The Department of Medicinal Chemistry and Molecular Pharmacology’s laboratory has adopted four “bench top” bioassays which do not require higher animals to screen and direct the fractionation of botanical extracts in drug discovery efforts. These are: 1. The brine shrimp lethality test (BST) (a general bioassay), 2. The inhibition of crown gall tumors on discs of potato tubers (an antitumor bioassay), 3. The inhibition of frond proliferation in duckweed (a bioassay for herbicides and plant growth stimulants), and 4. The yellow fever mosquito larvae lethality test (a bioassay for pesticides). The materials and procedures for those bioassays will be briefly described. The authors’ results in applying these simple methods in the discovery of Annonaceous acetogenins will be presented to illustrate their successful use. The BST is especially suggested as an inexpensive, simple, and rapid means of standardization of bioactivity in heterogeneous botanical products.

Extracts from the seeds of Annona muricata yielded three new Annonaceous acetogenins: muricatetrocin A ( = (5S)-3-{(2R)-2-hydroxy-9-{(2R,5S)-tetrahydro-5-[(1S,4S,5S)-1,4,5-trihydroxyheptadecyl]furan-2-yl}nonyl}-5-methylfuran-2(5H)-one; 1), muricatetrocin B ( = (5S)-{(2R)-2-hydroxy-9-{(2S,5S)-tetrahydro-5-[(1S,4S,5S)-1,4,5-trihydroxyheptadecyl]furan-2-yl}nonyl}-5-methylfuran-2(5H)-one; 2), and gigantetrocin B ( = (5S)-3-{(2R)-2-hydroxy-7-{(2S, 5S)-tetrahydro-5-[(1S,4R, 5R)-1,4,5-trihydroxynonadecyl]furan-2-yl}heptyl}-5-methyl-furan-2(5H)-one; 3). Their C-skeletons were deduced by mass spectrometry. Configurations were determined by H-1-NMR of ketal derivatives and 2D-NMR experiments utilizing Mosher esters. A previously described compound, gigantetrocin A ( = (5S)-3-{(2R)-2-hydroxy-7-{(2S,5S)-tetrahydro-5-[(1S,4S,5S)-1,4,5-trihydroxynona-decyl]furan-2-yl}heptyl}-5-methylfuran-2-(5H)one; 4), was also isolated and is new to this species. Compounds 1-4 were all selectively cytotoxic for the HT-29 human colon-tumor cell line with potencies at least 10 times that of adriamycin.

Two novel eight-membered-ring lactones, gonioheptolides A (1 and B (3), have been isolated from the bark of Goniothalamus giganteus. The structures were elucidated by IR, MS, 1H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra. 1 and 3 showed marginal cytotoxicities to certain human solid tumor cells in culture. A biogenetic pathway of the 14 styryllactones found in G. giganteus is proposed.

The EtOH extract of Asimina parviflora (Annonaceae), when subjected to activity-directed fractionation using lethality to brine shrimp, led to the isolation and identification of five bioactive compounds: asimicilone [1], which is a new 2-quinolone alkaloid, 6-cis-docosenamide [2], which is a new amide of a long hydrocarbon chain fatty acid, and three known compounds, asimicin, (+)-syringaresinol, and beta-sitosterol-beta-D- glucopyranoside. The structure determination of the new alkaloid was performed by extensive nmr analyses, including HMQC and HMBC. Selective cytotoxic activities of these compounds in three human solid tumor cell lines are also reported.

Brine shrimp lethality-directed fractionation of the 95% EtOH extract of ripe berries from Lindera benzoin led to the isolation of three new C21 alkane-alkene gamma-lactones designated isolinderanolide, isolinderenolide, and linderanolide as well as the known series of C17 and C19 obtusilactones (isoobtusilactone A, obtusilactone A, isoobtusilactone, and obtusilactone) previously isolated from Lindera obtusiloba. The novel (6Z,9Z,12Z)-pentadecatrien-2-one, the known (6Z,9Z)-pentadecadien-2-one, and the known (+)-(Z)-nerolidol were also isolated as bioactive compounds. The structural elucidation and biological activities of these compounds are reported.

Giganenin, a novel monotetrahydrofuran acetogenin with a double bond along the hydrocarbon chain, and 4-deoxygigantecin, a new nonadjacent bistetrahydrofuran acetogenin, have been isolated from the bark of Goniothalamus giganteus (Annonaceae) by the use of brine shrimp lethality for bioactivity-directed fractionation. The structures were elucidated based on spectral and chemical methods. Giganenin, which shows selective and highly potent cytotoxicities to human tumor cells in culture (ED50 values as low as 5.80 x 10(-8)-mu-g/ml) and toxicity to brine shrimp, is the most cytotoxic of the monotetrahydrofuran acetogenins reported thus far. 4-Deoxygigantecin is also cytotoxic to human tumor cells and toxic to brine shrimp.

Three additional new neolignans, dysodanthins D, E, and F, and one known neolignan, megaphyllone acetate, have been isolated from the ethanol extract of the roots of Endlicheria dysodantha (Lauraceae). Their structures were solved using spectral data. Results of biological testing suggest further evaluation of megaphyllone acetate as an antitumor agent.

Gigantetronenin [1] and gigantrionenin [6], two new monotetrahydrofuran Annonaceous acetogenins each possessing a double bond along the hydrocarbon chain, have been isolated from the bark of Goniothalamus giganteus by the use of brine shrimp lethality for bioactivity-directed fractionation. The structures were elucidated based on spectroscopic and chemical methods. Compounds 1 and 6 both show selective and potent cytotoxicities to human tumor cells in culture as well as toxicity to brine shrimp. A known cytotoxic acetogenin, annomontacin [11], was also isolated from this plant. The biogenetic pathway of the acetogenins from G. giganteus is discussed.

Three new styryllactones, goniobutenolides A (1) and B (3) and goniofupyrone (5), have been isolated from the bark of Goniothalamusgiganteus (Annonaceae). The structures were elucidated by ir, ms, 1H nmr, 13C nmr, 1H-1H COSY, nOe difference, and NOESY spectra. These compounds are marginally cytotoxic to human tumor cells in culture.

Fractionation of the bioactive constituents of the roots of endlicheria dysodantha mez. (lauraceae) using the brine shrimp lethality bioassay led to the isolation of four bioactive derivatives of benzyl benzoate: benzyl 2-hydroxybenzoate (1), benzyl 2-hydroxy-6-methoxybenzoate (2), benzyl 2,6-dimethoxybenzoate (3), and benzyl 2,5-dimethoxybenzoate (4). the structures of these compounds were identified by spectroscopic methods. this is the first report of these benzyl benzoates being isolated from the genus endlicheria.

Two new styryl lactones, 9-deoxygoniopypyrone [1] and 7-epi-goniofufurone [3], and a known styryl lactone, goniodiol [5], were isolated from the stem bark of Goniothalamus giganteus. The structures were elucidated by ir, ms, 1H-nmr, 13C-nmr, and 1H-1H COSY spectra; the relative configurations were determined by X-ray crystallographic analysis. Unlike goniopypyrone [2] and goniofufurone [4], neither of the new styryl lactones 1 and 3 showed significant bioactivities to human tumor cells. However, goniodiol [5] showed significant and selective cytotoxicity against human lung tumor cells (A-549).

A new bioactive monotetrahydrofuran acetogenin, reticulatacin, containing 37 carbons, has been isolated from a 95% EtOH extract of the bark of A. reticulata by directing the fractionation with brine shrimp lethality. Another previously reported highly potent antitumor, adjacent ring bistetrahydrofuran acetogenin, bullatacin, has also been isolated in large quantity. Two known diterpenes, (−)-kau-16-en-19-oic acid and methyl 16β,17-dihydro-(−)-kauran-19-oate and a known alkaloid, liriodenine, have also been isolated. Some of these compounds showed selective cytotoxic activities for certain human tumor cell lines.

Further fractionation of the EtOH extract of the bark of Annona bullata Rich. (Annonaceae) has led to the isolation of a new nonadjacent bis‐tetrahydrofuran acetogenin, bullatalicinone, and the known adjacent bis‐tetrahydrofuran acetogenin, squamocin. These two compounds showed potent and selective cytotoxicities for certain human tumor cell lines. Their structures have been elucidated by chemical and spectral methods. Bullatalicinone is the methylketo‐lactone derivative of bullatalicin which we found previously in this plant.

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Simple bench-top bioassays involving brine shrimp lethality, Lemna frond proliferation, and the inhibition of crown gall tumours on potato discs, as well as the human tumour cell lines A-549 lung carcinoma, MCF-7 breast carcinoma and HT-29 colon adenocarcinoma, were compared for their accuracy to detect known in vivo (P-388) active antitumour agents supplied by the National Cancer Institute. The potato disc assay was the best and showed excellent correlation to in vivo activity (p = 0.008). The brine shrimp assay (p = 0.033) proved to be superior or equally as accurate as the in vitro human solid tumour cell lines (p = 0.033-0.334). The Lemna assay (p = 0.708) showed the poorest correlation. The brine shrimp and potato disc assays are suggested as convenient in-house prescreens to existing cytotoxicity or antitumour assays.

AbstractGoniofufurone (I) and goniopypyrone (II) possess new natural skeletons.

From the antitumor-bioactive sap of Pogonopus speciosus, tubulosine [1] was isolated, by activity-directed fractionation using the brine shrimp lethality test, as the major antitumor constituent. 1H-nmr assignments, obtained from HETCOR and COSY, and X-ray crystallographic results are reported for the first time. Psychotrine [2] was also isolated, and its spectral data are also reported.

Crude extracts of the berries of Zanthoxylum americanum showed significant lethality to brine shrimp larvae and exhibited cytotoxicity to human tumor cells. Activity‐directed fractionation led to 5 furanocoumarins (cnidilin, imperatorin, isoimperatorin, psoralen, and xanthotoxin), the last three of which are bioactive in these bioassays. All were previously known as natural compounds but are new to this plant species.

Bullatalicin, a novel bioactive acetogenin having two nonadjacent tetrahydrofuran rings, has been isolated from the bark of Annona bullata (Annonaceae). Its structure has been elecidated from chemical and spectral data. This compound showed potent and selective cytotoxic activities for certain human tumor cell lines with ED50 values as low as 10−7 mcg/ml.

Screening of crude extracts of the bark of Annona bullata showed cytotoxic and pesticidal activities. By monitoring with brine-shrimp lethality, two novel, extremely potent acetogenins, bullatacin [1] and bullatacinone [2], were isolated. Spectral and chemical methods identified bullatacin as a diastereomer of asimicin. Bullatacinone represents bullatacin with the lactone cleaved and reformed at the 4-OH. Compounds 1 and 2 show selective cytotoxicities in human tumor cell lines, and certain susceptible cells give ED50 values as low as 10(-12)-10(-15) micrograms/ml. Bullatacin was pesticidal at concentrations as low as 1 ppm, but bullatacinone lacked pesticidal activities. The known compounds liriodenine and (-)-kaur-16-en-19-oic acid were also isolated and were lethal to brine shrimp but were not significantly cytotoxic.