Pyrimidine-2,4-diamines as antiplasmodial antifolates

Two series of substituted pyrimidine-2,4-diamines with a flexible side chain at either the 5-or 6-position of the pyrimdine ring were designed as potential inhibitors of P. falciparum dihydrofolate reductase (DHFR). The compounds were synthesised and evaluated for antiplasmodial activity in vitro against a cycloguanil-resistant strain of the P. falciparum parasite. 5-(3-(3,5-Dichlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine was identified as the most promising compound (IC 50 0.86 µM). In general, pyrimidine-2,4-diamines substituted at the 5-position of the pyrimidine ring showed better antiplasmodial activity (IC 50 0.86 - 26.55 µM ) than those bearing a modified side chain at the 6-position of the pyrimidine ring (IC 50


Introduction
In 2018, approximately half of the world's population was at risk of contracting malaria, a disease caused by parasitic protozoa of the genus Plasmodium. 1 According to the most recent World Malaria Report, 93% of the estimated 228 million malaria cases and 94% of the 405,000 deaths worldwide in 2018 were caused by Plasmodium falciparum, one of the Plasmodium species responsible for causing malaria in humans which is prevalent in sub-Saharan Africa.While malaria-prevention tools such as indoor residual spraying (IRS) of insecticides, the use of insecticide-treated nets (ITNs), and intermittent preventative therapy in pregnancy (IPTp) have played a major role in decreasing malaria incidence and mortality over the last five years, the WHO estimates that 43% of people at risk of contracting malaria in sub-Saharan Africa do not have access to these preventative tools. 1 Furthermore, although there has been a significant decline in the number of malaria fatalities over the last 15 years, the rate of decline has decreased, with mortality rates remaining at similar levels since 2014. 1 Folates are cellular cofactors that play an essential role in the life cycle of the malaria parasite. 2Antimalarial antifolates target two key parasitic enzymes, dihydropteroate synthase (DHPS) and dihydrofolate reductase, which, in the parasite, is a bifunctional dimer with thymidylate synthase (DHFR-TS).These enzymes are responsible for maintaining adequate cellular levels of folate derivatives either via a folate salvage pathway or a de novo synthetic pathway. 3The combination therapy of sulfadoxine-pyrimethamine, used in IPT both in pregnancy and in infants under the age of 5 years, targets these enzymes in the parasite, with pyrimethamine (1) targeting DHFR (Figure 1).5] Point mutations in the active site of the DHFR domains of DHFR-TS have been shown to be responsible for the development of resistance to these antifolates, which contain a rigid biaryl axis. 6Related compounds with inherent flexibility, such as WR99210 (3) and P65 (4) (Figure 1), however, have been shown to maintain a high binding affinity to all mutant forms of P. falciparum DHFR. 7These compounds contain a flexible linker between the two rings, which enables the compounds to avoid unfavourable contacts with mutant amino acid residues in the DHFR active site.Based on these findings, we reported the synthesis of a series of novel, flexible analogues of cycloguanil bearing a 4-atom linker between the two rings (see general structure (5), Figure 2), with in vitro antimalarial activity in the low nanomolar range against both drug-sensitive and drug-resistant strains of P. falciparum. 8The compounds were shown to inhibit parasitic DHFR, and were not cytotoxic.Despite the promising biological activity of these compounds; however, they were isolated as racemic mixtures in generally low yields.Furthermore, the synthesis proved challenging 9 and could not readily be adapted for asymmetric synthesis.Herein, we report the synthesis of pyrimidine analogues of general structure (6) of our dihydrotriazine series (5), and their biological activity in a whole cell P. falciparum assay.We also embarked on the synthesis of analogues of P65, of general structure (7), bearing a longer side chain moved to the 6-position of the pyrimidine ring in order to assess the effect of this on biological activity in vitro.6) and (7) prepared in this work.

Results and Discussion
The dihydrotriazines of general structure (5) that we have prepared previously 8 contained an atypical phenyl substituent at the 6-position of the dihydrotriazine ring.The majority of known antifolates targeting DHFR (such as those shown in Figure 1) contain a simple alkyl substituent at this position.Owing to the difficulties associated with the synthesis of the dihydrotriazines (5) prepared previously, [8][9] and the fact that they were isolated as racemic mixtures, we planned to prepare the fully aromatic pyrimidine equivalents which lack the stereogenic centre.
Our approach to the pyrimidine analogues of general structure (6) began with simple alkylation of commercially available phenols (8a-g) with 1,4-dibromobutane to afford the ethers (9a-g) in good yields (Scheme 1).Functional-group interconversions by reacting ethers (9a-g) with potassium cyanide gave pentanenitriles (10a-g) in reasonable yields.Reaction of 10a-g with ethyl benzoate in the presence of base afforded α-cyanoketones (11a-g), once again in good yields.
At this stage, we envisaged utilising methodology reported for the synthesis of 5-arylpyrimidines 10 on our substrates (11a-g) to afford the desired 5-alkylpyrimidines.This methodology involved formation of an intermediate enol ether, followed by reaction with guanidine hydrochloride to afford the desired pyrimidine products.Formation of the intermediate enol ethers was achieved by reaction with diazomethane generated in situ from diazald (N-methyl-N-nitroso-p-toluenesulfonamide) and potassium hydroxide.In general, the enol ethers were used as the crude products in the subsequent reactions with guanidine hydrochloride, after confirmation of the formation of the enol ether by 1 H NMR spectroscopy.
Unfortunately, the final reaction with guanidine hydrochloride either afforded the products in low yields or was unsuccessful, possibly due to the lack of an aromatic substituent at the α-position of our substrates 11a-g.Of the seven enol ethers prepared, only four were successfully converted into pyrimidine products (6a-d), however, in disappointingly low yields.Nonetheless, we subjected compounds (6a-d) to an initial in vitro assessment of antiplasmodial activity in a whole cell P. falciparum assay, at a single concentration of 20 μM.The compounds were screened against a cycloguanil-resistant strain (Gambian FCR-3), for direct comparison with our original series of dihydrotriazine compounds (of general structure (5) 8 ) which had been screened against this strain.At this single concentration, pyrimidines (6a-d) inhibited parasitaemia by more than 45% in each case (Table 1).IC 50 values were then determined for the compounds from the log sigmoid dose response curves generated by GraphPad Prism  software.Disappointingly, only one of the compounds, 5-(3-(3,5-dichlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine (6c) (entry 3, Table 1, IC 50 0.86 µM) showed activity in the nanomolar range comparable with the dihydrotriazines 8 prepared previously.The remainder of the compounds displayed only moderate activity in the low micromolar range.This could be attributed to the inflexibility of the biaryl axis now present between the 6-phenyl substituent and the pyrimidine ring, which was not present in the original series of dihydrotriazines, where the phenyl substituent was bonded to an sp 3 hybridised carbon with tetrahedral geometry.Notably, the DHFR inhibitor methotrexate also displayed antiplasmodial activity in the low micromolar range against the cycloguanil-resistant strain (entry 7, Table 1, IC 50 1.71 µM).In another aspect of this work, we wanted to assess the effect of moving the flexible side chain from C-5 to C-6 on the pyrimidine ring.This would eliminate the biaryl axis created by the presence of a phenyl substituent at C-6 as was present in 6a-d, and preliminary modelling studies suggested that the longer flexible chain could place the aromatic substituent at the correct binding position in the DHFR active site. 11he preparation of analogues of general structure (7) bearing a modified side chain could be achieved by simple substitution of a 4-hydroxypyrimidine.As shown in Scheme 2, commercially available phenols (8a-d) were alkylated with 1,3-dibromopropane, using the method described previously to afford bromoethers (12ad).2,4-Diaminopyrimidin-6-ol was then reacted with bromoethers (12a-d) to give pyrimidines (7g-j) bearing a 5-atom linker at the 6-position of the pyrimidine ring in moderate yields.2,4-Diaminopyrimidin-6-ol was also treated with previously-prepared bromoethers (9a-f) to afford pyrimidines (7a-f) bearing a 6-atom linker at the 6-position of the pyrimidine ring.Pyrimidines (7a-j) prepared by this method were then assessed in the whole cell P. falciparum screen described previously (Table 2).Scheme 2. Reagents, conditions, and yields: (a) For 12 (n = 1); 1,3-dibromopropane, K 2 CO 3 , CH 3 CN, reflux, 20 h, 87 -95%; for 9 (n = 2); 1,4-dibromobutane, reflux, 20 h, 63 -98%; (b) 2,4-diaminopyrimidin-6-ol, K 2 CO 3 , CH 3 CN, reflux, 20 h, 33 -55%.The results presented in Table 2 show that moving the side chain, albeit a modified side chain, to the 6position of the pyrimidine ring, has a detrimental effect on antiplasmodial activity in vitro, with the majority of pyrimidines (7a-j) being less active than pyrimidines (6a-d) against the drug-resistant strain of the P. falciparum parasite.Although from our initial docking studies it appeared that analogues bearing a longer side chain at the 6-position of the pyrimidine ring could adopt a suitable conformation for binding in the DHFR active site, the biological data suggest that this could be energetically unfavourable.

Conclusions
In summary, we have prepared two series of substituted pyrimidine-2,4-diamines with a flexible side chain at either the 5-or 6-position of the pyrimdine ring as potential inhibitors of P. falciparum dihydrofolate reductase (DHFR).The synthesis of the first series of compounds involved construction of the pyrimidine ring by reaction of the enol ether of a suitably substituted α-cyanoketone with guanidine hydrochloride while the second series was prepared by substitution of the commercially available 2,4-diaminopyrimidin-6-ol. The compounds prepared were evaluated for antiplasmodial activity in vitro against a cycloguanil-resistant strain of the P. falciparum parasite.Compounds bearing a substituent at the 5-position of the pyrimidine ring showed better activity, in general, than those substituted at the 6-position, with 5-(3-(3,5dichlorophenoxy)propyl)-6-phenylpyrimidine-2,4-diamine identified as the most active compound in the series (IC 50 0.86 µM).The remaining pyrimidine-2,4-diamines showed antiplasmodial activity in the micromolar range.

Experimental Section
Chemistry General.Reagents purchased from Sigma-Aldrich (Steinheim, Germany) were of reagent grade and used without any further purification unless specified.Ethyl acetate (EtOAc) and hexane used for chromatography or extractions were distilled prior to use.Dimethyl sulfoxide (DMSO) was distilled and stored over 4 Å molecular sieves.Acetonitrile (CH 3 CN) was distilled from calcium hydride and tetrahydrofuran (THF) was distilled from sodium prior to use.Reactions were monitored by thin-layer chromatography (TLC) using precoated aluminium-backed plates (Merck silica gel 60 F254) and visualised under UV light (λ = 254 nm).Intermediates and final compounds were purified by column chromatography on Fluka silica gel 60 (70-230 mesh).NMR spectra were acquired on a Bruker 300 or 500 MHz spectrometer at room temperature, using the specified deuterated solvent.For those compounds soluble in deuterated chloroform (CDCl 3 ), the solvent contained tetramethylsilane (TMS, 0.05% v/v) as internal standard.For others, the residual solvent signal was used for referencing (MeOD: 3.310 ppm; DMSO-d 6 : 2.500 ppm).Data processing was done using MestreNova Software under license from Mestrelab Research, CA, USA.Infra-red spectra were recorded on a Bruker Tensor-27 Fourier Transform spectrometer.Mass Spectra (High Resolution) were recorded on a SYNAPT G2 HDMS mass spectrometer (ESI) at Stellenbosch University.Melting points were determined on a Stuart SMP10 melting point apparatus and are uncorrected.

General procedure for the synthesis of 2-benzoyl-5-phenoxypentanenitriles (11).
To a solution of the substituted 5-phenoxypentanenitrile 10 (1 eq) in dry THF was added potassium tert-butoxide (3 eq) and ethyl benzoate (4 eq).The reaction mixture was stirred at room temperature under a nitrogen atmosphere overnight.After consumption of the starting material, the reaction was quenched with sat.aq.NH 4 Cl and THF removed under reduced pressure.The remaining aqueous residue was washed with EtOAc (3 × 100 ml) and the organic layers were combined, dried over MgSO 4 and filtered through celite.Crude products were purified by column chromatography (EtOAc/hexane 20:80).