Key interactions of pyrimethamine derivatives specific to wild-type and mutant P. falciparum dihydrofolate reductase based on 3D-QSAR, MD simulations and quantum chemical calculations.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target enzyme in malarial chemotherapy. An understanding of how novel inhibitors interact with wild-type (wtPfDHFR), quadruple-mutant (qmPfDHFR), and human (hDHFR) enzymes is required for the development of these compounds as antimalarials. This study is focused on a series of des-Cl and m-Cl phenyl analogs of pyrimethamine with various flexible 6-substituents. The interactions of these compounds with DHFR enzymes were investigated by 3 D-QSAR, MD simulations, MM-PBSA, and DFT calculations. CoMFA and CoMSIA models were developed with good predictive abilities for wtPfDHFR and qmPfDHFR. For hDHFR, CoMSIA models combined with clogP descriptor were successfully derived. Binding free energy using MM-PBSA and comparison of per residue decomposition energy analyses with the DFT method at M06-2X/6-31G ++(d,p) level of theory indicated that Asp54 and Phe58 play important roles in the binding of the most potent compound in the series (compound 27) with both wtPfDHFR and qmPfDHFR, whereas Arg59 and Arg122 were additionally found to interact with this inhibitor in qmPfDHFR. For hDHFR, the residues Glu30 and Phe34 but not Arg70, equivalent to Asp54, Phe58, and Arg122 in PfDHFR, also play role in compound 27 binding through strong hydrophobic interactions (Phe34) and hydrogen bond network with Glu30, Ile7, and Val115. From the key interactions identified in the DHFR-inhibitor complexes, a general scheme is proposed for designing new inhibitors selective for PfDHFR that is important for the development of novel antifolate antimalarials.Communicated by Ramaswamy H. Sarma.

Dihydrofolate Reductase Inhibitors: The Pharmacophore as a Guide for Co-Crystal Screening.

In this work, co-crystal screening was carried out for two important dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP) and pyrimethamine (PMA), and for 2,4-diaminopyrimidine (DAP), which is the pharmacophore of these active pharmaceutical ingredients (API). The isomeric pyridinecarboxamides and two xanthines, theophylline (THEO) and caffeine (CAF), were used as co-formers in the same experimental conditions, in order to evaluate the potential for the pharmacophore to be used as a guide in the screening process. In silico co-crystal screening was carried out using BIOVIA COSMOquick and experimental screening was performed by mechanochemistry and supported by (solid + liquid) binary phase diagrams, infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD). The in silico prediction of low propensities for DAP, TMP and PMA to co-crystallize with pyridinecarboxamides was confirmed: a successful outcome was only observed for DAP + nicotinamide. Successful synthesis of multicomponent solid forms was achieved for all three target molecules with theophylline, with DAP co-crystals revealing a greater variety of stoichiometries. The crystalline structures of a (1:2) TMP:THEO co-crystal and of a (1:2:1) DAP:THEO:ethyl acetate solvate were solved. This work demonstrated the possible use of the pharmacophore of DHFR inhibitors as a guide for co-crystal screening, recognizing some similar trends in the outcome of association in the solid state and in the molecular aggregation in the co-crystals, characterized by the same supramolecular synthons.

Discovery of novel STAT3 DNA binding domain inhibitors.

Background: STAT3 is a pro-oncogenic transcription factor. Pyrimethamine (PYM) is a STAT3 inhibitor that suppresses the proliferation of some cancer cells through downregulation of STAT3 target proteins. Methodology & Results: We have used structure-based tools to design novel PYM-based compounds. Intracellular target validation studies revealed that representative compounds 11b-d and 15a downregulate STAT3 downstream proteins and inhibit STAT3 DNA binding domain (DBD). Relative to PYM, a cohort of these compounds are >100-fold more cytotoxic to cancer cells with constitutively active (high pSTAT3) and basal (low pSTAT3) STAT3 signaling, suggesting that STAT3 DBD inhibition is deleterious to the proliferation of cancer cells with low and high pSTAT3 levels. Conclusion: These are promising leads for further preclinical evaluation as therapeutic agents for STAT3-dependent cancers.

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening.

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 µM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

Anticancer Activity of Pyrimethamine via Ubiquitin Mediated Degradation of AIMP2-DX2.

While aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a tumor suppressor, its exon 2-depleted splice variant (AIMP2-DX2 or shortly DX2) is highly expressed in human lung cancer, and the ratio of DX2 to AIMP2 increases according to the progression of lung cancer. In this study, pyrimethamine inhibited the level of DX2 (IC50 = 0.73 µM) in A549 cells expressing nanoluciferase-tagged DX2. In a panel of 5 lung cancer cell lines with various DX2 levels, pyrimethamine most potently suppressed the growth of H460 cells, which express high levels of DX2 (GI50 = 0.01 µM). An immunoblot assay in H460 cells showed that pyrimethamine decreased the DX2 level dose-dependently but did not affect the AIMP2 level. Further experiments confirmed that pyrimethamine resulted in ubiquitination-mediated DX2 degradation. In an in vivo mouse xenograft assay using H460 cells, intraperitoneal administration of pyrimethamine significantly reduced the tumor size and weight, comparable with the effects of taxol, without affecting body weight. Analysis of tumor tissue showed a considerably high concentration of pyrimethamine with a decreased levels of DX2. These results suggest that pyrimethamine, currently used as anti-parasite drug, could be repurposed to treat lung cancer patients expressing high level of DX2.

Pyrimethamine conjugated histone deacetylase inhibitors: Design, synthesis and evidence for triple negative breast cancer selective cytotoxicity.

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor which has been recognized as a promising cancer therapeutic target. Small molecule pyrimethamine (PYM) is a known direct inhibitor of activated STAT3 and it is currently under clinical trial. Also, histone deacetylase (HDAC) inhibition has been shown to indirectly attenuate STAT3 signaling through inhibition of STAT3 activation. Herein we described the design and biological profiling of two classes of PYM-conjugated HDAC inhibitors (HDACi). We observed that the class I PYM-HDACi compounds 12a-c potently inhibited HDACs 1 and 6 in cell free assays while a lead class II PYM-HDACi compound 23 showed a strong HDAC 6 selective inhibition. In a cell-based assay, 12a-c are preferentially cytotoxic to MDA-MB-231, a TNBC cell line that is highly STAT3-dependent, while 23 showed no such selective toxicity. Subsequent target validation studies revealed that a representative class I PYM-HDACi compound 12c elicited a signature of HDAC and STAT3 pathway inhibition intracellularly. Collectively, these data suggest that PYM-HDACi compounds are promising leads to develop targeted therapy for TNBC.

Understanding the Pyrimethamine Drug Resistance Mechanism via Combined Molecular Dynamics and Dynamic Residue Network Analysis.

In this era of precision medicine, insights into the resistance mechanism of drugs are integral for the development of potent therapeutics. Here, we sought to understand the contribution of four point mutations (N51I, C59R, S108N, and I164L) within the active site of the malaria parasite enzyme dihydrofolate reductase (DHFR) towards the resistance of the antimalarial drug pyrimethamine. Homology modeling was used to obtain full-length models of wild type (WT) and mutant DHFR. Molecular docking was employed to dock pyrimethamine onto the generated structures. Subsequent all-atom molecular dynamics (MD) simulations and binding free-energy computations highlighted that pyrimethamine's stability and affinity inversely relates to the number of mutations within its binding site and, hence, resistance severity. Generally, mutations led to reduced binding affinity to pyrimethamine and increased conformational plasticity of DHFR. Next, dynamic residue network analysis (DRN) was applied to determine the impact of mutations and pyrimethamine binding on communication dispositions of DHFR residues. DRN revealed residues with distinctive communication profiles, distinguishing WT from drug-resistant mutants as well as pyrimethamine-bound from pyrimethamine-free models. Our results provide a new perspective on the understanding of mutation-induced drug resistance.

Synthesis and in vitro anti-Toxoplasma gondii activity of a new series of aryloxyacetophenone thiosemicarbazones.

A new series of aryloxyacetophenone thiosemicarbazones 4a-q have been synthesized as anti-Toxoplasma gondii agents. All compounds showed significant inhibitory activity against T. gondii-infected cells (IC50 values 1.09-25.19 µg/mL). The 4-fluorophenoxy derivative (4l) was the most potent compound with the highest selectivity toward host cells (SI = 19), being better than standard drug pyrimethamine. SAR study indicated that the concurrence of proper substituents on both aryl ring of phenoxyacetophenone is important for potency and safety profile. Further in vitro experiments with the representative compounds 4l and 4p revealed that these compounds at the concentration of 5 µg/mL can significantly reduce the viability of T. gondii tachyzoites, as well as their infectivity rate and intracellular proliferation, comparable to those of pyrimethamine.

A new effective antiplasmodial compound: Nanoformulated pyrimethamine.

OBJECTIVES: The aim of this study was to evaluate the efficacy of pyrimethamine-loaded poloxamer 407 nanomicelles on Plasmodium berghei strain NICD in vivo. METHODS: Pyrimethamine-loaded nanomicelles were prepared and their zeta potential, particle size and polydispersity index were measured. For antiplasmodial assessment, 54 mice were randomly divided into six groups. Four groups were infected intraperitoneally with P. berghei, whereas the two remaining groups did not receive the parasite (negative controls). Three of the P. berghei-infected groups received treatment with either pyrimethamine-loaded nanomicelles (2 mg/kg), pyrimethamine (2 mg/kg) or empty nanomicelles (2 mg/kg); the fourth group remained untreated (positive control). The parasitaemia rate, survival rate and histopathological changes in the liver, spleen and kidneys were examined and were compared with the negative and positive control groups. RESULTS: The mean parasitaemia rate differed significantly between the nanoformulated pyrimethamine group and each of the other groups (P<0.05). Moreover, the survival rate of mice in the nanoformulated pyrimethamine group (7/9; 78%) was significantly higher compared with each of the other groups (P<0.01). The main histopathological changes, including hepatic necrosis in the liver, lymphoid hypoplasia in the spleen, and tubular nephrosis and perivascular and interstitial lymphocytic infiltration in the kidneys, were considerably lower in the nanoformulated pyrimethamine group than in the pyrimethamine and positive control groups. CONCLUSION: Pyrimethamine-loaded nanomicelles showed potent antimalarial activity and can be considered as a potential candidate for further examination of their suitability as an antimalarial drug.

Multispectral and molecular docking investigations on the interaction of primethamine/trimethoprim with BSA/HSA.

Primethamine (PMA) and trimethoprim (TMP) were investigated as traditional coccidiostats on the binding of bovine serum albumin (BSA) and human serum albumin (HSA) by multispectral and molecular docking techniques. The Stern-Volmer plots and time-resolved fluorescence measurement declared that PMA/TMP quenching the intrinsic fluorescence of BSA/HSA was static quenching process. The binding constants (Ka) and binding sites (n) were calculated at different temperatures. Meanwhile, thermodynamic parameters showed electrostatic forces played a leading role in the interaction of PMA/TMP with BSA/HSA. Some metal ions such as K+, Mg2+, Cu2+, Ca2+, Zn2+ and Fe3+ had no effects on the binding system. The UV-vis absorption spectra confirmed that the interaction between PMA/TMP and BSA/HSA did happen. The analyses of synchronous fluorescence, FT-IR and circular dichroism spectra illustrated that PMA/TMP changed the secondary structures of BSA/HSA. According to Förster non-radiative energy transfer theory, the binding distance between PMA/TMP and BSA/HSA was calculated. The binding location of PMA/TMP to BSA/HSA was identified as sub-domain IIA, which was further confirmed by molecular docking.Communicated by Ramaswamy H. Sarma.

CNT-based nanocarrier loaded with pyrimethamine for adipose mesenchymal stem cells differentiation and cancer treatment: The computational and experimental methods.

Pyrimethamine is an effective drug in the cancer cell treatment and is a dihydrofolate reductase inhibitor. In this work, the amount of drug loading up on CNT and its cytotoxicity effect upon MCF-7 cell lines was surveyed. The novel applications of some drugs and nanocarriers can induce the differentiation of adipose mesenchymal cells into nerve cells. Hence carbon nanotube-pyrimethamine was used to differentiate mesenchymal stem cells into the neural category, for the first time. The results of NSE and NFM gene expression level were evaluated using the real-time PCR. A detailed study on the interaction between pyrimethamine anticancer drug and (6, 0) zigzag single-walled carbon nanotube was performed by DFT/B3LYP and DFT/M06-2X with 6-31G* basis set calculations in gas phase and in solvent using the PCM. Different configurations of the adsorbed pyrimethamine onto the CNT surface were studied. Based on the results, the process of pyrimethamine adsorption on diff ;erent sites on the outer wall of the nanotube was exothermic and configurations were stable. The adsorption energy values indicated that the pyrimethamine molecule could be physically adsorbed on the external surface of the SWCNT. The QTAIM was used for characterizing the nature of the interactions between the pyrimethamine and the selected nanocarrier.

6-Hydrophobic aromatic substituent pyrimethamine analogues as potential antimalarials for pyrimethamine-resistant Plasmodium falciparum.

The series of des-Cl (unsubstituted) and m-Cl phenyl analogues of PYR with various flexible 6-substituents were synthesized and studied for the binding affinities with highly resistant quadruple mutant (QM) DHFR. The derivatives carrying 4 atoms linker with a terminal carboxyl substituted on the aromatic ring exhibited good inhibition to the QM enzyme and also showed effective antimalarial activities against resistant P. falciparum bearing the mutant enzymes with relatively low cytotoxicity to mammalian cells. The X-ray crystallographic analysis of the enzyme-inhibitor complexes suggested that the hydrophobic substituent at 6-position was accommodated well in the hydrophobic pocket and the optimal length of the flexible linker could effectively promote the binding of the terminal carboxyl group to the key amino acid residues, Arg59 and Arg122.

Antimalarial Drug Pyrimethamine Plays a Dual Role in Antitumor Proliferation and Metastasis through Targeting DHFR and TP.

Pyrimethamine (Pyr), an antimalarial drug that targeting plasmodium dihydrofolate reductase (pDHFR), has been proved to have antitumor activity. However, its direct target on cancer cells remains unclear. Methotrexate (MTX) is a widely used anticancer drug that blocks human dihydrofolate reductase (hDHFR). In this work, we examined the anticancer effects of Pyr in vitro and in vivo Our results showed that hDHFR and pDHFR have similar secondary and three-dimensional structures and that Pyr can inhibit the activity of hDHFR in lung cancer cells. Although Pyr and MTX can inhibit the proliferation of lung cancer cells by targeting DHFR, only Pyr can inhibit the epithelial-mesenchymal transition (EMT), metastasis and invasion of lung cancer cells. These results indicated that hDHFR is not the only target of Pyr. We further found that thymidine phosphorylase (TP), an enzyme that is closely associated with the EMT of cancer cells, is also a target protein of Pyr. The data retrieved from the Cancer Genome Atlas (TCGA) database revealed that TP overexpression is associated with poor prognosis of patients with lung cancer. In conclusion, Pyr plays a dual role in antitumor proliferation and metastasis by targeting DHFR and TP. Pyr may have potential clinical applications for the treatment of lung cancer.

Supramolecular hydrogen-bonding patterns in salts of the antifolate drugs trimethoprim and pyrimethamine.

Nine salts of the antifolate drugs trimethoprim and pyrimethamine, namely, trimethoprimium [or 2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidin-1-ium] 2,5-dichlorothiophene-3-carboxylate monohydrate (TMPDCTPC, 1:1), C14H19N4O3+·C5HCl2O2S-, (I), trimethoprimium 3-bromothiophene-2-carboxylate monohydrate, (TMPBTPC, 1:1:1), C14H19N4O3+·C5H2BrO2S-·H2O, (II), trimethoprimium 3-chlorothiophene-2-carboxylate monohydrate (TMPCTPC, 1:1:1), C14H19N4O3+·C5H2ClO2S-·H2O, (III), trimethoprimium 5-methylthiophene-2-carboxylate monohydrate (TMPMTPC, 1:1:1), C14H19N4O3+·C6H5O2S-·H2O, (IV), trimethoprimium anthracene-9-carboxylate sesquihydrate (TMPAC, 2:2:3), C14H19N4O3+·C15H9O2-·1.5H2O, (V), pyrimethaminium [or 2,4-diamino-5-(4-chlorophenyl)-6-ethylpyrimidin-1-ium] 2,5-dichlorothiophene-3-carboxylate (PMNDCTPC, 1:1), C12H14ClN4+·C5HCl2O2S-, (VI), pyrimethaminium 5-bromothiophene-2-carboxylate (PMNBTPC, 1:1), C12H14ClN4+·C5H2BrO2S-, (VII), pyrimethaminium anthracene-9-carboxylate ethanol monosolvate monohydrate (PMNAC, 1:1:1:1), C12H14ClN4+·C15H9O2-·C2H5OH·H2O, (VIII), and bis(pyrimethaminium) naphthalene-1,5-disulfonate (PMNNSA, 2:1), 2C12H14ClN4+·C10H6O6S22-, (IX), have been prepared and characterized by single-crystal X-ray diffraction. In all the crystal structures, the pyrimidine N1 atom is protonated. In salts (I)-(III) and (VI)-(IX), the 2-aminopyrimidinium cation interacts with the corresponding anion via a pair of N-H...O hydrogen bonds, generating the robust R22(8) supramolecular heterosynthon. In salt (IV), instead of forming the R22(8) heterosynthon, the carboxylate group bridges two pyrimidinium cations via N-H...O hydrogen bonds. In salt (V), one of the carboxylate O atoms bridges the N1-H group and a 2-amino H atom of the pyrimidinium cation to form a smaller R21(6) ring instead of the R22(8) ring. In salt (IX), the sulfonate O atoms mimic the role of carboxylate O atoms in forming an R22(8) ring motif. In salts (II)-(IX), the pyrimidinium cation forms base pairs via a pair of N-H...N hydrogen bonds, generating a ring motif [R22(8) homosynthon]. Compounds (II) and (III) are isomorphous. The quadruple DDAA (D = hydrogen-bond donor and A = hydrogen-bond acceptor) array is observed in (I). In salts (II)-(IV) and (VI)-(IX), quadruple DADA arrays are present. In salts (VI) and (VII), both DADA and DDAA arrays co-exist. The crystal structures are further stabilized by π-π stacking interactions [in (I), (V) and (VII)-(IX)], C-H...π interactions [in (IV)-(V) and (VII)-(IX)], C-Br...π interactions [in (II)] and C-Cl...π interactions [in (I), (III) and (VI)]. Cl...O and Cl...Cl halogen-bond interactions are present in (I) and (VI), with distances and angles of 3.0020 (18) and 3.5159 (16) Å, and 165.56 (10) and 154.81 (11)°, respectively.

In vitro activity of the interaction between taxifolin (dihydroquercetin) and pyrimethamine against Toxoplasma gondii.

Toxoplasmosis is one of the most neglected zoonotic foodborne parasitic diseases that cause public health and socioeconomic concern worldwide. The current drugs used for the treatment of toxoplasmosis have been identified to have clinical limitations. Hence, new drugs are urgently needed to eradicate T.gondii infections globally. Here, an in vitro anti-Toxoplasma gondii activity of taxifolin (dihydroquercetin) and dihydrofolate inhibitor (pyrimethamine) alone and in combination with a fixed concentration of pyrimethamine were investigated against the rapidly proliferating T.gondii RH strain at 48 hr using colorimetric assay. Pyrimethamine showed the highest anti-T. gondii activity with IC50P of 0.84 µg/ml (p > .05), respectively. The combination of pyrimethamine with dihydroquercetin gave a significant inhibitory activity against tachyzoites in in vitro with IC50p of 1.39 µg/ml (p < .05). The IC50p ranges obtained for the individual and the combination of taxifolin with pyrimethamine inhibition of parasite growth were not cytotoxic to the infected HFF and Hek-293 cell lines used. These compounds combination should be investigated further using in vivo model of toxoplasmosis.

Identification of compounds that suppress Toxoplasma gondii tachyzoites and bradyzoites.

Drug treatment for toxoplasmosis is problematic, because current drugs cannot eradicate latent infection with Toxoplasma gondii and can cause bone marrow toxicity. Because latent infection remains after treatment, relapse of infection is a problem in both infections in immunocompromised patients and in congenitally infected patients. To identify lead compounds for novel drugs against Toxoplasma gondii, we screened a chemical compound library for anti-Toxoplasma activity, host cell cytotoxicity, and effect on bradyzoites. Of 878 compounds screened, 83 demonstrated >90% parasite growth inhibition. After excluding compounds that affected host cell viability, we further characterized two compounds, tanshinone IIA and hydroxyzine, which had IC50 values for parasite growth of 2.5 µM and 1.0 µM, respectively, and had no effect on host cell viability at 25 µM. Both tanshinone IIA and hydroxyzine inhibited parasite replication after invasion and both reduced the number of in vitro-induced bradyzoites, whereas, pyrimethamine, the current therapy, had no effect on bradyzoites. Both tanshinone IIA and hydroxyzine are potent lead compounds for further medicinal chemistry. The method presented for evaluating compounds for bradyzoite efficacy represents a new approach to the development of anti-Toxoplasma drugs to eliminate latency and treat acute infection.

A novel prediction approach for antimalarial activities of Trimethoprim, Pyrimethamine, and Cycloguanil analogues using extremely randomized trees.

Malaria is still one of the most serious diseases in tropical regions. This is due in part to the high resistance against available drugs for the inhibition of parasites, Plasmodium, the cause of the disease. New potent compounds with high clinical utility are urgently needed. In this work, we created a novel model using a regression tree to study structure-activity relationships and predict the inhibition constant, Ki of three different antimalarial analogues (Trimethoprim, Pyrimethamine, and Cycloguanil) based on their molecular descriptors. To the best of our knowledge, this work is the first attempt to study the structure-activity relationships of all three analogues combined. The most relevant descriptors and appropriate parameters of the regression tree are harvested using extremely randomized trees. These descriptors are water accessible surface area, Log of the aqueous solubility, total hydrophobic van der Waals surface area, and molecular refractivity. Out of all possible combinations of these selected parameters and descriptors, the tree with the strongest coefficient of determination is selected to be our prediction model. Predicted Ki values from the proposed model show a strong coefficient of determination, R2=0.996, to experimental Ki values. From the structure of the regression tree, compounds with high accessible surface area of all hydrophobic atoms (ASA_H) and low aqueous solubility of inhibitors (Log S) generally possess low Ki values. Our prediction model can also be utilized as a screening test for new antimalarial drug compounds which may reduce the time and expenses for new drug development. New compounds with high predicted Ki should be excluded from further drug development. It is also our inference that a threshold of ASA_H greater than 575.80 and Log S less than or equal to -4.36 is a sufficient condition for a new compound to possess a low Ki.

Synthesis and evaluation of naphthyl bearing 1,2,3-triazole analogs as antiplasmodial agents, cytotoxicity and docking studies.

Novel series of naphthyl bearing 1,2,3-triazoles (4a-t) were synthesized and evaluated for their in vitro antiplasmodial activity against pyrimethamine (Pyr)-sensitive and resistant strains of Plasmodium falciparum. The synthesized compounds were assessed for their cytotoxicity employing human embryonic kidney cell line (HEK-293), and none of them was found to be toxic. Among them 4j, 4k, 4l, 4m, 4n, 4t exhibited significant antiplasmodial activity in both strains, of which compounds 4m, 4n and 4t (∼3.0-fold) displayed superior activity to Pyr against resistant strain. Pyr and selected compounds (4n, 4p and 4t) that repressed parasite development also inhibited PfDHFR activity of the soluble parasite extract, suggesting that anti-parasitic activity of these compounds is a result of inhibition of the parasite DHFR. In silico studies suggest that activity of these compounds might be enhanced due to π-π stacking.

Development of a simple, rapid, and robust liquid chromatographic method for the simultaneous determination of sulfalene, sulfadoxine, and pyrimethamine in tablets.

A simple, cost effective, accurate, and precise RP-HPLC method was developed for the simultaneous determination of sulfalene and sulfadoxine in fixed dose dual combinations with pyrimethamine together with their related substances. Proprietary products containing these combinations are often being prescribed in malaria endemic countries. Quantification of the active compounds and impurity profiling was achieved using two standard C18 columns with a mobile phase being composed of 60% (v/v) of a 0.05M KH2PO4 buffer solution (pH=2.6) and 40% (v/v) of methanol, applying an isocratic elution mode and a detection wavelength of 215nm. The method allows a quick quantitative determination of sulfadoxine and sulfalene and the separation of the respective impurities within a total runtime of approximately 15min and was validated with respect to specificity, linearity, precision, accuracy, limits of detection and quantification, robustness, and stability of the standard and sample solutions. The method is simpler than the corresponding method described in the International Pharmacopoeia and the United States Pharmacopoeia in terms of being easy to apply, being less time consuming, and utilizing reagents and chemicals which are cost efficient.

Antimalarial Activity of Small-Molecule Benzothiazole Hydrazones.

We synthesized a new series of conjugated hydrazones that were found to be active against malaria parasite in vitro, as well as in vivo in a murine model. These hydrazones concentration-dependently chelated free iron and offered antimalarial activity. Upon screening of the synthesized hydrazones, compound 5f was found to be the most active iron chelator, as well as antiplasmodial. Compound 5f also interacted with free heme (KD [equilibrium dissociation constant] = 1.17 ± 0.8 µM), an iron-containing tetrapyrrole released after hemoglobin digestion by the parasite, and inhibited heme polymerization by parasite lysate. Structure-activity relationship studies indicated that a nitrogen- and sulfur-substituted five-membered aromatic ring present within the benzothiazole hydrazones might be responsible for their antimalarial activity. The dose-dependent antimalarial and heme polymerization inhibitory activities of the lead compound 5f were further validated by following [(3)H]hypoxanthine incorporation and hemozoin formation in parasite, respectively. It is worth mentioning that compound 5f exhibited antiplasmodial activity in vitro against a chloroquine/pyrimethamine-resistant strain of Plasmodium falciparum (K1). We also evaluated in vivo antimalarial activity of compound 5f in a murine model where a lethal multiple-drug-resistant strain of Plasmodium yoelii was used to infect Swiss albino mice. Compound 5f significantly suppressed the growth of parasite, and the infected mice experienced longer life spans upon treatment with this compound. During in vitro and in vivo toxicity assays, compound 5f showed minimal alteration in biochemical and hematological parameters compared to control. In conclusion, we identified a new class of hydrazone with therapeutic potential against malaria.