Pestic. Sci. 1997, 49, 372È378

Comparative Evaluations ofSAR AnnonaceousAcetogenins for Pesticidal ActivityKan He, Lu Zeng, Qing Ye, Guoen Shi, Nicholas H. Oberlies, Geng-Xian Zhao, C.Jesse Njoku & Jerry L. McLaughlin*

Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and PharmacalSciences, Purdue University, West Lafayette, Indiana 47907 USA

(Received 10 June 1996 ; revised version received 25 August 1996 ; accepted 29 November 1996)

Abstract : The activities of 44 Annonaceous acetogenins, which were originallyisolated by monitoring plant fractionations with the brine shrimp lethality test(BST), were evaluated in the yellow fever mosquito larvae microtiter plate (YFM)assay. The results clearly demonstrate that most acetogenins have pesticidalproperties. The structureÈactivity relationships indicate that the compoundsbearing adjacent bis-THF (tetrahydrofuran) rings with three hydroxyl groups arethe most potent. Bullatacin (1) and trilobin (7) gave the best activities againstYFM with values of 0É10 and 0É67 mg litre~1, respectively. CompoundsLC50showing values below 1É0 mg litre~1 in this assay are usually consideredLC50signiÐcant as new lead candidates for pesticidal development. In the BST, thecorresponding values were 1É6 ] 10~3 (1) and 9É7 ] 10~3 (7) mg litre~1.LC50This is the Ðrst report of pesticidal structureÈactivity relationships for a series ofAnnonaceous acetogenins which are known to act, at least in part, as potentinhibitors of mitochondrial NADH: ubiquinone oxidoreductase.

Key words : Annonaceous acetogenins, biopesticide, structureÈactivity relation-ship, brine shrimp lethality test, yellow fever mosquito larvae microtiter plateassay, plant extracts, mitochondrial enzyme inhibitors

1 INTRODUCTION

The Annonaceous acetogenins are naturally occurringlong-chain fatty acid derivatives possessing uniquestructures and powerful antitumor and pesticidal activ-ities. They are only found in the plant family, Annona-ceae. In India, preparations from seeds, leaves, bark orfruit of Annona muricata L., A. reticulata L. and A. squa-mosa L. are widely employed by local people for emetic,astringent, insecticidal, pesticidal, anthelminthic andantidysenteric purposes.1 The report of the Ðrst acetoge-nin, uvaricin, in 1982,2 stimulated interest in the dis-covery of additional new bioactive compounds of thistype. To date, more than 220 acetogenins have been iso-lated from 26 Annonaceous species by bioassay-directedfractionation and phytochemical isolation methods.3These Annonaceous acetogenins have exhibited a diver-sity of bioactivities including antitumor, pesticidal,insect antifeedant, antimalarial, T-cell suppressant, anti-parasitic and antimicrobial properties.4h9

* To whom correspondence should be addressed.

The chemical structures of the acetogenins are con-sidered to be derived from C-32 or C-34 long-chainfatty acids which are combined with a 2-propanol unitat C-2 to form an a,b-unsaturated c-lactone ring. In thebiogenesis, double bonds along the fatty acid chainseem to epoxidize and cyclize to form tetrahydrofuran(THF) rings. According to the number and positions ofthe THF rings, the linear acetogenins are classiÐed inmono-THF, adjacent bis-THF, non-adjacent bis-THFand tri-THF ring systems. One compound (mucocin, 21)has recently been found which has a tetrahydropyran(THP) ring as well as a non-adjacent THF ring.3 Thesources, biogenesis, isolation, chemistry, synthesis andbiological activities of Annonaceous acetogenins havebeen extensively reviewed.3,10h13

The remarkable pesticidal, insecticidal and anti-feedant properties of the Annonaceous acetogenins havebeen reported14h16 and patented.5h7 A standardizedextract of Asimina triloba Dunal, which is rich in aceto-genins, shows strong pesticidal activities. Asimicin (11),a major biologically active component isolated fromthis species, demonstrated promising activities against

372Pestic. Sci. 0031-613X/97/$17.50 1997 SCI. Printed in Great Britain(

SAR evaluations of Annonaceous acetogenins for pesticidal activity 373

Fig. 1. Bis-THF ring acetogenins.

Mexican been beetles, Epilachna varvestis Muls., melonaphids, Aphis gossypii Glov., mosquito larvae, Aedesaegypti L., the nematode, Caenorhabditis elegansMaupas, blowÑy larvae, Calliphora vicina R. & D. andstriped cucumber beetles, Acalymma vittata F.16 Thepesticidal e†ect of the acetogenins is exerted, at least inpart, via inhibition of complex I (NADH: ubiquinoneoxidoreductase) in mitochondrial electron transportsystems ; this inhibition results in the deprivation ofATP at cellular levels.16h18

Examination of a series of structurally diverseAnnonaceous acetogenins in an assay with yellow fevermosquito (Ae. aegypti) larvae, enables us to evaluate therelative potencies of their bioactivities as well as theirpesticidal structureÈactivity relationships. Acetogeninswith pesticidal properties used in this study possessseveral common structural features : they contain one ortwo THF (or THP) rings with one or two Ñankinghydroxyl groups ; the bis-THF rings can be either adja-cent or non-adjacent. A c-lactone ring is at one termin-

Fig. 2. Non-adjacent bis-THF ring, adjacent bis-THF ringwith one Ñanking hydroxyl and non-adjacent tetrahydropyran

and mono-THF ring acetogenins.

Fig. 3. Mono-THF ring acetogenins with two Ñankinghydroxyls.

374 Kan He et al.

Fig. 4. Mono-THF ring acetogenins with one Ñankinghydroxyl.

al, and other functional groups include hydroxyls,acetoxyls and double bonds (Figs 1È4).

2 EXPERIMENTAL

2.1 Isolation of acetogenins

All the acetogenins used in this study were isolated fromeither Annona muricata L., Annona bullata Rich.,Asimina longifolia Kral, Asimina triloba Dunal, Gonio-thalamus giganteus Hook. f. and Thomas, or Rolliniamucosa Baill. by our research group. The isolations werebioassay-directed using the brine shrimp (Artemia salinaL.) lethality test (BST).19,20 The plant extracts containcomplex mixtures of acetogenins with some compoundspresent in very small concentrations ; thus, repeatedchromatographic methods are required to separate theirmixtures. In our laboratory, Alkofahi et al.14 havereported a general scheme for the isolation whichinvolves extraction with ethanol of the selected plant

materials (usually bark, leaves or seeds) to obtain theresidue of the ethanol extract (F001). F001 is then sub-jected to partitioning between chloroform (ordichloromethane) and water to give the water-soluble(F002), chloroform-soluble (F003) and insoluble inter-face residues (F004). F003 is further partitioned betweenhexane and 90% aqueous methanol to producemethanol-soluble (F005) and hexane-soluble (F006) resi-dues. The pure acetogenins are obtained from F005 byrepeated chromatography over open columns (silicagel), chromatotrons and/or HPLC (normal and reversedphases). The BST is routinely employed to monitor theextraction and isolation of the bioactive compounds,and the results correlate well with a series of pesticidaltests.14 The values in the BST for the acetogeninsLC50are listed in Table 1 for the purpose of comparison withthe results of the YFM test. The values of rote-LC50none are included as a positive control. The purities andcharacterizations of each isolated acetogenin wereevaluated by TLC, HPLC, [1H]NMR and [13C]NMR,and their sources and appropriate reference pub-lications are cited in our published reviews.3,10h12

2.2 Yellow fever mosquito microtiter plate assay

The yellow fever mosquito larvae microtiter plate assay(YFM)21 was used to determine the relative pesticidalactivities of the series of acetogenins (Table 1). Theyellow fever mosquito (Ae. aegypti) eggs were providedby Dr Steve Sackett (New Orleans Mosquito ControlBoard, New Orleans, Louisiana 70126). The eggs wereplaced into 200È300 ml of water and were allowed tohatch overnight. The larvae were then transferred to a400-ml beaker containing bovine liver powder (ICNBiochemicals) at a concentration of 2È4 g litre~1 andallowed to develop for four days. Harvested livinglarvae were transferred with a 10-ml plastic transferpipette into a 20-ml scintillation vial Ðlled with MES (2-[N-morpholino]ethanesulfonic acid,) bu†er (5 mM, pH6É5 ; 10 ml) ; the larvae were then ready for the followingexperiment. A Vaccupette/96 was used to Ðll 96-well Ñatbottom assay plates with 240 kl of 5 mM MES per well.A single larva was added per well by using a Model edpmicroliter repipettor (Rainin Instrument), and the testacetogenins in methanol (5 kl) were subsequently intro-duced to each well. Rotenone was used as a positivecontrol and a 20 ml litre~1 methanol solution was runas a negative control in each experiment. Dilutions(1 : 10) of test compounds in methanol were preparedstarting at 500 mg litre~1 or 50 mg litre~1, dependingupon the available quantities of each sample. Two com-pounds per plate and eight replicates per dose wereused. After adding the solutions of the test compounds,the plates were covered and incubated in the darkat 25È28¡C for four days. The plate was then scoredfor dead larvae. values and 95% conÐdenceLC50intervals in mg litre~1 were calculated using a Finney

SAR evaluations of Annonaceous acetogenins for pesticidal activity 375

TABLE 1Results of Brine Shrimp Lethality (BST) and Yellow Fever Mosquito (YFM) Larvae Tests for Annonaceous

Acetogenins

Compounds BST activities (L C50 mg litre~1)a Y FM activities (L C50 mg litre~1)a

Bullatacin (1) 1É6 ] 10~3 (0É8 ] 10~3È12É4 ] 10~2) 1É0 ] 10~1 (2É0 ] 10~2È3É9 ] 10~1)Trilobin (7) 9É7 ] 10~3 (5É3 ] 10~3È1É6 ] 10~2) 6É7 ] 10~1 (3É7 ] 10~2È2É1 ] 10~1)Trilobacin (8) 8É7 ] 10~3 (5É2 ] 10~3È14É4 ] 10~3) 1É6 (5É0 ] 10~1È5É0)Asiminacin (9) 5É7 ] 10~3 (3É5 ] 10~3È9É0 ] 10~3) 1É6 (5É0 ] 10~1È5É0)Asimicin (11) 2É6 ] 10~2 (1É5 ] 10~2È5É3 ] 10~2) 2É7 (8É1 ] 10~1È8É7)Motrilin (3) 1É0 ] 10~2 (0É6 ] 10~3È1É9 ] 10~2) 4É7 (1É6È14É1)Rollitacin (6) 4É7 (1É6È14É1)Bullatetrocin (2) 3É1 ] 10~1 (2É0 ] 10~1È4É6 ] 10~1) 6É2 (1É9È18É9)12-OH Bullatacin A (4) 8É0 (2É0È27É7)12-OH Bullatacin B (5) 8É0 (2É0È27É7)Trilobacinone (9) 3É1 ] 10~1 (1É6 ] 10~1È5É3 ] 10~1) 10É4 (1É4È28É9)10-OH Asimicin (12) 4É3 ] 10~1 (2É8 ] 10~1È6É7 ] 10~1) 15É2 (4É8È30É9)Longimicin D (10) 4É6 (1É3È9É2) 11É5 (3É5È31É5)Longimicin B (14) 7É3 (4É0È15É7) 27É3 (8É1È87É3)Longimicin C (15) 9É4 (6É6È18É4) 65É7 (18É6È171É8)Sylvaticin (16) 8É0 ] 10~1 (0É34È1É7) 1É6 (5É0 ] 10~1È5É0)cis-Sylvaticin (17) 1É1 (6É0 ] 10~1È2É7) 6É0 (1É7È23É4)Bullatalicin (18) 1É5 ] 10~1 (5É1 ] 10~2È2É9 ] 10~1) 9É0 (2É4È57É1)Rollidecin A (19) 4É2 ] 10~1 (2É6 ] 10~1È6É7 ] 10~1) 11É2 (3É5È31É3)Rollidecin B (20) 2É8 ] 10~1 (1É4 ] 10~1È4É3 ] 10~1) [ 50Mucosin (21) 1É8 (1É0È1É8) 2É1 (6É4 ] 10~1È6É8)Annonacin (22) 3É1 ] 10~1 (1É7È7É4) 3É0 (8É0 ] 10~1È6É7)Xylomaticin 4-OAc (26) 34É1 (11É8È53É2) 6É2 (1É9È18É9)Annonacin 4-OAc (24) 21É9 (15É3È40É1) 6É2 (1É9È18É9)Giganenin (29) 89É0 (65É0È101É4) 7É8 (2É0È26É1)Annonacin A (23) 8É3 ] 10~1 (4É8 ] 10~1È1É4) 10É8 (1É4È28É9)Goniothalamicin B (25) 13É3 (3É6È29É7)Rollinecin A (27) 3É6 ] 10~1 (5É3 ] 10~2È1É5 ] 10~1) [ 50Rollinecin B (28) 1É3 ] 10~1 (6É6 ] 10~2È2É2 ] 10~1) [ 50Longifolicin (31) 3É5 (1É8È6É3) 249 (50É3È600)Longicoricin (30) 1É6 (0É3È2É7) 58É0 (12É1È101É6)Muricatetrocin B (38) 1É8 (8É5 ] 10~1È3É8) 2É2 (5É7 ] 10~1È7É3)Gigantetrocinone (32) 1É3 ] 10~1 (1É0 ] 10h1È2É0 ] 10~1) 2É2 (5É7 ] 10~1È7É3)Gigantrionenin (33) 13É9 (5É3È31É5) 4É0 (1É3È13É2)Gigantetroneninone (35) 1É1 ] 10~1 (1É7 ] 10~2È2É3 ] 10~1) 8É3 (2É8È14É8)cis-Gigantrionenin (34) 2É5 (1É3È4É3) 8É6 (2É2È33É7)Gigantetrocin A (37) 6É0 ] 10~1 (3É5 ] 10~1È1É9) 10É4 (1É4È28É9)Gigantriocin (36) 5É6 (2É0È10É0) 18É5 (4É1È44É9)Muricatetrocin C (39) 7É6 ] 10~1 (2É5 ] 10~1È1É4) [ 50Annopentocin A (41) 8É9 (4É5È20É5) [50Annopentocin B (42) 11É2 (6É3È21É3) [50Annopentocin C (40) 13É8 (5É4È31É2) [50Murihexocin A (43) 28É6 (15É5È62É1) [50Murihexocin B (44) 33É7 (18É7È70É8) [50Rotenone (positive control) 4É9 ] 10~2 (2É3 ] 10~2È9É3 ] 10~2) 1É2 (1É2 ] 10~1È4É1)

a 95% conÐdence intervals in parentheses

Probit analysis computer program,20 and the relativeactivities of each acetogenin were subsequently evalu-ated (Table 1).

3 RESULTS AND DISCUSSION

Based on the results of the YFM assay, as shown inTable 1, several general conclusions about the pesticidal

structureÈactivity relationships (SAR) of Annonaceousacetogenins can be drawn: (i) most acetogenins tested inthis study are toxic to the yellow fever mosquito larvaeat concentrations of less than 50 mg litre~1, (ii) theadjacent or non-adjacent bis-THF ring acetogeninsshow higher potencies of activity than the mono-THFring compounds which possess the same number ofhydroxyl groups ; and (iii) the potency of activity is

376 Kan He et al.

related to the number and the positions of the hydroxylgroups and the positions of the THF rings.

Bullatacin (1) and trilobin (7) exhibited activitiessuperior to those of the other acetogenins. The highpotencies of bullatacin and trilobin are not restricted tothe YFM assay. They are also found to be two of themost powerful acetogenins in human solid tumor cellcytotoxicity and rat liver mitochondrial inhibitionassays,12,22h25 and they are among the four most potentacetogenins in the BST (Table 1). The two compoundshave similar levels of bioactive potencies. A positionalshift of the third hydroxyl group, as well as the stereo-conÐguration of the bis-THF ring system, seems toinÑuence the potencies, e.g. in the cases of motrilin (3),trilobacin (8), asiminacin (10) and asimicin (11).

The importance of the polarities of the molecules ina†ecting the biological activities of acetogenins has beenrecognized for some time. The large potency di†erence(a factor of 10) between bullatacin and bullatetrocin (2),12-hydroxy bullatacin A (4), 12-hydroxy bullatacin B (5)and rollitacin (6), which all possess four hydroxylgroups, indicates the strong inÑuence of the number ofhydroxyl groups. The extra hydroxyl group in thesefour-hydroxylated bullatacin type acetogenins increasesthe polarity of the molecules but decreases their bio-activities. A similar situation is observed between asimi-cin and 10-hydroxy asimicin (12) in which asimicin, withone less hydroxyl, has enhanced potency. It appearsthat, to obtain optimum activities, three hydroxylgroups are required in adjacent bis-THF acetogeninmolecules.

We also note that the position of the THF rings inthe acetogenin molecules has a remarkable e†ect on thespeciÐc structural requirements for activity, i.e. a suit-able chain length between the THF ring and c-lactonering is required. Bullatacin (1), trilobin (7), asiminacin(10), asimicin (11), trilobacin (8) and motrilin (3) all havethe THF rings located at C-15 to C-24 relative to theterminal c-lactone ring, and all show highly potent bio-activities against yellow fever mosquito larvae, rangingfrom 0É10 to 5É0 mg litre~1. Among the several acetoge-nins of the asimicin type, asiminacin (10) and asimicin(11) are active compounds in the YFM assay with LC50values of 1É6 and 2É7 mg litre~1, respectively. Longimi-cins B (14), C (15) and D (13) are structurally di†erentfrom asiminacin and asimicin in that their THF ringsystems are shifted along the chain toward the c-lactone. Longimicin D (13, THF rings at C-13 to C-22and the third hydroxyl at C-10 instead of C-4) andlongimicin B (14, THF rings at C-11 to C-20) show adecreasing trend of activities, with values of 11É5LC50and 27É3 mg litre~1, respectively ; while longimicin C(15), with the ring position further shifted to C-9 toC-18, has its activity further reduced to 65É7 mgLC50litre~1.

The three non-adjacent THF ring acetogenins, cis-and trans-sylvaticin (17 and 16) and bullatalicin (18),

exhibited comparable toxicity to the mosquito larvae,although they contain four hydroxyl groups. It is, there-fore, suggested that a THF ring at C-20 might beimportant for maximal bioactivity. The recently dis-covered acetogenin, mucocin (21), with a tetra-hydropyran (THP) ring at C-20 and a THF ring atC-12, showed very good activity at 2É1 mg litre~1.LC50This is the Ðrst reported acetogenin with a THP ring.26

The mono-THF ring acetogenins, while less potent,still displayed similar SAR trends to those with thebis-THF ring systems. Both the hydroxyl numbers andthe positions of the THF rings are important in deter-mining their relative potencies. The most active mono-THF ring compounds tested in this study were thosemolecules with four hydroxyl groups and with the THFring positioned at C-15 to C-20, such as in annonacin(22) and annonacin A (23). With the shifts of the THFring to C-17, such as in rollinecins A (27, LC50[ 50 mglitre~1) and B (28, litre~1) or to C-13,LC50 [ 50 mgand/or the loss of one of the four hydroxyl groups, sig-niÐcant decreases in the activities were observed, e.g. inlongicoricin (30, 58É0 mg litre~1) and in longifoli-LC50cin (31, 249 mg litre~1), respectively. However, aLC50close structural relative of longifolicin is giganenin (29) ;giganenin exhibited activity 20-fold higher than that oflongifolicin, suggesting that the double bond in gigan-enin plays an important role in biopotency.

The class of mono-THF ring acetogenins having oneÑanking hydroxyl group gives maximum activities withthree or four hydroxyl groups, e.g., gigantetrocinone(32), gigantrionenin (33), gigantetroneninone (35), cis-gigantrionenin (34) and gigantetrocin A (37). Three Ðve-hydroxyl (40È42) and two six-hydroxyl (43 and 44)substituted mono-THF acetogenins were found to beinactive at concentrations less than 50 mg litre~1.When the THF ring was shifted from C-9 to C-11(muricatetrocin C, 39), activity against YFM but notBST was lost at concentrations of \50 mg litre~1.Muricatetrocin B (38), a C-20 epimer of muricatetrocinC, exhibited signiÐcant activity over its isomer thatcould only be explained by stereoselectivity. A similarexample is exhibited between the C-24 epimers, rollide-cins A (19) and B (20), where A showed activity at LC5011É2 mg litre~1, while no activity was observed at con-centrations of \50 mg litre~1 in rollidecin B. Onceagain, although activity against YFM was lost in goingfrom rollidecin A to B, activity in the BST was not lostbut increased slightly in going from 19 to 20.

4 CONCLUSIONS

The pesticidal properties of the Annonaceous acetoge-nins have been known for several years.6 The pesticidalSARs indicate that, of all the acetogenins tested so far,the adjacent bis-THF ring molecules with threehydroxyl groups are the most potent. Bullatacin (1) andtrilobin (7) are the strongest and give valuesLC50

SAR evaluations of Annonaceous acetogenins for pesticidal activity 377

against YFM of 0É10 and 0É67 mg litre~1, respectively ;while rotenone, as a positive control gave 1É2 mgLC50litre~1. Usually, compounds showing valuesLC50below 1É0 mg litre~1 in the YFM test are consideredsigniÐcant as new leads for pesticidal development.

Acetogenins show a tremendous potential for devel-opment as new natural pesticides due to their potentbioactivities, their known mechanism of action, theirnatural origin and their expected low oral toxicities inhigher animals (they are emetic).27 The use of naturalacetogenin mixtures in plant extracts is predicted as aneconomical and practicable means for pest control. Forexample, two Annonaceous species, Asimina trilobaDunal (paw paw) and Annona muricata L. (sour sop,guanabana), are, respectively, abundant as fruit trees intemperate eastern North America and in the tropicsworldwide. Both of these species have now yielded avariety of new acetogenins (over 30 acetogenins each),and their crude extracts exhibit potent pesticidal e†ects.These extracts, which can be easily prepared, can beemployed as safe, e†ective, economical and environ-mentally friendly pesticides with an emphasis on thehome garden, ornamental, greenhouse and producemarkets, pending regulatory approval.27

ACKNOWLEDGEMENTS

We gratefully acknowledge Ðnancial support from theNational Cancer Institute, National Institutes of Health(R01 grant no. CA 30909). Thanks are also due to con-tract support from FMC Corporation, Princeton, NewJersey, and Xenova Limited, Berkshire, England.

REFERENCES

1. Chopra, R. N., Nayar, S. L. & Chopra, I. C., Glossary ofIndian Medicinal Plants. Council of ScientiÐc and Indus-trial Research, New Delhi, 1956, pp. 19È20.

2. Jolad, S. D., Ho†mann, J. J., Schram, K. H., Cole, J. R.,Tempesta, M. S., Kriek, G. R. & Bates, R. B., Uvaricin, anew antitumor agent from Uvaria accuminata(Annonaceae). J. Org. Chem., 47 (1982) 3151È3.

3. Zeng, L., Ye, Q., Oberlies, N. H., Shi, G., Gu, Z.-M., He,K. & McLaughlin, J. L., Recent advances in Annonaceousacetogenins. Nat. Prod. Rep., 13 (1996) 275È306.

4. Moeschler, H. F. PÑuger, W. & Wendish, D., Pureannonin and process for the preparation. US Patent 4 689232, 1987.

5. Moeschler, H. F., PÑuger, W. & Wendish, D., Annonininsecticide. German Patent DE 3 438 763 A1, 1986.

6. Mikolajczak, K. L., McLaughlin, J. L. & Rupprecht, J. K.,Control of pests with Annonaceous acetogenins. USPatent 4 721 727, 1988.

7. Mikolajczak, K. L., McLaughlin, J. L. & Rupprecht, J. K.,Control of pests with Annonaceous acetogenins. USPatent 4 855 319, 1989.

8. Cave, A., Hocquemiller, R., & Laprevote, O., UtilisationdÏacetogenins en therapeutique en tant que substancesantiparasitaires. French Patent 1048 No. 88 09 674.

9. Oberlies, N. H., Jones, J. L., Corbett, T. H., Fotopoulos, S.S., & McLaughlin, J. L., Tumor cell growth inhibition byseveral Annonaceous acetogenins in an in-vitro disk di†u-sion assay. Cancer L ett., 96 (1995) 55È62.

10. Rupprecht, J. K., Hui, Y.-H. & McLaughlin, J. L., Annon-aceous acetogenins : A review. J. Nat. Prod., 53 (1990)237È78.

11. Fang, X.-P., Rieser, M. J., Gu, X.-M., Zhao, G.-X. &McLaughlin, J. L., Annonaceous acetogenins : an updatedreview. Phytochem. Anal., 4 (1993) 49È67.

12. Gu, Z.-M., Zhao, G.-X., Oberlies, N. H., Zeng, L. &McLaughlin, J. L., Annonaceous acetogenins : Potentmitochondrial inhibitors with diverse applications. InRecent Advances In Phytochemistry, ed. J. T. Arnason, R.Mata & J. T. Romeo. Plenum Press, New York, 1995, pp.249È310.

13. Cave, A., Acetogenins from Annonaceae. In Phyto-chemistry of Plants Used in T raditional Medicine, ed. K.Hostettmann, A. Marston, M. Maillard & M. Hamburger.Clarendon Press, Oxford, 1995, pp. 227È48.

14. Alkofahi, A., Rupprecht, J. K., Anderson, J. E., McLaugh-lin, J. L., Mikolajczak, K. L. & Scott, B. A., Search forNew Pesticides from Higher Plants. In Insecticides ofPlant Origin, ed. J. T. Arnason, B. J. R. Philogene & P.Morand. American Chemical Society, Washington, DC.,1989, pp. 25È43.

15. Ratnayake, S., Rupprecht, J. K., Potter, W. M. &McLaughlin, J. L., Evaluation of various parts of the PawPaw Tree, Asimina T riloba (Annonaceae), as commercialsources of the pesticidal Annonaceous acetogenins. J.Econ. Entomol., 85 (1992) 2353È6.

16. Lewis, M. A., Arnason, J. T., Philogene, B. J., Rupprecht,J. K. & McLaughlin, J. L., Inhibition of respiration at siteI by asimicin, an insecticidal acetogenin of the Paw Paw,Asimina triloba (Annonaceae). Pestic. Biochem. Physiol., 45(1993) 15È23.

17. Ahammadsahib, K. I., Hollingworth, R. M., McGovern, P.J., Hui, Y.-H. & McLaughlin, J. L., Mode of action ofbullatacin : A potent antitumor and pesticidal Annona-ceous acetogenin. L ife Sci., 53 (1993) 1113È20.

18. Londerhausen, M., Liecht, W., Leib, F. & Moeschler, H.,Molecular mode of action of annonins. Pestic. Sci., 33(1991) 427È38.

19. Meyer, B. M., Ferrigni, N. R., Putnam, J. E., Jacobsen, L.B., Nichols, D. E. & McLaughlin, J. L., Brine shrimp: Aconvenient general bioassay for active plant constituents.Planta Med., 45 (1982) 31È4.

20. McLaughlin, J. L., Crown gall tumors on potato discs andbrine shrimp lethality : two simple bioassays for higherplant screening and fractionation. In Methods in PlantBiochemistry, ed. K. Hostettmann. Academic Press,London, 1991, Vol. 6., pp. 1È32.

21. Anonymous, Instructions for determining the suscepti-bility or resistance of mosquito larvae to insecticides. W ld.Hlth Org. Rep. Ser. (1970) No. 443, 66È79.

22. Gu, Z.-M., Zeng, L., Schwelder, J. T., Wood, K. V. &McLaughlin, J. L., New bioactive adjacent bis-THFAnnonaceous acetogenins From Annona bullata. Phyto-chemistry, 40 (1995) 467È77.

23. Zhao, G.-X., Gu, Z.-M., Chao, J.-F., Kozlowski, J. F.,Wood, K. V. & McLaughlin, J. L., The absolute conÐgu-ration of trilobacin and trilobin, a novel highly potentacetogenin from the stem bark of Asimina triloba(Annonaceae). T etrahedron, 51 (1995) 7149È60.

24. Alfonso, D., Johnson, H. A., Colman-Saizarbitoria, T.,Presley, C. P., McCabe, G. P. & McLaughlin, J. L., SARsof Annonaceous acetogenins in rat liver mitochondria.Nat. T oxins, 4 (1996) 181È188.

378 Kan He et al.

25. Landolt, J. L., Ahammadsahib, K. I., Hollingworth, R. M.,Barr, R., Crane, F. L., Buerck, N. L., McCabe, G. P. &McLaughlin, J. L., Determination of structureÈactivityrelationships of Annonaceous acetogenins by inhibition ofoxygen uptake in rat liver mitochondria. Chemico-Biol.Interact., 98 (1995) 1È13.

26. Shi, G., Alfonso, D., Fatope, M. O., Zeng, L., Gu, Z.-M.,Zhao, G.-X., He, K., MacDougal, J. M. & McLaughlin, J.L., Mucocin : a new Annonaceous acetogenin bearing a

tetrahydropyran ring. J. Am. Chem. Soc., 117 (1995)10409È10.

27. McLaughlin, J. L., Zeng, L., Oberlies, N. H., Alfonso, D.,Johnson, H. A. & Cummings, B. A., Annonaceous aceto-genins as new natural pesticides : recent progress. InPhytochemical Pest Control Agents, ed. P. Hedin, R. Hol-lingworth, J. Mujamoto, E. Masler & D. Thompson.American Chemical Society, Washington, DC., 1996,pp. 117È133.