was artemisinin (8, Figure 1),
a sesquiterpene lactone, isolated from Artemisia annua.[13] Because
of its low solubility in water and lipids, several derivatives have
been developed including artemether and artesunate (9–10,
Figure 1).[13,14] Now, these agents are recommended worldwide in
combination with other antimalarials (ACTs) to cure P. falciparum
infections. Unfortunately, because of the development of
resistances, there is an urgent need for innovations in the field of
We decided to work on these polyphenols, despite their known
PAINS (pan‐assay interference compounds) potential, for various
reasons.[30,43] First, in the case of antiplasmodial research, these in-
terferences could be easily identified and controlled by microscopy
counting with the Giemsa stain.[20] Second, in our opinion, even these
panacea compounds exist and are responsible for bias during in vitro
assays, you cannot decently discard all the molecules from a same
family for all their potential uses, just because some of them are
guilty for bias during some studies.[31] Moreover, some of the me-
chanisms responsible for these interferences are the same as those
for biological effects.[43] Thus, we have considered the PAINS
[6–8]
antimalarials.
Therefore, we have selected gallic acid (GA) (11, Figure 2) as
a promising candidate for optimization. Indeed, this structure is a
common secondary metabolite in plants and commercially available.
FIGURE 1 Examples of antimalarial drugs accepted for treatment against Plasmodium falciparum

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TABLE 1 Previously reported cytotoxicity of gallic acid (11)
Cell lines
L1210
IC50 (µM)
>50
Reference
[18]
CEM
>50
[18]
HCT15
Lymphocytes
HepG2
MCF‐7
564
[19]
>588
265
[19]
[21]
470
[21]
MDA‐MB231
HT29
441
[21]
411
[21]
MCF10A
Caco‐2
L929
>3527
>235
>235
Nontoxic
[21]
[22]
[22]
U937
[22]
FIGURE 2 Structures of polyphenolic compounds selected for
pharmacomodulation
the Plasmodium growth inhibition. Owing to a protocol adapted from
Fischer's esterification, small esters of GA were obtained and all have
shown a similar range of activity against the 3D7 strain of Pf (IC50
~20 µM, Table 2).[32] Similarly, all the other benzoic acid derivatives
were converted into their corresponding methyl esters (21–23,
Figure 3).
character as a “Pay‐Attention” flag, but not a sufficient reason to
ignore this potential class of compounds.
Consequently, we have synthesized various compounds whose
structures are inspired by the gallate scaffold. This focused chemical
library was then mainly evaluated to identify new antiplasmodial
agents. Moreover, we have also tested other hydroxybenzoic mole-
cules (12–14, Figure 2) to better understand the features responsible
for this activity.
These reactions resulted in a slight increase in the lipophilicity as
could have been anticipated (LogP increased from 0.53 to 0.77 for
11–17, respectively).[33] After that, we introduced two different kinds
of protective groups for the phenolic functions. Indeed, they are
usually reported as being essential for the biological effects, for ex-
ample, the antiplasmodial activity of caffeic acid derivatives or the
antibacterial effect of gallate derivatives.[27,32]
We have also followed the impact of the pharmacomodulations
on water solubility, a key parameter for drug development. In con-
clusion, the main objective of our study was to obtain a compound
with a good to promising activity against Pf (IC50 < 15 µM), without
significantly affecting the water solubility and cytotoxicity.
Therefore, easily reversible protective groups were inserted
through an acetylation reaction (24–27, Figure 3) while phenol me-
thylation was performed, resulting in a less‐labile protective group
(31–34, Figure 3).[32,34] Moreover, to show if these O‐alkyl sub-
stituents were sufficient to increase the antiplasmodial activity of our
hit compounds, we also prepared their corresponding acid derivatives
(28–30 and 35–37, Figure 3). Consequently, acetylated prodrugs
were obtained after reaction with acetic anhydride (Ac2O) and sul-
furic acid, following the process described by Gokcen et al.,[34] except
for 1,2,3‐triacetoxybenzene (27), which required the action of Ac2O
and pyridine.[34,35]
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
A library of hydroxybenzoic structures was selected for evaluations
and pharmacomodulations during this study to better understand the
structure–activity relationships responsible for the antiplasmodial
effect. The target compounds were mainly derivatives of 11 as well
as 3,4‐, 3,5‐, and 2,5‐dihydroxybenzoic acids (12–14, Figure 3). In
addition, benzoic acid and pyrogallol (15–16, Figure 3) were also used
to explore the impact of the phenolic and carboxylic functions on
pharmacological activity. Therefore, as reported in Scheme 1, various
synthetic pathways were used to obtain the desired poly-
hydroxybenzoic acid derivatives. First, we synthesized various esters
of 11 (17–20, Figure 3) to observe the impact of the O‐alkyl chain on
In terms of the methoxylated analogs, the reaction of the hy-
droxybenzoic acids (31–34) with dimethylsulfate (DMS) was followed
by basic saponification by means of LiOH to cleave the methyl ester
(35–37, Figure 3).[32,36]
Finally, as 11 was the most active among the selected hydro-
xybenzoic acids, we decided to explore this scaffold with some bigger
substituents to increase its lipophilicity in a more marked way.
Therefore, we followed the protocol of para‐substitution described by

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FIGURE 3 Structures of the derivatives from polyhydroxybenzoic acids screened for antiplasmodial activities
Pearson et al.[35] to obtain methyl 4‐benzyloxy‐3,5‐diacetoxybenzoate
(38) and then methyl 4‐benzyloxy‐3,5‐dihydroxybenzoate (39) after
saponification (Scheme 2). A superior homolog of 39, with a naph-
thylmethyl substituent (40, Figure 4), was obtained using another
protocol.[36] The steric hindrance of the naphthylmethoxy substituent
could explain why this second protocol could not be applied for the
introduction of benzyl. Indeed, the substitution could occur on the
other phenols (3‐ and 5‐OH) with benzyl bromide and not so easily for
the 2‐(bromomethyl)naphthalene.
hydroxybenzoic acid scaffolds on the selected 3D7 strain of P. falci-
parum (chloroquine‐sensitive). Their effect was compared to the ac-
tivity shown by two reference antimalarial drugs: quinine (1) and
artemisinin (8). These standards were used to confirm the validity of
the assays. Indeed, their IC50 values were similar to the values reported
previously: 600 12 nM and 9.2 5.2 nM, respectively.[37,38]
We observed that 11 showed a modest inhibitory effect
against the malaria parasite, with an IC50 value of around 70 µM,
and a quite similar activity for 16, which has an IC50 value of
58 38 µM. The activity obtained with 11 was similar to previously
reported data, where the IC50 value was found to be
71.53 8.96 µM.[28]
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2.2
Pharmacology/biology
On the contrary, 12–15 were not active up to 100 µg/ml (IC50
>
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2.2.1
In vitro antiplasmodial assay
700 µM). These results were different from that reported by Ndjonka
et al.,[28] for example, 14, for which the IC50 value was quantified as
49.76 0.32 µM. The explanation for these differences may be the
revelation method, which consisted of a [3H]‐hypoxanthine incorpora-
tion assay in that paper, in the test protocol, as they employed a 24h‐
Plasmodium lactate dehydrogenase
All the compounds were then assessed for their antiplasmodial po-
tential. However, only the active molecules on Pf are reported in
Table 2 and further explored. First, we evaluated the different
[39]
exposure, compared to 48h in our case. Similarly, Aldulaimi et al.

DEGOTTE ET AL.
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demonstrated greater activity of 11 and 14 on P. falciparum, with IC50
values of around 27 and 84 µM, respectively. These significant differ-
ences in the concentration range could be due to the Dd2 strain used,
known to be resistant compared to the sensitive 3D7 strain that we
used. In addition, the protocol used was also slightly different, with
Plasmodium DNA detection.
As highlighted by previous studies with caffeic acid derivatives,
esters are known to be more efficient than their corresponding
acids in antiplasmodial assays.[32] This trend could be partially ex-
plained by higher lipophilicity, which could increase the ability of the
compounds to reach the cytoplasm of red blood cells (RBC) and then
inhibit the growth of the asexual stages of Pf.[40] This was high-
lighted in the study of Zahrani et al.,[41] who summarized the ob-
servations made about alkyl gallates. Indeed, several studies on
anticancer properties have shown more favorable activities of the
esters compared to their corresponding acid. This was attributed to
the higher lipophilicity caused by a long alkyl chain and responsible
for improved cancer cell permeability. However, this increase in li-
pophilicity was not always linear with the activity. Indeed, this en-
hancement of the anticancer effect seemed limited to a specific
range of LogP values. Therefore, the optimum compounds had a
partition coefficient between 3.32 and 6.09 against leukemia
cells.[18] In contrast, an acidic molecule is not often recognized as a
promising structure to cross the intestinal barrier, even if some
transporters exist on enterocytes, for example, the monocarboxylic
acid transporter.[17]
SCHEME 1 Synthetic pathways used on hydroxybenzoic acid
scaffolds. Reagents and conditions: (i) MeOH, H2SO4; (ii) Ac2O,
H2SO4; (iii) DMS, K2CO3; and (iv) LiOH, MeOH/H2O
TABLE 2 Biological activities of the reported structures
Products
1
Plasmodium falciparuma
0.60 0.12
0.0092 0.0052
68 20
HUVEC (SI)b
Solubility (mM)
cLogPc
2.48
3.17
0.53
0.06
0.77[33]
1.27
1.78
1.62
1.36
1.20
1.54
0.204
0.94
3.62
2.32
3.50
‐
‐
8
‐
‐
11
16
17
18
19
20
21
24
25
27
28
38
39
40
>294 (4)
70.2 8.2
58 38
‐
‐
27 1.8
171 43 (6)
74.3 7.7
22 0.7
‐
205 26
34 11
‐
130 23
29 13
‐
121 7.4
99 22
‐
‐
9.3 3.0
>161 (17)
‐
0.59 0.75
113 22
‐
24 3.0
>198 (8)
‐
‐
203 34
‐
56 2.2
>140 (3)
>182 (6)
140 4.3 (8)
0.46 0.22
3.72 0.79
0.005 0.001
33 5.4
17
4
aIC50 value in µM of the malaria agent Plasmodium falciparum 3D7‐strain.
bIC50 values in µM of human umbilical vein endothelial cells (HUVEC) (selectivity index).
cCalculated LogP using ChemDraw 12.0.

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SCHEME 2 Synthetic pathways for para‐
substituted gallate derivatives. Reagents and
conditions: (i) MeOH, H2SO4; (ii) Ac2O, H2SO4; (iii)
BnBr, K2CO3, KI; (iv) K2CO3, MeOH/H2O; and (v)
2‐(bromomethyl)naphthalene, K2CO3
Table 2). In contrast, all the esters of the other hydroxybenzoic
molecules (21–23) were mostly inactive.
The effect of 18 was similar to that reported previously in Jansen
et al.,[29] where the IC50 value was found to be 32.3 5.0 µM. In
contrast, in the studies of Arsianti et al.,[20] as well as Aldulaimi
et al.,[39] multiple esters of 11 were synthesized for antiplasmodial
purposes and showed a greater effect and even reached the micro-
molar range of activity. The difference in our results could again be
partially explained by the different experimental protocols used.
Therefore, as only 17–20 were quite active and as the methyl ester is
known to be more resistant to hydrolysis, we chose 17 as the lead
compound to continue our pharmacomodulation.
As mentioned before, phenolic functions are often considered
essential for the pharmacological activities of polyphenols.[27,32]
Therefore, we protected the phenolic groups with methoxy or acet-
oxy substituents to observe the impact of hiding the former on the
growth‐inhibitory effect of our compounds.
The antiplasmodial assay led to two totally different observations.
First, none of the methoxy derivatives (31–37) were efficient against
the malarial agent, even for the 3,4,5‐ or the 3,4‐dihydroxybenzoic
scaffolds, whose esters were previously active.
In contrast, some of the acetylated derivatives (24–25 and
28–29) showed, respectively,
a low to good inhibitory effect
FIGURE 4 Structures of the para‐substituted gallate
(IC50 = 9.3–203 µM), especially 24, which was the most active com-
pound identified in this paper (IC50 = 9.3 µM). In addition, 27 was also
a marked inhibitor of the 3D7 strain of Pf, with IC50 = 24.0 3.0 µM.
Thus, the increase in the activity of our phenolic scaffolds, especially
11 and 12, may be explained by the increase in lipophilicity, as pre-
viously described for caffeic acid derivatives.[32]
This trend prompted us to prepare esters of the selected benzoic
scaffolds 11–15 and to explore the impact of the alkyl chain on the in
vitro activity against 3D7 (17–23). Surprisingly, we observed a similar
range of activity for all the structures related to 11 (IC50 ~28 µM,

DEGOTTE ET AL.
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On the contrary, the introduction of a stable protective group,
the estimated IC50 values were in the same range as the values ob-
tained using the main revelation method.[45] This confirmed the ef-
ficacy of our structures on the malaria agent and their potential as
antiplasmodial lead compounds.
methyl ether, impeded the antiplasmodial effect. This decrease in
efficiency could be explained by the loss of the free phenol groups. In
contrast, a labile acetoxy substituent seemed mostly beneficial for
the anti‐infectious effect. Indeed, the acetyl moiety was often used in
drug development to increase the lipophilicity in a reversible manner,
for example, acetylsalicylic acid. Consequently, acetylated structures
such as 24 are often considered prodrugs, and not responsible for the
observed efficacy. In our case, these results highlighted the need for
the phenolic moieties for the antiplasmodial effect.
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2.2.2
Hemolysis
However, to establish whether the antiplasmodial effect could be due
to a factor other than an inhibitory effect on the malarial agent, we
also tested our molecules in a hemolysis assay. Indeed, as Pf is mostly
an intracellular parasite, the destruction of RBCs can impede its
multiplication. Therefore, a hemolytic product could bias the eva-
luation of antiplasmodial effects by a toxic effect on RBC.
Finally, as among the polyhydroxybenzoic acids (11–15), the
gallate moiety was the most active one, with an IC50 value of around
28 µM (16–20); we attempted to increase this activity to reach the
micromolar range (IC50 < 5 µM).
For this purpose and guided by the increase in activity linked to
higher lipophilicity, we introduced some bulky hydrophobic chains on
the phenolic functions, and more precisely, on the para‐position
(Scheme 2). Therefore, a benzyl or a naphthyl substituent was grafted
on the 4‐position of the trihydroxy scaffold (38–40, Figure 4) and
tested against the 3D7 strain. Unfortunately, none of them reached
the desired range of activity (<5 µM) and were often less active than
17 or 24 (IC50 > 25 µM), except for 40 (IC50 = 17 4.0 µM).
This trend confirmed again the importance of the phenolic
functions (a catechol moiety) because the increase in lipophilicity was
not followed by a similar enhancement of the activity. This could be
due to the phenoxy groups, which could remain partially hidden in
comparison with 24. However, as opposed to the methoxy scaffolds,
the graft of these bulky and nonversatile substituents did not lead to
a complete loss of activity. This could be partially explained by the
fact that some phenols could remain free but also by the establish-
ment of new interactions with the biological target.[42]
None of our compounds showed any hemolytic potential. In-
deed, at 100 µg/ml, none reached a percentage of hemolysis superior
to 1%. Therefore, the observed effect on Pf (3D7) could not be ex-
plained by the destruction of erythrocytes.
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2.2.3
Cytotoxicity
As some of our compounds were quite toxic against the protozoon P.
falciparum (3D7), we performed an evaluation of cytotoxicity to
confirm if this effect was selective. Therefore, we assessed the most
active ones on a healthy cell line. As the action site of antimalarial
agents is mainly blood vessels, we selected human umbilical vein
endothelial cells (HUVEC) as the most interesting model to evaluate
the selectivity of our derivatives (Table 2).
Finally, none of the selected molecules was highly toxic on the
HUVEC. Therefore, it seemed that our compounds showed good
selectivity for Plasmodium 3D7 (SI > 3). This selectivity was previously
described for alkyl derivatives on HepG2. Indeed, most of the com-
pounds were slightly toxic on this cell line despite significant anti-
plasmodial effects on Dd2 strains.[39] However, evaluations on other
cell lines could be beneficial to confirm this lack of cytotoxicity.
Interestingly, the increase in the lipophilicity that has been pre-
viously reported to increase the cancerous cell toxicity or the anti‐
Plasmodium activity by higher membrane permeability did not lead to
increased cytotoxicity on the chosen healthy cell line.[18,39]
In conclusion, we can consider that the most essential part of the
hydroxybenzoic structure for the antiplasmodial effect was the pyr-
ogallol moiety (16), considering the low efficacy of the other benzoic
acid scaffolds, as highlighted by previous studies.[39] Therefore, the
pyrogallol moiety was essential for the effect but not only, as 12 or
21 had no significant inhibition of Plasmodium.
Microscopy
In addition, we used a second revelation method to confirm the ab-
sence of PAINS behavior during our in vitro assays on Pf. Indeed, as
mentioned before, polyphenols are often reported as panacea com-
pounds.[43,44] Thus, it was necessary to confirm that the reduction in
parasitemia was not the result of interference between the revelation
reagents and our products.
|
2.2.4
Solubility
One of the main problems with highly active molecules in vitro was
often their low water solubility, which could negatively impact the
ADME profile, for example, low oral bioavailability. Thus, it was im-
portant to follow the impact of our pharmacomodulation on this
critical property. Considering the results from the in vitro assays, we
have decided to quantify the maximal water solubility at room tem-
perature (RT; Cmax, Table 2) for some derivatives owing to a protocol
adapted from Bala et al., in a kind of ADME‐early vision.[46] There-
fore, as reported before, 11 was a highly soluble scaffold, with a Cmax
Consequently, a microscopy measure was performed for 24, 38,
and 40. Thus, after the 48‐h incubation, the culture was centrifuged
at 2000 rpm to concentrate the erythrocytes. Then, a thin blood
smear was prepared and stained with Giemsa as reported before.[20]
Finally, 1000 RBCs were counted to establish the percentage of
parasitemia at each concentration.
Fortunately, all the test samples showed
a
significant
concentration‐dependent reduction in parasite growth. Moreover,

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DEGOTTE ET AL.
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value of around 10 mg/ml at RT~25°C.[15,16] This was confirmed by
our UV spectrophotometric method, with Cmax value of
of hydrosolubility (5 µM to 205 mM), which was considered a critical
parameter here for the pharmacokinetic profile.
a
11.9 1.4 mg/ml. Moreover, 17–20 showed similar behavior in wa-
ter, with a high Cmax value, for example, of 13.7 1.4 mg/ml for 17.
Thus, the esterification of 11 with a small alkyl chain, like methanol,
did not impact the water solubility of this scaffold negatively. How-
ever, the solubility of the ethyl and (iso)propyl esters (18–20) was up
to 3‐fold higher than that of 11 or 17, despite increased lipophilicity
(cLogP). This could be explained by the experimental conditions,
especially the temperature, which has a major influence on this
parameter as demonstrated before.[15] However, it could also be a
result of how the molecules interact with each other in the solvents.
Indeed, ellagic acid, a GA dimer, is a highly hydrophilic structure
(cLogP 1.05), but scarcely soluble in water (Cmax = 9.7 µg/ml) because
of the strong inter‐ and intramolecular bonds, enabled by its flat
structure.[46,47] Therefore, a similar phenomenon could explain why
18 was more soluble than its corresponding acid, despite its higher
LogP value (1.27 vs. 0.53, respectively).
In conclusion, 39 seemed to be the most promising compound
from our library as it showed good activity, with no signs of cyto-
toxicity (SI > 5.6) or hemolysis (<1%). Moreover, it showed an inter-
esting solubility (3.72 mM) in contrast to other highly active
compounds. Consequently, for the first time, this study highlighted
the potential of para‐benzyl gallates as promising antiplasmodials and
the next avenue for future antimalarial research.
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All the chemicals were purchased from Fluorochem, Sigma‐Aldrich,
FisherScience, or VWR. MilliQ water was obtained from the Milli‐Q
Reference A+ system. Melting points were determined on the Stuart
SMP3 capillary apparatus and were uncorrected. Products were
purified by the Buchi Reveleris® prep on an irregular silica cartridge,
4‐80 g. The 1H and 13C NMR spectra (see the Supporting Informa-
tion) were recorded on a Bruker Advance (500 MHz for 1H; 125 MHz
for 13C) instrument using deuterated dimethyl sulfoxide (DMSO‐d6)
or deuterated chloroform (CDCl3) as a solvent with tetramethylsilane
(TMS) as the internal standard; chemical shifts are reported in δ va-
lues (ppm) relative to that of internal TMS. Elemental analyses (C, H,
N, and S) were carried out on a Thermo Scientific Flash EA1112
elemental analyzer and were within 0.4% of the theoretical values
for carbon, hydrogen, and nitrogen. This analytical method confirmed
a purity of 95% or above for each tested compound. All reactions
were routinely checked by thin‐layer chromatography (TLC) on a si-
lica gel Merck 60 F254. The UV spectra were obtained using a Hi-
tachi U‐3010 UV/Vis spectrophotometer model.
However, the substitution of the phenol functions with moieties
like acetyl (24) or the para‐substitution (38–40) has a greater impact
on this parameter. Indeed, we observed a marked reduction in the
Cmax value (<1 mM), which was consistent with the reduction of the
H‐bond capacity and the increase of the lipophilicity (cLogP).
In contrast, the deprotection of some phenol groups, as observed
with 38 and 39, led to an increase (eightfold) in the Cmax value, for
example, 165 80 µg/ml for 38 to 1.02 0.22 mg/ml for 39. Surpris-
ingly, 40 did not show a similar behavior. Indeed, the water solubility of
the naphthyl derivative was even lower (95‐ to 120‐fold) than that
measured for 24 or 38. This trend could be partially explained by the
steric hindrance of the substituent and the high lipophilicity of this
compound (cLogP = 3.5). In conclusion, 39 seems to be the best of the
substituted derivatives in terms of the lipophilicity–hydrosolubility
balance, with Cmax = 3.72 mM and cLogP = 2.32. Indeed, it is necessary
to control both parameters because of their major influence on the
bioavailability and thus, the pharmacokinetic component, in addition to
the influence on the pharmacodynamics.[41,48]
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as the Supporting Information.
|
3
CONCLUSIONS
|
4.1.2
General procedure for the synthesis of ester
We selected various hydroxybenzoic acids, based on the activity of
derivatives
11 on Pf combined with its interesting ADME profile. Our pharma-
comodulation aimed to gain
a
better understanding of the
Hydroxybenzoic acid (1 g) was dissolved in 50 ml of the appropriate
alcohol (mostly MeOH) and a few drops of concentrated H2SO4 were
cautiously added. The mixture was heated under reflux until the end
of the reaction (monitored by TLC, ~6 h). The reaction mixture was
cooled before a part of the organic solvent was eliminated by eva-
poration under vacuum. After that, water (25 ml) was added and the
solution was extracted three times with ethyl acetate (50 ml). Then,
the organic phase was washed with brine (50 ml) and dried over
anhydrous magnesium sulfate (MgSO4). The suspension was filtered
and the solvent was removed by evaporation under vacuum. The
solid was placed overnight in a stove (30°C).
structure–activity relationships and the synthesis of potential new
antimalarials.
The in vitro assay against Pf showed that the important functions
were served by the phenolic groups, especially a trihydroxy scaffold.
Moreover, the increase in lipophilicity led to a greater effect, certainly
as a result of a higher penetration rate in the erythrocytes. Thus, the
introduction of a strong and bulky chain on the 4‐position (39–40)
improved the activity without higher cell toxicity (SI > 3). Indeed, these
derivatives were selective as none demonstrated any toxicity against
healthy cells in humans. Moreover, they demonstrated various ranges

DEGOTTE ET AL.
9 of 13
|
Methyl gallate (17)
observed and after the entire solid dissolved, the stirring was con-
tinued for 1 or 2 h. Then, water (100 ml) was added and the solution
was stirred for another 1 h to remove any excess acetic anhydride
left. The solid precipitate was filtered on G3, washed with water
(3 × 50 ml), and dried under vacuum.
White solid, yield 88%, melting point (MP): 257°C (decomp.)/Lit.
240–242°C[49]; UV: 271 nm, 1H‐NMR (in DMSO) δ 9.26 (2H, s), 8.93
(1H, s), 6.94 (2H, s), and 3.75 (3H, s); 13C (in DMSO) δ 166.78 (s),
146.04 (s), 138.87 (s), 119.75 (s), 108.95 (s), and 52.05 (s); theorical
elemental analysis (EA Th): C, 52.18%; H, 4.38%; Found: C, 52.30%;
H, 4.38%.
Methyl 3,4,5‐triacetoxybenzoate (24)
White solid, yield 92%, MP: 126°C/Lit. 126–128°C[35]
; UV:
Ethyl gallate (18)
White solid, yield 77%, MP: 145°C (decomp.)/Lit. 148–150°C[48]
1H‐NMR (in DMSO) δ 6.94 (2H, s), 4.19 (2H, q), and 1.27 (3H, t); 13C
(in DMSO) δ 166.28 (s), 146.01 (s), 138.79 (s), 120.03 (s), 108.91 (s),
60.45 (s), and 14.73 (s); EA Th: C, 54.55%; H, 5.09%; Found: C,
53.95%; H, 5.07%.
232 nm, 1H‐NMR (in DMSO) δ 7.80 (2H, s), 3.88 (3H, s), 2.34 (3H,
s), and 2.30 (6H, s); 13C (in DMSO) δ 168.48 (s), 167.39 (s), 164.86
(s), 143.83 (s), 139.15 (s), 128.05 (s), 122.40 (s), 53.17 (s), 20.86 (s),
and 20.32 (s); EA Th: C, 54.20%; H, 4.55%; Found: C, 54.16%;
H, 4.69%.
;
Methyl 3,4‐diacetoxybenzoate (25)
Isopropyl gallate (19)
;
White solid, yield 93%, MP: 124.5°C/Lit. 126–128°C[50] 1H‐NMR (in
Colorless oil, yield 81% (after flash chromatography n‐hexane/EtOAc:
1:0–3:2), 1H‐NMR (in CDCl3) δ 7.95 (1H, dd), 7.87 (1H, d), 7.28 (1H,
d), 3.91 (3H, s), and 2.31 (6H, s); 13C (in CDCl3) δ 168.01 (s), 167.71
(s), 165.54 (s), 145.95 (s), 141.99 (s), 128.73 (s), 128.12 (s), 125.08 (s),
123.50 (s), 52.40 (s), 20.69 (s), and 20.58 (s).
DMSO) δ 9.22 (2H, s), 8.89 (1H, s), 6.93 (2H, s), 5.03 (1H, m), and 1.27
(6H, d); 13C (in DMSO) δ 165.77 (s), 145.98 (s), 138.71 (s), 120.39 (s),
108.89 (s), 67.59 (s), and 22.25 (s); EA Th: C, 56.60%; H, 5.70%;
Found: C, 56.20%; H, 5.90%.
Methyl 3,5‐diacetoxybenzoate (26)
Propyl gallate (20)
White solid, yield 86%, MP: 145°C/Lit. 145–146°C[48]
DMSO) δ 6.95 (2H, s), 4.12 (2H, t), 1.68 (2H, m), and 0.95 (3H, t); 13
Colorless oil, yield 66% (after flash chromatography n‐hexane/EtOAc:
1:0–3:2), 1H‐NMR (in CDCl3) δ 7.66 (2H, s), 7.14 (1H, s), 3.91 (3H, s),
and 2.31 (6H, s); 13C (in CDCl3) δ 168.77 (s), 165.36 (s), 150.95 (s),
132.23 (s), 120.29 (s), 120.09 (s), 52.51 (s), and 21.02 (s).
;
1H‐NMR (in
C
(in DMSO) δ 166.33 (s), 146.02 (s), 138.81 (s), 120.02 (s), 108.91 (s),
65.89 (s), 22.16 (s), and 10.86 (s); EA Th: C, 56.60%; H, 5.70%; Found:
C, 56.93%; H, 6.10%.
3,4,5‐Triacetoxybenzene (27)
White solid, yield 24%, MP: 167°C; 1H‐NMR (in CDCl3) δ 7.25 (1H, t),
7.12 (1H, s), 7.10 (1H, s), 2.29 (3H, s), and 2.28 (6H, s); 13C (in CDCl3)
δ 167.90 (s), 167.01 (s), 143.55 (s), 134.69 (s), 125.98 (s), 120.74 (s),
20.68 (s), and 20.20 (s); EA Th: C, 57.14%; H, 4.80%; Found: C,
56.94%; H, 4.96%. This solid was obtained through the acetylation
protocol proposed by Pearson et al.[35]
Methyl 3,4‐dihydroxybenzoate (21)
;
White solid, yield 93%, MP: 137°C/Lit. 137–139°C[51] 1H‐NMR (in
DMSO) δ 9.77 (1H, s), 9.36 (1H, s), 7.35 (1H, s), 7.30 (1H, s), 6.79 (1H,
s), and 3.76 (3H, s); 13C (in DMSO) δ 166.62 (s), 150.88 (s), 145.54 (s),
122.22 (s), 120.95 (s),116.72 (s), 115.78 (s), and 52.06 (s); EA Th: C,
57.14%; H, 4.80%; Found: C, 57.05%; H, 4.87%.
3,4,5‐Triacetoxybenzoic acid (28)
Methyl 3,5‐dihydroxybenzoate (22)
White solid, yield 98%, MP: 166°C/Lit. 169–170°C[52]
DMSO) δ 9.62 (2H, s), 6.81 (2H, s), 6.44 (1H, s), and 3.79 (3H, s); 13C
(in DMSO) δ 166.73 (s), 159.01 (s), 131.75 (s), 107.63 (s), and 52.47
(s); EA Th: C, 57.14%; H, 4.80%; Found: C, 57.21%; H, 4.91%.
White solid, yield 99%, MP: 159°C/Lit. 166–168°C[53] 1H‐NMR (in
;
;
1H‐NMR (in
CDCl3): δ 7.86 (2H, s) and 2.31 (9H, s); 13C (in CDCl3) δ 168.78 (s),
167.58 (s), 166.38 (s), 143.52 (s), 139.33 (s), 127.32 (s), 122.84 (s),
20.60 (s), and 20.20 (s); EA Th: C, 52.71%; H, 4.08%; Found: C,
52.74%; H, 4.14%.
Methyl benzoate (23)
3,4‐Diacetoxybenzoic acid (29)
;
White solid, yield 59%, MP: 150°C/Lit. 157–158°C[54] 1H‐NMR
Colorless oil, yield 76%, 1H‐NMR (in CDCl3) δ 8.04 (2H, d), 7.56 (1H,
t), 7.44 (2H, t), and 3.92 (3H, s); 13C (in CDCl3) δ 166.13 (s), 132.92 (s),
130.18 (s), 129.58 (s), 128.37 (s), and 52.11 (s).
(in CDCl3): δ 8.03 (1H, dd), 7.94 (1H, d), 7.33 (1H, d), and 2.33
(6H, s); 13C (in CDCl3) δ 169.74 (s), 167.98 (s), 167.65 (s), 146.70
(s), 142.10 (s), 128.78 (s), 127.74 (s), 125.71 (s), 123.69 (s), 20.70
(s), and 20.58 (s); EA Th: C, 55.47%; H, 4.23%; Found: C, 55.21%;
H, 4.28%.
|
4.1.3
General procedure for acetylated derivatives
To a stirred solution of hydroxybenzoic acid or the corresponding
ester (20 mmol) in acetic anhydride (120 mmol), a few drops of con-
centrated H2SO4 were added. A fast increase in temperature was
3,5‐Diacetoxybenzoic acid (30)
White solid, yield 76%, MP: 156°C/Lit. 161°C[55]
CDCl3): δ 7.72 (2H, d), 7.21 (1H, t), and 2.32 (6H, s); 13C (in CDCl3) δ
;
1H‐NMR (in

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DEGOTTE ET AL.
|
168.86 (s), 168.73 (s), 151.04 (s), 131.17 (s), 120.84 (s), and 21.04 (s);
EA Th: C, 55.47%; H, 4.23%; Found: C, 55.30%; H, 4.19%.
The resulting organic phase was washed with brine (25 ml) and dried
over anhydrous MgSO4. The solid was filtered and the solvents were
removed under vacuum. The resulting solid compound was kept
under vacuum overnight.
|
4.1.4
General procedure for methoxy derivatives
3,4,5‐Trimethoxybenzoic acid (35)
The appropriate hydroxybenzoic acid (6.5 mmol) was dissolved in
acetonitrile (60 ml) and supplemented with potassium carbonate
(K2CO3, 19.5 mmol). The mixture was stirred at 40°C for 30 min and
then, dimethylsulfate (DMS; 19.5 mmol) was added. The stirred so-
lution was heated under reflux for 5 h. Potassium carbonate was
removed by filtration; the organic solvents and DMS were distilled
under vacuum and the resulting solid was dried under vacuum
overnight.
White solid, yield 81%, MP: 169°C/Lit. 166–167°C[60] 1H‐NMR (in
;
CDCl3) δ 7.37 (2H, s), 3.94 (3H, s), and 3.93 (6H, s); 13C (in CDCl3) δ
170.49 (s), 153.01 (s), 142.97 (s), 123.96 (s), 107.43 (s), 60.98 (s), and
56.28 (s); EA Th: C, 56.60%; H, 5.70%; Found: C, 56.51%; H, 5.81%.
3,4‐Dimethoxybenzoic acid (36)
White solid, yield 98%, MP: 181°C/176–178°C[61]
;
1H‐NMR (in
CDCl3) δ 7.78 (1H, dd), 7.61 (1H, d), 6.93 (1H, d), and 3.96 (6H, d); 13
C
(in CDCl3) δ 171.19 (s), 153.68 (s), 148.69 (s), 124.56 (s), 121.74 (s),
112.32 (s), 110.33 (s), 56.09 (s), and 56.03; EA Th: C, 59.34%; H,
5.53%; Found: C, 59.22%; H, 5.65%.
Methyl 3,4,5‐trimethoxybenzoate (31)
;
White solid, yield 54 (%), MP: 81°C/Lit. 81–83°C[56] 1H‐NMR (in
CDCl3): δ 7.30 (2H, s) and 3.91 (12H, s); 13C (in CDCl3) δ 166.74 (s),
152.95 (s), 142.17 (s), 125.16 (s), 106.80 (s), 60.94 (s), 56.25 (s), and
52.25 (s); EA Th: C, 58.40%; H, 6.24%; Found: C, 58.45%; H, 6.32%.
3,5‐Dimethoxybenzoic acid (37)
White solid, yield 93%, MP: 185°C; 1H‐NMR (in CDCl3) δ 7.26 (2H, d),
6.70 (1H, t), and 3.85 (6H, d); 13C (in CDCl3) δ 170.58 (s), 160.73 (s),
130.92 (s), 107.68 (s), 106.65 (s), and 55.62 (s); EA Th: C, 59.34%; H,
5.53%; Found: C, 59.54%; H, 5.72%.
Methyl 3,4‐dimethoxybenzoate (32)
White solid, yield 88% (after recrystallization in EtOH/H2O on ice),
MP: 58°C/Lit. 57–58°C[57] 1H‐NMR (in CDCl3): δ 7.69 (1H, dd), 7.55
;
(1H, d), 6.89 (1H, d), 3.94 (6H, s), and 3.90 (3H, s); 13C (in CDCl3) δ
166.91 (s), 152.96 (s), 148.61 (s), 123.59 (s), 122.70 (s), 111.97 (s),
110.26 (s), 56.02 (s), and 52.01 (s); EA Th: C, 61.22%; H, 6.17%;
Found: C, 61.27%; H, 6.18%.
|
4.1.6
General procedure for para‐substitution
Methyl 3,4,5‐triacetoxybenzoate (2.00 g, 6.45 mmol) was dissolved in
dry acetone (100 ml) with potassium carbonate (2.70 g, 19.35 mmol)
and potassium iodide (0.165 g, 0.97 mmol). Benzyl chloride was
cautiously added (1.63 g, 12.90 mmol), and the mixture was heated
under reflux overnight. After that, K2CO3 was removed by filtration
and acetone was distilled under vacuum. The product was re-
crystallized with EtOH at 0°C, filtered on G3, and washed thoroughly
with EtOH. The resulting white solid was dried under vacuum
overnight.[35]
Methyl 3,5‐dimethoxybenzoate (33)
White solid, yield 84%, MP: 43°C/Lit. 42–44°C[58]
:
1H‐NMR (in
CDCl3): δ 7.19 (2H, d), 6.65 (1H, t), 3.91 (3H, s), and 3.83 (6H, s); 13
C
(in CDCl3) δ 166.88 (s), 160.65 (s), 130.02 (s), 107.13 (s), 105.80 (s),
55.59 (s), and 52.26 (s); EA Th: C, 61.22%; H, 6.17%; Found: C,
61.34%; H, 6.26%.
3,4,5‐Trimethoxybenzene (34)
A white solid was obtained after 3 days of agitation at RT before
Methyl 4‐benzoxy‐3,5‐diacetoxybenzoate (38)
treatment, yield 23% (after flash chromatography n‐hexane/EtOAc),
;
MP: 45.5°C/Lit. 43–45°C[59] 1H‐NMR (in CDCl3): δ 6.99 (1H, t), 6.59
(2H, d), 3.87 (6H, s), and 3.86 (3H, s); 13C (in CDCl3) δ 153.55 (s),
138.15 (s), 123.65 (s), 105.23 (s), 60.85 (s), and 56.09 (s); EA Th: C,
64.27%; H, 7.19%; Found: C, 64.36%; H, 7.34%.
White product, yield 85%, MP: 106°C/Lit. 94–96°C[35]; UV; 254 nm,
1H‐NMR (in DMSO) δ 7.69 (2H, s), 7.38 (5H, m), 5.03 (2H, s), 3.85
(3H, s), and 2.25 (6H, s); 13C (in DMSO) δ 169.04 (s), 165.05 (s),
147.50 (s), 144.34 (s), 136.85 (s), 128.92 (s), 128.82 (s), 128.49 (s),
125.20 (s), 122.78 (s), 75.71 (s), 52.96 (s), and 20.97 (s); EA Th: C,
63.68%; H, 5.06%; Found: C, 63.75%; H, 5.19%.
|
4.1.5
General procedure for saponification
|
4.1.7
General procedure for deacetylation
To the stirred solution of the methoxy derivatives (1 g) in methanol
(30 ml) was added a solution of lithium hydroxide in demineralized
water (0.61 g/4 ml). The mixture was heated under reflux until the
end of the reaction (monitored by TLC, ~2 h) and then acidified with
6 N HCl. Methanol was partially removed under vacuum and
the aqueous phase was extracted with ethyl acetate (3 × 30 ml).
The acetylated product (4.5 mmol) was dissolved in methanol
(80 ml) at raw temperature. A solution of potassium carbonate in
water (4.07 g, 40 ml) was added to the stirred mixture. After that,
the solution was acidified to pH 3 with 12 N hydrochloric acid
(HCl). The aqueous solution was extracted with ethyl acetate

DEGOTTE ET AL.
11 of 13
|
(3 × 100 ml). The organic phase was washed with 75 ml of water
and brine and then dried over anhydrous magnesium sulfate. The
drying agent was removed by filtration and the solvents were
evaporated under vacuum.[35] The resulting solid was dried under
vacuum overnight.
DMSO is known to be toxic for Pf. Thus, the maximal amount
during the in vitro assay was 1% for the initial concentrations. Finally,
each sample was evaluated in a series of eight twofold dilutions in
triplicate (n = 3). The assay was performed as previously described.[32]
The main method used was the measure of Plasmodium lactate
dehydrogenase as reported by Makler et al.[45] In addition, a visual
evaluation of parasitemia reduction was also performed with a
Giemsa stain.[20]
Methyl 4‐benzoxy‐3,5‐dihydroxybenzoate (39)
White solid, yield 96%, MP: 129°C/Lit. 133–134°C[35]; UV: 263 nm,
1H‐NMR (in DMSO) δ 9.56 (2H,s), 7.50 (2H, d), 7.35 (3H, m), 6.97
(2H,s), 5.05 (2H, s), and 3.77 (3H, s); 13C (in DMSO) δ 166.49 (s),
151.35 (s), 138.91 (s), 138.28 (s), 128.54 (s), 128.50 (s), 128.18 (s),
124.84 (s), 109.02 (s), 73.54 (s), and 52.37 (s); EA Th: C, 65.69%; H,
5.15%; Found: C, 65.94%; H, 5.31%.
|
4.2.2
Hemolysis induction
Hemolytic potential was evaluated for all the tested compounds
based on a reported procedure.[62] Consequently, a 10% red blood
cell suspension in phosphate‐buffered saline (PBS) (v/v) (A+ or O+)
was incubated with compounds at 100 µg/ml in duplicate. The po-
sitive control was Triton X‐100 1% (v/v) (corresponding to 100%
lysis) and PBS was used as the negative control (corresponding to 0%
lysis).
|
4.1.8
General procedure for the synthesis of
compound 40
Methyl gallate (17, 1.09 mmol) was dissolved in DMF (30 ml) with
potassium carbonate (2.2 mmol), potassium iodide (6.52 µmol),
and (2‐bromomethyl)naphtalene (1.30 mmol) under vacuum. The
mixture was stirred for 24 h at RT. Then, the solids were removed
by filtering on Celite° and water was added to the filtrate (20 ml).
The solution was extracted with EtOAc (3 × 50 ml). The organic
phase was treated with aq. NaOH 1 N. The aqueous layer was
|
4.2.3
Maximal water solubility (Cmax)
The maximum concentration of the compounds in H2O was
determined using the classical shaking flask method using an ultra‐
violet (UV) spectrophotometric method in milliQ water.[46] Therefore,
the maximal wavelength was determined by a UV scan between 190
and 400 nm to obtain the UV‐absorption spectra in a 1‐cm quartz
cuvette. After that, a calibration curve was constructed by three di-
lutions of a standard solution prepared by dissolving an accurately
weighed quantity of products in milliQ water. Linearity was evaluated
by linear regression analysis (R2 > 0.95).
acidified with HCl 1 N to pH
5 and extracted with EtOAc
(3 × 50 ml). Ethyl acetate was washed with water and brine. After
the general drying procedure (MgSO4), the solvent was evapo-
rated under vacuum. The crude product was dissolved in acetone
and recrystallized with n‐hexane. After that, the acetone was
evaporated and purified with DCVC (n‐hexane/EtOAc, 1:0 to 3:7)
to obtain a white solid.[36]
Next, the saturated solutions were prepared by overnight agi-
tation of an excess of the product in milliQ water at raw temperature
(around 25°C). After that, it was filtered on a 0.45 µM filter (Chro-
mafil XTra PVDF‐45/25; Filter Service) to remove the nondissolved
product and diluted with milliQ water. The absorbance was measured
at the selected wavelength. This experiment was performed for three
consecutive days. Then, the maximal water concentration was cal-
culated by the calibration curve.
Methyl 4‐naphtyl‐3,5‐dihydroxybenzoate (40)
White solid, yield 70%, MP: 199°C/Lit. 203–205°C[36] UV: 225 nm,
1H‐NMR (in DMSO) δ 7.97 (1H,s), 7.90 (3H, d), 7.70 (1H, dd), 7.51
(2H, m), 6.97 (2H,s), 5.22 (2H, s), and 3.76 (3H, s); 13C (in DMSO) δ
166.48 (s), 151.42 (s), 138.90 (s), 135.95 (s), 133.12 (s), 133.04 (s),
128.27 (s), 128.03 (s), 127.05 (s), 126.79 (s), 126.60 (s), 126.46 (s),
124.90 (s), 109.01 (s), 73.64 (s), and 52.38 (s); EA Th: C, 70.36%; H,
4.97%; Found: C, 69.96%; H, 5.06%.
|
4.2.4
In vitro cytotoxicity
|
4.2
Pharmacological/biological assays
The tested compounds were evaluated on HUVECs, supplied by
Lonza®. These cells were maintained in vitro in EBM‐2 medium,
supplemented with EGM‐2 SingleQuots® from Lonza®.
|
4.2.1
In vitro antiplasmodial activity
The tested strain was obtained through BEI Resources, NIAID, NIH:
Pf, strain 3D7, MRA‐102, contributed by Daniel J. Carucci. The in-
traerythrocytic forms of Pf were continuously grown in vitro using a
modified method of Trager and Jensen.[37] Tested products were
dissolved in DMSO at 10 mg/ml; artemisinin and quinine (Sigma‐
Aldrich) were used as positive standards.
The samples were dissolved in DMSO at 10 mg/ml. As DMSO
is known to be toxic to cells, the maximal tested concentration was
0.5% in EBM‐2. For the assay, 100 µl (104 cells) was seeded in a
96‐well plate and allowed to stand for 24 h at 37°C. After that,
50 µl of the test solutions were added (three wells for every six
concentrations) before the incubation for 48 h. Finally, the

12 of 13
DEGOTTE ET AL.
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supernatant was removed and replaced by 10‐fold diluted Presto
Blue®. The plate was incubated at 37°C for 2 h before reading in
fluorescence mode (560–590 nm). The growth was compared
between treated and untreated cells. Each compound was tested
in triplicate.
[16] A. Daneshfar, H. S. Ghaziaskar, N. Homayoun, J. Chem. Eng. Data
2008, 53, 776.
[17] Y. Konishi, Y. Hitomi, E. Yoshioka, J. Agric. Food Chem. 2004, 52, 2527.
[18] C. Locatelli, R. Rosso, M. C. Santos‐Silva, C. A. de Souza, M. A. Licínio,
P. Leal, M. L. Bazzo, R. A. Yunes, T. B. Creczynski–Pasa, Bioorg. Med.
Chem. 2008, 16, 3791.
[19] Y. P. Devi, A. Uma, M. L. Narasu, C. Kalyani, Res. J. Pharm. Biol.
Chem. Sci. 2015, 6, 460.
ACKNOWLEDGMENTS
[20] A. Arsianti, H. Astuti, F. Fadilah, D. Martin Simadibrata,
Z. Marie Adyasa, D. Amartya, A. Bahtiar, H. Tanimoto, K. Kakiuchi,
Orient. J. Chem. 2018, 34, 655.
The authors would like to thank the FRS‐FNRS (FC23283) and the
Fondation Léon Frédéricq for their financial support. The authors are
also grateful to P. Desdemoustier for her expertise in the in vitro
evaluations and to S. Counerotte for the elemental analysis.
[21] A. Hassani, M. M. S. Azarian, W. N. Ibrahim, S. A. Hussain, Sci. Rep.
2020, 10, 1.
[22] F. Truzzi, M. C. Valerii, C. Tibaldi, Y. Zhang, V. Abduazizova,
E. Spisni, G. Dinelli, Nutrients 2020, 12, 1.
CONFLICT OF INTERESTS
[23] Z. J. Li, M. Liu, G. Dawuti, Q. Dou, Y. Ma, H. G. Liu, S. Aibai, Phyther.
Res 2017, 31, 1039.
The authors declare that there are no conflicts of interests.
[24] S. Choubey, L. R. Varughese, V. Kumar, V. Beniwal, Pharm. Pat. Anal.
2015, 4, 305.
ORCID
[25] T. Koide, M. Nose, M. Inoue, Y. Ogihara, Y. Yabu, N. Ohta, Planta
Med. 1998, 64, 27.
Gilles Degotte
http://orcid.org/0000-0001-6318-4626
https://orcid.org/0000-0001-8251-8257
https://orcid.org/0000-0002-0770-9990
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SUPPORTING INFORMATION
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How to cite this article: G. Degotte, B. Pirotte, M. Frédérich,
P. Francotte. Polyhydroxybenzoic acid derivatives as potential
new antimalarial agents. Arch. Pharm. 2021; e2100190.
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