In vivo transformations of artemisinic acid in Artemisia annua plants

Artemisinic acid labeled with both ¹³C and ²H at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid—as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation—closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Δ^4,5 double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species.     

Simultaneous isolation of artemisinin and its precursors from Artemisia annua L. by preparative RP-HPLC

It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C(18) column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner.     

Metabolic fingerprinting investigation of Artemisia annua L. in different stages of development by gas chromatography and gas chromatography-mass spectrometry

Artemisia annua L. is an annual herb native of Asia and this plant has been famous for the discovery of the anti-malarial drug artemisinin since 1971. In this work, to investigate variety of whole metabolites, metabolic fingerprinting analysis of A. annua L. was carried out by GC and GC-MS coupled with trimethylsilyl derivatisation. Principal component analysis and partial least squares discriminant analysis were employed to classify GC data of A. annua L. samples at five developmental stages. The results indicated that there was no distinct difference of metabolites between control (001) and transgenic strain (F4) from the tender seedling stage to adult seedling stage, but clear differences were detected at pre-flower budding stage, flower budding stage and full flowering stage. Three precursors of artemisinin biosynthesis were studied at five developmental stages and found that a possible bottleneck exists in the conversion from artemisinic acid or dihydroartemisinic acid to artemisinin.     

Biomimetic synthesis of arteannuin h and the 3,2-rearrangement of allylic hydroperoxides

The acyl endoperoxide arteannuin H, recently reported as a novel natural product from Artemtsia annua, has been obtained in two steps from the photooxidation of dihydroartemisinic acid, thereby confirming biogenetic speculation regarding its derivation from a secondary allylic hydroperoxide. The little studied 3,2-rearrangement reaction of such allylic hydroperoxides is also discussed.     

Simultaneous densitometric determination of artemisinin, artemisinic acid and arteannuin-B in Artemisia annua using reversed-phase thin layer chromatography

A rapid and simple RP-TLC method for simultaneous quantification of pharmacologically important sesquiterpene artemisinin (AM) together with its precursors arteannuin-B (AB) and artemisinic acid (AA) in the inflorescence part of Artemisia annua plant has been developed. The RP-TLC of sesquiterpenes was performed on RP-18 F254 S thin-layer chromatographic plates by developing in mobile phase, containing 0.2% TFA in water/ACN (35:65, v/v). The densitometric determination of AM, AB and AA was carried out after derivatization with anisaldehyde reagent at 426 nm in absorption-reflectance mode.     

Autoxidation of 4-amorphen-11-ol and the biogenesis of nor- and seco-amorphane sesquiterpenes from fabiana imbricata

Photooxidation of 4-amorphen-11-ol (1), recently reported as one of the major sesquiterpene natural products from the medicinal plant Fabiana imbricata, results in three allylic hydroperoxides 6, 9 and 10, which are expected from the “ene-type” reaction of molecular oxygen with the tri-substituted double bond in 1. The tertiary allylic hydroperoxide 6 undergoes carbon-carbon bond cleavage and a second autoxidation reaction to yield the more highly oxygenated seco-amorphane 11 under very mild conditions. In acid, this compound may then undergo either a second carbon-carbon bond cleavage reaction to yield nor-sesquiterpenes 2 and 3 (reported as bona fide natural products from F. imbricata, or cyclize to the sesquiterpene peroxofabianane (5), which is a presumed precursor to the natural product fabianane (4). Some mechanistic investigations concerning the two chemical processes: viz:- carbon-carbon bond cleavage and autoxidation which would account for the formation of natural products 2, 3 and 4 from 1 are reported. Tertiary allylic hydroperoxide 32, which lacks the 11-hydroxyl functional group present in 1 undergoes only C-4/C-5 carbon-carbon bond cleavage under more forcing conditions, suggesting a role for this functional group in assisting the autoxidation reactions of 4-amorphen-11-ol.     

A Continuous‐Flow Process for the Synthesis of Artemisinin

Isolation of the most effective antimalarial drug, artemisinin, from the plant sweet wormwood, does not yield sufficient quantities to provide the more than 300 million treatments needed each year. The high prices for the drug are a consequence of the unreliable and often insufficient supply of artemisinin. Large quantities of ineffective fake drugs find a market in Africa. Semisynthesis of artemisinin from inactive biological precursors, either dihydroartemisinic acid (DHAA) or artemisinic acid, offers a potentially attractive route to increase artemisinin production. Conversion of the plant waste product, DHAA, into artemisinin requires use of photochemically generated singlet oxygen at large scale. We met this challenge by developing a one‐pot photochemical continuous‐flow process for the semisynthesis of artemisinin from DHAA that yields 65 % product. Careful optimization resulted in a process characterized by short residence times. A method to extract DHAA from the mother liquor accumulated during commercial artemisinin extractions, a material that is currently discarded as waste, is also reported. The synthetic continuous‐flow process described here is an effective means to supplement the limited availability of artemisinin and ensure increased supplies of the drug for those in need.     

In vivo transformations of dihydroartemisinic acid in Artemisia annua plants

[15-¹³C²H₃]-dihydroartemisinic acid (2a) and [15-C²H₃]-dihydroartemisinic acid (2b) have been fed via the root to intact Artemisia annua plants and their transformations studied in vivo by one-dimensional ²H NMR spectroscopy and two-dimensional ¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY). Labelled dihydroartemisinic acid was transformed into 16 12-carboxy-amorphane and cadinane sesquiterpenes within a few days in the aerial parts of A. annua, although transformations in the root were much slower and more limited. Fifteen of these 16 metabolites have been reported previously as natural products from A. annua. Evidence is presented that the first step in the transformation of dihydroartemisinic acid in vivo is the formation of allylic hydroperoxides by the reaction of molecular oxygen with the Δ^4,5-double bond in this compound. The origin of all 16 secondary metabolites might then be explained by the known further reactions of such hydroperoxides. The qualitative pattern for the transformations of dihydroartemisinic acid in vivo was essentially unaltered when a comparison was made between plants, which had been kept alive and plants which were allowed to die after feeding of the labelled precursor. This, coupled with the observation that the pattern of transformations of 2 in vivo demonstrated very close parallels with the spontaneous autoxidation chemistry for 2, which we have recently demonstrated in vitro, has lead us to conclude that the main ‘metabolic route’ for dihydroartemisinic acid in A. annua involves its spontaneous autoxidation and the subsequent spontaneous reactions of allylic hydroperoxides which are derived from 2. There may be no need to invoke the participation of enzymes in any of the later biogenetic steps leading to all 16 of the labelled 11,13-dihydro-amorphane sesquiterpenes which are found in A. annua as natural products.     

Determination of artemisinin and artemisinic acid by capillary and packed supercritical fluid chromatography

Artemisinin (an antimalarial compound) and its bioprecursor artemisinic acid, present in the plant Atemisia annua L., were analyzed by supercritical fluid chromatography (SFS) using capillary and packed columns, coupled respectively with a flame ionization detector (FID) and an evaporative light scattering detector (ELSD). Both methods were optimized and validated with columns of different polarity in order to separate artemisinin and artemisinic acid. Analytical results were comparable, but the paced SFC-ELSD method was faster. Indeed, artemisinin and artemisinic acid were separated with an aminopropyl silica column in less than 8 minutes instead of about 25 minutes by capillary SFS. Contrary to conventional gas and liquid chromatography coupled to an UV-visible detector, SFS methods determined both compounds directly, without degradation and/or derivatization in the concentration range expected in the plant material. Results obtained on plant extracts by capillary SFS-FID and packed SFS-ELSD were confirmed by GC-MS.     

A new sesquiterpene and other constituents from Saussurea lappa root

Five sesquiterpenes, dehydrocostus lactone (1), santamarine (5), beta-cyclocostunolide (6), 4alpha-hydroxy-4beta-methyldihydrocostol (7) and 10alpha-hydroxyl-artemisinic acid (9), along with four other compounds, beta-sitosterol (2), daucosterol (3), 5-hydroxymethyl-furaldehyde (4), and trans-syingin (8), were isolated and identified from the roots of Saussurea lappa (Compositae). Based on previous reports and our study, sesquiterpene derivatives are common and characteristic constituents of the genus Saussurea. Among the nine compounds obtained, 9 is a new sesquiterpene. It is an artemisinic acid derivative, whose structural skeleton has not been reported for Saussurea species before, but artemisinic acid is a common compound in another Compositae species, Artemisia annua. Dehydrocostus lactone (1) is present in high-content and is a possible bioprecursor of 10alpha-hydroxyartemisinic acid (9).     

1,2,5,10,11,14-hexaoxadispiro[5.2.5.2]hexadecanes: novel spirofused bis-trioxane peroxides

A set of new bis-spirofused 1,2,4-trioxanes 4a-d was obtained from the reaction of cyclohexane-1,4-dione with allylic hydroperoxides 2a-d, bearing an additional hydroxy group in the homoallylic position, by diastereoselective photooxygenation of allylic alcohols 1a-d and subsequent BF(3)-catalyzed peroxyacetalization with the diketone. From the reaction of a monoprotected cyclohexane-1,4-dione 5 with the allylic hydroperoxide 6 derived from the singlet oxygenation of methyl hydroxytiglate, one monospiro compound was obtained, the 1,2,4-trioxane ketone 7, as well as a mixture of the diastereoisomeric syn- and anti bis-1,2,4-trioxanes 8. The structures of bis-1,2,4-trioxanes were examined theoretically by DFT methods and compared with X-ray structural data in order to evaluate the preferential trioxane ring conformational orientation.     

Direct analysis of artemisinin from Artemisia annua L. using high-performance liquid chromatography with evaporative light scattering detector, and gas chromatography with flame ionization detector

Since the isolation of artemisinin 32 years ago, it has been analyzed by different chromatographic techniques. This work compared the analysis of artemisinin from crude plant samples by GC with flame ionization detection (GC-FID) and HPLC with evaporative light scattering detector (HPLC-ELSD). Data is also presented indicating that GC is suitable for the quantification of two of artemisinin precursors (arteannuin B and artemisinic acid) if a mass spectrometer is available. GC-FID and HPLC-ELSD were chosen because of their low cost compared to other detection methods, their ease of operation compared to HPLC with electrochemical detection, and because neither require artemisinin derivatization. Both GC-FID and HPLC-ELSD provided sensitive (ng level) and reproducible results for the analysis of artemisinin from field plants, with a correlation coefficient of r(2)=0.86 between the two methods. Both methods could be easily adapted to the analysis of pharmaceutical-grade artemisinin.     

Continuous Flow Reduction of Artemisinic Acid Utilizing Multi‐Injection Strategies—Closing the Gap Towards a Fully Continuous Synthesis of Antimalarial Drugs

One of the rare alternative reagents for the reduction of carbon–carbon double bonds is diimide (HN?NH), which can be generated in situ from hydrazine hydrate (N₂H₄ ? H₂O) and O₂. Although this selective method is extremely clean and powerful, it is rarely used, as the rate‐determining oxidation of hydrazine in the absence of a catalyst is relatively slow using conventional batch protocols. A continuous high‐temperature/high‐pressure methodology dramatically enhances the initial oxidation step, at the same time allowing for a safe and scalable processing of the hazardous reaction mixture. Simple alkenes can be selectively reduced within 10–20 min at 100–120 °C and 20 bar O₂ pressure. The development of a multi‐injection reactor platform for the periodic addition of N₂H₄ ? H₂O enables the reduction of less reactive olefins even at lower reaction temperatures. This concept was utilized for the highly selective reduction of artemisinic acid to dihydroartemisinic acid, the precursor molecule for the semisynthesis of the antimalarial drug artemisinin. The industrially relevant reduction was achieved by using four consecutive liquid feeds (of N₂H₄ ? H₂O) and residence time units resulting in a highly selective reduction within approximately 40 min at 60 °C and 20 bar O₂ pressure, providing dihydroartemisinic acid in ≥93 % yield and ≥95 % selectivity.     

Artemisinin

Artemisinin was first extracted in the 1970s from sweet wormwood (Artemisia annua), which was used for the treatment of malaria in traditional Chinese Medicine for more than 1600 years. Meanwhile the biosynthetic pathway of artemisinin, which derives from terpene‐metabolism is well established. In the past 30 years numerous total syntheses have been published, which impressively illustrate the achievements in modern organic chemistry. Nowadays, on industrial scale artemisinin is produced by extraction from wormwood and by fermentation and partial synthesis.     

Identification of radical species derived from allergenic 15-hydroperoxyabietic acid and insights into the behaviour of cyclic tertiary allylic hydroperoxides in Fe(II)/Fe(III) systems

To understand the skin sensitization mechanism of 15-hydroperoxyabietic acid, the major allergen in colophony, we first examined the formation of potential reactive radicals derived from its reaction with light, heat and TPP-Fe³⁺. Trapping with 1,1,3,3-tetramethylisoindolin-2-yloxyl nitroxide confirmed the formation of carbon-centred radicals derived from allyloxyl/allylperoxyl radicals as a consequence of the hydroperoxide scission. Particular interest was further given to the reactivity with Fe(II)/Fe(III) due to the biological importance of haem containing enzymes. Using a monocyclic 15-hydroperoxyabietic acid-like compound as a model of allergenic allylic hydroperoxides, we evidenced, by the ESR spin-trapping technique, the competition between carbon and oxygen-centred radicals formed in the presence of Fe(II)/Fe(III) in organic/aqueous media. We complemented the study by showing the possibility of formation, via a radical mechanism induced by ferric chloride, of an adduct between the allylic hydroperoxide and N-acetyl-cysteine ethyl ester. The results gave new knowledge on the possible generation of highly reactive radicals that could lead to the formation of antigenic structures.     

Conversion of antimalarial drug artemisinin to a new series of tricyclic 1,2,4-trioxanes1

A highly efficient route for the conversion of the antimalarial drug artemisinin to a novel hydroxy-functionalized tricyclic 1,2,4-trioxane 6 is reported. Neither the trioxane 6 nor its derivatives 14-16, all of which lack the hydrolytically unstable acetal-lactone linkage, show antimalarial activity comparable with that of artemisinin.     

Artemisinin evaluation in Romanian Artemisia annua wild plants using a new HPLC/MS method

Artemisinin, a sesquiterpene lactone from Artemisia annua L., has received considerable attention in the last few decades as a potent antimalarial drug. Artemisinin has rather low toxicity; it is effective against drug-resistant Plasmodium species and against cerebral malaria. This study reports the development of a rapid and sensitive assay for the quantification of artemisinin in A. annua by reversed phase HPLC/MS. In the selected optimal experimental conditions, artemisinin exhibited a well-defined chromatographic peak with a retention time of 2 ± 0.2 min. The chromatographic signal shows a linear dependence with artemisinin concentration, enabling the use of this signal for artemisinin quantification according to the following regression equation: y = 2665.40x - 14697.61. The correlation coefficient (R(2)) was 0.9989. For every concentration within the range of the standard curve (0.1-2 μg mL(-1)), accuracy was between 95 and 104%. Artemisinin content in Romanian A. annua wild plants varies between 0.17 and 0.21% (dry weight basis).     

Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae)

A new sesquiterpene glycoside, artemisinic acid 3-β-O-β-D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3-β-hydroxyartemisinic acid (2, 36.69%) and 3-β-hydroxyartemisinic acid β-D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC?? value of 0.56?μmol?mL?1.     

Development of a sensitive monoclonalantibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate–bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC₅₀) and the working range of the icELISA were 1.3 and 0.2–5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 μg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R ²) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials. Figure  Artemisia annua plant and antimalarial drugs derived from artemisinin     

Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway

Many terpenes used as fragrance compounds autoxidize when exposed to air, forming allylic hydroperoxides that have the potential to be skin contact allergens. To trigger the immunotoxicity process that characterizes contact allergy, these hydroperoxides are supposed to bind covalently to proteins in the skin via radical pathways. We investigated the formation of reactive radical intermediates from 7-hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol and 2-hydroperoxylimonene, responsible for the sensitizing potential acquired by autoxidized linalool and limonene. Both compounds were synthesized through new short and reproducible synthetic pathways. The hydroperoxide decomposition catalyzed by Fe(II)/Fe(III) redox systems, playing a key role in degradating peroxides in vivo, was examined by spin-trapping-EPR spectroscopy. Alkoxyl and carbon-centered free radicals derived from the hydroperoxides were successfully trapped by the spin-trap 5,5-dimethyl-1-pyrroline N-oxide, whereas peroxyl radicals were characterized by spin-trapping studies with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. Using liquid chromatography combined with mass spectrometry, we demonstrated the formation of adducts, via radical mechanisms induced by Fe(II)/Fe(III), between the hydroperoxides and N-acetylhistidine methyl ester, a model amino acid that is prone to radical reactions. Free radicals derived from these hydroperoxides can thus induce amino acid chemical modifications via radical mechanisms. The study of these mechanisms will help to understand the sensitizing potential of hydroperoxides.