Activity of Fluorinated Curcuminoids against Leishmania major and Toxoplasma gondii Parasites

Article abstract of DOI:10.1002/cbdv.202100381

A new 3,4-difluorobenzylidene analog of curcumin, CDF, was recently reported, which demonstrated significantly enhanced bioavailability and in vivo anticancer activity compared with curcumin. For highlighting the antiparasitic behavior of CDF, we tested this compound together with its new O-methylated analog MeCDF against Leishmania major and Toxoplasma gondii parasites. Both CDF and MeCDF were tested in vitro against L. major and T. gondii. In addition, the in vitro cytotoxicity against Vero cells and macrophages was determined and selectivity indices were calculated. The DPPH radical scavenging activity assay was carried out in order to determine the antioxidant activity of the test compounds. Both compounds showed high activities against both parasite forms with EC₅₀ values in the (sub-)micromolar range (0.35 to 0.8 μM for CDF, 0.31 to 1.2 μM for MeCDF). The higher activity of CDF against L. major amastigotes when compared with MeCDF can in parts be attributed to the antioxidant activity of CDF while MeCDF lacking any antioxidant activity was more active than CDF against T. gondii parasites. In conclusion, CDF and MeCDF are promising antiparasitic drug candidates due to their high activities against L. major and T. gondii parasites.

Full text of DOI:10.1002/cbdv.202100381

doi.org/10.1002/cbdv.202100381
FULL PAPER
Activity of Fluorinated Curcuminoids against Leishmania major and
Toxoplasma gondii Parasites
Tariq A. Khan,a Waleed S. Koko,b Ibrahim S. Al Nasr,b, c Rainer Schobert,d and Bernhard Biersack*d
a
Department of Clinical Nutrition, College of Applied Health Sciences, Qassim University, Ar Rass, Saudi Arabia
b
Department of Science Laboratories, College of Science and Arts, Qassim University, Ar Rass, Saudi Arabia
c
Department of Biology, College of Science and Arts, Qassim University, Unaizah, Saudi Arabia
Organic Chemistry I, University of Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany,
d
e-mail: bernhard.biersack@yahoo.com; bernhard.biersack@uni-bayreuth.de
© 2021 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG. This is an open access article under the terms of the
Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
A new 3,4-difluorobenzylidene analog of curcumin, CDF, was recently reported, which demonstrated
significantly enhanced bioavailability and in vivo anticancer activity compared with curcumin. For highlighting
the antiparasitic behavior of CDF, we tested this compound together with its new O-methylated analog MeCDF
against Leishmania major and Toxoplasma gondii parasites. Both CDF and MeCDF were tested in vitro against L.
major and T. gondii. In addition, the in vitro cytotoxicity against Vero cells and macrophages was determined and
selectivity indices were calculated. The DPPH radical scavenging activity assay was carried out in order to
determine the antioxidant activity of the test compounds. Both compounds showed high activities against both
parasite forms with EC50 values in the (sub-)micromolar range (0.35 to 0.8 μM for CDF, 0.31 to 1.2 μM for MeCDF).
The higher activity of CDF against L. major amastigotes when compared with MeCDF can in parts be attributed
to the antioxidant activity of CDF while MeCDF lacking any antioxidant activity was more active than CDF against
T. gondii parasites. In conclusion, CDF and MeCDF are promising antiparasitic drug candidates due to their high
activities against L. major and T. gondii parasites.
Keywords: curcumin, CDF, neglected tropical diseases, Leishmania major, Toxoplasma gondii.
comprise toxic antimonials, miltefosine, amphotericin
Introduction
and pentamidine.[2] The emergence of drug-resistant
Leishmaniasis is classified as a neglected tropical parasite forms as well as the systemic toxicity of
disease (NTD) and it is clinically subdivided into clinically applied drugs such as pentavalent antimoni-
cutaneous leishmaniasis (CL), mucocutaneous leishma- als pose severe problems to the clinician and new
niasis (MCL), and visceral leishmaniasis (VL). The CL potent antiparasitic drugs against leishmaniasis are
form is triggered by several Leishmania species such as sought. Toxoplasmosis is another infection disease
L. major, L. tropica, L. mexicana, and L. amazonensis. CL brought about by the globally occurring protozoal
is the dominant form of leishmaniasis, which is Toxoplasma gondii parasite. In particular, immune-
responsible for up to 1 million patients every year and compromised people are in danger of grave complica-
affecting mainly young persons.[1,2] CL often leads to tions upon infection with T. gondii, and, thus, the
severe and disfiguring skin lesions which poses the development of new drugs for the treatment of
problem of stigmatization depressing especially young toxoplasmosis is necessary, too.[5] Currently, both CL
patients.[3,4] Current treatment options for CL patients and toxoplasmosis are endemic diseases in countries
of the Middle East.[6]
Curcumin
[(1E,6E)-1,7-bis(4-hydroxy-3-methoxy-
phenyl)hepta-1,6-diene-3,5-dione] (Figure 1) is a bio-
logically active constituent of turmeric, a spice made
Supporting information for this article is available on the
WWW under https://doi.org/10.1002/cbdv.202100381
Chem. Biodiversity 2021, 18, e2100381
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Chem. Biodiversity 2021, 18, e2100381
pared with parent curcumin.[20] Fluorinated curcu-
minoids like EF24 were reported to be superior to
curcumin in the treatment of various cancers.[21,22]
Padhye and coworkers developed the difluoroben-
zylidene derivative (1E,6E)-4-[(3,4-difluorophenyl)-
methylidene]-1,7-bis(4-hydroxy-3-methoxyphenyl)
hepta-1,6-diene-3,5-dione, a.k.a. CDF (Figure 1).[23]
CDF exhibited improved bioavailability and distinct
activity against various tumors including prostate
and colon carcinomas.[24–26] It was well tolerated by
mice and, thus, is an auspicious drug candidate for
other human diseases such as leishmaniasis and
toxoplasmosis, too. In the present report we
disclose first promising results of CDF and of its
new O-methylated analog MeCDF (Figure 1) con-
cerning their activity against L. major and T. gondii
parasites, which may be of relevance for the design
of new potent drugs against these parasites.
Figure 1. Structures of curcumin and its semi-synthetic deriva-
tives CDF and MeCDF (the new methoxy groups of MeCDF are
highlighted by spheres).
Results and Discussion
CDF was prepared from curcumin and 3,4-difluoroben-
zaldehyde according to
a
literature procedure
(Scheme 1).[23] The new compound MeCDF was pre-
pared from CDF and methyl iodide via Williamson
from the roots of Curcuma longa which is widely etherification in high yield (96%). MeCDF was ob-
applied in Indian cuisine and traditional Indian tained as a yellow solid and analyzed by NMR
medicine.[7] Curcumin has shown significant anti- spectroscopy and mass spectrometry. Four CH3 groups
cancer activity by addressing various cellular targets according to four methoxy groups of MeCDF were
1
involved in cancer cell survival and metastasis identified in the H-NMR spectrum. The asymmetric
including inhibition or down-regulation of NF-кB, structure of MeCDF due to the 3,4-difluorobenzylidene
COX-2, STAT-3, Akt, and MMP-2.[8–12] A phase II trial in residue led to distinctly different shifts for each
patients with advanced pancreatic cancer revealed
high tolerability of curcumin in all patients and
efficacy of curcumin, orally administered as a mono-
therapy, in some patients with pancreatic cancer.[13]
As a natural product of a tropical plant, curcumin has
great potential as a drug against infections and
tropical diseases.[14,15] Curcumin was tested against
various protozoal parasites and it showed activity
against Leishmania, Plasmodium and Toxoplasma
species.[16,17] Curcumin and its metal complexes
displayed activity against Leishmania major.[18] In
addition, curcumin inhibited glyoxalase 1 of Toxoplas-
ma gondii and the proliferation of this parasite
in vitro.[19]
The efficacy of curcumin as a therapeutic agent
for cancer diseases is often hampered by its low
bioavailability. Modified curcumin derivatives with
improved bioavailability frequently show a signifi-
cantly enhanced in vitro and in vivo activity com-
Scheme 1. Reagents and conditions: (i) 3,4-Difluorobenzalde-
hyde, piperidine, MeOH, r.t., 48 h; (ii) MeI, K2CO3, DMF, r.t., 24H,
96%.
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Chem. Biodiversity 2021, 18, e2100381
carbonyl signal in the 13C-NMR spectrum (δ=186.4
and 197.3 ppm, Δδ=10.9 ppm).
CDF and MeCDF were initially tested against
Toxoplasma gondii parasites and non-malignant Vero
kidney epithelial cells (Table 1). Their activities were
compared with the activity of curcumin and the
known antiparasitic drug atovaquone (ATO) (Table 1).
Both CDF and MeCDF showed activities against
T. gondii, with sub-micromolar EC50 concentrations,
exceeding that of curcumin. MeCDF was especially
active here with an EC50 value below 0.4 μM and it
was more than twice as active as the first-generation
derivative CDF. Tests against L. major parasites re-
vealed high activities of CDF and MeCDF against
promastigotes (EC50 <0.4 μM). CDF also exhibited a
high activity against L. major amastigotes while MeCDF
was less active here (Table 1). CDF surpassed the
activity of the approved antileishmanial drug ampho-
tericin B (AmB) and showed reasonable selectivities for
L. major promastigotes and amastigotes (SI>2.4). We
also detected an antioxidant activity for CDF (IC50 =
52.2 μM) while MeCDF showed no antioxidant activity
(IC50 >300 μM). These results clearly show that the
activities of CDF and MeCDF against L. major promas-
tigotes and T. gondii are superior those of curcumin by
far. The antiparasitic activities of CDF and MeCDF
match the efficacy of approved antiparasitic drugs
such as AmB and ATO in these parasites. Although the
observed selectivities of CDF and MeCDF are lower
than the selectivity of ATO for T. gondii and AmB for
L. major, the many other positive effects and proper-
ties of CDF suggest it to be a promising, well tolerated,
and effective drug candidate for parasitic diseases. The
in vivo activities of MeCDF remain to be elucidated.
The secondary antioxidant effect seems to play only a
minor role for the antiparasitic activity of CDF and
none at all for MeCDF.
CDF and MeCDF were also tested for activity
against macrophages (Table 2). MeCDF showed a
distinctly lower activity against macrophages than
against L. major promastigotes with an SI value of 4.4.
MeCDF displayed a lower activity against macrophages
than against Vero cells, while CDF showed virtually the
same toxicity to both cell types. Hence, the SI values of
CDF based on Vero cells and on macrophages are
roughly the same. The approved reference drugs ATO
and AmB were less toxic to the macrophages than
were CDF and MeCDF. However, the SI value of AmB
taking the promastigotes into account (SI=9.3) was
only twice as high as the corresponding SI value of
MeCDF (SI=4.4). As mentioned above, in vivo activ-
ities of MeCDF remain to be elucidated. But since it
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Chem. Biodiversity 2021, 18, e2100381
Table 2. Inhibitory concentrations IC50 (in μM)[a] of test compounds when applied to macrophages, selectivity indices (SI).[b]
Compounds
Macrophages
SI (promastigotes)
SI (amastigotes)
CDF
MeCDF
ATO
1.02
1.35
10.1
8.1
2.8
4.4
–
2.9
1.7
–
AmB
9.3
17.2
[a] Values are the means of at least three independent experiments (�SD<15%). They were derived from concentration-response
curves obtained by measuring the percentage of vital cells relative to untreated controls after 72 h. [b] Selectivity index (IC50/EC50)
calculated from the corresponding IC50 values for macrophages and the EC50 values for L. major (Table 1).
showed similar or higher selectivities than CDF for the protozoal parasites will probably identify further
mentioned parasites, it is possible that MeCDF will also possible fields of application.
be well tolerated and highly bioavailable in laboratory
animals.
Experimental Section
General
Conclusions
Column chromatography: silica gel 60 (230–
The antiparasitic activities of the semi-synthetic curcu- 400 mesh). Melting points (uncorrected), Electrother-
minoids CDF and MeCDF were characterized by EC50 mal 9100; NMR spectra, Bruker Avance 300 spectrom-
values in the low micromolar range. Both compounds eter (see also Supporting Information); chemical shifts
can be considered as highly active against T. gondii are given in parts per million (δ) downfield from
and L. major parasites. When compared with the tetramethylsilane as internal standard; Mass spectra,
parent curcumin, its analogs CDF and MeCDF were Thermo Finnigan MAT 8500 (EI). Elemental analysis,
much more active against these parasites. Both CDF PerkinElmer 2400 CHN elemental analyzer. The prepa-
and MeCDF were also more active than the approved ration of CDF was reported previously.[23,27] All tested
drug AmB against L. major promastigotes. It is also compounds were >98% pure by elementary analysis.
remarkable that CDF and MeCDF were similarly or All starting compounds were purchased from the usual
more active against L. major promastigotes than retailers and used without further purification.
against L. major amastigotes. This is remarkable since
AmB showed a higher activity against amastigotes
than against promastigotes. Based on these findings, methylidene]-1,7-bis(3,4-dimethoxyphenyl)hepta-
future studies will show if a combination of CDF or 1,6-diene-3,5-dione (MeCDF) CDF (266 mg,
MeCDF with AmB can lead to synergy effects. 0.54 mmol) was stirred with K2CO3 (746 mg,
Synthesis
of
(1E,6E)-4-[(3,4-Difluorophenyl)
The antioxidant activity of CDF can be correlated 5.4 mmol) and iodomethane (3.38 mL, 54 mmol) in
with its phenolic hydroxy groups because the DMF (5 mL) at room temperature for 24 h. Ethyl
tetramethyl ether MeCDF showed no antioxidant acetate was added and the mixture was washed with
activity. However, the antioxidant activity of CDF was water. The organic phase was dried over Na2SO4,
only moderate and, thus, the antiparasitic activity of filtered and the filtrate was concentrated in vacuum.
CDF is likely based on other mechanisms, which The residue was purified by column chromatography
remain to be elucidated.
(silica gel 60, ethyl acetate/hexane 1:1, v/v). Yield:
Both CDF and MeCDF can be suitable drug 268 mg (0.52 mmol, 96%); Rf =0.41 (ethyl acetate/
°
candidates for infection diseases caused by these hexane, 1:1); yellow solid of m.p. 94–96 C; νmax
parasites and further in-depth investigations of their (ATR) cmÀ 1 3002, 2953, 29363, 2838, 1575, 1508,
potential as anti-parasitic drugs are warranted given 1464, 1441, 1422, 1340, 1306, 1260, 1232, 1158, 1137,
the fact that CDF has already been tested in vivo 1118, 1020, 977, 806, 763, 713; 1H-NMR (300 MHz,
where it was well tolerated and showed higher CDCl3) δ 3.83 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 3.85
bioavailability than curcumin.[26] In addition, tests of (3H, s), 6.7–7.3 (11H, m), 7.42 (1H, d, J=16.2), 7.66
these promising curcumin derivatives against other (1H, s), 7.73 (1H, d, J=15.4); 13C-NMR (75.5 MHz,
CDCl3) δ 55.8, 55.9, 109.9, 110.4, 110.9, 111.0, 117.5,
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Chem. Biodiversity 2021, 18, e2100381
117.8, 118.5, 118.7, 119.5, 123.5, 123.8, 125.1, 126.6, concentrations (50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, and
126.8, 126.9, 127.3, 130.5, 130.6, 137.5, 141.5, 145.8, 0.37 μgmLÀ 1). Negative control wells contained cul-
147.9, 148.3, 148.5, 149.1, 149.4, 149.5, 151.7, 151.8, tures with DMSO (1%) only. Plates were incubated at
152.1, 152.9, 186.4, 197.3; EI-MS: m/z 520 (36) [M+], 26 C for 72 h to evaluate the antiproliferative effect.
°
300 (68), 299 (100), 191 (53), 151 (37).
Colorimetric MTT method was used to count viable
promastigotes via microplate absorbance spectropho-
tometer (xMark, Bio-Rad, USA) at 570 nm. The experi-
ment was done in triplicate.[30]
Toxoplasma gondii culture conditions
Tachyzoites of T. gondii RH strain were cultivated in
Vero cells according to a published method.[28] Com-
Macrophages from the peritoneal of female BALB/c
plete RPMI 1640 medium (Invitrogen, USA) with 10% mice (6–8 weeks old) were used for culturing amasti-
fetal bovine serum (FBS, Invitrogen, USA) was used for gote stages of L. major in order to assess the activity of
culturing Vero cells in 96-well plates (5×103 cells/well test compounds against amastigotes. Briefly, 1 mL of
°
in 200 μL medium), which were incubated at 37 C 3% Brewer thioglycollate medium/mouse was injected
supplied with 5% CO2 for 24 h followed by washing into the peritoneal cavity. After 4 days, the abdominal
with phosphate buffered saline (PBS) in order to skin was removed to expose the peritoneal wall. 3 mL
remove the medium. Then, RPMI 1640 medium with of RPMI 1640 medium was injected before cells were
2% FBS containing T. gondii tachyzoites (RH strain) at a collected by aspiration, whereupon 96-wells plates
ratio of 5 (parasite): 1 (Vero cells) was added. After containing RPMI 1640 medium with 10% FBS were
incubation at 37 C and 5% CO2 for 5 h, the cells were used for culturing 5×104 cells/well, which were incu-
°
°
washed with PBS and then overlaid with medium bated at 37 C for 4 h in 5% CO2. After washing with
containing test compounds or atovaquone (ATO, PBS, RPMI 1640 medium containing 5×105 promasti-
concentrations of 50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, gotes were added to each well. Then, the cells were
and 0.37 μgmLÀ 1).
incubated at 37 C in humidified 5% CO2 for 24 h to
enhance infection and differentiation of amastigotes.
°
°
After incubation at 37 C and 5% CO2 for 72 h, the Washing with PBS for several times removed free
cells were stained with 1% toluidine prior to the promastigotes. Fresh complete RPMI 1640 medium
examination under an inverted photomicroscope containing test compounds or AmB at final concen-
(MCD-400, Leica, Japan) in order to determine the trations (50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, and
À 1
°
infection index (number of infected cells from 200 0.37 μgmL ) was added and incubated at 37 C in
tested cells) of T. gondii. The experiment was done in humidified 5% CO2 atmosphere for 72 h. DMSO (1%)
triplicate.[27]
only in complete RPMI media was used as negative
control. The percentage of infected macrophages was
assessed microscopically after the removal of the
medium, followed by washing with PBS, fixation with
Leishmania major promastigotes and amastigotes
Promastigote stages of L. major were isolated and methanol and staining with Giemsa. The reading was
maintained according to a described method.[29] done in triplicate.[30] The handling of the laboratory
BALB/c mice were injected at hind footpads with 1× animals followed the instructions and rules of the
106 metacyclic promastigotes of L. major. Amastigote committee of research ethics, Deanship of Scientific
stage cells of L. major were collected from infected Research, Qassim University, permission number 20-
mice. After 8-weeks from inoculation, Schneider’s 03-20.
medium (Invitrogen, USA) containing antibiotics and
10% FBS was used to transform isolated amastigotes
Cytotoxicity Assay
°
to promastigotes by incubation at 26 C. Amastigote-
derived promastigotes with less than five in vitro MTT colorimetric assay was conducted for assessing
passages were only used for infection.
the cytotoxicity of the compounds according to a
method described previously.[31] Briefly, Vero cells or
For assessing the activity of the test compounds macrophages were cultured in 96-well plates (5×
against L. major promastigotes, 106 promastigotes/mL 103 cells/well/200 μL) in RPMI 1640 medium with 10%
°
were cultured in 96-well plates in Schneider’s medium FBS and 5% CO2 at 37 C for 24 h. Thereafter, PBS was
with 10% FBS. Then, the test compounds or ampho- used for washing the cells. Then, the compounds in
tericin B (AmB) were added to obtain the final RPMI complete medium with 10% FBS (at varying
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Chem. Biodiversity 2021, 18, e2100381
concentrations of 50, 25, 12.5, 6.25, 3.13, 1.65, 0.75, We are grateful to Prof. Subhash Padhye (Interdiscipli-
and 0.37 μgmLÀ 1) were added to 96-well plates and nary Science & Technology Research Academy, Abeda
incubated for 72 h. Cells treated with medium contain- Inamdar College, Pune, India) for the kind provision of
ing only 1% DMSO were used as negative control. CDF. Open access funding enabled and organized by
After washing, MTT (1 mgmLÀ 1 in RPMI 1640 medium) Projekt DEAL.
was added to each well. The cells were incubated for
4 h followed by the removal of the supernatant. Then,
150 μL DMSO was added and a microplate absorbance
spectrophotometer was applied for colorimetric analy-
Author Contribution Statement
sis (λ=540 nm). Cytotoxic effects were expressed by T. A. K. contributed to the writing of the manuscript
IC50 values (concentration that caused a 50% reduc- and supervised the study, W. S. K. designed and
tion in viable cells). The experiment was done in supervised the biological studies, I. S. A. N. carried out
triplicate.[27]
the biological assays, R. S. contributed to the writing
of the manuscript and supervised the study, B. B.
prepared the new compound MeCDF and wrote the
article.
DPPH Radical Scavenging Activity Assay
The technique was done according to a published
method with slight modifications.[32,33] A solution of
2,2-diphenyl-1-picrylhydrazyl (DPPH) (1 mg/25 mL
ethanol, 100 μL) was added to 5 μL of the compound
solution with different concentrations (200, 100, 50,
25, 12.5, 6.25, 3.13, 1.65, 0.75, 3.13 μgmLÀ 1). The
mixture was shaken for 15 min. A blank sample of the
same volume was prepared from ethanol and DPPH.
Then, a spectrophotometer at a wavelength of 517 nm
was used to determine the absorbance values. The
test was performed in triplicate. The scavenging
activities of the tested samples were expressed as
percentage of inhibition according to the following
equation:
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Acknowledgements
We thank Qassim University and the Deanship of
Scientific Research for material support of this study.
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Received May 18, 2021
Accepted July 1, 2021
[22] D. Subramaniam, R. May, S. M. Sureban, K. B. Lee, R.
George, P. Kuppusamy, R. P. Ramanujam, K. Hideg, B. K.
www.cb.wiley.com (7 of 7) e2100381 © 2021 The Authors. Chemistry & Biodiversity Published by Wiley-VHCA AG