Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity

A series of derivatives of a triazole analogue of thiamine has been synthesised. When tested as inhibitors of porcine pyruvate dehydrogenase, the benzoyl ester derivatives proved to be potent thiamine pyrophosphate (TPP) competitive inhibitors, with the affinity of the most potent analogue (Ki = 54 nM) almost matching the affinity of TPP itself. When tested as antiplasmodials, most of the derivatives showed modest activity (IC50 value >60 μM), except for a 4′-N-benzyl derivative, which has an IC50 value in the low micromolar range. This activity was not affected by increasing the extracellular concentration of thiamine in the culture medium for any of the compounds (except a modest increase in the IC50 for the unfunctionalized benzoyl ester), nor by overexpressing thiamine pyrophosphokinase in the parasite, making it unlikely to be due to an effect on thiamine transport or metabolism.

Unless otherwise noted, all chemicals and reagents were purchased from commercial suppliers and used without further purification. Analytical TLC was carried out on Merck glass plates with silica Kieselgel 60 F254 of thickness 0.25 mm and visualised under UV or KMnO4 dip. Silica chromatography was carried out in the indicated solvent system using prepacked silica gel cartridges for use on the Biotage Purification System. All solvents were removed under reduced pressure using a Büchi rotary evaporator with dry ice traps. Yields of all reactions refer to the purified products. 1 H NMR spectra were recorded at 400 MHz or 700 MHz, in CDCl3 or CD3OD solution on a Bruker 400 MHz or 700 MHz spectrometer, and chemical shifts were recorded in parts per million (ppm) and were referenced to the residual solvent signal. 13 C NMR spectra were recorded on either a Bruker 400 MHz or 700 MHz spectrometer. Mass spectra used electrospray ionisation (ESI). Melting points of compounds were measured using a Reichert machine and are uncorrected. The synthesis and characterisation data for compounds 6 and 11 have been previously described. 1 General procedure for preparation of compounds 12a-g: To a stirred solution of carboxylic acid (1.3 equiv.) and DCC (3 equiv.) in dry DMF (0.2 M) under nitrogen at 0 °C was added DMAP (1.3 equiv.) and compound 6 (1 equiv.). The reaction mixture was stirred at room temperature for two days, then diluted with CH2Cl2 and filtered through cotton wool. The filtrate was treated with aqueous phosphate buffer (pH 7) and extracted with CH2Cl2. The combined organic phases were washed with the phosphate buffer, then dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by silica flash chromatography (5% to 20% MeOH in CH2Cl2) to yield the ester product.  6, 165.4, 162.0, 155.2, 144.6, 139.1, 130.8, 128.5, 128.4, 122.6, 108.6, 63.7, 47.4, 24.9, 23.8 General procedure for preparation of compounds 13a, b: To a stirred solution of 2(or 4)-fluoro-1-nitrobenzene (1 equiv.) and compound 6 (1.1 equiv.) in dry NMP (0.2 M) under nitrogen at 0 °C was added KHMDS (1 M in THF, 1.1 equiv.) dropwise. The reaction mixture was stirred at room temperature overnight, then treated with saturated aqueous NaHCO3 and extracted with EtOAc. The combined organic phases were dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by silica flash chromatography (5% to 10% MeOH in CH2Cl2) to yield the aryl ether. 2-(1-((4-((4-Bromobenzyl)amino)-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)ethan-1-ol (15): To a stirred solution of 6 (129 mg, 0.55 mmol) in dry NMP (3 mL) under nitrogen at 0 °C was added KHMDS (1 M in THF, 0.5 mL, 0.5 mmol) dropwise. The solution was allowed to warm to room temperature. After 15 min, a solution of 4-bromobenzyl bromide (150 mg, 0.6 mmol) in dry NMP (1 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 30 min and then at 45 °C overnight, then treated with saturated aqueous NaHCO3 (10 mL) and extracted with EtOAc (3 x 30 mL). The combined organic phases were washed with brine (10 mL

Maintenance of P. falciparum parasites
The human malaria parasite P. falciparum strain 3D7 (chloroquine-sensitive) and the same strain expressing an extra copy of TPK with a GFP-tag (PfTPK-GFP) were maintained in the intraerythrocytic stage essentially as described previously by Allen and Kirk. 3 Briefly, RPMI 1640 medium supplemented with 11 mM glucose, 200 µM hypoxanthine, 24 g/mL gentamicin and 6 g/L Albumax II was used to maintain the parasites. Fresh human erythrocytes (blood type O + ) were added every two days (when parasites were in the trophozoite stage). The parasite cultures were maintained at 37 °C inside a shaking incubator and under an atmosphere of 1% oxygen, 3% carbon dioxide and 96% nitrogen.
Antiplasmodial activity assay Compounds were tested at different highest final concentrations (between 25 M and 350 M) depending on their solubility. Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO) followed by dilution in RPMI 1640 medium in the absence of thiamine or in the presence of 2.97 M (the concentration normally present in RPMI 1640) or 297 M thiamine. The final concentration of DMSO that the parasites were exposed to never exceeded 0.05%, a concentration that has no effect on parasite proliferation. 4 To investigate whether compound 15 inhibits parasite proliferation by interfering with folate metabolism, the compound was tested in normal RPMI 1640 medium, which contains 2.2 M folate, and in the same medium with 220 M folate. Sulfadoxine (an established antimalarial that targets folate metabolism) 5 was used as a positive control because its effect can be antagonised by increasing the concentration of folate in the culture medium. 6 Two-fold serial dilutions were performed, with each concentration tested in triplicate. The assay was performed as described by Tjhin et al. with some modifications. 7 Experiments were initiated with parasites in the ring-stage, a parasitemia level of 0.5% and a haematocrit of 2%. Chloroquine (0.5 M) was used as the positive control (i.e. complete inhibition of parasite proliferation), and parasites maintained in the absence of any inhibitor represented 100% parasite proliferation. The final volume in each well was 200 L. Plates were incubated at 37 °C under an atmosphere of 96% nitrogen, 3% carbon dioxide and 1% oxygen.
Parasite proliferation was measured using the SYBR-Safe assay 8 , which correlates fluorescence intensity to parasite DNA. Compound 15 appeared to be incompatible with this fluorescence-based assay (there were inconsistent fluorescence intensity readings at some concentrations). The malstat assay was therefore used instead for this compound. The malstat assay correlates parasite lactate dehydrogenase activity with parasite proliferation during the 96-hour incubation period. 9 The concentration at which the compound suppresses parasite proliferation by 50% (i.e. IC50) was determined from non-linear regression plots using GraphPad Prism. The data were averaged from three independent experiments. The antiplasmodial activity of compounds (12a, 15, and oxythiamine) that appeared to be affected under certain experimental conditions was analyzed using Student's ttest.

Cytotoxicity evaluation of selected compounds
Cytotoxicity testing of selected compounds was conducted using HFF cells (human foreskin fibroblasts) as described by Howieson et al. with some modifications. 10 Briefly, the HFF cells were seeded in 96well plates at a density of approximately 25 x 10 4 cells/mL. Cycloheximide (10 M; a protein synthesis inhibitor) was used as a control to indicate complete inhibition of HFF cell proliferation. Plates were incubated at 37 °C in a humidified 5% carbon dioxide incubator for 96 h. A sample of the supernatant (150 L) was then carefully aspirated from each well and discarded. The plates were then stored at -80 °C. SYBR-Safe assay was used to measure cell proliferation. Briefly, the plates were thawed, SYBR-Safe lysis solution (150 L) was added to each well and mixed via pipetting to ensure the HFF cells were detached from the plate and lysed. The plates were then processed as described for the antiplasmodial assay.

Generation of parasites expressing PfTPK-GFP
The P. falciparum TPK gene (PF3D7_0924300) was amplified from gDNA by PCR using the following primers: 5'-CTCGAGATGAAAAAAAAGTACCATATATATTTAAAATGATTTC-3' (forward), XhoI restriction site underlined, and 5'-GGTACCAAATTCCTCATTTTTTAATTGCGAATTC-3' (reverse), KpnI restriction site underlined. The amplified product was then ligated into pGlux-1 plasmid. 11 Ligation was carried out using NEB T4 DNA ligase overnight. The ligation product was transformed into the PMC103 E. coli strain. Transformants were confirmed by PCR screening and the purified plasmid sequenced at the Biomolecular Resource Facility, Australian National University. The plasmid (60 g) was then transfected into P. falciparum parasites (strain 3D7) as described previously by Rug and Maier. 12 The transgenic parasites were selected and maintained using WR99210 (10 nM). Expression of PfTPK-GFP was confirmed by western blot using anti-GFP antibodies as described previously. 11 S12 Fig. S8. Anti-GFP western blot of lysates prepared from P. falciparum 3D7 parasites (left lane) and transgenic parasites expressing PfTPK-GFP (right lane). The size of PfTPK-GFP is approximately 74 kDa. S13 Fig. S9. In vitro antiplasmodial activity of compounds 12a, 12c, 15, and oxythiamine against P. falciparum 3D7 parasites (black circles), 3D7 parasites transfected with an empty plasmid (black triangles), and 3D7 parasites expressing PfTPK-GFP (white circles) in the thiamine-free medium (leftside panels), and medium containing 2.97 M thiamine (right-side panels). Data are averaged from three independent experiments, each carried out in triplicate. Error bars represent SEM and, where not visible, are smaller than the symbols. The antiplasmodial activity of compound 15 tested in 2.97 M thiamine was statistically not affected by overexpression of TPK in the parasites, despite the apparent leftward shift in the dose-response curve (Student's t-test, p = 0.16). As reported previously, 13 the antiplasmodial activity of oxythiamine was enhanced by TPK overexpression (bottom two panels; p  0.01 comparing the IC50 values of wild-type parasites to TPK-overexpressing parasites, Student's t-test).