Limonoids from Cipadessa baccifera.

Eighteen new limonoids, including eight methyl angolensates (1-8) and 10 cipadesins (9-18), were isolated from the leaves of Cipadessa baccifera. Their structures were characterized by means of spectroscopic data analyses, single-crystal X-ray diffraction, and quantum chemistry computational methods. The C-6 configurations in those compounds possessing a C-6 hydroxy group were all assigned as S regardless of the magnitude of J5,6, and the C-2' configuration in those bearing a 2-methylbutyryl residue was defined by single-crystal X-ray diffraction and NMR data. Compounds 1, 5, 6, 7, 11, and 12 showed moderate antimalarial activities with IC50 values ranging from 12 to 28 µM.

Antimalarial diterpenoids from Vitex rotundifolia: Isolation, structure elucidation, and in vitro antiplasmodial activity.

Vitex rotundifolia is an important medicinal plant frequently employed in traditional medicines for the treatment of various ailments. Although this plant species has been under exploration for its constituents by various research groups including our own group, no reports were found regarding the antimalarial potential of this plant or of its purified phytochemicals. Phytochemical investigation of this plant yielded three new (1-3) and five known (4-8) diterpenoids. These compounds were purified by modern chromatographic techniques and their structures were determined by advanced spectroscopic techniques such as nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The in vitro antiplasmodial activities were encouraging, as compounds 2, 6, and 8 were found to have significant IC50 values of 1.2, 1.3 and 11.0 µM, respectively against Plasmodium falciparum.

Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum.

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/ß hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

Isolation and characterization of antiplasmodial constituents from the marine sponge Coscinoderma sp.

Six known compounds, namely two halisulfates 1 and 2 and four epidioxy sterols 3-6, were isolated from the marine sponge Coscinoderma sp. The structures of these compounds were confirmed by nuclear magnetic resonance (1H and 13C NMR) spectroscopy, and their antiplasmodial activities were determined against the chloroquine-resistant Dd2 strain of Plasmodium falciparum. The epidioxy steroids 3-6 all showed moderate to weak antiplasmodial activity, with IC50 values of 2.7 µM for (24S)-5α,8α-epidioxy-24-methylcholesta-6-en-3ß-ol (3), 11.6 µM for 5α,8α-epidioxycholesta-6,24(28)-dien-3ß-o1 (4), 2.33 µM for 5α,8α-epidioxy-24-methylcholesta-6,9(11)-24(28)-trien-3ß-ol (5), and between 12 and 24 µM for 5α,8α-epidioxycholesta-6-en-3ß-ol (6). In contrast, halisulfate 2 (1) was inactive, and halisulfate 1 (2) had an of IC50 value of about 24 µM.

Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter.

Plasmodium falciparum multidrug resistance constitutes a major obstacle to the global malaria elimination campaign. Specific mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) mediate resistance to the 4-aminoquinoline drug chloroquine and impact parasite susceptibility to several partner agents used in current artemisinin-based combination therapies, including amodiaquine. By examining gene-edited parasites, we report that the ability of the wide-spread Dd2 PfCRT isoform to mediate chloroquine and amodiaquine resistance is substantially reduced by the addition of the PfCRT L272F mutation, which arose under blasticidin selection. We also provide evidence that L272F confers a significant fitness cost to asexual blood stage parasites. Studies with amino acid-restricted media identify this mutant as a methionine auxotroph. Metabolomic analysis also reveals an accumulation of short, hemoglobin-derived peptides in the Dd2 + L272F and Dd2 isoforms, compared with parasites expressing wild-type PfCRT. Physiologic studies with the ionophores monensin and nigericin support an impact of PfCRT isoforms on Ca2+ release, with substantially reduced Ca2+ levels observed in Dd2 + L272F parasites. Our data reveal a central role for PfCRT in regulating hemoglobin catabolism, amino acid availability, and ionic balance in P. falciparum, in addition to its role in determining parasite susceptibility to heme-binding 4-aminoquinoline drugs.

Two cap residues in the S1 subsite of a Plasmodium falciparum M1-family aminopeptidase promote broad specificity and enhance catalysis.

The aminopeptidase PfA-M1 is a key contributor to peptide catabolism in the human malaria parasite Plasmodium falciparum. PfA-M1 substrate specificity is shaped by the cylindrical S1 subsite, which accommodates the sidechain of the substrate P1 residue. At the top of the S1 subsite are two "cap" residues, E572 and M1034, that are positioned to influence S1 subsite specificity. In this study, we have mutated the cap residues, individually and together, and have evaluated the effects on PfA-M1 specificity and catalytic efficiency. When the P1 residue was too small to engage the cap residues, the mutations had no effect on catalysis. Hydrolysis of dipeptide substrates with a basic P1 residue was significantly impaired in the E572A mutant, most likely due to the loss of a stabilizing salt bridge between E572 and the P1 sidechain. With M1034A, a substantial reduction in catalytic efficiency was observed when the P1 sidechain was large and non-polar. The double E572A/M1034A exhibited significant decreases in catalytic efficiency for most substrates. This effect was not reversed with the polar substitutions E572N/M1034Q, which replaced the PfA-M1 cap residues with those of Escherichia coli aminopeptidase N. Both E572 and M1034 contributed to the binding of the competitive aminopeptidase inhibitor bestatin.

Bioactive Neolignans and Other Compounds from Magnolia grandiflora L.: Isolation and Antiplasmodial Activity.

Bioassay-guided fractionation of a methanol extract of Magnolia grandiflora against Plasmodium falciparum yielded two new (1 and 2) and six known (3 - 8) bioactive compounds. The structures of the new compounds were assigned by mass spectrometric and 1D- and 2D-NMR data. Known compounds were identified by comparison of 1 H-NMR and MS data with literature data. The two known neolignans 3 and 4 showed moderate antiplasmodial activity with the IC50 values of 2.8 ± 0.1 and 3.4 ± 0.1 µm, respectively. Weak antiplasmodial activity was recorded for compounds 1, 2, 5, 6, 7, and 8, with the IC50 values of 38 ± 2, 23 ± 2, 16.5 ± 0.2, 86 ± 1, 44 ± 4, and 114 ± 9 µm, respectively.

Isolation, structure elucidation, and synthesis of antiplasmodial quinolones from Crinum firmifolium.

Antiplasmodial bioassay guided fractionation of a Madagascar collection of Crinum firmifolium led to the isolation of seven compounds. Five of the seven compounds were determined to be 2-alkylquinolin-4(1H)-ones with varying side chains. Compounds 1 and 4 were determined to be known compounds with reported antiplasmodial activities, while 5 was believed to be a new branched 2-alkylquinolin-4(1H)-one, however, it was isolated in limited quantities and in admixture and therefore was synthesized to confirm its structure as a new antiplasmodial compound. Along with 5, two other new and branched compounds 6 and 7 were synthesized as well. Accompanying the five quinolones were two known compounds 2 and 3 which are inactive against Plasmodium falciparum. The isolation, structure elucidation, total synthesis, and biological evaluation of these compounds are discussed in this article.

Antiplasmodial Sesquiterpenoid Lactones from Trichospira verticillata: Structure Elucidation by Spectroscopic Methods and Comparison of Experimental and Calculated ECD Data.

A dichloromethane extract of Trichospira verticillata from the Natural Products Discovery Institute was discovered to have good antiplasmodial activity (IC50 ∼5 µg/mL). After purification by liquid-liquid partition and C18 reversed-phase HPLC, four new germacranolide-type sesquiterpenoid lactones named trichospirolides A-D (1-4) were isolated. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and MS data. The relative and absolute configurations were assigned based on a comparison of calculated and experimental ECD and UV spectra, specific rotations, internuclear distances, and coupling constants for all possible diastereomers for each compound. Among these four compounds, the conjugated dienone 1 displayed the most potent antiplasmodial activity, with an IC50 value of 1.5 µM.

Fortunoids A-C, Three Sesquiterpenoid Dimers with Different Carbon Skeletons from Chloranthus fortunei.

Three dimeric sesquiterpenoids (1-3), fortunoid A (1) possessing a new carbon skeleton of rearranged lindenane dimer and fortunoids B (2) and C (3) representing the first example of the dimers of a lindenane and a eudesmane sesquiterpene, were isolated from Chloranthus fortunei. Their structures with absolute configurations were established by spectroscopic data and electric circular dichroism analysis. Their biosynthetic origins were also proposed. Compounds 1 and 2 showed moderate antimalarial activities.

Nanomolar Antimalarial Agents against Chloroquine-Resistant Plasmodium falciparum from Medicinal Plants and Their Structure-Activity Relationships.

Inspired by the discovery of the antimalarial drug artemisinin from a traditional Chinese medicine (TCM), a natural product library of 44 lindenane-type sesquiterpenoids was assessed for activities against the Dd2 chloroquine-resistant strain of the malaria parasite Plasmodium falciparum. These compounds were mainly isolated from plants of the Chloranthus genus, many species of which are named "Sikuaiwa" in TCM and have long been used to treat malaria. The compounds consisted of 41 sesquiterpenoid dimers and three monomers, including the 12 new dimers 1-12 isolated from Chloranthus fortunei. The results showed that 16 dimers exhibited potent antiplasmodial activities (<100 nM); in particular, compounds 1, 14, and 19 exhibited low nanomolar activities with IC50 values ranging from 1 to 7 nM, which is comparable to the potency of artemisinin, and selectivity index values toward mammalian cells greater than 500. A comprehensive structure-activity relationship study indicated that three functional groups are essential and two motifs can be modified.

New potently bioactive alkaloids from Crinum erubescens.

Antimalarial bioassay-guided fractionation of the swamp lily Crinum erubescens led to the isolation of four compounds with potent antiplasmodial activity. Compounds 1 and 2 were determined from their spectroscopic data to be the known pesticidal compound cripowellin A and the known pesticidal and antiproliferative compound cripowellin B. 1D and 2D-NMR techniques were used to determine the identities of 3 and 4 as the new compounds cripowellin C and D. A fifth compound was identified as the known alkaloid hippadine, which was inactive against Plasmodium falciparum. The antiplasmodial IC50 values of compounds 1-4 were determined to be 30±2, 180±20, 26±2, and 260±20nM, respectively, and their antiproliferative IC50 values against the A2780 human ovarian cancer cell line were 11.1±0.4, 16.4±0.1, 25±2, and 28±1nM.

Parallel inhibition of amino acid efflux and growth of erythrocytic Plasmodium falciparum by mefloquine and non-piperidine analogs: Implication for the mechanism of antimalarial action.

Despite the troubling psychiatric side-effects it causes in some patients, mefloquine (MQ) has been used for malaria prophylaxis and therapy, due to its activity against all Plasmodium species, its ease of dosing, and its relative safety in children and pregnant women. Yet at present there is no consensus on the mechanism of antimalarial action of MQ. Two leading hypotheses for the mechanism of MQ are inhibition of heme crystallization and inhibition of host cell hemoglobin endocytosis. In this report we show that MQ is a potent and rapid inhibitor of amino acid efflux from intact parasitized erythrocytes, which is a measure of the in vivo rate of host hemoglobin endocytosis and catabolism. To further explore the mechanism of action of MQ, we have compared the effects of MQ and 18 non-piperidine analogs on amino acid efflux and parasite growth. Among these closely related compounds, an excellent correlation over nearly 4 log units is seen for 50% inhibition concentration (IC50) values for parasite growth and leucine efflux. These data and other observations are consistent with the hypothesis that the antimalarial action of these compounds derives from inhibition of hemoglobin endocytosis.

New Antiplasmodial Diterpenes from Gutierrezia sarothrae.

Bioassay guided fractionation of the MeOH extract of the plant Gutierrezia sarothrae (Asteraceae) using an assay for antiplasmodial activity against the drug-resistant Dd2 strain of Plasmodium falciparum led to the isolation of the two new diterpenes 3α-angeloyloxy-15-hydroxylabda-7,13-dien-16,15-olid-18-oic acid (1) and 3α-angeloyloxy-15-methoxylabda-7,13-dien-16,15-olid-18-oic acid (2). The structures of 1 and 2 were elucidated by interpretation of 1D and 2D NMR spectroscopic data, mass spectrometry, and comparison with the data of related compounds reported in the literature. Compound 1 exhibited moderate antiplasmodial activity with an IC50 values of 10.4 ± 4.3 µM.

Euphorbesulins A-P, Structurally Diverse Diterpenoids from Euphorbia esula.

Aqueous ethanol extracts of powdered twigs of Euphorbia esula afforded 16 new diterpenoids, named euphorbesulins A-P. These euphorbesulins included presegetane (1-3), jatrophane (4-14), paraliane (15), and isopimarane (16) diterpenoids as well as six known analogues. Compounds 1-3 represent a rare type of presegetane diterpenoid. Their structures were determined by analysis of the spectroscopic data, and the absolute configuration of 1 was established by X-ray crystallography. Diterpenoid 7 showed low nanomolar antimalarial activity, while the remaining compounds showed only moderate or no antimalarial activity.

Synthesis and Antimalarial Activity of Mallatojaponin C and Related Compounds.

The phloroglucinol mallotojaponin C (1) from Mallotus oppositifolius, which was previously shown by us to have both antiplasmodial and cytocidal activities against the malaria parasite Plasmodium falciparum, was synthesized in three steps from 2',4',6'-trihydroxyacetophenone, and various derivatives were synthesized in an attempt to improve the bioactivity of this class of compounds. Two derivatives, the simple prenylated phloroglucinols 12 and 13, were found to have comparable antiplasmodial activities to that of mallotojaponin C.

Antiplasmodial phloroglucinol derivatives from Syncarpia glomulifera.

Bioassay guided fractionation of a MeOH extract of the stem bark of Syncarpia glomulifera (Myrtaceae) led to the isolation of the two new phloroglucinol derivatives (±)-rhodomyrtosone F (1) and (±)-calliviminone I (2), the three known triterpenes, betulinic acid (3), ursolic acid-3-acetate (4), and ursolic acid (5), and 1-(2,4,6-trihydroxyphenyl)-1-hexanone (6). Compound 1 exhibited strong antiplasmodial activity, while compounds 2-4 were moderately active and 5 and 6 were inactive in this assay. The structures of 1 and 2 were elucidated based on analyses of their mass spectrometric data, 1D and 2D NMR spectra, and comparison with related compounds.

Antimalarial diterpenoid dimers of a new carbon skeleton from Aphanamixis grandifolia.

Chemical investigation into the minor constituents of Aphanamixis grandifolia yielded three new diterpenoid dimers, aphadilactones E-G (1-3) featuring a new carbon skeleton. Their structures and absolute configurations were fully established by comprehensive spectroscopic data analysis and ECD calculation. Discovery of another two new dimers (4 and 5) suggested the structure of recently reported aphanamene A to be re-investigated. Compounds 1-5 showed moderate antimalarial activities with low micromolar IC50 values.