| Summary: | Malaria, a widespread disease caused by the protozoan parasite of the genus Plasmodium, was
responsible for an estimated 216 million infections and 655 000 deaths worldwide in 2010, with
the vast majority occurring in Africa (91%) in children under 5 years of age. Parasitic resistance
greatly undermines the current combination therapy treatment regimen such that the synthesis
of new antimalarial drugs with high e cacy and low toxicity is crucial for future hopes of
combating the disease. This is especially true for the antifolates, which remain one of the most
important areas for antimalarial drug discovery today. The malarial parasite relies heavily on
folate derivatives such as cellular cofactors for the biosynthesis of purines, pyrimidines and
certain amino acids, which in turn are required for cellular replication and protein synthesis
respectively. Folate derivatives can be produced by the folate salvage pathway or alternatively
by the de novo pathway, a route unique to the parasite.
In this project, novel antifolates targeting malarial folate metabolism were developed using
two approaches, both of which aimed to alleviate the costs of drug development, an important
consideration in antimalarial research.
In the rst instance, compounds based on already existing drugs, namely the well-known anticancer
drugs methotrexate (MTX) and pemetrexed, were synthesised. Cancerous cells are
associated with abnormal and frequent mitotic divisions and thus require folate derivatives for
cellular growth and replication in a similar manner to rapidly dividing malarial cells. These
are acquired via a salvage folate pathway, which contains many of the enzymes common to
the parasite's own folate salvage pathway, and hence can also be targeted with antifolate therapy.
Despite this, chemotherapeutic drugs and, methotrexate in particular, are not used in
the treatment of malaria as they are considered too toxic. We aimed to address the issue of
toxicity by synthesising precursors of methotrexate and pemetrexed which lack a glutamic acid
residue. These molecules, which contain a terminal aromatic carboxylic acid, could potentially
be glutamated in situ by enzymes of the parasite's de novo pathway to a ord the active drugs,
methotrexate and pemetrexed. In this way, the concentration of the active compound present
in the bloodstream would be kept to a minimum. Furthermore, the drug would only be formed
in infected cells as the de novo synthetic pathway is not present in humans. To this end, precursors
of both methotrexate 4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzoic acid
and pemetrexed 4-(2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl)benzoic
acid were synthesised over 7 and 6 steps in overall yields of 2.2% and 3.2% respectively. Antimalarial
testing was conducted in vitro in a whole cell P. falciparum screen against a cycloguanil resistant/CQ sensitive Gambian FCR-3 strain. The methotrexate precursor was found to exhibit
antimalarial activity (IC50 2.478 M) whereas the pemetrexed precursor was inactive and
did not inhibit parasitic growth.
The second approach for the synthesis of novel antimalarial antifolates made use of chemical
modi cation of molecular sca olds of known antimalarial activity. Using this strategy, a series
of
exible pyrimidine compounds were designed by the structural simpli cation of a dihydrotriazine
antifolate hit compound generated from a previous study. Disconnection of the pyrimidine
ring into its corresponding -cyano ketone and guanidine compounds indicated a possible
route towards their synthesis. Initially, a control study was conducted in which the more rigid
pyrimethamine analogue: 5,6-diphenylpyrimidine-2,4-diamine was synthesised over three steps
in an overall yield of 46%. We then applied this methodology to the synthesis of the targeted
pyrimidine compounds. Starting from the appropriately substituted phenol, the bromoether of
each analogue was successfully synthesised by alkylation with 1,4-dibromobutane. Functional
group interconversion of the bromide to the nitrile group, followed by the addition of ethyl benzoate
at the -cyano position a orded a series of -cyano ketone compounds in yields of 9-68%.
The enol ether intermediates were formed using diazomethane instead of the experimental conditions employed in the control study (using triethylorthoformate and an acid catalyst) which had resulted in low yields and the formation of alternative products. By contrast, the high yielding, relatively clean methylation reaction using diazomethane allowed for an attempt of the nal step such that the pyrimidine ring was formed on reaction with guanidine in DMSO, a ording the 5-(3-(4-chlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine over ve steps, in an overall yield of 5%. In this way, we had proved the viability of the synthetic route towards the targeted series of
exible, pyrimidine analogues.
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