Design, synthesis and Anti-Plasmodial activity of Mortiamide-Lugdunin conjugates.

In this study, two linear and corresponding cyclic heptapeptide versions of mortiamide A-lugdunin hybrids were designed and synthesized by integrating an anti-malarial peptide epitope derived from Mortiamide A, combined with four residues known for their membrane interactions. Using this synthetic strategy, the sequence of mortiamide A was partly re-engineered with an epitope sequence of lugdunin along with an amino acid replacement using all-L and D/L configurations. Importantly, the re-engineered cyclic mortiamides with all-L (3) and D/L (4) configurations exhibited promising anti-malarial activities against the P. falciparum drug-sensitive TM4/8 strain with half-maximal inhibitory concentration (IC50) values of 6.2 ± 0.5 and 4.8 ± 0.1 µM, respectively. Additionally, they exhibited anti-malarial activities against the P. falciparum multidrug-resistant V1/S strain with IC50 values of 5.0 ± 2.6 and 3.7 ± 0.7 µM, respectively. Interestingly, a linear re-engineered mortiamide with D/L configuration (2) exhibited promising anti-malarial activities, surpassing those of the re-engineered cyclic mortiamides (3 and 4), against both the P. falciparum sensitive TM4/8 and multidrug-resistant V1/S strains with IC50 values of 3.6 ± 0.5 and 2.8 ± 0.7 µM (IC50 of Mortiamide A = 7.85 ± 0.97, 5.31 ± 0.24 µM against 3D7 and Dd2 strains) without any cytotoxicity at >100 µM. The presence of D/L forms in a linear structure significantly impacted the anti-malarial activity against both the P. falciparum sensitive TM4/8 strain and the multidrug-resistant V1/S strain.

Discovery of rigid biphenyl Plasmodium falciparum DHFR inhibitors using a fragment linking strategy.

Plasmodium falciparum dihydrofolate reductase (PfDHFR), a historical target for antimalarials, has been considered compromised due to resistance inducing mutations caused by pyrimethamine (PYR) overexposure. The clinical candidate P218 has demonstrated that inhibitors could efficiently target both PYR-sensitive and PYR-resistant parasites through careful drug design. Yet, P218 clinical development has been limited by its pharmacokinetic profile, incompatible with single dose regimen. Herein, we report the design of new PfDHFR inhibitors using fragment-based design, aiming at improved lipophilicity and overall drug-like properties. Fragment-based screening identified hits binding in the pABA site of the enzyme. Using structure-guided design, hits were elaborated into leads by fragment linking with 2,4-diaminopyrimidine. Resulting compounds display nM range inhibition of both drug-sensitive and resistant PfDHFR, high selectivity against the human isoform, drug-like lipophilicity and metabolic stability. Compound 4 and its ester derivative 3 kill blood stage TM4/8.2 parasite at nM concentrations while showing no toxicity against Vero cells.

N-Containing α-Mangostin Analogs via Smiles Rearrangement as the Promising Cytotoxic, Antitrypanosomal, and SARS-CoV-2 Main Protease Inhibitory Agents.

New N-containing xanthone analogs of α-mangostin were synthesized via one-pot Smiles rearrangement. Using cesium carbonate in the presence of 2-chloroacetamide and catalytic potassium iodide, α-mangostin (1) was subsequently transformed in three steps to provide ether 2, amide 3, and amine 4 in good yields at an optimum ratio of 1:3:3, respectively. The evaluation of the biological activities of α-mangostin and analogs 2-4 was described. Amine 4 showed promising cytotoxicity against the non-small-cell lung cancer H460 cell line fourfold more potent than that of cisplatin. Both compounds 3 and 4 possessed antitrypanosomal properties against Trypanosoma brucei rhodesiense at a potency threefold stronger than that of α-mangostin. Furthermore, ether 2 gave potent SARS-CoV-2 main protease inhibition by suppressing 3-chymotrypsinlike protease (3CLpro) activity approximately threefold better than that of 1. Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CLpro active site regarding an additional ether moiety. Thus, the series of N-containing α-mangostin analogs prospectively enhance druglike properties based on isosteric replacement and would be further studied as potential biotically active chemical entries, particularly for anti-lung-cancer, antitrypanosomal, and anti-SARS-CoV-2 main protease applications.

Molecular Docking Studies, Synthesis and Biological Evaluation of Substituted Pyrimidine-2,4-diamines as Inhibitors of Plasmodium falciparum Dihydrofolate Reductase.

A series of 5-[(phenethylamino)methyl]pyrimidine-2,4-diamines were assessed in silico as potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), synthesised and tested for inhibitory activity against PfDHFR in vitro. The compounds displayed promising inhibitory activity against both wild-type (Ki 1.3-243 nM) and quadruple mutant (Ki 13-208 nM) PfDHFR in the biochemical enzyme assay, but were less potent in the whole-cell P. falciparum assay (IC50 (TM4/8.2) 0.4-28 µM; IC50 (V1S) 3.7-54 µM). Further investigation into the pharmacokinetic properties of these compounds may guide the development of more potent analogues.

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds' binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.

Semi-Synthesis of N-Aryl Amide Analogs of Piperine from Piper nigrum and Evaluation of Their Antitrypanosomal, Antimalarial, and Anti-SARS-CoV-2 Main Protease Activities.

Piper nigrum, or black pepper, produces piperine, an alkaloid that has diverse pharmacological activities. In this study, N-aryl amide piperine analogs were prepared by semi-synthesis involving the saponification of piperine (1) to yield piperic acid (2) followed by esterification to obtain compounds 3, 4, and 5. The compounds were examined for their antitrypanosomal, antimalarial, and anti-SARS-CoV-2 main protease activities. The new 2,5-dimethoxy-substituted phenyl piperamide 5 exhibited the most robust biological activities with no cytotoxicity against mammalian cell lines, Vero and Vero E6, as compared to the other compounds in this series. Its half-maximal inhibitory concentration (IC50) for antitrypanosomal activity against Trypanosoma brucei rhodesiense was 15.46 ± 3.09 µM, and its antimalarial activity against the 3D7 strain of Plasmodium falciparum was 24.55 ± 1.91 µM, which were fourfold and fivefold more potent, respectively, than the activities of piperine. Interestingly, compound 5 inhibited the activity of 3C-like main protease (3CLPro) toward anti-SARS-CoV-2 activity at the IC50 of 106.9 ± 1.2 µM, which was threefold more potent than the activity of rutin. Docking and molecular dynamic simulation indicated that the potential binding of 5 in the 3CLpro active site had the improved binding interaction and stability. Therefore, new aryl amide analogs of piperine 5 should be investigated further as a promising anti-infective agent against human African trypanosomiasis, malaria, and COVID-19.

Synthesis and evaluation of tetrahydroisoquinoline derivatives against Trypanosoma brucei rhodesiense.

Human African Trypanosomiasis (HAT) is a neglected tropical disease caused by the parasitic protozoan Trypanosoma brucei (T. b.), and affects communities in sub-Saharan Africa. Previously, analogues of a tetrahydroisoquinoline scaffold were reported as having in vitro activity (IC50 = 0.25-70.5 µM) against T. b. rhodesiense. In this study the synthesis and antitrypanosomal activity of 80 compounds based around a core tetrahydroisoquinoline scaffold are reported. A detailed structure activity relationship was revealed, and five derivatives (two of which have been previously reported) with inhibition of T. b. rhodesiense growth in the sub-micromolar range were identified. Four of these (3c, 12b, 17b and 26a) were also found to have good selectivity over mammalian cells (SI > 50). Calculated logD values and preliminary ADME studies predict that these compounds are likely to have good absorption and metabolic stability, with the ability to passively permeate the blood brain barrier. This makes them excellent leads for a blood-brain barrier permeable antitrypanosomal scaffold.

Isolation of bioactive compounds from medicinal plants used in traditional medicine: Rautandiol B, a potential lead compound against Plasmodium falciparum.

BACKGROUND: Neorautanenia mitis, Hydnora abyssinica, and Senna surattensis are medicinal plants with a variety of traditional uses. In this study, we sought to isolate the bioactive compounds responsible for some of these activities, and to uncover their other potential medicinal properties. METHODS: The DCM and ethanol extracts of the roots of N. mitis and H. abyssinica, and the leaves of S. surattensis were prepared and their phytochemical components were isolated and purified using chromatographic methods. These extracts and their pure phytochemical components were evaluated in in-vitro models for their inhibitory activities against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Mycobacterium tuberculosis, α-amylase (AA), and α-glucosidase (AG). RESULTS: Rautandiol B had significant inhibitory activities against two strains of Plasmodium falciparum showing a high safety ratio (SR) and IC50 values of 0.40 ± 0.07 µM (SR - 108) and 0.74 ± 0.29 µM (SR - 133) against TM4/8.2 and K1CB1, respectively. While (-)-2-isopentenyl-3-hydroxy-8-9-methylenedioxypterocarpan showed the highest inhibitory activity against T. brucei rhodesiense with an IC50 value of 4.87 ± 0.49 µM (SR > 5.83). All crude extracts showed inhibitory activities against AA and AG, with three of the most active phytochemical components; rautandiol A, catechin, and dolineon, having only modest activities against AG with IC50 values of 0.28 mM, 0.36 mM and 0.66 mM, respectively. CONCLUSION: These studies have led to the identification of lead compounds with potential for future drug development, including Rautandiol B, as a potential lead compound against Plasmodium falciparum. The relatively higher inhibitory activities of the crude extracts against AG and AA over their isolated components could be due to the synergistic effects between their phytochemical components. These crude extracts could potentially serve as alternative inhibitors of AG and AA and as therapeutics for diabetes.

A new xanthone and a biphenyl from the flower and twig extracts of Garcinia mckeaniana.

The phytochemical investigation of the flower and twig extracts of Garcinia mckeaniana yielded a new xanthone, mckeanianone F (1) and a new biphenyl, mckeaniabiphenyl (2) together with 15 known compounds. The isolated compounds were characterized using spectroscopic techniques and mass spectrometry. Some of the isolated compounds from the twigs exhibited antimalarial and cytotoxic activities.

(R)-3-(8'-Hydroxyfarnesyl)-indole and other chemical constituents from the flowers of Anomianthus dulcis and their antimalarial and cytotoxic activities.

A new farnesylindole, (R)-3-(8'-hydroxyfarnesyl)-indole (1), as a scalemic mixture (33% ee) along with nine known compounds (2-10), including one farnesylindole, three flavanones, three flavone derivatives and two chalcone derivatives were isolated from the methanolic crude extract of the flowers from Anomianthus dulcis. All compounds were purified by appropriate chromatographic techniques and their structures elucidated by spectroscopic methods. Compounds 1, 2 and 8 showed moderate antiplasmodial activities against TM4/8.Two and K1CB1 strains of which compound 2 displayed the best activity with IC50 values of 27.9 ± 2.57 and 21.4 ± 1.68 µM, respectively. In addition, compound 1 also presented modest cytotoxicity against a KB cell line with an IC50 value of 22.3 ± 0.39 µM. None of these compounds showed cytotoxicity against Vero cells.

Unique polyacetylenic ester-neolignan derivatives from Mitrephora tomentosa and their antimalarial activities.

The phytochemical investigation of the methanol extracts of the leaves and twigs of Mitrephora tomentosa Hook. f. & Thomson resulted in the isolation and identification of undescribed polyacetylenic ester-neolignan derivatives, along with six known compounds. These six undescribed natural products were named as mitrephentosins A-F. The structures of these compounds were determined by spectroscopic techniques including UV, IR, NMR, and mass spectrometric analyses. The absolute configurations of mitrephentosins A-F were determined based on specific rotations values and ECD spectral data by comparisons made with the known parent neoligan compound mitredrusin. Mitrephentosins C, E, and F showed moderate antimalarial activities (IC50 values of 13.3-24.6 µM) against the Plasmodium falciparum strains TM4/8.2 and K1CB1 and were not toxic to Vero cells, while the other isolated compounds were not active against these P. falciparum strains.

Isolation of CFTR and TMEM16A inhibitors from Neorautanenia mitis (A. Rich) Verdcourt: Potential lead compounds for treatment of secretory diarrhea.

A phytochemical study on the root extracts of Neorautanenia mitis, a Nigerian medicinal plant used in the management of diarrhea, led to the isolation of one new and 19 known natural products. These compounds and crude extracts were evaluated for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel and calcium-activated Cl- channel (TMEM16A) inhibitory activities in T84 and Calu-3 cells, respectively. Four compounds namely dolineon, neodulin, pachyrrhizine, and neotenone inhibited cAMP-induced Cl- secretion across T84 cell monolayers with IC50 values of ~0.81 µM, ~2.42 µM, ~2.87 µM, and ~4.66 µM, respectively. Dolineon having the highest inhibitory activity also inhibited a Ca + activated Cl- channel (TMEM16A) with an IC50 value of ~4.38 µM. The in vitro antidiarrheal activity of dolineon was evaluated on cholera toxin (CT) induced chloride secretion in T84 cells, where it inhibited CT-induced chloride secretion by >70% at 100 µM. Dolineon also inhibited CT-induced fluid secretion by ~70% in an in vivo mouse closed loop model at a dose of 16.9 µg/loop. The cytotoxicity of the extracts and compounds was evaluated on KB, Vero and BHK21 cells, dolineon showed low cytotoxicity of >29.6 µM and 57.30 + 6.77 µM against Vero and BHK21 cells, respectively. Our study revealed that several compounds isolated from N. mitis showed antidiarrheal activity. The most active compound dolineon can potentially serve as a lead compound towards the development of CFTR and TMEM16A inhibitors as future therapeutics for secretory diarrhea.

Flexible diaminodihydrotriazine inhibitors of Plasmodium falciparum dihydrofolate reductase: Binding strengths, modes of binding and their antimalarial activities.

A series of flexible diaminodihydrotriazines or cycloguanil (Cyc) analogues are developed and shown to inhibit P. falciparum dihydrofolate reductase (PfDHFR) of the wild type or those carrying either single (S108N), double (C59R + S108N and A16V + S108T), triple (N51I + C59R + S108N and C59R + S108N + I164L) or quadruple (N51I + C59R + S108N + I164L) mutations, responsible for antifolate resistance. The flexibility of the side chain at position N1 has been included in the design so as to avoid unfavourable steric interaction with the side chain of residue 108 of the resistant mutants. The inhibition constants of many inhibitors for the mutant enzymes are in the low nanomolar region. Regaining of drug binding efficacies was achieved with both A16V and S108N series of mutants. X-ray studies of some enzyme-inhibitor complexes designed for optimal interaction with the mutant enzymes reveal the modes of binding in line with the Ki values. A number of these compounds show excellent antimalarial activities against resistant P. falciparum bearing the mutant enzymes, and exhibit low cytotoxicity to mammalian cells, making them good candidates for further development as antimalarial drugs.

Antibacterial and cytotoxic activities of phenolic constituents from the stem extracts of Spatholobus parviflorus.

A new 2-arylbenzofuran, spathobenzofuran (1), together with ten known compounds including a 2-arylbenzofuran, three pterocarpans and six isoflavones were isolated from the acetone crude extract of the stems of Spatholobus parviflorus. All compounds were characterised by spectroscopic methods. Compound 4 was active (MIC 8 µg/mL) against Gram-negative Pseudomonas aeruginosa TISTR 781 while compound 2 had modest activity against Gram-positive Staphylococcus aureus TISTR 1466 with a MIC value of 16 µg/mL. All isolated compounds showed no cytotoxicity against Vero and KB cells.

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.

Polyoxygenated Cyclohexenes and Their Chlorinated Derivatives from the Leaves of Uvaria cherrevensis.

The chemical study of leaf extracts from Uvaria cherrevensis resulted in the identification of 11 new polyoxygenated cyclohexenes, cherrevenols A-K (1-11), and a new seco-cyclohexene derivative, cherrevenol L (12). Nine known compounds (13-21) were also isolated. Three of the isolated compounds are chlorinated polyoxygenated cyclohexenes. The structures of these compounds were determined using spectroscopic methods and, in some cases (compounds 2, 6, 8, and 10), single-crystal X-ray crystallographic structural analysis or chemical correlation (compounds 6 and 7). Compounds 6 and 7 were both isolated as scalemic mixtures (ee 23-24%).

Four new C-benzyl flavonoids from the fruit of Uvaria cherrevensis.

The phytochemical investigation of the fruit extracts of Uvaria cherrevensis led to the isolation and characterization of four new C-benzyl flavonoids; cherrevenones A-D (1-4) together with 11 known compounds. The isolated compounds were characterized using spectroscopic techniques. Compounds 1, 3, 5 and 11 showed moderate inhibitory activities against the P. falciparum strains TM4/8.2 and K1CB1 with IC50 values ranging from 21.0 ±â€¯3.10 - 33.7 ±â€¯7.69 and 21.0 ±â€¯5.44 - 43.5 ±â€¯11.9 µM, respectively. Compounds 1, 2, 5, 10 and 11 exhibited strong cytotoxic activities against KB cells with IC50 values ranging from 0.60 ±â€¯0.17 - 4.91 ±â€¯2.69 µM which were similar to their cytotoxic activities found against Vero cells, except for compound 5, which was non-toxic to Vero cells.

Hybrid Inhibitors of Malarial Dihydrofolate Reductase with Dual Binding Modes That Can Forestall Resistance.

The S108N mutation of dihydrofolate reductase (DHFR) renders Plasmodium falciparum malaria parasites resistant to pyrimethamine through steric clash with the rigid side chain of the inhibitor. Inhibitors with flexible side chains can avoid this clash and retain effectiveness against the mutant. However, other mutations such as N108S reversion confer resistance to flexible inhibitors. We designed and synthesized hybrid inhibitors with two structural types in a single molecule, which are effective against both wild-type and multiple mutants of P. falciparum through their selective target binding, as demonstrated by X-ray crystallography. Furthermore, the hybrid inhibitors can forestall the emergence of new resistant mutants, as shown by selection of mutants resistant to hybrid compound BT1 from a diverse PfDHFR random mutant library expressed in a surrogate bacterial system. These results show that it is possible to develop effective antifolate antimalarials to which the range of parasite resistance mutations is greatly reduced.

2-Phenylnaphthalenes and a polyoxygenated cyclohexene from the stem and root extracts of Uvaria cherrevensis (Annonaceae).

Three new 2-phenylnaphthalene derivatives, cherrevenaphthalenes A-C (1-3), and a new polyoxygenated cyclohexene derivative, (-)-uvaribonol F (4) together with six known compounds, 5-10, were isolated from the stem and root extracts of Uvaria cherrevensis (Annonaceae). The structures of all isolated compounds were elucidated by spectroscopic analysis. The structures of 3 and 4 were further confirmed by single crystal X-ray diffraction methods. Compound 2 exhibited modest antiplasmodial activity against the P. falciparum stains TM4/8.2 and K1CB1 with IC50 values of 18.8±3.63 and 23.4±4.08µM, respectively, and weak cytotoxicity to a Vero cell line. Furthermore, compound 4 displayed cytotoxic activity against a KB cell line with an IC50 value of 22.1±0.42µM but was non-cytotoxic to the Vero cell line. Compound 5 revealed stronger cytotoxicity towards the KB cell line, with an IC50 value of 5.05±0.86µM and was nearly equally cytotoxic to the Vero cell line.

Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick.

The significant strides made in reducing global malaria burden over the past decades are being threatened by the emergence of multi-drug resistant malaria. Mechanisms of resistance to several classes of antimalarial drugs have been linked to key mutations in the Plasmodium falciparum genes. Pyrimethamine targets the dihydrofolate reductase of the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS), and specific point mutations in the dhfr-ts gene have been assigned to resistant phenotypes. Several molecular methods are available to detect the mutant genotypes including DNA sequencing and PCR-based methods. In this study, we report the development of PfSNP-LAMP to detect nucleotide polymorphism in the dhfr gene associated with N51I mutation and antifolate resistance. The PfSNP-LAMP method was validated with genomic DNA samples and parasite lysates prepared from sensitive and pyrimethamine resistant strains of P. falciparum.