Geoffrey W. Kilbee

Regioselective ether cleavages of rotenoids: spiro-ether formation and stereoselective isotopic labelling of (E)- or (Z)-prenyl methyl groups in (6aS, 12aS)-rot-2′-enonic acid

Treated with boron tribromide (–)-(6aS,12aS,5′R)-rotenone is converted first into a primary allylic bromide by ring-E cleavage, then into the 2-de-O-methyl and finally the 2,3-dide-O-methyl derivatives. With (6aS,12aS,5′R)-6′7′-dihydrorotenone and (6aS,12aS)-isorotenone, ring-E cleavage does not take place. The main reaction is 2-, followed by 2,3-demethylation: this supports a stereospecific pericyclic mechanism for the rotenone ring-E cleavage. Treatment of the geometrically pure (E)-bromide with cyanoboro-deuteride or -tritide leads to (E)-4′-labelled (6aS,12aS)-rot-2′-enonic acid without reduction of the 12-carbonyl group. By using [7′-13C or -14C]-rotenone, (E)-[4′-13C- or -14C-]rot-2′-enonic acid is accessible. Trimethylsilyl iodide can cleave the 2-methoxygroup of rotenone without rupturing ring E, and remethylation with [2H]- or [3H]-diazomethane represents a convenient method for preparing a general tracer molecule.On treatment with sodium hydride, 3-de-O-methylisorotenone (but not the 2-isomer) rearranges into a spiroether, thus confirming the position of initial de-O-methylation as deduced from 1H and 13C n.m.r, data. Because of this rearrangement, methylenation (NaH–CH2I2) of 2,3-dide-O-methylisorotenone gives mainly the methylenedioxy-spiro-ether, with small yields of methylenedioxy-rotenoid.Deuteriogenolysis of (–)-rotenone over palladium catalyst in (2H5)pyridine gives (E)-[4′-2H]rot-2′-enonic acid, but experiments using [7′-13C]rotenone indicate stereoselectivity rather than stereospecificity, ca. 12% of (Z)-[5′-13C]- accompanying the major (E)-product. A similar specimen of [4′-14C]rotenonic acid has been prepared. A hydrogenolysis route from amorphigenin, via[8′-2H]rotenone, to (Z)-[5′-2H]rot-2′-enonic acid is described.

Synthesis of 4″-carboxylated cannabinoids: Stereospecific processes involving ethylidenemalonic ester

Abstract 4″-Carboxylated-cannabidiol,-Δ 1 -and-Δ 1,6 -tetrahydrocannabinols and - cannabinol are synthesised. Condensation between aromatic aldehydes and ethylidenemalonic ester gives a 2 E ,4 E -half ester stereospecifically, a reaction which can be used to make 2 E -4 E - or 2 Z ,4 E -pentadienoates.

Carbon-13 magnetic resonance spectra of natural rotenoids and their relatives

Natural abundance 13C n.m.r. spectra of six rotenoids and eighteen related structures have been measured by pulse-Fourier transform techniques. From use of noise-decoupling, and single frequency off-resonance decoupling, a self-consistent series of assignments has emerged.

Biosynthesis of rotenone and amorphigenin. Study of the origins of isopropenyl-substituted dihydrofuran E-rings using isotopically labelled late precursors

Whilst epoxidation of rot-2′-enonic acid is the most likely source of dalpanol in Amorpha fruticosa seedlings, administration of (5′R,6′S)-[7′-3H]dalpanol shows that it is not an intermediate on the path to rotenone and amorphigenin. Labelled 4′-hydroxy- or 5′-hydroxy-rot-2′-enonic acid also do not qualify as intermediates in rotenone biosynthesis, but they are each converted into amorphigenin with chemospecific attack on the methyl group. By administration and re-isolation of [8′-14C]amorphigenin from A. fruticosa seedlings, our earlier conclusion that hydroxylation of rotenone to form amorphigenin proceeds with even label scrambling between C-7′ and C-8′, probably via an allylic radical, is confirmed. Competitive double-labelling experiments are employed to support a scheme in which rotenone derives directly from rot-2′-enonic acid by an enzyme-induced radical-type reaction without the intervention of an hydroxylated intermediate, and the two labelled hydroxyrot-2′-enonic acids are similarly cyclised using their methyl groups. The incorporations into amorphigenin of labelled 4- and 5-hydroxyrot-2′-enonic acids, both of which are shown to occur naturally in A. fruticosa, are similar, but only about one sixth that of rotenone.This, and our related biosynthetic work, rests on an extensive programme of isotopic labelling and reconstructive synthesis. Our earlier method for making [7′-14C]-rotenone has been improved, and similar procedures adapted for [7′-13C]- and [7′-14C]-amorphigenin, 8′-Labelled rotenones are made by a positional interchange using addition of benzeneselenenyl chloride and elimination of the selenoxide, whilst [8′-14C]amorphigenin is made via addition of phenylselenophthalimida. Unlabelled amorphigenin can be isotopically labelled by oxidation to the aldehyde and reduction using sodium borodeuteride or borotritide and a method additional to those we have described earlier is given for tritium labelling of rot-2′-enonic acid. [13C]- and [14C]-Labelling in the 5′-position of 4′-and 5′-hydroxyrot-2′-enonic acids can be attained through the catalytic hydrogenolysis of [7′-14C]amorphigenin though special methods must be used to scrub the samples totally free from the latter. Methods based on the hydrolysis of labelled 4′-bromorot-2′-enonic acid are also described, and 4′-tritium-labelled 4′-hydroxyrot-2′-enonic acid is made from unlabelled material, or from rot-2′-enonic acid, by simple oxidation/reduction methods.

Biosynthesis of rotenoids by Amorpha fruticosa: sequence, specificity, and stereochemistry in the development of the hemiterpenoid segment

By isolation and radiochemical methods rot-2′-enonic acid, dalpanol, rotenone, and amorphigenin have been identified in Amorpha fruticosa seedlings, and ordered in biosynthetic sequence. Its specific activity shows that 12aβ-hydroxyamorphigenin has origins other than direct 12aβ-hydroxylation of amorphigenin: its occurrence and labelling establish it as a true natural product.Except for isopentenyl alcohol, the potential hemiterpene precursors mevalonic acid, 3-hydroxy-3-methylglutaric acid, and dimethylallyl alcohol are poor precursors for amorphigenin. By employing the already prenylated (E)-[4′-14C]rot-2′-enonic acid, it is shown that the 4′-C of this compound becomes 7′-C of rotenone. By assuming normal rear-side attack on an intermediate epoxide, and utilising the known absolute configurations at position 5′ of dalpanol and rotenone, a stereochemical sequence can be written. (E)-4′-Labelled rot-2′-enonic acid leads to a (2′S,3′S)-epoxide, which on intramolecular attack by phenolate anion would give (5′R,6′S)-dalpanol, dehydration to rotenone then involving the labelled (pro-S)-7′-methyl group of dalpanol.Neither (6′R)- nor (6′S)-amorphigenol is a precursor of amorphigenin. Administration of [7′-14C]rotenone to A. fruticosa seedlings has led, in three experiments designed to avoid inadvertent chemical scrambling of the allylic label, to amorphigenin having even label distribution between C-7′ and C-8′. Possible interpretations are considered.

Formation and dehydration of a prochiral 2-hydroxyisopropyl centre during biosynthesis: the rot-2′-enonic acid–rotenone transformation in Amorpha fruticosa

Rot-2′-enonic acid (2), dalpanol (4), and rotenone (5) are formed during the conversion of 9-demethylmunduserone (1) into amorphigenin (6) by Amorpha fruticosa seedlings: the 4′-E-methyl of (2) becomes predominantly the 7′-methylene of (5), leading to stereochemical proposals for a formation scheme.

Synthesis of [7′-14C]- and [7′-13C]-rotenone, and [4′-14C]- and [4′-13C]-rot-2′-enonic acid: 1,4-allylic hydrogenolysis showing substantial stereoselectivity

C-Trimethylsilyloxy protection of the B/C-ring junction allows regenerative synthesis of (–)-rotenone isotopically labelled at the C-7′-methylene, without loss of chirality; 1,4-hydrogenolysis of (–)-rotenone labelled in this way proceeds stereoselectively with transference of 88% of the label to the 4′-E-methyl of (–)-rot-2′-enonic acid.

Identification and isotopic labelling of the diastereotopic methyl groups of the prenyl-derived 2-hydroxyisopropyl residues of natural products: the rotenoid dalpanol

The 2-hydroxyisopropyl (1 -hydroxy-1 -methylethyl) segment derived from a prenyl residue occurs in a number of natural products, including the rotenoid dalpanol, and the aim of this work is the identification and isotopic labelling of the (pro-R) and (pro-S)-methyls of the latter compound. The identification sequence involves linkage between (4′-E)-labelled rot-2′-enonic acid and the labelled (5′S,6′R)-stereoisomer of dalpanol (5′-epidalpanol)via the (2′R,3′R)-epoxide of absolute configuration known from X-ray analysis. Dalpanol of the natural (5′R)-series is labelled in the (6′S)-methyl with deuterium by this sequence and the procedure is applicable to 3H, 13C- and 14C labelling since such 7′-labelled specimens of rotenone, the precursors of 4′-labelled rot-2′-enonic acids, are available from our earlier work.A second labelling method employs the separable rotenone (5′R,6′S)- and (5′R,6′R)-epoxides, which are reduced with lithium aluminium deuteride, followed by reoxidation at C-12a to give both 2H-labelled (5′R,6′S)- and (5′R,6′R)-dalpanols. Oxymercuriation at the 6′,7′-double bond of rotenone is selective, though not specific, for the si-face, leading mainly to the (6′R)-mercuriated product. Purification of the latter, followed by reduction with sodium borotritide, provides an excellent means for the preparation of specifically radiolabelled (5′R,6′S)-dalpanol. Monitoring was by 3H NMR spectroscopy, which is more revealing than the deuterium equivalent.

Hydroxylation of rotenone to amorphigenin in Amorpha fruticosa seedlings

Rotenone (2), but neither of the 6′-epimers of amorphigenol (3), is an excellent precursor for amorphigenin (1) in Amorpha fruticosa; using [7′-14C]rotenone and three different work-up techniques, the 14C-label was found to be equivalently distributed between the 7′-methylene and 8′-hydroxymethylene of amorphigenin.