Synthesis and biological evaluation of 2-substituted vinylgembisphosphonates against Plasmodium falciparum and Trypanosoma brucei

Article abstract of DOI:10.1080/10426507.2020.1728757

A series of 2-substituted vinylidene-1,1-bisphosphonate esters and their acids were synthesized and tested in?vitro for activity against Plasmodium falciparum and Trypanosoma brucei. For each compound, % parasite viability in treated wells was calculated relative to untreated controls for both P. falciparum and T. brucei. Fifty percentage inhibitory concentration (IC₅₀) was also determined for the compounds. Chloroquine and pentamidine were used as positive control drug standards for activity against P. falciparum and T. brucei, respectively. The esters had better antiparasitic activity compared to their corresponding acids. Some of the compounds reduced % parasite viability to as low as 24.3% for P. falciparum and down to 0.602% for T. brucei. Tetraethyl-2-(o-tolyl)-ethene-1,1-bisphosphonate (3b) recorded the best IC₅₀ against T. brucei which was 0.0345 μmol/mL.

Full text of DOI:10.1080/10426507.2020.1728757

Phosphorus, Sulfur, and Silicon and the Related Elements
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Synthesis and biological evaluation of 2-
substituted vinylgembisphosphonates against
Plasmodium falciparum and Trypanosoma brucei
Siphamandla B. Simelane, Monisola I. Ikhile, Derek T. Ndinteh & Xavier Y.
Mbianda
To cite this article: Siphamandla B. Simelane, Monisola I. Ikhile, Derek T. Ndinteh & Xavier Y.
Mbianda (2020): Synthesis and biological evaluation of 2-substituted vinylgembisphosphonates
against Plasmodiumꢀfalciparum and Trypanosomaꢀbrucei, Phosphorus, Sulfur, and Silicon and the
Related Elements, DOI: 10.1080/10426507.2020.1728757
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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
https://doi.org/10.1080/10426507.2020.1728757
Synthesis and biological evaluation of 2-substituted vinylgembisphosphonates
against Plasmodium falciparum and Trypanosoma brucei
Siphamandla B. Simelane, Monisola I. Ikhile, Derek T. Ndinteh, and Xavier Y. Mbianda
Department of Applied Chemistry, University of Johannesburg, Doornfontein, South Africa
ABSTRACT
ARTICLE HISTORY
Received 20 May 2019
Accepted 5 February 2020
A series of 2-substituted vinylidene-1,1-bisphosphonate esters and their acids were synthesized
and tested in vitro for activity against Plasmodium falciparum and Trypanosoma brucei. For each
compound, % parasite viability in treated wells was calculated relative to untreated controls for
both P. falciparum and T. brucei. Fifty percentage inhibitory concentration (IC50) was also
determined for the compounds. Chloroquine and pentamidine were used as positive control drug
standards for activity against P. falciparum and T. brucei, respectively. The esters had better
antiparasitic activity compared to their corresponding acids. Some of the compounds reduced %
parasite viability to as low as 24.3% for P. falciparum and down to 0.602% for T. brucei. Tetraethyl-
2-(o-tolyl)-ethene-1,1-bisphosphonate (3b) recorded the best IC50 against T. brucei which was
0.0345 mmol/mL.
KEYWORDS
Substituted vinyl
gem-bisphosphonic acids;
antimalarial activity;
antitrypasomal activity;
drug resistance
GRAPHICAL ABSTRACT
Introduction
have a great potential in pharmaceutical applications.[6]
Initially, BPs were used as antiscaling and anticorrosive
agents as well as complexing agents in the textile, fertilizer,
and oil industries. Their potential for the treatment of cal-
cium and bone associated disorders was discovered in the
1960s.[8] BPs are now clinically used to treat diseases such
as arthritis, Paget’s disease, and osteoporosis in women.
Etidronate and elandronate are also clinically used in the
prevention of osteoporosis.[7] They treat calcium associated
disorders by binding to the mineral surfaces in bone and
prevent the dissolution of calcium phosphate crystals
thereby inhibiting bone resorption.[9]
Malaria and Human African Trypanosomiasis (HAT) are
diseases caused by protozoan parasites of the genera
Plasmodium and Trypanosoma, respectively.[1,2] Malaria is
responsible for about 1 million deaths each year whilst
approximately half a million cases of HAT have been
reported in Sub-Saharan Africa between 1990 and pre-
sent.[3,4] Over the years, the deadly species causing malaria,
Plasmodium falciparum, has gained resistance against first
line drugs such as chloroquine.[5] Due to lack of vaccines
and constant emergence of drug-resistant strains, it is neces-
sary to develop improved drugs candidates.
Even though BPs are popular for the treatment of these
calcium disorders, recent studies have demonstrated that
they have a huge potential for other pharmaceutical applica-
tions. Several studies have reported that BPs also have anti-
cancer, antimalarial, and antibacterial properties.[10–12] They
also activate ÇT cells of the immune system which help the
body fight infections as well as cancer.[6,13] BPs can be con-
jugated with other molecules (drugs and imaging agents) to
Bisphosphonates (BPs) are synthetic organic compounds
characterized by a P-C-P backbone structure. They are
metabolically stable structural analogues of the inorganic
pyrophosphate that has a P-O-P backbone.[6] BPs were dis-
covered in the 19th century and their first synthesis was
reported in 1865.[7] These compounds are also analogous to
many units in molecules found in living organisms such as
deoxyribonucleic acids (DNA), ribonucleic acids (RNA),
adenosine triphosphate (ATP), and so on. Therefore, they target bone tissue as delivery systems because they have
CONTACT Derek T. Ndinteh
dndinteh@uj.ac.za
Department of Applied Chemistry, University of Johannesburg, Doornfontein 2028, South Africa.
Supplemental data for this article is available online at https://doi.org/10.1080/10426507.2020.1728757.
ß 2020 Taylor & Francis Group, LLC

2
S. B. SIMELANE ET AL.
high affinity for hydroxyapatite, the main component of
bone mineral.[14] Many studies have revealed that BPs are
potent inhibitors of protozoan parasites and also have anti-
tumor properties.[11] Martin et al. showed that BPs can also
inhibit the growth of Trypanosoma brucei and P. falciparum
which cause HAT and malaria, respectively.[15] The chal-
lenge with BPs currently available for clinical applications is
their strong affinity for bone mineral which makes them
unsuitable when targeting other tissues.[10] Vinyl gem-BPs,
unlike most of the clinical BPs, do not have the hydroxyl
group in the a-position, which reduces affinity toward bone
mineral. To the best of our knowledge there are no studies
in literature that have reported the biological evaluation of
substituted vinyl gem-BPs without reducing the double
bond. In this paper, we report the synthesis and biological
evaluation of vinyl gem-BPs esters and their acids against T.
brucei and P. falciparum.
Results and discussion
Synthesis
Substituted vinyl gem-BPs were successfully synthesized by
allowing to react aldehydes and tetraethyl methylenebi-
sphosphonate 2 in the presence of titanium tetrachloride
(TiCl4) and N-methylmorpholine as shown in Scheme 1.[16,17]
Nuclear magnetic resonance (NMR) results were in agreement
with those reported in literature.[18]
1H NMR spectra (Figure 1) of the products showed two dis-
tinct triplets for CH3 protons (accounting for 6 protons each)
at about 1.2 and 1.4 ppm, whereas their signal appears as one
triplet in 2. This indicates that there is no rotation in the mol-
ecule, which confirms the presence of the double bond. Two
separate signals were also observed for the OCH2 protons at
about 3.80 and 4.30 ppm. The presence of the double bond
was also confirmed by the appearance of a doublet of doublets
(accounting for one proton) corresponding to the vinylic pro-
ton at about 8.25 ppm. The cis- and trans-coupling constants,
2JPH, were 29.1 and 47.7 Hz, respectively.
The 31P NMR spectrum (Figure 2) showed two doublets
at 12 and 17.5 ppm with a coupling constant 2Jpp around
50 Hz. The two phosphorus atoms appeared at different
chemical shifts because there is no free rotation around the
double bond, thus the two phosphorus atoms are in differ-
ent magnetic environments.
Our first attempt to hydrolyze vinyl gem-BPs 3 was by
refluxing with hydrochloric acid, as it is a fast and simple
method. 31P NMR analysis showed major peaks as singlets
at 1.4, ꢀ1.5, and ꢀ1.8 ppm which are not consistent with
the expected bisphosphonic acid 4. These peaks correspond
to phosphate groups which indicate that the vinyl BP was
destroyed under these conditions.
Scheme 1. Synthesis of vinyl bisphosphonate esters.
Figure 1. 1H NMR spectrum of tetraethyl 2-phenyl-ethene-1,1-PB 3a.

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 2. 31P NMR spectrum of tetraethyl 2-phenylethene-1,1-BP 3a.
Purification of the product 4 to remove the morpholi-
nium salt required multiple recrystallizations which resulted
in poor yield. The reaction was therefore repeated without
adding N-methylmorpholine (Scheme 2). After complete
silylation and methanolysis, the solvent was removed under
reduced pressure and the product washed with acetone.
1
Results of H NMR analysis were consistent with the sub-
stituted vinyl gem-bisphosphonic acids 4. The spectra
showed the absence of ethoxy groups, which confirmed
complete dealkylation of the vinyl BPs. The NMR signal of
3
H-2 appeared at 8 ppm as a doublet of doublets with JPH
3
(cis) of 29 Hz and JPH (trans) of 49 Hz. These values are
similar to those observed for the vinyl BP esters which con-
firmed that the double bond was not destroyed during
dealkylation.
Scheme 2. TMSBr assisted dealkylation of vinyl gem-BPs 3.
Table 1. Biological evaluation of compounds 3 and 4.
Antiplasmodial activity
% Parasite and cell viability
Table 1 shows that the activity of vinyl BP esters against P.
falciparum (strain 3D7) is significantly higher than that of
the corresponding acids. Compounds 3a–c reduced percent-
age parasite viability to 24.3%, 34.2%, and 27.0%, respect-
ively. On the wells treated with the vinyl bisphosphonic
acids (compounds 4a–c) percentage parasite viability
remained above 100% indicating that they were actually
growing. The activity of compounds 3a and 3c could be
caused by the general cytotoxicity of the compounds. As can
be seen in Table 1 they reduced percentage cell viability to
47.5% and 16.0%, respectively. Compound 3c had better
antiplasmodial activity and lower cytotoxicity compared to
the other esters. Saito et al. reported that BPs interfere with
protein isoprenylation by targeting farnesyl pyrophosphate
synthase (FPPS) thereby killing the parasite.[21] Hwan et al.
suggested that geranyl pyrophosphate synthase could be
another target for antimalarial drugs.[10] One structural dif-
ference between 3b and the other two esters is the electron
donating methyl substituent at the phenyl ring. The poor
activity of the acids could be due to poor lipophilicity hence
Antimalarial activity
Antitrypanosomal activity
Cytotoxicity
3a
3ba
3c
4a
4b
4c
24.3
34.2
27.0
119.9
117.5
113.9
0.602
0.711
0.895
100.4
104.8
91.8
47.5
73.7
16.0
91.8
83.1
79.9
aIC50 calculated for compounds with high potency and low cytotoxicity.
IC50 for compound 2 – 0.0345 mmol/mL.IC50 for Pentamidine – 0.009612 uM.
We then turned our attention to TMSBr assisted hydroly-
sis developed by McKenna with modification as shown in
Scheme 2.[19,20] 31P NMR analysis showed that the vinyl BPs
were converted to the silyl ester intermediate 5. The 31P
NMR spectra of 5 showed two doublets at 7.5 and 3.1 ppm
2
with JPP of about 69 Hz.
The silyl esters 5 were then stirred with methanol to give
the substituted vinyl gembisphosphonic acids 4. 31P NMR
spectrum of the bisphosphonic acid 4a showed two doublets
at about 14 and 9 ppm with a coupling constant of
about 56 Hz.

4
S. B. SIMELANE ET AL.
they cannot easily diffuse into the cells. This phenomenon stirred for 3 h at room temperature. The reaction was
has also been observed in other studies.[10]
quenched with water (50 mL) and extracted three times with
ethyl acetate (100 mL). The organic phase was dried with
anhydrous sodium sulfate and purified by column chroma-
tography (acetone:EtOAc, 1:2) to give the product.
Antitrypanosomal activity
Tetraethyl-2-(phenyl)-ethene-1,1-bisphosphonate (3a): Pale
yellow oil (56%): H NMR (400 MHz, CDCl3): d ¼ 1.16 (t,
The activity of the esters against T. brucei was also found to
be higher compared to the acids, as shown in Table 1.
However, in this case, all the esters reduced viability of T.
brucei to about 0%, which is very high compared to the
activity against P. falciparum. Many studies have reported
that the BPs kill the parasite by inhibiting T. brucei FPPS
enzyme.[22,23]. The lower activity of the esters against P. fal-
ciparum relative to T. brucei could be attributed to poor
uptake of the BPs into the parasite.[15] Fifty percentage
inhibitory concentration (IC50) was calculated for compound
3b because it had good antitrypanosomal activity and lower
cytotoxicity. It was found to be 0.0345 mmol/mL, which is
comparable to that of the standard drug pentamidine.
1
J ¼ 6.9 Hz, 6H, CH3), 1.39 (t, J ¼ 7.3 Hz 6H, CH3),
4.00 ꢀ 4.06 (m, 4H, CH2), 4.17 ꢀ 4.25 (m, 4H, CH2),
7.30 ꢀ 7.41 (m, 3H, arom-H), 7.50 ꢀ 7.76 (m, 2H, arom-H),
8.32 (dd, J ¼ 47.7 Hz, 29.1 Hz, 1H, CH). 13C NMR
(100 MHz, CDCl3): d ¼ 15.1 (d, J ¼ 7.0 Hz, CH3); 16.1 (d,
J ¼ 6.0 Hz, CH3); 61.7 (d, J ¼ 6.3 Hz, CH2), 61.8 (d,
J ¼ 5.3 Hz, CH2), 119.3 (t, J ¼ 169.3 Hz, C1); 127.1 (s, C-
arom.); 129.5 (s, C-arom.); 129.8 (s, C-arom.); 133.9 (dd,
J ¼ 22.6 Hz, 8.6 Hz, Cipso); 159.5 (s, C2). 31P NMR (160 MHz,
CDCl3): d ¼ 12.0 (d, J ¼ 49.6 Hz), 17.3 (d, J ¼ 49.6).
Tetraethyl-2-(o-tolyl)-ethene-1,1-bisphosphonate (3b): Pale
1
yellow oil (80%). H NMR (400 MHz, CDCl3): d ¼ 1.10 (t,
It was also noted in Table 1 that the vinyl gem-BPs with
electron-withdrawing substituents at the phenyl ring (3a and
3c) have higher cytotoxicity and better antiparasitic activity
compared to the one with electron donating substituents 3b.
J ¼ 7.1 Hz, 6H, CH3), 1.40 (t, J ¼ 7.1 Hz, 6H, CH3), 2.30 (s,
3H, CH3), 3.83 ꢀ 4.00 (m, 4H, CH2), 4.16 ꢀ 4.23 (m, 4H,
CH2), 7.17 ꢀ 7.30 (m, 3H, arom-H), 7.58 ꢀ 7.70 (m, 1H,
arom-H), 8.35 (dd, J ¼ 47.5 Hz, 28.0 Hz, 1H, CH). 13C NMR
(100 MHz, CDCl3): d ¼ 15.1 (d, J ¼ 7.0 Hz, CH3); 15.5 (d,
J ¼ 3.0 Hz, CH3); 19.1 (s, CH3); 61.6 (d, J ¼ 6.0 Hz, CH2);
61.8 (d, J ¼ 6.0 Hz, CH2); 124.7 (t, J ¼ 170.4 Hz, C1); 124.4
(s, C-arom.); 128.2 (s, C-arom.); 129.0 (s, C-arom.); 134.2
(dd, J ¼ 20.5 Hz, 8.3 Hz, Cipso); 135.0(s, C-arom.); 160.5 (s,
C2). 31P NMR (160 MHz, CDCl3): d ¼ 11.8 (d, J ¼ 52.8 Hz),
16.5 (d, J ¼ 52.8 Hz).
Conclusions
The results presented show that the BPs described have a
potential in treating diseases caused by protozoan parasites.
The results also showed that esters have better potency than
the corresponding acids. This suggests that lipophilicity of
the BPs plays a role in the efficiency of the compounds.
One of the most effective compounds (3b) against T. brucei
showed minimal cytotoxicity against human cells while
reducing parasite viability to almost 0%.
Tetraethyl-2-(p-fluorophenyl)-ethene-1,1-bisphosphonate (3c):
1
Clear oil (60%). H NMR (400 MHz, CDCl3): d ¼ 8.25 (dd,
J ¼ 47.6 Hz, 29.1 Hz, 1H, –CH¼); 7.83 (m, 2H, arom-H);
7.09 (t, J ¼ 8.6 Hz, 2H, arom-H); 4.21 (m, 4H, CH2); 4.06
(m, 4H, CH2); 1.39 (t, J ¼ 6.9 Hz, 6H, CH3); 1.20 (t,
J ¼ 6.9 Hz, 6H, CH3). 13C NMR (CDCl3, 100 MHz):
Experimental
1
d ¼ 163.4 (d, JCF ¼ 266.9 Hz, C-F); 159.2 (s, –CH¼); 133.6
Synthesis
3
(t, J ¼ 8.0 Hz, Cipso); 136.4 (d, JCF ¼ 8.0 Hz, C-arom); 120.1
2
Starting materials were purchased from Sigma Aldrich or
Merck Chemicals unless specified otherwise. Solvents were
dried using standard procedures in organic chemistry. NMR
spectra were recorded with a Bruker Avance 400 MHz
instrument operating at 400 MHz (1H), 100 MHz (13C), and
160 MHz (31P) as well as with a Bruker 500 instrument
operating at 500 MHz (1H), 126 MHz (13C) and 200 MHz
(31P). Mass spectrometry analysis was obtained using the
(t, J ¼ 167.5 Hz, ¼C<); 114.4 (d, JCF ¼ 21.0 Hz, C-arom);
61.8 (d, J ¼ 6.0 Hz, CH2); 61.9 (d, J ¼ 7.0 Hz, CH2); 15.6
(d, J ¼ 7.1 Hz, CH3); 15.4 (d, J ¼ 7.1 Hz, CH3). 31P
NMR (160 MHz, CDCl3): d ¼ 17.3 (d, J ¼ 48.0 Hz); 12.5
(d, J ¼ 48.0 Hz).
General procedure for synthesis of vinyl bisphosphonic
acids: Bromotrimethylsilane (1.47 g, 9.6 mmol) was added to
a solution of tetraethyl 2-(phenyl)-ethene-1,1-bisphospho-
Bruker Compact Q-TOF mass spectrometer. Reactions were nate 3a (0.452 g, 1.20 mmol) in dichloromethane (5 mL) at
monitored by thin layer chromatography on silica gel 60 0 ꢁC and stirred for 1 h. The reaction mixture was then
(F254) plates. The Supplemental Materials contains sample allowed to warm up to room temperature and stirred over-
1H, 13C, and 31P NMR spectra of products
(Figures S1–S9).
General procedure for synthesis of vinyl BP esters:
Benzaldehyde (10 mmol) was dissolved in dry THF (80 mL)
and TiCl4 (3 mL) in toluene (20 mL) was added drop wise.
4
night. Methanol (5 mL) was added and stirring was contin-
ued for 4 h. The solvents were removed under reduced
pressure and the solid product was washed with acetone and
dried under vacuum.
2-(Phenyl)-ethene-1,1-bisphosphonic acid (4a): Off-white
The mixture was stirred for 5 min at 0 ꢁC. A solution of solid, 0.317 g (90%): 1H NMR (400 MHz, D2O): d ¼ 7.86
tetraethyl methylenebisphosphonate (12 mmol) and N-meth- (dd, J ¼ 46.9 Hz, 29.1 Hz, 1H, CH), 7.35 (d, J ¼ 4.4 Hz, 2H,
ylmorpholine (24 mmol) in dry THF (20 mL) was added arom-H), 7.16 (s, 3H, arom-H). 13C NMR (126 MHz, D2O):
drop wise. The mixture was stirred for 1 h at 0 ꢁC, and then d ¼ 157.0 (s, C2), 134.9 (dd, J ¼ 21.6 Hz, 9.0 Hz, Cipso), 130.2

PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
5
(s, C-arom.), 129.5 (s, C-arom.), 128.2 (s, C-arom.), 125.7 concentrations were determined by adding a resazurin-based
(dd, J ¼ 161.5 Hz, 158.5 Hz, C1). 31P NMR (162 MHz, D2O): reagent. For each compound concentration, % parasite via-
d ¼ 15.0 (d, J ¼ 53.7 Hz), 9.8 (d, J ¼ 53.7 Hz). HRMS (ESIþ): bility – the resorufin fluorescence in compound-treated wells
m/z ¼ 265.0030
¼ 264.9953).
(calcd.
[M þ Hþ]
for
C8H10O6P2 relative to untreated controls – was calculated. Compounds
were tested in duplicate wells, and a standard deviation (SD)
2-(o-Tolyl)-ethene-1,1-bisphosphonic acid (4b): White crys-
tals (83%). 1H NMR (500 MHz, D2O): d ¼ 8.08 (dd,
J ¼ 46.7 Hz, 28.0 Hz, 1H, CH); 7.32 (d, J ¼ 7.6 Hz, 1H, arom-
H); 7.20 (t, J ¼ 7.4 Hz, 1H, arom-H); 7.16 ꢀ 7.08 (m, 2H,
arom-H); 2.13 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3):
d ¼ 161.5 (s, C2); 151.5 (s, C-arom.); 140.0 (dd, J ¼ 22.5 Hz,
was derived. For each compound, percentage viability was
then plotted against log (compound concentration) and the
IC50 (50% inhibitory concentration) was obtained from the
resulting dose-response curve by non-linear regression.
Pentamidine was used as a positive control.
Cytotoxicity: Cytotoxicity of the compounds was deter-
9.1 Hz, Cipso); 139.3 (s, C-arom.); 132.8 (s, C-arom.), 129.3 mined by incubating HeLa (human cervix adenocarcinoma)
(dd, J ¼ 168.7 Hz, 164.0 Hz, C1), 128.4 (s, C-arom), 128.2 (s, cells with compounds at a concentration of 20 uM for 48 h.
C-arom), 20.1 (s, CH3). 31P NMR (202 MHz, D2O): d ¼ 14.0 Percentage cell viability was determined using the resazurin-
(d, J ¼ 56.3 Hz), 9.9 (d, J ¼ 56.3 Hz). HRMS (ESIþ): m/ based reagent and reading resorufin fluorescence in a multi-
z ¼ 279.0183 (calcd. [M þ Hþ] for C9H12O6P2 ¼ 279.0109).
2-(p-Fluorophenyl)-ethene-1,1-bisphosphonic acid (4c):
Light brown crystals (95%). 1H NMR (500 MHz, D2O):
d ¼ 7.93 (dd, J ¼ 46.1 Hz, 28.9 Hz, 1H, CH), 7.59 (m,
wall plate reader. Compounds were tested in duplicates and
the standard deviation was calculated. Emetine was used as
a positive control standard.
J ¼ 7.8 Hz, 6.0 Hz, 2H, arom-H), 7.05 (t, J ¼ 8.8 Hz, 2H,
Funding
arom-H). 13C NMR (125 MHz, D2O) d ¼ 163.5 (d, JCF
¼
1
3
248.4 Hz, C-F), 155.2 (s, –CH¼), 132.2 (d, JCF ¼ 8.7 Hz, C-
This project was supported by the South African National Research
Foundation (NRF) and the University of Johannesburg. It was also sup-
ported by the South African Medical Research (MRC) with funds from
National Treasury under its Economic Competitiveness and Support
Package, and Rhodes University Sandisa Imbewu.
2
arom), 131.3 (m, Cipso), 126.1 (m, ¼C<), 115.2 (d, JCF
¼
22.0 Hz, C-arom). 31P NMR (200 MHz, D2O): d ¼ 15.1 (d,
J ¼ 53.4 Hz), 9.33 (d, J ¼ 53.4 Hz). HRMS (ESIþ): m/
z ¼ 283.9933 (calcd. [M þ Hþ] for C8H9O6P2 ¼ 283.9858).
Biological evaluation
References
Antimalarial activity: To determine antimalarial potency,
compounds were added to parasite cultures (P. falciparum
strain 3D7) in 96-well plates and incubated for 48 h in a
37 ꢁC CO2 incubator. Twenty microliter of culture was
removed from each well and mixed with 125 mL of a mixture
of Malstat solution and NBT/PES solution in a fresh 96-well
plate. These solutions measure the activity of the parasite
lactate dehydrogenase (pLDH) enzyme in the cultures. A
purple product is formed when pLDH is present, and this
product can be quantified in a 96-well plate reader by
absorbance at 620 nm (Abs620). The Abs620 reading in each
well is thus an indication of the pLDH activity in that well
and also the number of parasites in that well. Compounds
were tested in a range extending from 100 to 0.006 uM (4
fold-dilutions). For each compound concentration, % para-
site viability – the pLDH activity in compound-treated wells
relative to untreated controls – was calculated. Compounds
were tested in duplicate wells, and a standard deviation (SD)
was derived. For each compound, percentage viability was
then plotted against log (compound concentration) and the
IC50 (50% inhibitory concentration) was obtained from the
resulting dose-response curve by non-linear regression. For
comparative purposes, chloroquine was used as a posi-
tive control.
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somal potency of test compounds, serial dilutions of the
compounds were added to in vitro cultures of T. brucei in
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