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Synthesis and Antimalarial Properties of Three Substituted Trinitro-Tribenzylamine
Junior Kindala1,4*, Ezekiel Afolabi1, Noel Wannang2, Ukpe Ajima1, Francis Agwom1, Titilayo Johnson3, Jean Kayembe4, Pius Mpiana4 and Kalulu Taba4
1Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, Nigeria
2Department of Pharmacology, Faculty of Pharmaceutical Sciences, University of Jos, Nigeria
3Department of Biochemistry, Faculty of Medical Sciences, University of Jos, Nigeria
4Department of Chemistry, Faculty of Sciences, University of Kinshasa, Congo-DR
Submission: September 29, 2016; Published: October 26, 2016
*Corresponding author: Junior Kindala, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos, Nigeria
How to cite this article: Junior K, Ezekiel A, Noel W, Ukpe A, Francis A, et al. Synthesis and Antimalarial Properties of Three Substituted Trinitro- Tribenzylamine. Organic & Medicinal Chem IJ. 2016; 1(3): 555561. DOI: 10.19080/OMCIJ.2016.01.555561
Malaria infection is a major health burdens in Tropical Africa and this is made worst with the emergence of Plasmodium falciparum resistance to the commonly used antimalarial drugs. There is therefore the need for new potent antimalarial drugs. Syntheses of o-trinitro tribenzylamine (2TBN), m-trinitro tribenzylamine (3TBN) and p-trinitro tribenzylamine (4TBN) were carried and yields of 38.49%, 41.01% and 39.67% respectively were obtained on reacting the respective o-, m-, and p-nitro benzaldehyde with formamide. The three synthesized trinitro-tribenzylamine derivatives were tested in vitro against Plasmodium falciparum isolates (3D7 strain) using parasite lactate dehydrogenase assay (pLDH). The result showed significant activity with IC50 ranging from 18 to 30 nM. The meta derivative (IC50:18.823 nM) showed better antiplasmodial activity than ortho (IC50: 30.287 nM) and para (IC50: 22.297 nM) derivatives, and even chloroquine phosphate (IC50: 22.777 nM).
Malaria remains one of the major causes of death in several parts of the world. Among the species of plasmodium that commonly infect humans, Plasmodium falciparum and plasmodium vivax are the most prevalent and plasmodium falciparum is the most dangerous [1-3]. The emergence of parasite resistance to commonly used antimalarial drugs, particularly in Plasmodium falciparum case, has increased the burden of malaria . According to WHO, the burden is heaviest in African Region, where an estimated 90% of all malaria deaths occur . The need for new antimalarial drug candidates is therefore very urgent.
Amine compounds are recognized for their polarity and basicity; they are usually incorporated into several drugs to enhance binding to different drug targets as well as to facilitate their absorption. Many antimalarial drugs such as quinine, chloroquine, mefloquine, amodiaquine, etc. are alkaloid, and their mechanism of action has been shown to also depend on the amine functional group present in their structures. The molecules are transported into the acidic food vacuole (pH 4.7) down the pH gradient and the amine gets protonated prior to inhibition of haem polymerization. Accumulation of the haem generated from the degradation of haemoglobin is lethal to the parasite due to the high toxicity of this waste product .
Despite decades of effort to eradicate malaria, it remains endemic in several parts of the world. The challenges associated with the treatment of malaria have led Medicinal chemists to keep working to discover potent antimalarial agents. This work aimed to synthesize, characterize and screen the antimalarial activity of some trinitro-tribenzylamine analogues. Also, to evaluate the effect of varying the position of the nitro group between the ortho, meta and para positions of the tribenzylamine moiety on the antimalarial activity.
Three trinitro-tribenzylamine analogues were obtained in
full synthesis using Leuckart reaction (Equation 1). In a 250
ml-round bottom flask fitted with reflux condenser is placed
0.05 Moles of nitro benzaldehyde derivative (o-, m-, and p-nitro
benzaldehyde) and 0.5 Moles of formamide. The contents of the
flask were heated and stirred to reflux in acetic acid; after eight
hours of refluxing and stirring, the solution was cooled to room
temperature. The solution was then neutralized with sodium
bicarbonate and extracted with diethyl ether, the ether extracts
were concentrated in vacuo to give a product. The structures of
synthetized compounds were determined by nuclear magnetic
resonance (1H-NMR and 13C-NMR), and FTIR techniques.
The human parasite Plasmodium falciparum strain used
in this study was Chloroquine sensitive (3D7), it was obtained
from Hematology Department of Jos University Teaching
Hospital, Plateau State-Nigeria. The O+ infected erythrocyte
(at 4% of hematocrit and 0.5% of parasitemia) was cultured in
vitro according to the method described by Trager and Jensen
with some modifications , in RPMI 1640 medium (Catalog #:
ABI-111-02), containing 2g/L of glucose, 1mg/L of L-glutamine,
2g/L of NaHCO3, Amino Acids, Vitamins, Inorganic Salts,
Phenol Red and supplemented with 0.01 mg/mL of gentamicin
(Sigma-aldrich), 25 of mMN-2-hydroxyethylpiperazine-N-2-
ethanesulfonic acid (HEPES) buffer (Sigma-aldrich), and 5% of
lipid-enriched bovine albumin preparation (Albumax®).
The synthesized compounds (o-, m-, and p-nitro
tribenzylamine) and standards drug: Dihydroartemisinin
(Sigma-Aldrich), chloroquine phosphate (Sigma-Aldrich) and
Piperaquine-dihydroartemisinin) were dissolved in DMSO
0.5% to produce a stock solution of 100 nM. The infected
erythrocyte was exposed to synthesized compounds and
standards drug in various concentrations (from 0.2 to 47 nM
for the synthesized compounds (2TBN, 3TBN and 4TBN), 0.1
to 24 nM for piperaquine-dihydroartemisinin (PIP-DHA), 0.2
to 48 nM for chloroquine phosphate (CQ), and 0.3 to 70 nM for dihydroartemisinin (DHA) in triplicate in 96-well culture
plates and incubated for 72 h at 37°C in an atmosphere of 92%
N2, 5% CO2 and 3% O2. For negative control wells, the infected
Erythrocytes were devoid of trinitro-tribenzylamine analogues
or standards drug. Synthesized compounds and standard drugs
were assessed for antiplasmodial activity in vitro using parasite
lactate dehydrogenase assay (pLDH).
After 72 h of incubation, 20 μL were removed and added to
100 μL of the Malstat reagent (Prepared by dissolving 0.11% of
Triton X -100, 115.7mM of L-lactate, 30.27mM of Tris buffer, and
0.62mM of 3-acetyl pyridine adenine dinucleotide in deionized
water, the pH was adjusted to 9.2 with hydrochloric acid) present
in a new flat-bottomed 96-well micro titer plate in triplicate. 25
μL of NTB/PES (prepared by dissolving 1.96 mM of Nitro Blue
Tetrazolium and 0.24mM of Phenazine Ethosulfate in deionized
water) were then added to each well, thereby initiating the
lactate dehydrogenase reaction. Absorbance was then measured
at 630nm using an ELISA plate reader.
The optical density values from negative control wells were
referred to as having 100% pLDH activity. The IC50-values of each
tribenzylamine analogues or standard drugs were obtained by
regression analysis, using a non-linear dose-response curvefitting
equation in Graph Pad Prism 7.0 software.
Parasite % survivals were plotted versus the decimal
logarithm of concentration in Graph Pad Prism 7.0 software to
determine the concentration inhibiting 50% of parasite growth
(IC50 value), and are presented in (Figures 1- 6). The IC50
presented in Table 1, were obtained from three independent
triplicate experiments and were expressed as mean ± SEM.
A series of trinitro-tribenzylamine derivatives (ortho-,
meta- and para) were prepared in good yields. The structures
of the synthetized compounds were confirmed based on the
data from IR, 1H-NMR and 13C-NMR spectral analysis and the
signals obtained were all in a good agreement with the proposed
structures. From the graph presented to the (Figures 1-6), showed
the decrease in parasite survival as signify by the negative curve
in all the synthesize compounds and standard drugs. The results
of this study showed that the three synthesized compounds
possess Plasmodium falciparum suppressive effect, with IC50
between 18 to 30 nM. From literature, a drug is potentially
interesting as antimalarial if its IC50 is below 10μM, 5μM is used
as a cut-off level to reduce the number of false positives .
Therefore the trinitro-tribenzylamine derivatives synthesized in
this study can be added to the growing list of compounds with
antiplasmodial activity against plasmodium falciparum, and
hence serves as new lead compounds for the development of
new antimalarial drugs.
The meta-trinitro tribenzylamine had a lower IC50 than
chloroquine phosphate indicating a better antimalarial activity;
the para substituted analogue is comparable to chloroquine
phosphate, while the ortho has lesser activity to chloroquine
phosphate. All the synthesized compounds showed lesser
activity compared to DHA, and dihydroartemisinin-piperaquine.
The IC50 results, summarized in (Table 1), indicated that the
introduction of nitro group into tribenzylamine skeletal in metaposition
enhanced bioactivity against Plasmodium falciparum
isolates by almost one point six-fold factor compared to the
ortho-derivative and one point two-fold factor compared to the
Ortho-, meta- and para-trinitro tribenzylamine derivatives
have been synthesized successfully through a simple one-step
reaction (Leuckart reaction). All the three compounds showed
good antiplasmodial activity against chloroquine sensitive strain
of Plasmodium falciparum (3D7). The substitution of nitro group
in meta-position into tribenzylamine moiety has shown to be the
most potent derivatives. These derivatives can represent a new
template for antimalarial agent development.