Psammaplysin derivatives from the Balinese marine sponge Aplysinella strongylata.

Twenty-one new psammaplysin derivatives (4-24) exhibiting a variety of side chains, as well as six previously known psammaplysins, were identified from the Indonesian marine sponge Aplysinella strongylata. The double bond on the side chain of the fatty acid-containing psammaplysins was located by GC-MS analysis of the fatty acid methyl esters and their pyrrolidide derivatives. HPLC and Mosher ester studies confirmed that the isolated metabolites possessing a 19-OH substituent were mixtures of diastereomers. Selected compounds (4, 5, 7, 8, 12, 18, and 22) were screened for in vitro activity against chloroquine-sensitive (3D7) P. falciparum malaria parasites. Of the new psammaplysins, 19-hydroxypsammaplysin E (4) showed the best antimalarial activity, with an IC(50) value of 6.4 µM.

Enzyme-substrate complementarity governs access to a cationic reaction manifold in the P450(BM3)-catalysed oxidation of cyclopropyl fatty acids.

The products of cytochrome P450(BM3)-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs.

Substrate analog studies of the ω-regiospecificity of Mycobacterium tuberculosis cholesterol metabolizing cytochrome P450 enzymes CYP124A1, CYP125A1 and CYP142A1.

We report the synthesis and evaluation of a series of cholesterol side-chain analogs as mechanistic probes of three important Mycobacterium tuberculosis cytochrome P450 enzymes that selectively oxidize the ω-position of the methyl-branched cholesterol side-chain. To probe the structural requirements for the thermodynamically disfavored ω-regiospecificity we compared the binding of these substrate analogs to each P450, determined the turnover rates, and characterized the enzymatic products. The results are discussed in the context of the structure-activity relationships of the enzymes and how their active sites enforce ω-oxidation.

Oxidative processes in the Australian marine sponge Plakinastrella clathrata: isolation of plakortolides with oxidatively modified side chains.

Sixteen new cyclic peroxides (1-16) with a plakortolide skeleton and the methyl ester derivative of a didehydroplakinic acid (17) were isolated from the Australian sponge Plakinastrella clathrata Kirkpatrick, 1900. Structural elucidation and configurational assignments were based on spectroscopic analysis and comparison with data for previously isolated plakortolides and revealed both phenyl- and methyl-terminating side chains attached to the plakortolide core. Plakortoperoxides A-D (5-8) each contained a second 1,2-dioxine ring; a cis configuration for the side chain endoperoxide ring was determined by a low-temperature NMR study and by comparison of chemical shift values with those of reported compounds. An enantioselective HPLC study compared natural plakortoperoxide A with a synthetic sample prepared by cyclization of plakortolide P with singlet oxygen and revealed that the natural sample was a mixture of cis diastereomers at C-15/C18. Four other cyclic peroxides (9-12) possessed a C(9)-truncated side chain terminating in a formyl or carboxylic acid functionality, suggesting that these metabolites may have been formed by oxidative cleavage of the Δ(9,10) bond of diene-functionalized plakortolides. A final group of four metabolites (13-16) with hydroxy or the rare hydroperoxy functionality unexpectedly revealed a C(8) side chain, while the ester (17) represents further structural variation within the growing family of cyclic peroxy sponge metabolites.

Crotalaria medicaginea associated with horse deaths in northern Australia: new pyrrolizidine alkaloids.

Crotalaria medicaginea has been implicated in horse poisoning in grazing regions of central-west Queensland, which resulted in the deaths of more than 35 horses from hepatotoxicosis in 2010. Liver pathology was suggestive of pyrrolizidine alkaloidosis, and we report here the isolation of two previously uncharacterized pyrrolizidine alkaloids from C. medicaginea plant specimens collected from pastures where the horses died. The first alkaloid was shown by mass spectometric and NMR analyses to be 1ß,2ß-epoxy-7ß-hydroxy-1α-methoxymethyl-8α-pyrrolizidine, which, like other alkaloids previously isolated from C. medicaginea, lacks the requisite functionality for hepatotoxcity. The second alkaloid isolated in this investigation was a new macrocyclic diester of otonecine, which we have named cromedine. The (1)H and (13)C NMR spectra of cromedine were fully assigned by 2D NMR techniques and allowed the constitution of the macrocyclic diester to be assigned unambiguously. C. medicaginea specimens implicated in this investigation do not belong to any of the three recognized Australian varieties (C. medicaginea var. neglecta, C. medicaginea var. medicaginea, and C. medicaginea var. linearis) and appear to be a local variant or form, referred to here as C. medicaginea (chemotype cromedine).

Revision of the absolute configurations of bethosides B and C and their aglycone.

The absolute stereochemistry of the steroidal saponins bethosides B and C was previously assigned as (22R,25R) on the basis of work that employed Horeau's method. Our studies of helosides A and B created doubt about both the original assignment and consequently our conclusion that relied upon it. The absolute configurations of bethosides B and C are revised to (22S,25R) following X-ray crystallographic analysis of their aglycone. Synthesis and full spectral characterization of both the 22R and 22S aglycones is reported to facilitate future stereochemical assignments in this series of saponins.

Polyunsaturated alkyl amides from Echinacea: synthesis of diynes, enynes, and dienes.

The synthesis of 20 alkyl amides, including 15 naturally occurring polyunsaturated alkyl amides previously identified from Echinacea spp. (1-13 and 62) or from Achilla sp. (55) and five previously unknown geometric isomers (23, 28, 67, 73, and 80), is described. Importantly, these amides include all of the major alkyl amides present in commercially used Echinacea extracts. The syntheses demonstrate methodology used for constructing alkyl amides containing conjugated diyne and isomerically pure enyne and diene moieties and may be adapted easily for the preparation of other alkyl amides present in Echinacea spp. Terminal-conjugated diynes were prepared by a Cadiot-Chodkiewitz coupling/deprotection sequence utilizing a protected bromoacetylene, and methyl-substituted diynes were made via a base-catalyzed rearrangement of terminal-skipped diynes. Conjugated dienes were prepared conveniently and with high stereoselectivity by the reduction of enynes or diynes with Rieke zinc. With the exception of 1-2 and 11-12, the alkyl amides are synthesized here for the first time, and their NMR data are consistent with that of the reported isolated natural compounds.

Daphnane- and tigliane-type diterpenoid esters and orthoesters from Pimelea elongata.

Investigation of Pimelea elongata ("Lakebed Pimelea") afforded 18 tigliane- and daphnane-type diterpenes (1-18). Eight of these were new compounds: four (1-3, 5) tigliane esters and four (7, 8, 10, 11) daphnane orthoesters. The 10 known compounds were 12-O-decanoylphorbol-13-acetate (4), P. simplex subtoxin B (6), wikstroelide E (9), pimelotides A and B (12, 13), gnidiglaucin (14), simplexin (15), huratoxin (16), kirkinine D (17), and 12-ß-acetoxyhuratoxin (18). The structures and relative configurations of the new compounds were determined by 1D and 2D NMR spectroscopic studies in combination with MS analyses.

Synthesis of the sponge-derived plakortone series of bioactive compounds.

The Caribbean sponges of the genus Plakortis, P. halichondrioides, and P. simplex have provided a series of biologically active furanolactones-the plakortones A-D (1-4) from the former sponge and B-F (2-6) from the latter. The defining motif of the plakortones is a sterically congested 2,6-dioxabicyclo[3.3.0]octan-3-one moiety, the emblematic furanolactone core. This core is efficiently accessed by a palladium(II) mediated hydroxycyclization-carbonylation-lactonization cascade with an appropriate ene-1,3-diol. Total syntheses of plakortones C (3) and F (6) are now described which settle constitutional and stereochemical features in this group of secondary metabolites. Acquisition of plakortone D (4), the most effective activator of SR-Ca(2+)-pumping ATPase, utilized stereodefined lactone cores that resulted from asymmetric dihydroxylation of protected homoallylic alcohol 29. A derived lactone aldehyde was then coupled with an independently generated, sulfone-activated side chain unit, 57. The 11,12-E-double bond, carried through the sequence as a protected, stereodefined diol, was released therefrom by stereospecific syn-elimination via an orthoester derivative. In this way, plakortone D (4) was demonstrated to possess the (3S,4S,6S,10R,11E) configuration. Racemic plakortone E (5) was also acquired by using the Pd(II) induced sequence, but in this case, the required, complete acyclic system 52 was assembled first. Plakortone C (3) resulted from a sequence commencing with (R)-(+)-3-hydroxy-2-methylpropionate, with a derived iodide 76 alkylating the enolate of the butyramide 77 generated from (1S,2S)-(+)-pseudoephedrine. The liberated primary alcohol 79 was converted by standard procedures to key enediol 89 which, with the Pd(II) protocol, afforded the major separable plakortones 90 and 91, with the former being identical with natural plakortone C (3). Very mild hydrogenation of 90 afforded a saturated plakortone, identical with natural plakortone F (6), thus establishing its structure and absolute stereochemistry. Available information on the stereoselective routes to plakortones E (5) and B (2) are also outlined, so that the constitution and absolute stereochemistry of plakortones B-F are now established.

Pimelotides A and B, diterpenoid ketal-lactone orthoesters with an unprecedented skeleton from Pimelea elongata.

A detailed investigation of the minor phytochemical components of Pimelea elongata foliage led to the discovery of two new diterpenoid daphnane ketal-lactone orthoesters with an unprecedented skeleton, pimelotides A (1) and B (2). Their structures and relative configurations were elucidated by NMR spectroscopy.

Steroidal saponins from the roots of Trillium erectum (Beth root).

Eleven steroidal saponins including three previously unreported saponins 1-3, two known ecdysteroids and one fatty acid, have been isolated from the roots of Trillium erectum (Beth root) by RP-HPLC and characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (LCMS) methods.

Spiroacetal biosynthesis in insects from Diptera to Hymenoptera: the Giant Ichneumon wasp Megarhyssa nortoni nortoni Cresson.

The volatile components of the mandibular gland secretion generated by the Giant Ichneumon parasitoid wasp Megarhyssa nortoni nortoni Cresson are mainly spiroacetals and methyl ketones, and all have an odd number of carbon atoms. A biosynthetic scheme rationalizing the formation of these diverse components is presented. This scheme is based on the results of incorporation studies using (2)H-labeled precursors and [(18)O]dioxygen. The key steps are postulated to be decarboxylation of beta-ketoacid equivalents, beta-oxidation (chain shortening), and monooxygenase-mediated hydroxylation leading to a putative ketodiol that cyclizes to spiroacetals. The generality of the role of monooxygenases in spiroacetal formation in insects is considered, and overall, a cohesive, internally consistent theory of spiroacetal generation by insects is presented, against which future hypotheses will have to be compared.

Steroidal saponins from the roots of Asparagus racemosus.

Five steroidal saponins, shatavarins VI-X, together with five known saponins, shatavarin I (or asparoside B), shatavarin IV (or asparinin B), shatavarin V, immunoside and schidigerasaponin D5 (or asparanin A), have been isolated from the roots of Asparagus racemosus by RP-HPLC and characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (LCMS) methods.

Complete (1)H and (13)C assignments of the four major saponins from Dioscorea villosa (wild yam).

Complete (1)H and (13)C spectral assignments for the four major steroidal saponins isolated by methanolic extraction of the roots of Dioscorea villosa, collected in North Carolina, United States (in summer and autumn), are presented in this paper. The structures were determined by a combination of (1)H, (13)C and 2D NMR techniques and were found to be ((3beta,25R)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl-O-beta-D-glucopyranosyl-(1 --> 3)-O-beta-D-glucopyranosyl-(1 --> 4)-O-[alpha-L-rhamnopyranosyl-(1 --> 2)]-beta-D-glucopyranoside (1) (or methyl parvifloside), ((3beta,25R)-26-(beta-D-glucopyranosyloxy)-22 methoxyfurost-5-en-3-yl-O-alpha-L-rhamnopyranosyl-(1 --> 2)-O-[beta-D-gluco- pyranosyl-(1 --> 4)]-beta-D-glucopyranoside (2) (or methyl protodeltonin), (3beta,25R)-spirost-5-en-3-yl-O-beta-D-glucopy ranosyl-(1 --> 3)-O-beta-D-glucopyranosyl-(1 --> 4)-O-[alpha-L-rhamnopyranosyl-(1 --> 2)]-beta-D-glucopyranoside (3) (or Zingiberensis saponin I) and (3beta,25R)-spirost-5-en-3-yl-O-alpha-L-rhamnopyranosyl-(1 --> 2)-O-[beta-Ds-glucopyranosyl -(1 --> 4)]-beta-D-glucopyranoside (4) (or deltonin).

Synthesis and absolute configuration of a constitutionally-new [5.6] spiroacetal from B. tryoni (Queensland fruit fly).

A novel spiroacetal, (2S,6R,8S)-2-methyl-8-ethyl-1,7-dioxaspiro[5.6]dodecane (1), has been identified from the volatile secretions of female B. tryoni by mass spectral analysis and synthesis of an authentic, enantioenriched sample.

RP-HPLC detection of a sulphiting-induced artefact from paeoniflorin in dried roots of Paeonia lactiflora.

Paeoniflorin is one of the bioactive ingredients of the roots of Paeonia lactiflora (Paeoniaceae). A comparative study of processed and non-processed commercial samples of dried roots of P. lactiflora indicated a very low level of paeoniflorin in the processed sample and the formation of a new more polar component, sodium paeoniflorin sulphonate, during treatment of the roots with sulphiting agents.

Polycationic lipophilic-core dendrons as penetration enhancers for the oral administration of low molecular weight heparin.

Two polycationic lipophilic-core carbohydrate-based dendrons 2a-b and five polycationic lipophilic-core peptide dendrons 3-6, containing four arginine or lysine terminal residues, were synthesized and then tested in rats as penetration enhancers for the oral delivery of low molecular weight heparin. Better results were obtained with dendrons containing terminal lysine residues than terminal arginine. A significant anti-factor Xa activity was obtained when low molecular weight heparin was coadministered with dendron 5.

Flexible synthesis of enantiomerically pure 2,8-dialkyl-1,7-dioxaspiro[5.5]undecanes and 2,7-dialkyl-1,6-dioxaspiro[4.5]decanes from propargylic and homopropargylic alcohols.

A new approach to enantiomerically pure 2,8-dialkyl-1,7-dioxaspiro[5.5]undecanes and 2,7-dialkyl-1,6-dioxaspiro[4.5]decanes is described and utilizes enantiomerically pure homopropargylic alcohols obtained from lithium acetylide opening of enantiomerically pure epoxides, which are, in turn, acquired by hydrolytic kinetic resolution of the corresponding racemic epoxides. Alkyne carboxylation and conversion to the Weinreb amide may be followed by triple-bond manipulation prior to reaction with a second alkynyllithium derived from a homo- or propargylic alcohol. In this way, the two ring components of the spiroacetal are individually constructed, with deprotection and cyclization affording the spiroacetal. The procedure is illustrated by acquisition of (2S,5R,7S) and (2R,5R,7S)-2-n-butyl-7-methyl-1,6-dioxaspiro[4.5]-decanes (1), (2S,6R,8S)-2-methyl-8-n-pentyl-1,7-dioxaspiro[5.5]undecane (2), and (2S,6R,8S)-2-methyl-8-n-propyl-1,7-dioxaspiro[5.5]undecane (3). The widely distributed insect component, (2S,6R,8S)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane (4), was acquired by linking two identical alkyne precursors via ethyl formate. In addition, [(2)H(4)]-regioisomers, 10,10,11,11-[(2)H(4)] and 4,4,5,5-[(2)H(4)] of 3 and 4,4,5,5-[(2)H(4)]-4, were acquired by triple-bond deuteration, using deuterium gas and Wilkinson's catalyst. This alkyne-based approach is, in principle, applicable to more complex spiroacetal systems not only by use of more elaborate alkynes but also by triple-bond functionalization during the general sequence.

Insect chemistry and chirality.

Examination of the chemistry of a number of Australian insect species provided examples of unusual structures and encouraged determinations of their absolute stereochemistry by stereocontrolled syntheses and chromatographic comparisons. Inter alia, studies with the fruit-spotting bug (Amblypelta nitida), certain parasitic wasps (Biosteres sp.), the aposematic shield bug (Cantao parentum), and various species of scarab grubs are summarized. The determination of enantiomeric excesses (ee's) for component epoxides, lactones, spiroacetals, and allenes are described. Stereochemical and related aspects of the biosynthesis of spiroacetals in certain fruit-fly species (Bactrocerae sp.) are also presented.

Total synthesis and absolute stereochemistry of plakortone D.

The first total synthesis of plakortone D is described and thereby establishes the structure and absolute stereochemistry of the most biologically active member of the marine-derived plakortone family. The sterically congested bicyclic lactone core results from a Pd(II)-induced hydroxycyclization-carbonylation-lactonization sequence on an enediol whose chirality was installed by AD-technology. Attachment of the side chain, also constructed using AD-methodology, was achieved by using a modified Julia coupling. The described approach enables acquisition of other plakortones and analogues, in the correct (natural) stereochemical series.