US007820209B2
(12) United States Patent (10) Patent No.: US 7820,209 B2 Spooner-Hart et al. (45) Date of Patent: Oct. 26, 2010
(54) METHODS AND COMPOSITIONS FOR 6,054,137 A 4/2000 Breton et al. CONTROLLING PESTS 6,180,568 B1 1/2001 Takahashi et al. 6,218,579 B1 4/2001 Jones et al. (75) Inventors: Robert Neil Spooner-Hart, Kurrajong 6,291,400 B1 9, 2001 Barton et al. (AU); Albert Habib Basta, Glenwood 6,352,727 B1 3/2002 Takahashi (AU) 6,444,216 B2 9, 2002 Reifenrath FOREIGN PATENT DOCUMENTS (73) Assignees: Bioprospect Limited, Perth, Western Australia (AU); University of Western WO WO 93,2444.6 A1 12, 1993 Sydney, South Penrith DC, New South WO WO99, 188O2 A1 4f1999 Wales (AU) WO WOOOf 27206 A1 5, 2000 OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 Rangahau, M., Essential oil production from manuku and kanuka, Crop and Food Research, 116, Jul. 2000.* U.S.C. 154(b) by 0 days. Morrow, P.A., Eucalyptus Oils in the Defensive Oral discharge of Australian Sawfly Larvae, Oecologia 24, 193-206.* (21) Appl. No.: 12/134,035 Rubinov, D.B., 2-acylcycloalkane-1,3-Diones, occurrence in nature, biological activity, Biogenesis, Chemical Synthesis, Chemistry of (22) Filed: Jun. 5, 2008 natural compounds, vol. 31, No. 5, 1995, 537-559.* T. Greene, Antiparasitic behaviour in New Zealand parakeets (65) Prior Publication Data (cyanoramphus species). Notornis 36 (4) (1989): 322-323.* US 2008/O23438.6 A1 Sep. 25, 2008 Bignell, C.M. et al. 1997 Flav Frag Journal 12:177-183. Boland, D. et al. 1993 American Chemical Society 525:72-87. Brophy, J.J. et al. 1990J Essent Oil Res 2:87–90. Related U.S. Application Data Brophy, J.J. et al. 1995J Essent Oil Res 7:237-254. (63) Continuation of application No. 10/477.057, filed as Hellyer, R. 1968 Aust.J Chem 21 (11) 2825-2828. application No. PCT/AU02/00569 on May 8, 2002, Southwell, I.A. et al. 2000J Essent Oil Res 12:267-278. now abandoned. * cited by examiner (30) Foreign Application Priority Data Primary Examiner Johann R Richter Assistant Examiner Danielle Sullivan May 8, 2001 (AU) ...... PR4842 (74) Attorney, Agent, or Firm Knobbe Martens Olson & (51) Int. Cl. Bear LLP A6 IK 36/6 (2006.01) AOIN 35/00 (2006.01) (57) ABSTRACT (52) U.S. Cl...... 424/742: 514/675 The present invention is directed to pest-controlling compo (58) Field of Classification Search ...... 514/675; sitions comprising as active ingredients one or more f3-di 424/742 ones, particularly B-diketones and B-triketones, and to the use See application file for complete search history. of these compositions inter alia for preventing, eradicating, destroying, repelling or mitigating harmful, annoying or (56) References Cited undesired pests including insects, arachnids, helminths, mol U.S. PATENT DOCUMENTS luscs, protozoa and viruses. The present invention is further directed to processes of preparing B-diones by de novo syn 4,202,840 A 5/1980 Gray et al. 4,780,127 A 10/1988 Michaely et al. thesis or from natural Sources Such as Volatile oil-bearing 4,797,150 A 1, 1989 Carter et al. plants from families including Alliaceae, Apiaceae, Aster 4,853,028 A 8, 1989 Carter et al. aceae, Cannabinaceae, Lamiaceae, Pteridaceae, Myrtaceae, 5,411,728 A 5, 1995 Joulain et al. Myoporaceae, Proteaceae, Rutaceae and Zingiberaceae. 5,416,061 A 5, 1995 Hewett et al. 5.426,091 A 6, 1995 Barton et al. 8 Claims, 4 Drawing Sheets U.S. Patent Oct. 26, 2010 Sheet 1 of 4 US 7820,209 B2
O O O O O O O O O O O O
O OCH3 tasman One angustione dehydroangs utione flawes One
O O O O O O O O O O O O
OHC CHO OCH3 O O O leptosperm one isoleptOS perm One jensen one agglomerone
O O O O O O O O O O O O
O CH O O 3 XanthoStem one papuanone grandiflorone lateritic One
HO HO O O HCO OCH O O H3CO OCH3 3. 3 H3CO OCH3 H3C CH HC CH H3C CH3 OCH 3 3 O CH OCH3 3 OCH 3
torquatone isolateriticone
O O O O HO OCH HCO OCH H3CO OCH3 O O
H3C H3C CH3 OCH3 OCH3 OCH O
baecke Ol conglomerone
FIGURE 1 U.S. Patent Oct. 26, 2010 Sheet 2 of 4 US 7820,209 B2
Abundance
TC: 96O1 OO1D 18OOOOO 17ooooo. 2 16ooooo. 15ooooo. 14OOOOO
13OOOOO i f 12OOOOO! 11 OOOOO 1 OOOOOO 9 OOOOO a Ooooo 7ooooo 6Ooooo 1. 5Ooooo; : 4ooooo 9 3ooooo; 2Ooooo. 3 1ooooo...... ------4------l A-, all--bidsci------.r 8. OD1 oloo 2OO4. OO16 OO18 OO2O)22.OO24O26 OO28 OOBOOO32. OO34. OO Time--- FIGURE 2 U.S. Patent Oct. 26, 2010 Sheet 3 of 4 US 7820,209 B2
H NMR Data (CDCl3, 500MHz)
H (see below) Signal (ppm) Integral Signal (ppm) Integral A 19.18 0.89 1845 0.48
3.98 1.09 3.85 0.55
3.96 2.75 3.87 155
1.97 3.00 1.92 1.72
1.34 6.43 1.46 3.52
1.15 6.13 1.17 3.48
OCH
MNOR
FIGURE 3 U.S. Patent Oct. 26, 2010 Sheet 4 of 4 US 7820,209 B2
O CH O CH O CH
FIGURE 4 US 7,820,209 B2 1. 2 METHODS AND COMPOSITIONS FOR helminthicidal and/or molluscicidal activity. This discovery CONTROLLING PESTS has been reduced to practice in novel pest-controlling com positions and methods for their preparation and use, as CROSS-REFERENCE TO RELATED described hereinafter. APPLICATIONS BRIEF DESCRIPTION OF THE DRAWINGS This application is a continuation of application Ser. No. 10/477.057, filed Sep. 23, 2004, which is the U.S. National FIG. 1 shows the structures relating to the major constitu Phase under 35 U.S.C. S371 of International Application No. ents of the published Myrtaceae essential oils. PCT/AU02/00569, filed May 8, 2002 designating the U.S. 10 FIG. 2 is a representation of a GC-MS trace of E. cloeziana and published in English as WO 02/089587, which claims oil. priority to Australian Patent Application PR 4842, filed May FIG.3 is a tabular and graphical representation showing "H 8, 2001 the entire disclosures of which are incorporated NMR data recorded on a fraction (F4) obtained from silica gel herein by reference in their entireties. chromatography of E. cloeziana oil and the structure of the 15 major and minor isomers of the compound deduced from FIELD OF THE INVENTION these data. This invention relates generally to methods and composi FIG. 4 is a diagrammatic representation showing various tions for controlling pests. More particularly, the present tautomeric forms of an isolated 3-triketone compound in invention relates to pest-controlling compositions compris solution (CDC1). ing as active ingredients one or more B-diones, particularly 3-diketones and B-triketones, and to the use of these compo DETAILED DESCRIPTION OF THE INVENTION sitions inter alia for preventing, eradicating, destroying, repelling or mitigating harmful, annoying or undesired pests One aspect of the present invention contemplates the use of including insects, arachnids, helminths, molluscs, protozoa 25 a 3-dione compound, particularly a B-diketone or a B-trike and viruses. The present invention further relates to processes tone compound, in the preparation of a composition for con of preparing B-diones by de novo synthesis or from natural trolling harmful, annoying or undesired pests, said compound Sources such as Volatile oil-bearing plants from families being represented by the general formula (I) including Alliaceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae, Pteridaceae, Myrtaceae, Myoporaceae, Pro 30 teaceae, Rutaceae and Zingiberaceae. Bibliographic details (I) of various publications referred to in this specification are collected at the end of the description.
BACKGROUND OF THE INVENTION 35 Triketones have been used for many years as herbicides for the control of undesired vegetation. Herbicidal triketones wherein have been described, for example, in EP-A-33.8992, EP-A- A is (C=O)R. (C=S)R, OR, SR (CRNRRs), 336898, U.S. Pat. No. 4,869,748, EP-A-186118, EP-A- 40 C(R), OR, NRRs (C—N Ra)R, N=O, N(EO), 186119, EP-A-186120, U.S. Pat. No. 4,202,840, U.S. Pat. NROR or SOR: No. 4,695,673, U.S. Pat. No. 4,780,127, U.S. Pat. No. 4,921, R is selected from H. C-Co alkyl, C-C arylalkyl, 526, U.S. Pat. No. 5,006,150, U.S. Pat. No. 5,545,607, U.S. C-C cycloalkyl, C-Co alkenyl, C-Co heteroarylalkyl, Pat. No. 5,925,795, U.S. Pat. No. 5,990,046, U.S. Pat. No. C-Co haloalkyl, C-Co dihaloalkyl, C-Co trihaloalkyl, 6,218,579, EP-A-249.150, EP-A-137963, EP-A-394889, EP 45 C-Co haloalkoxy, C-Co hydroxyalkyl, C-Co thioalkyl A-506907 or EP-B-135191. Examples of herbicidal trike tones are interalia Sulcotrione (MIKADOR) whose chemical and C-C, nitroalkyl, OR, SR (CRNRRs), NRRs, designation is 2-(2-chloro-4-methanesulfonylbenzoyl)-1,3- (C=N-R)R N=O, N(-O), NROR, or SOR: cyclohexandione, 2-(4-methylsulfonyloxy-2-nitrobenzoyl)- R is selected from H. C-Co alkyl, C-C arylalkyl, 4,4,6,6-tetramethyl-1,3-cyclohexane dione: 3-(4-methylsul 50 C-C cycloalkyl, C-C alkenyl, C-C heteroarylalkyl, fonyloxy-2-nitrobenzoyl)-bicyclo-3.2.1]octane-2,4-dione; C-Co haloalkyl, C-Co dihaloalkyl, C-Co trihaloalkyl, 3-(4-methylsulfonyl-2-nitrobenzoyl)-bicyclo-3.2.1]octane (CRNRRs), NRRs (C=N R)R. N=O, N(=O), or 2,4-dione: 4-(4-chloro-2-nitrobenzoyl)-2,6,6-trimethyl-2H NROR; 1.2-oxazine-3,5(4H, 6H)-dione: 3-(4-methylthio-2-nitroben R is selected from H. C-Co alkyl, C-C arylalkyl, Zoyl)-bicyclo3.2.1]octane-2,4-dione; 4-(2-nitro-4- 55 C-C cycloalkyl, C-C alkenyl, C-C heteroarylalkyl, trifluoromethoxybenzoyl)-2,6,6-trimethyl-2H-1,2-oxazine C-Co haloalkyl, C-Co dihaloalkyl, C-Co trihaloalkyl, 3.5(4H, 6H)-dione. C-Co haloalkoxy, OR7, SR 7, (CRNRRs), NRRs. (C=N-R)R N=O, N(-O), NROR, or SOR: SUMMARY OF THE INVENTION RandRs are independently selected from H. C-Coalkyl, 60 C-Clo arylalkyl, C-C cycloalkyl, C-Coalkenyl, C-Co The instant invention is predicated in part on the discovery heteroarylalkyl, C-Co haloalkyl, C-Co dihaloalkyl, that B-diones, particularly 3-diketones and 3-triketones, such C-C trihaloalkyl, OR, or SR-7; as those obtainable from Volatile oil-bearing plants including R is selected from H. C-C alkyl, C-C arylalkyl, plants from the families Alliaceae, Apiaceae, Asteraceae, C-C cycloalkyl, C-Co alkenyl, C-Co heteroarylalkyl, Cannabinaceae, Lamiaceae, Pteridaceae, Myrtaceae, Myo 65 C-Co haloalkyl, C-Co dihaloalkyl, C-Co trihaloalkyl, poraceae, Proteaceae, Rutaceae and Zingiberaceae, exhibit C-Co haloalkoxy, OR7, SR-7, (CRNRR), NRoRo or significant pesticidal, especially insecticidal, arachnicidal, NROR; US 7,820,209 B2 3 4 R is selected from H. C-Co alkyl, C-Co arylalkyl, A preferred carbocyclic ring formed by Q is an optionally C-C cycloalkyl, C-C alkenyl, C-C heteroarylalkyl, Substituted cyclohexanedione. C-Cohaloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl; A preferred Subgroup of compounds of formula (I) is rep Rs is selected from H. C-Co alkyl, C-Co arylalkyl, resented by formula (II) C-C cycloalkyl, C-Co alkenyl, C-Co heteroarylalkyl, 5 C-Co haloalkyl, C-Co dihaloalkyl, C-Co trihaloalkyl, OR, SR or NRRo: (II) Ro and Rio are independently selected from H. C-Co alkyl, C-C arylalkyl, C-C cycloalkyl, C-C alkenyl, C-Co heteroarylalkyl, C-Co haloalkyl, C-Co diha 10 loalkyl, C-Co trihaloalkyl, OR or SR; R is selected from H. C-C alkyl, C-C arylalkyl, C-C cycloalkyl, C-Co alkenyl, C-Co heteroarylalkyl, C-Cohaloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl: R is selected from H. C-C alkyl, C-C arylalkyl, 15 Such compounds may exist in a number of tautomeric C-C cycloalkyl, C-C alkenyl, C-C heteroarylalkyl, forms. For example, in the case wherein X and Y are each C-Cohaloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl; oxygen, and B is hydrogen, then the compounds of formula II B is H. C-C alkyl, C-Coalkenyl, aryl or heteroaryl; may exist as one or more of the structural formulae shown X and Y are independently selected from oxygen, Sulfur, below. —N-R, and Q completes a 5-8-member Saturated or unsaturated car bocyclic or heterocyclic ring in which optionally one or more O R O R HO R members comprise —C(=X)—, and wherein Q is optionally substituted with one or more substituents selected from C-Clo alkyl, C-C cycloalkyl, C2-Cio alkenyl, Co-Co 25 haloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl, C-Co haloalkoxy, OR, SR (CRNRRs), NRRs (C—N—R) Q Q Q R, N=O, N(-O), NROR, SOR, C-C 1-arylalkyl, C-Co 2-arylalkyl or (C=X)R. It is intended that all such tautomeric structures are Heterocyclic systems can be optionally attached to a moi 30 included within the scope of the present invention. ety other than those set forth above via a carbon atom or a It should also be appreciated that some of the compounds heteroatom of R to R. of formula (I) are capable of existing as different geometric Preferred compounds represented by formula (I) are isomers and diastereomers. The invention thus includes both 3-diketones and especially preferred are 3-triketones. the individual isomers and mixtures of Such isomers. As used herein, the term “alkyl refers to linear or branched 35 Another preferred Subgroup of compounds of formula (I) is chains. The term “haloalkyl refers to an alkyl group substi represented by formula (III) tuted by at least one halogen. Similarly the term “haloalkoxy” refers to an alkoxy group Substituted by at least one halogen. (III) As used herein the term “halogen refers to fluorine, chlorine, A B bromine and iodine. 40 As used herein the term “aryl refers to aromatic carbocy X Y clic ring systems such as phenyl or naphthyl, anthracenyl, especially phenyl. Suitably, aryl is C-C with mono, di, tri. F C tetra and penta substitution containing OR, F, Cl, Br, I., NO, E D CF, COR, NRRs, SOR, SR. 45 As used herein the terms "heterocycle”, “heterocyclic”. Z "heterocyclic systems’” and the like refer to a saturated, unsat urated, or aromatic carbocyclic group having a single ring, multiple fused rings (for example, bicyclic, tricyclic, or other wherein similar bridged ring systems or Substituents), or multiple 50 X, Y and Z are each independently selected from oxygen, condensed rings, and having at least one heteroatom such as sulfur, —N R or one of C=X, C=Y or C=Z is CH: nitrogen, oxygen, or Sulfur within at least one of the rings. A is (C=O)R. (C=S)R, OR, SR (CRNRRs), This term also includes "heteroaryl which refers to a hetero C(R), OR, NRRs (C=N R)R, N=O, N(-O). cycle in which at least one ring is aromatic. Any heterocyclic NROR or SOR: or heteroaryl group can be unsubstituted or optionally Substi 55 B is as defined above; tuted with one or more groups, as defined above. Further, bi C, D, E and F are each independently selected from H. or tricyclic heteroaryl moieties may comprise at least one C-Clo alkyl, C-C cycloalkyl, C2-Cio alkenyl, Co-Co ring, which is either completely, or partially, Saturated. Suit haloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl, OR, SR, able heteroaryl moieties include, but are not limited to (CRNRRs), NRRs (C—N-R)R, N=O, N(-O), oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl. 60 NROR, SOR, C-C 1-arylalkyl, C-C 2-arylalkyl or 2H-pyrrolyl. N-pyrrolyl, imidazolyl pyrazolyl, isothiazolyl, (C—X)R; and isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, R. R. R. R. and Rs are as defined above. indolizinyl, isoindolyl, indoyl, indolyl, purinyl, phthalazinyl. Preferred f-diones represented by formula (III) are flave The articles “a” and “an are used herein to refer to one or sone (1-isobutyroyl-3,3,5,5-tetramethylcyclohexan-2,4,6- to more than one (i.e., to at least one) of the grammatical 65 trione), isoleptospermone (1-isovaleroyl-3,3,5,5-tetrameth object of the article. By way of example, “an element’ means ylcyclohexan-2,4,6-trione), leptospermone (1-Valeroyl-3.3, one element or more than one element. 5.5-tetramethylcyclohexan-2,4,6-trione), papuanone US 7,820,209 B2 5 6 (1-pentoyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione), mulae, wherein the structural formula of isoleptospermone is grandiflorone (1-(2-phenylethyl)-3,3,5,5-tetramethylcyclo shown for comparative purposes: hexan-2,4,6-trione) andjensenone (1-Valeroyl-3,5-dicarbon ylcyclohexan-2,4,6-trione), including analogues and deriva tives thereof. By way of example, flavesone analogues contemplated by the present invention include, but are not restricted to, com O HO HN pounds having the following structural formulae, wherein the structural formula of flavesone is shown for comparative pur- 10 O O O O O O poses:
O O O O HO HN 15
CHBr O O O O O O
2O
O O O Flavesone CHBr CHCI CHOH 25 O O O
O O O O O O Isoleptospermone 30
C HCI C H2OH O O O CHF CF O O 35 O O S O O O O O O O O S S
40
O O O O O CHF CF H NN 1s HC NN 1. HO NN 1. 45 O O O O O O O O
O O O O 50
O O O
O O 55
S H NN H3C NN 60 S S
Non-limiting examples of isoleptospermone analogues 6s contemplated by the present invention include, but are not O O O restricted to, compounds having the following structural for US 7,820,209 B2
-continued -continued CH2SH CH2OH CHF
HO n N l? O 5 O O
O O O O
10
O O CF CH2SH 15 O O
O O O O 2O
O O 25 CHNO Non-limiting examples of leptospermone analogues con- HO templated by the present invention include, but are not O N restricted to, compounds having the following structural for
mulae, wherein the structural formula of leptospermone is so O O O shown for comparative purposes:
35 O O O HO HN Non-limiting examples of jensenone analogues contem O O O O O O plated by the present invention include, but are not restricted 40 to, compounds having the following structural formulae, wherein the structural formula of jensenone is shown for comparative purposes: O O O Lepteptospermone 45 CHBr O HO O S H YN O O O O 50 O O S S O O
OHC CHO OHC CHO
O O O O O 55 Jensenone CHCI CHBr . "n O O HN O 60 O O O O O O O O O O
OHC CHO OHC CHO 65 O O O O O US 7,820,209 B2 9 10 Another preferred Subgroup of compounds of formula (I) is -continued represented by formula (IV) CHCI CH2OH (IV) O O A B X Y O O O O F
10 E C OHC CHO OHC CHO D O O CHF CF 15 wherein O O X and Y are each independently selected from oxygen, sulfur - N R or one of C=X or C=Y is CH: O O O O A is (C=O)R. (C=S)R, OR, SR (CRNRRs), C(R) OR, NRRs, (C—N—R)R. N—O, N(=O), NROR or SOR: OHC CHO OHC CHO B is as defined above; O O C, D, E and F are each independently selected from H. C-C alkyl, C-C arylalkyl, C-C cycloalkyl, C-Coalk 25 enyl, C-Coheteroarylalkyl, C-Cohaloalkyl, C-Co diha loalkyl, C-C trihaloalkyl, C-C haloalkoxy, OR, SR, S H NN r (CRNRRs), NRRs (C—N-R)R, N=O, N(-O), NROR, SOR: and S S O O R. R. R. R. and Rs are as defined above. 30 Preferred B-diones represented by formula (IV) are tas manone (1-isobutroyl-4-methoxy-3.5.5-trimethylcyclohex OHC CHO OHC CHO 3-en-2,6-dione), agglomerone (1-isobutroyl-4-methoxy-5.5- O O dimethylcyclohex-3-en-2,6-dione), lateriticone (1-valeroyl 4-methoxy-3.5.5-trimethylcyclohex-3-en-2,6-dione), 35 isolateriticone (1-isovaleroyl-4-methoxy-3, 5,5-trimethylcy HC r clohex-3-en-2,6-dione and platyphyllol (6,6-dimethyl-2- NN HN N CO acetyl-5-methoxycyclohex-4-ene-1,3-dione), including ana logues and derivatives thereof. O O O O 40 Non-limiting examples of tasmanone analogues contem plated by the present invention include, but are not restricted to, compounds having the following structural formulae, OHC CHO OHC CHO wherein the structural formula of tasmanone is shown for O O comparative purposes: CHSH CHNO 45
O O O HO HN
O O O O 50 O O O O O O
OHC CHO OHC CHO
O O 55 OCH OCH OCH Tasmanone HO V CHBr CHCI CH2OH NS O O O
O O 60 O O O O O O
OHC CHO
O 65 OCH OCH OCH US 7,820,209 B2 12 Preferred B-diones represented by formula (V) are angus -continued tione (1-acetyl-3,5,5-trimethylcyclohex-2,6-dione), dehy CHF CF droangustione (1-acetyl-3,5,5-trimethylcyclohex-3-en-2,6- O O S dione) and Xanthostemone (1-isobutroyl-5,5- dimethylcyclohex-3-en-2,6-dione), including their O O O O S S analogues and derivatives. By way of example, angustione analogues contemplated by the present invention include, but are not restricted to, 10 compounds having the following structural formulae, wherein the structural formula of angustione is shown for OCH OCH OCH comparative purposs: H NN 1s HC NN 1. HO NN 1. 15 O HO HN O O O O O O
r r r OCH CHSH Angustione O O 25 O CHBr O CHCl2 O CH2OH O O O O r O r O r 30 OCH OCH OCH
Another preferred Subgroup of compounds of formula (I) is O CHF O CF S represented by formula (V) 35 OH r S SH A B (V)
X Y 40 HO H C HN 1. N N 1. NN 1.
G D F E 45 O OH r r wherein X and Y are independently selected from oxygen, sulfur or O CHSH O CHNO —N-R, and A is (C=O)R. (C=S)R, OR, SR (CRNRRs), 50 C(R), OR, NRRs (C—N—R)R, N=O, N(EO), NROR or SOR: r r B is as defined above; C, D, E, F, G and H are each independently selected from N H. C-C alkyl, C-Co arylalkyl, C-C cycloalkyl, C-Co 55 Ho1 n alkenyl, C-Co heteroarylalkyl, C-Co haloalkyl, C-Co dihaloalkyl, C-C trihaloalkyl, C-Co haloalkoxy, OR, O OH SR (CRNRRs), NRRs (C=N-R)R, N=O, N (=O), NROR or SOR: and 60 R. R. R. R. and Rs are as defined above. More specifically unsaturation, epoxides and thioex poxides may exist at positions designated by H (or G) con nected to F (or E) or F (or E) connected to C (or D). A Non-limiting examples of dehydroangustione analogues four-membered ring forming a part of a bicyclic structure 65 include, but are not restricted to, compounds having the fol may exist at positions designated by H (or G) connected to C lowing structural formulae, wherein the structural formula of (or D). dehydroangustione is shown for comparative purposes: US 7,820,209 B2 13
O HO HN O HO HN 5 O O O O O O O O
Dehydroangustione 10 Xanthostemone O CHBr O CHCl2 CHBr CHCI CHOH O O O O O 15 O O O O O O
O CH2OH O CHF CHF CF
HO O 25
S HN1 30 HO 2 O HS 2 S
35
N N1 HO n N1
O 40
45 O CHSH O CHNO
HO O HO O Derivatives of the above compounds include, but are not restricted to, ethoxylate derivatives, propoxylate derivatives, 50 hydrates, aldehyde derivatives, ester derivatives, ether deriva tives, alcohol derivatives, phenol derivatives, amine deriva tives, other biologically or chemically equivalent Substances, and any combination of two or more of the foregoing. N 55 Similarly effective as pesticides are salts of the above com Ho1 n pounds, including mono-valent salts (e.g., Sodium, potas sium) and di-Valent metal salts (e.g., calcium, magnesium, HO O iron or copper) and ammonium salts (e.g., isopropyl ammo nium, trialkyl and tetraalkylammonium salts). 60 The compounds according to any one of formulae (I)-(V) can be prepared according to methods analogous to those known in the art for the preparation of B-diones. Exemplary methods are disclosed for example in EP-A-33.8992, EP-A- Non-limiting examples of Xanthostemone analogues 336898, U.S. Pat. No. 4,202,840, U.S. Pat. No. 4,869,748, include, but are not restricted to, compounds having the fol 65 EP-A-186118, EP-A-186119, EP-A-186120, U.S. Pat. No. lowing structural formulae, wherein the structural formula of 4,695,673, U.S. Pat. No. 4,780,127, U.S. Pat. No. 4,921,526, Xanthostemone is shown for comparative purposes: U.S. Pat. No. 5,006,150, U.S. Pat. No. 5,545,607, U.S. Pat. US 7,820,209 B2 15 16 No. 5,925,795, U.S. Pat. No. 5,990,046, U.S. Pat. No. 6,218, under an atmosphere of nitrogen. The mixture is cooled to 0° 579, EP-A-249.150, EP-A-137963, EP-A-394889 EP-A- C. and lithium hydride (1.1 mole eq) (60% in mineral oil) is 506907 or EP-B-1351.91. added in portions. After addition the mixture is stirred for a More particularly, compounds according to formulae (III)- further 10 mins before the addition of benzoyl cyanide 5 (V) can be synthesised using the representative procedures 5 R—CO CN, R is depicted above (1.1 mole eq). The mix outlined below. ture is allowed to warm to room temperature over 12 hat For compounds according to formula (III), 1,3,5-trihy which time the reaction is quenched with water and parti droxybenzene 1 is reacted with CHCN in the presence of tioned. The ether layer is dried (NaSO) and evaporated Zinc chloride and hydrochloric acid, according to A. H. Blatt affording crude 1-benzoyl-3-methoxy-2,4,4-trimethylcyclo (1943, Org. Synth. Col. II, 522-523), affording 1-acetyl-2,4, 10 hex-2-en-1,5-dione 6 which is purified by SiO, column chro 6-trihydroxybenzene 2 (phloroacetophenone) (R=Me) matography (hexane/ethyl acetate, gradient) (Scheme 3). (Scheme 1).
Scheme 3 15 OH OH O - 2O O -- He HO OH HO OH O C’s 4 5
Rgroups other than methyl are depicted above. Reaction of 25 O O 1-acetyl-2,4,6-trihydroxybenzene 2 affording 1-acetyl-3,3,5, 5-tetramethylcyclohexan-2,4,6-trione 3 is a representative 1. C procedure for all compounds according to formula (III) (R.A. Gray et al., U.S. Pat. No. 4,202,840) (Scheme 2). Anhydrous MeI (6 eq) is slowly added, at room temperature under an 30 O O atmosphere of nitrogen, to a mechanically stirred solution of 1-acetyl-2,4,6-trihydroxybenzene 2 (1 eq) and sodium ethox ide (6 eq) in anhydrous methanol. The mixture is refluxed for Alternatively, compounds of formula (IV) can be prepared 4 hours. On cooling the mixture is concentrated under vacuo, according to a second representative procedure, as follows: providing a residue, which is diluted with water and acidified 35 with 2 Mhydrochloric acid. Diethylether extracts are washed 3-methoxy-2,4,4-trimethylcyclohex-2-en-1,5-dione 4 (1 with saturated sodium sulfite solution, water and then dried mole eq) (commercially available) and benzoyl cyanide are (NaSO). Evaporation of the diethylether provides the dissolved in anhydrous dichloromethane and cooled to 0°C. desired product 3. under an atmosphere of nitrogen. To the cooled solution is 40 added anhydrous finely powdered zinc chloride (1.1 mole eq.) followed by slow addition of triethylamine (1.2 mole eq). The reaction mixture is stirred at room temperature for 5-6 hand then poured into 2 M hydrochloric acid. The mixture is par OH O titioned and the dichloromethane layer is washed with 5% 45 Sodium carbonate. The aqueous carbonate phase is then acidi fied with hydrochloric acid and extracted with methylene chloride and dried (NaSO). The solvent is removed and the residue subjected to SiO2 column chromatography (hexane/ HO OH ethyl acetate) affording 1-benzoyl-3-methoxy-2,4,4-trimeth 50 ylcyclohex-2-en-1,5-dione 6 (W. J. Michaely and G. W. Kraatz, EP-B-135191). Compounds of formula (V) can be prepared according to a For mono, di, tri and tetra B, C, D, E, F substitution pat first representative procedure, as follows: 4,4-dimethylcyclo terns, reactions between one and seven mole equivalents of hexane-1,3-dione 7 (1 mole eq) (commercially available) is R—I and Sodium ethoxide are used. Lawasson’s reagent is 55 dissolved in anhydrous diethylether and hexamethylphos used for conversion of oxygen into Sulfur groups and sodium phoramide (solvent ratio, 20:1 respectively) under an atmo borohydride or sodium cyanoborohydride is used to reduce sphere of nitrogen. The mixture is cooled to 0°C. and lithium ketone, thioketone and imino groups. When additional car diisopropylamide (2.1 mole eq) is added dropwise over 40 bonyl groups are introduced into the cyclohexane ring system mins. The mixture is then stirred for a further 10 mins before the procedure of Crow is utilised (M. L. Bolte et al., 1985, 60 the addition of methyl iodide (1 mole eq). The mixture is Agric. Biol. Chem., 49, 761). stirred for 12 hand then benzoyl cyanide (2 mole eq) is added Compounds of formula (IV) can be prepared according to and the mixture stirred for a further 24 h. The reaction was a first representative procedure, as follows: 3-methoxy-2,4,4- quenched with water and the ether layer partitioned and dried trimethylcyclohex-2-en-1,5-dione 4 (1 mole eq), prepared (NaSO). The solvent was removed and the residue sub according to Herzig (J. Herzig, and F. Wenzel, Monatsh, 65 jected to SiO2 column chromatography (hexane/ethyl 1903, 24, 101), is dissolved in anhydrous diethylether and acetate) affording 1-benzoyl-3,3,5-trimethylcyclohexan-2,6- hexamethylphosphoramide (solvent ratio, 20:1 respectively) dione 8 (Scheme 4). US 7,820,209 B2 17 18 The present inventors have discovered that the B-diones of the invention can be obtained from natural Sources and, in Scheme 4 particular, from Volatile oil-bearing organisms. Accordingly, in another aspect, the present invention encompasses the use 2O 5 ofa B-dione compound, particularly a B-diketone or a B-trike 1) LiNiCH) tone compound, obtainable from a volatile oil-bearing organ He 2) MeI ism, including an analogue or derivative thereof, in the prepa 3) 7 ration of a pesticidal composition for controlling harmful, annoying or undesired pests. O O O 10 The present invention contemplates the use of any Volatile 7 8 oil-bearing organism that produces B-diones, preferably the 3-diones according to any one of formulae (I)-(V), and espe Alternatively, the compounds of formula (V) can be pre cially B-diketones and/or B-triketones, for the preparation of pared according to a second representative procedure, as fol 15 the pesticidal compositions of the invention. Preferred vola lows: 4,4-dimethyl-1,3-cyclohexanedione 7 (1 mole eq) tile oil-bearing organisms are volatile oil-bearing plants (commercially available) and benzoyl cyanide are dissolved including, but not restricted to, plants from the families Alli in anhydrous dichloromethane and cooled to 0°C. under an aceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae, atmosphere of nitrogen. To the cooled solution is added anhy Pteridaceae, Myrtaceae, Myoporaceae, Proteaceae, Rutaceae drous finely powdered zinc chloride (1.1 mole eq) followed and Zingiberaceae. Preferably, the volatile oil-bearing plant by slow addition of triethylamine (1.2 mole eq). The reaction is selected from genera of the Myrtaceae family including, mixture is stirred at room temperature for 5-6 hand then but not limited to, Angophora, Austromyrtus, Backhousia, poured into 2 Mhydrochloric acid. The mixture is partitioned Baeckea, Callistemon, Corymbia, Darwinia, Eucalyptus, and the dichloromethane layer washed with 5% sodium car Kunzea, Leptospermum, Melaleuca, Syzygium and Xanthos bonate. The aqueous carbonate phase is then acidified with 25 hydrochloric acid and extracted with methylene chloride and teinOn. dried (Na2SO). The solvent is removed and the residue sub Thus, the compositions of the present invention may con jected to SiO2 column chromatography (hexane/ethyl tain as active ingredients Substantially purified B-diones or acetate) affording 1-benzoyl-3,3,5-trimethylcyclohexan-2,6- crude B-dione-containing extracts obtained from a volatile dione 8. (W. J. Michaely and G. W. Kraatz, EP-B-135191). 30 oil-bearing organism, preferably a Volatile oil-bearing plant. Dehydroangustione and Xanthostemone derivatives are Volatile oils, also known in the art as essential oils, typically simply derived from dehydrogenation of angustione deriva comprise a volatile mixture of esters, aldehydes, alcohols, tives, for example, by treatment of 1-benzoyl-3,3,5-trimeth ketones and terpenes, which can be prepared from botanical materials or plant cell biomass from cell culture. Volatile oils ylcyclohexan-2,6-dione 8 with palladium on charcoal in can be prepared by Subjecting botanical materials to a distil methanol, which thereby affords 1-benzoyl-3,5,5-trimethyl 35 lation process, for example. A number of different procedures cyclohex-3-en-2,6-dione 9 (Scheme 5). can be used for distillation. For example, plant matter (e.g., foliage, stems, roots, seeds, bark etc) of a Volatile oil-bearing plant is placed in a Suitable still and steam distillation is used to break down the cells of the plant to release the oil. The 40 steam is then condensed and the oil phase is separated from the aqueous phase to obtain the volatile oil. It will be appre ciated that other methods of Volatile oil extraction (e.g., Sol vent extraction) are known to those of skill in the art and it will be understood, in this regard, that the present invention is not 45 limited to the use or practice of any one particular method of extracting Volatile oils. Suitably, the compositions comprise naturally-occurring compounds derived from a volatile oil-bearing organism. 50 Thus, in a preferred embodiment, the pesticidal composition of the invention comprises one or more B-dione active com pounds, particularly B-diketone- and/or B-triketone-active compounds, that are derived from the volatile oil of a volatile oil-bearing organism. In this embodiment, the composition 55 may optionally contain a naturally-occurring carrier and/or other naturally-occurring additives. Naturally-occurring additives contemplated by the present Metal salts (enolates) of the above compounds can be invention include natural antioxidants, which can be used prepared by the reaction of triketone derivatives with the advantageously to reduce the effect of oxidation of the com corresponding metal hydroxides Suspended in methanol or 60 pounds of the invention. An example of a Suitable naturally ethanol. Trialkylammonium salts can be prepared by the reac occurring antioxidant is C.-tocopherol. Other additives, such tion of triketone derivatives (e.g., 3) with trialkylamines in a as naturally-occurring stabilisers, are also contemplated, chlorinated solvent such as dichloromethane. Tetraalkylam which may desirably be added to improve the stability and monium salts can be prepared by adding a halogenated tet shelf life of the composition. Examples of suitable natural raalkylammonium salt to a metal salt in dichloromethane, 65 stabilisers include gum arabic, guar gum, Sodium caseinate, which precipitates the metalhalide removed by filtration. The polyvinyl alcohol, locust bean gum, Xanthan gum, kelgum, pure material is obtained by evaporation of the filtrate. and mixtures thereof. US 7,820,209 B2 19 20 In an alternate embodiment, the naturally-occurring com that varies by as much as 30%, preferably by as much as 20%, pounds derived from a volatile oil may be modified or deri and more preferably by as much as 10% to a reference quan vatised to improve, for instance, their shelf-life, stability, tity, level, value or amount. activity and/or bioavailability. Where the compounds are in the form of B-dione-contain The compounds of the present invention are useful for ing extracts, the formulations will usually comprise as their controlling harmful, annoying or undesired pests. They may principal active ingredient from about 0.0001% to about 90%, be used singularly or in combination with other pest-control preferably from about 0.0001% to about 50%, more prefer ling compounds of the invention. By “controlling is meant ably from about 0.0005% to about 10%, even more preferably preventing, combating, eradicating, destroying, repelling, or from about 0.0005% to about 5%, even more preferably from 10 about 0.0005% to about 1% and still even more preferably mitigating pests or increasing the mortality or inhibiting the from about 0.001% to about 0.5% by weight of the extract. growth and/or development of pests. The term "pest” is used Alternatively, where the compounds are in the form of herein in its broadest sense and includes within its scope Substantially purified preparation of B-diones, the formula insects, arachnids (e.g., acari, spiders), helminths (e.g., nema tions will usually comprise as their principal active ingredient todes), molluscs, protozoa (e.g., Plasmodium sp. Parame 15 from about 0.00005% to about 90%, preferably from about cium sp.), viruses (e.g., herpesviruses) and the like. Suitable 0.0001% to about 50%, more preferably from about 0.0005% applications for Such control include, but are not limited to, to about 10%, even more preferably from about 0.001% to combating and/or eradicating infestations by pests in animals about 5% and still even more preferably from about 0.001% (including humans) and/or plants (including trees) and/or to about 1% by weight of the substantially purified f-dione. stored products, which includes the administration to the By “substantially purified’ is meanta compound which has animal or site of an effective quantity of a compound of the been separated from components that naturally accompany it. invention. Typically, a compound is substantially pure when at least By “effective amount' is meant the administration or appli 60%, more preferably at least 75%, more preferably at least cation of that amount of active compound, either in a single 90%, and most preferably at least 99% of the total material dose or as part of a series, that is effective for controlling a 25 (by volume, by wet or dry weight, or by mole percent or mole significant number of pests. Thus, for example, a "pesticid fraction) in a sample is the compound of interest. Purity can ally-effective” amount is the amount of active compound that be measured by any appropriate method, e.g., by chromatog is effective for increasing the mortality or decreasing the raphy or HPLC analysis. growth of a significant number of pests. Alternatively, a "pest Examples of Solid carriers useful in preparing the formu repelling effective amount is the amount of active compound 30 lations are clays including kaolin clay, diatomite, water-con that is noxious to, and/or induces behavioural changes in, a taining synthetic silicon oxide, bentonite, Fubasami clay, and significant number of pests. The effective amount will vary acid clay; talcs; ceramics; inorganic minerals such as Celite, depending upon the taxonomic group of pest exposed to the quartz, Sulfur, active carbon, calcium carbonate and hydrated active compound, the formulation of the composition, and silica; and chemical fertilisers such as ammonium sulfate, other relevant factors. It is expected that the amount will fall 35 ammonium phosphate, ammonium nitrate, urea and ammo in a relatively broad range that can be determined through nium chloride, these solid carriers being finely divided or routine trials. granular. Examples of useful liquid carriers are water, alco hols such as methanol and ethanol, ketones Such as acetone Accordingly, the compounds of the present invention can and methyl ethyl ketone, aromatic hydrocarbons such as ben be used as pesticides, such as but not limited to insecticides, 40 Zene, toluene, Xylene, ethylbenzene and methylnaphthalene, arachnicides, anti-helminthics, molluscicides antivirals, anti aliphatic hydrocarbons such as hexane, cyclohexane, kero protozoals and the like, or as pest repellents including repel sene and light oil, esters such as ethyl acetate and butyl lents of insects, arachnids, helminths, molluscs, protozoa and acetate, nitriles such as acetonitrile and isobutyronitrile, viruses. In especially preferred embodiments, the compounds ethers such as diisopropyl and dioxane, acid amides such as of the present invention are used in the control of insects, 45 N,N-dimethylformamide and N,N-dimethylacetamide, halo arachnids, helminths or molluscs. In practice, the compounds genated hydrocarbons such as dichloromethane, trichloroet can be applied as formulations containing the various adju hane and carbon tetrachloride, dimethyl sulfoxide, and fish vants and carriers known to or used in the industry for facili oils, mineral oils, plant derived oils such as canola oil, cotton tating bioavailability, stability and dispersion. The choice of seed oil, soybean oil and sesame oil as well as essential oils formulation and mode of application for any given compound 50 Such as lavender oil, eucalyptus oil, tea tree oil, citrus oil etc. may affect its activity, and selection will be made accordingly. Solid or liquid carriers can be used alone or in combination. In general, a pest-controlling compound of the invention Examples of gas carriers, i.e., those of propellants, are butane can be compounded with appropriate inert carriers and addi gas, LPG (liquefied petroleum gas), dimethyl ether, fluoro tives in an appropriate ratio by means of dissolving, separat carbons and carbon dioxide gas. ing, Suspending, mixing, impregnating, adsorbing or precipi 55 Examples of surfactants are alkylsulfuric acid esters, alkyl tating operation to formulate into oil formulations, Sulfonic acid salts, alkylarylsulfonic acid salts, alkyl aryl emulsifiable concentrates, wettable powders, flowables, ethers and polyoxyethylene adducts thereof, polyethylene granules, powders, dusts, Solutions, Suspensions, emulsions, glycol ethers, polyhydric alcohol esters, Sugar alcohol deriva controlled-release forms such as microcapsules, aerosols or tives, Sorbitane monolaurate, alkylallyl Sorbitane monolau fumigants. Typically, the compounds of the present invention 60 rate, alkylbenzene Sulfonate, alkylnaphthalene Sulfonate, lig can be mixed with a solid carrier, liquid carrier orgas carrier, nin Sulfonate, and Sulfuric acid ester salts of higher alcohols. optionally together with a Surfactant and other adjuvants use These Surfactants may be used alone or in combination. ful for such formulations. Examples of adjuvants for the formulations, such as bind The compounds of the invention can be used in an amount ers and dispersants, are casein, gelatin, polysaccharides Such from about 0.00005% to about 90% by weight as contained in 65 as starch, gum arabic, cellulose derivatives and alginic acid, these formulations as their active component. As used herein, lignin derivatives, bentonite, Sugars and water-soluble Syn the term “about” refers to a quantity, level, value or amount thetic high-molecular-weight Substances such as polyvinyl US 7,820,209 B2 21 22 alcohol, polyvinyl pyrrolidone and polyacrylic acids. pharmaceuticals, which are usually applied to human bodies Examples of stabilisers are PAP (acid isopropyl phosphate), or animals. They can be formulated in, for example, lotions, BHT (2,6-di-tert-butyl-4-methylphenol), BHA (mixture of aerosols, milky lotions, creams or the like. These compounds 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methox can be further incorporated with other insect repellents, anti yphenol), Synergists such as piperonyl butoxide, vegetable oxidants, UV-absorbers, humectants or other additives. oils, mineral oils, fish oils, Surfactants and fatty acids or esters The above compounds or the above-prepared compositions thereof. of the present invention can be applied directly to human Emulsifying agents that may be used are suitably one or bodies or animals. Besides, Substrates. Such as sheets, films, more of those selected from non-ionic oranionic emulsifying nets, timber or the like, which have preliminarily been treated agents. Examples of non-ionic emulsifying agents include, 10 with the above compounds or compositions by means of but are not restricted to, polyoxyethylenealkylphenylether, application, impregnation or blending, can also be used. polyoxyethylenealkylether, polyethyleneglycol fatty ester, The quantity of the above compounds to be formulated in sorbitan fatty ester, polyoxyethylene sorbitan fatty ester, the noxious-insect repellents depends upon the dosage form, polyoxyethylenesorbitol fatty ester, polyoxyethylenepolyox usage or other conditions. Suitable dosages may be selected ypropylenealkylether. Examples of anionic emulsifying 15 from about 0.1% to about 90% by weight. agents include alkyl Sulphates, polyoxyethylenealkylether Thus, in another aspect of the present invention there is Sulphates, SulfoSuccinates, taurine derivatives, sarcosine provided a method for controlling harmful, annoying or derivatives, phosphoric esters, alkylbenzenesulfonates and undesired pests, said method comprising exposing said pests the like. A mixture consisting of polyoxyethylenestyrylphe to a pest-controlling effective amount of a composition com nylether and calcium allylbenzenesulfonate is preferred. prising a B-dione compound as broadly described above. These emulsifying agents may be used in an amount of 5 to 20 Preferred embodiments of this type include exposing said weight parts per 100 weight parts of the compositions of the pests to a pesticidally effective amount or a pest-repelling present invention. effective amount of said composition. Formulation thus obtained can be used solus or diluted, for The terms “comprise”, “comprises' and “comprising and example, with water or other diluent. The formulations can be 25 the like refer, unless the context requires otherwise, to the used also as admixtures with other pesticides Such as insec inclusion of a stated Step or element or group of steps or ticides, arachnids, anti-helminthics, molluscicides, herbi elements but not the exclusion of any other step or element or cides, plant growth regulators, synergists, soil improvers, group of steps or elements. baits and the like, or can be used simultaneously with Such The compositions and methods of the present invention agents without mixing. For example, the pest-controlling 30 may be applied to pests including, but not restricted to, compounds of the invention can be combined with other insects, arachnids, helminths, molluscs, protozoa and viruses. naturally derived bioactive compounds or extracts such as For example, suitable insects that fall within the scope of the neem or its components, derris, pyrethrum; microbial extracts present invention include those: Such as avermectins or streptomycins; with synthetic insecti (a) from the order of the lepidopterans (Lepidoptera), for cides, acaricides, molluscicides, anti-helminthics; anti-proto 35 example, Adoxophyes Orana, Agrotis ypsilon, Agrotis seg Zoals, antivirals or with microorganisms having insecticidal, etum, Alabama argillacea, Anticarsia gemmatalis, Argy acaricidal, molluscicidal, anti-helminthic anti-protozoal or resthia conjugella, Autographa gamma, Cacoecia murinama, antiviral activity e.g., bacteria Such as Bacillus thuringiensis, Capua reticulana, Choristoneura filmiferana, Chilo partel Bacillus popillae, entomogenous fungi such as Metarhizium lus, Choristoneura Occidentalis, Cirphis unipuncta, Cnapha spp., Verticillium lecanii, nematodes such as Steinernema spp 40 locrocis medinalis, Crocidolomia binotalis, Cydia and Heterorhabditis. Alternatively, or in addition, the pest pomonella, Dendrolimus pini, Diaphania initidalis, Diatraea controlling compounds of the invention can be combined grandiosella, Earias insulana, Elasmopalpus lignosellus, with synergists such as piperonyl butoxide, and with ultra Eupoecilia ambiguella, Feltia subterranea, Grapholitha violet Screening compounds of natural or synthetic origin. finebrana, Grapholitha molesta, Heliothis armigera, Helio When used as an agricultural pesticide, the compound of 45 this virescens, Heliothis zea, Hellula undalis, Hibernia defo the invention is preferably applied usually in an amount of liaria, Hyphantria cunea, Hyponomeuta malinellus, Keiferia 0.01 to 500 g/100 m. When an emulsifiable concentrate, lycopersicella, Lambdina fiscellaria, Laphygma exigua, Leu wettable powder or flowables are used as diluted with water, coptera scitella, Lithocolletis blancardella, Lobesia botrana, the compound is applied usually at a concentration of 0.1 to Loxostege Sticticalis, Lymantria dispar; Lymantria monacha, 1000 ppm, preferably 1 to 500 ppm. The granular or dust can 50 Lyonetia clerkella, Manduca sexta, Malacosoma neustria, be applied without dilution. Mamestra brassicae, Mocis repanda, Operophthera bru The amount or concentration of application, although mata, Orgvia pseudotsugata, Ostrinia nubilalis, Pandemis exemplified above, can be suitably increased or reduced heparana, Panolis flammea, Pectinophora gossypiella, according to the type of preparation, time, place, method of Phthorimaea operculella, Phyllocnistis citrella, Pieris bras application, kind of pest and extent of harm or annoyance 55 sicae, Plathypena scabra, Platynota Stultana, Plutella xylos suffered. tella, Prays citri, Prays oleae, Prodenia Sunia, Prodenia orni The invention also contemplates the use of the above thogalli, Pseudoplusia includens, Rhyacionia frustrana, described B-dione compounds in pest repellent, particularly Scrobipalpula absoluta, Sesamia inferens, Sparganothis pil insect repellent, compositions. Repellent compositions con leriana, Spodoptera frugiperda, Spodoptera littoralis, templated by the present invention include those that are 60 Spodoptera litura, Syllepta derogata, Synanthedon myopae noxious to, and/or induce behavioural changes in, a pest. The formis, Thaumatopoea pity'Ocampa, Tortrix viridana, Tri latter compositions suitably comprise an activity including, choplusia ni, Tryporyza incertulas, Zeiraphera Canadensis, but not restricted to, an antifeedant activity, an oviposition (b) furthermore Galleria mellonella and Sitotroga cere deterrent activity and an insect growth regulatory activity. alella, Ephestia cautella, Tineola bisselliella, Insect repellent compositions in various dosage forms can be 65 (c) from the order of the beetles (Coleoptera), for example, prepared by blending the above-described B-dione com Anthonomus grandis, Anthononus pomorum, Apion vorax, pounds as active ingredients with a base of cosmetics or Atomaria linearis, Blastophagus piniperda, Cassida nebu US 7,820,209 B2 23 24 losa, Cerotoma trifurcata, Ceuthorhynchus assimilis, Sitobion avenae, Sogatella fircifera, Toxoptera citricida, Tri Ceuthorhynchus mapi, Chaetocnema tibialis, Conoderus ves aleurodes abutilonea, Trialeurodes vaporariorum, Viteus pertinus, Crioceris asparagi, Dendroctonus refipennis, vitifolii; Diabrotica longicornis, Diabrotica 12-punctata, Diabrotica (k) from the order of the termites (Isoptera), for example, virgifera, Epilachna varivestis, Epitrix hirtipennis, Eutinobo 5 Calotermes flavicollis, Coptotermes spp., Leucotermesfiavi thrus brasiliensis, Hylobius abietis, Hypera brunneipennis, pes, Macrotermes subhyalinus, Nasutitermes spp. Such as Hypera postica, Ips typographus, Lema bilineata, Lema mel Nasutitermes walkeri, Odontotermes formosanus, Reticuli anopus, Leptinotarsa decemlineata, Limonius Californicus, termes lucifiugus, Termes natalensis, Lissorhoptrus Oryzophilus, Melanotus communis, Melligethes (1) from the order of the orthopterans (Orthoptera), for aeneus, Melolontha hippocastani, Melolontha melolontha, 10 example, Gryllotalpa gryllotalpa, Locusta migratoria, Mel Oulema oryzae, Ortiorrhynchus sulcatus, Otiorrhynchus anoplus bivittatus, Melanoplus femur-rubrum, Melanoplus ovatus, Phaedon cochleariae, Phyllopertha horticola, Phyl mexicanus, Melanoplus sanguinipes, Melanoplus spretus, lophaga sp., Phyllotreta chrysocephala, Phyllotreta nem Nomadacris Septemfasciata, Schistocerca americana, Schis orum, Phyllotreta Striolata, Popilliaiaponica, Psylliodes tocerca peregrina, Stauronotus maroccanus, Schistocerca mapi, Scolytus intricatus, Sitona lineatus, 15 gregaria, (d) furthermore Bruchus rufimanus, Bruchus pisorum, (m) furthermore Acheta domestica, Blatta Orientalis, Blat Bruchus lentis, Sitophilus granaria, Lasioderma serricorne, tella germanica, Periplaneta americana, Oryzaephilus Surinamensis, Rhyzopertha dominica, Sitophi (n) from the order of the phthirapterans (Phthiraptera), for lus Oryzae, Tribolium castaneum, Trogoderma granarium, example, Mallophaga. Such as Damalina spp., and Anoplura Zabrotes subfasciatus, Such as Linognathus and Haematopinus spp.; (e) from the order of the dipterans (Diptera), for example, (o) from the order of the hemipterans (Hemiptera), for Anastrepha ludens, Ceratitis capitata, Contarinia sorghi example, Aphis, Bemisia, Phorodon, Aeneolamia, Empoasca, cola, Dacus cucurbitae, Dacus oleae, Dasineura brassicae, Parkinsiella, Pyrilla, Aonidiella, Coccus, Pseudococcus, Delia coarctata, Delia radicum, Hydrellia griseola, Hyl Helopeltis, Lygus, Dysdercus, Oxycarenus, Nezara, Aley emyia platura, Liriomyza sativae, Liriomyza trifolii, May 25 rodes, Triatoma, Psylla, Myzus, Megoura, Phylloxera, Adel etiola destructor, Orseolia Oryzae, Oscinella frit, Pegomya ges, Nilaparvata, Nephotettix or Cimw.x spp.; hyoscyami, Phorbia antiqua, Phorbia brassicae, Phorbia coarctata, Rhagoletis cerasi, Rhagoletis pomonella, (p) from the order of the siphonapterans (Siphonaptera), (f) furthermore Aedes aegypti, Aedes vexans, Anopheles for example, Ctenocephalides or Pulex spp.; maculipennis, Chrysomya bezziana, Chrysomya hominivo 30 (q) from the order of the thysanurans (Thysanura), for rax, Chrysomya macellaria, Cordylobia anthropophaga, example, Lepisma spp.; Culex pipiens, Fannia canicularis, Gasterophilus intestina (r) from the order of the dermapterans (Dermaptera), for lis, Glossina morsitans, Haematobia irritans, Haplodiplosis example, Forficula spp.; and equestris, Hypoderma lineata, Lucilia caprina, Lucilia cup (s) from the order of the psocopterans (Psocoptera), for rina, Lucilia sericata, Musca domestica, Muscina stabulans, 35 example, Peripsocus spp. Oestrus ovis, Tabanus bovinus, Simulium damnosum, Arachnids contemplated by the present invention include, (g) from the order of the thrips (Thysanoptera), for but are not limited to, Spiders and Scorpions and especially example, Frankliniella fisca, Frankliniella Occidentalis, mites such as phytophagous mites (Acari). Such as Aculops Frankliniella tritici, Haplothrips tritici, Heliothrips haemor lycopersicae, Aculops pelekassi, Aculus Schlechtendali, rhoidalis, Scirtothrips citri, Thrips oryzae, Thrips palmi, 40 Brevipalpus phoenicis, Bryobia praetiosa, Eotetranychus Thrips tabaci, carpini, Eutetranychus banksii, Eriophyes Sheldoni, Oligo (h) from the order of the hymenopterans (Hymenoptera), nychus pratensis, Panonychus ulmi, Panonychus citri, Phyl for example, Athalia rosae, Atta cephalotes, Atta sexdens, locoptruta oleivora, Polyphagotarisonemus latus, Tarson Atta texana, Hoplocampa minuta, Hoplocampa testudinea, emus pallidus, Tetranychus cinnabarinus, Tetranychus Iridomyrmes humilis, Iridomyrmex purpureus, Monomorium 45 kanzawai, Tetranchus pacificus, Tetranychus urticae, ticks, pharaonis, Solenopsis geminata, Solenopsis invicta, Sole Such as Amblyomma americanum, Amblyomma variegatum, nopsis richteri, Technomyrmex albipes, Argas persicus, Boophilus annulatus, Boophilus decoloratus, (i) from the order of the heteropterans (Heteroptera), for Boophilus microplus, Dermacentor Silvarum, Hvalomma example, Acrosternum hilare, Blissus leucopterus, Cyrtopel truncatum, Ixodes ricinus, Ixodes rubicundus, Ornithodorus tis notatus, Dysdercus cingulatus, Dysdercus intermedius, 50 moubata, Otobius megnini, Rhipicephalus appendiculatus Eurygaster integriceps, Euschistus impictiventris, Leptoglo and Rhipicephalus evertsi, and animal-parasitic mites. Such SSus phyllopus, Lygus hesperus, Lygus lineolaris, Lygus prat as Dermanyssus gallinae, Psoroptes Ovis and Sarcoptes sca ensis, Nezara viridula, Piesma quadrata, Solubea insularis, biei. Thyanta perditor, Helminths falling within the scope of the present invention () from the order of the homopterans (Homoptera), for 55 may be selected from cestodes such as fish tapeworm, pork example, Acyrthosiphon Onobrychis, Acyrthosiphon pisum, tapeworm, beef tapeworm, and dwarf tapeworm, trematodes Adelges laricis, Aonidiella aurantii, Aphidula masturtii, Such as from the genera Metagonimus and Heterophyes; and Aphis fabae, Aphis gossypii, Aphis pomi, Aulacorthum Solani, nematodes such as but not limited to filariid, ascarid, Bemisia tabaci, Brachycaudus Cardui, Brevicoryne brassi strongyle and trichoStrongyle nematodes of the genera Acan cae, Dalbulus maidis, Dreyfusia nordmannianae, Dreyfusia 60 thocheilonema, Aelurostrongylus, Ancylostoma, Angios piceae, Dysaphis radicola, Empoasca fabae, Eriosoma lani trongylus, Ascaris, Brugia, Bunostomum, Dictyocaulus, Dio gerum, Laodelphax striatella, Macrosiphum avenae, Macro ctophyme, Dipetalonema, Dirofilaria, Dracunculus, siphum euphorbiae, Macrosiphon rosae, Megoura viciae, Filaroides, Lagochilascaris, Loa, Mansonella, Muellerius, Metopolophium dirhodium, Myzus persicae, Myzus cerasi, Necator. Onchocerca, Parafilaria, Parascaris, Protostrongy Nephotettix cincticeps, Nilaparvata lugens, Perkinsiella sac 65 lus, Setaria, Stephanofilaria, Strongyloides, Strongylus, charicida, Phorodon humuli, Psylla mali, Psylla piri, Psylla Thelazia, Toxascaris, Toxocara, Trichinella, Uncinaria and pyricola, Rhopalosiphum maidis, Schizaphis graminum, Wuchereria. US 7,820,209 B2 25 26 Suitable molluscs include those of the Gastropoda class the progeny of the cross; and (3) growing B-dione-producing/ examples of which include Snails, slugs, conchs, and whelks. pest-resistant plants from the reproductive material. Where Protozoa may be selected for example from Plasmodia, desirable or necessary, the agronomic characteristics of the Toxoplasma, Leishmania, Trypanosoma, Giardia, Entam Susceptible taxon can be substantially preserved by expand Oeba, Acanthamoeba, Nagleria, Hartmanella, Balantidium, ing this method to include the further steps of repetitively: (1) Babesia, Cryptosporidium, Isospora, Microsporidium, Tri backcrossing the pest-resistant progeny with pest-Susceptible chomonas or Pneumocystis species. plants from the Susceptible taxon; and (2) selecting for Viruses may be selected from RNA viruses or DNA expression of a B-dione(oran associated marker gene) among viruses, which include but are not limited to Human Immu the progeny of the backcross, until the desired percentage of nodeficiency Virus (HIV), Poliovirus, Influenza virus, Rous 10 the characteristics of the Susceptible taxon are present in the Sarcoma virus, Flaviviruses such as Japanese encephalitis, progeny along with the gene or genes imparting 3-dione Influenza virus, Respiratory Syncytial Virus, Hepatitis virus, activity. Parvovirus, Rotavirus, Coronavirus, Adenovirus and Herpes By the term “taxon herein is meant a unit of botanical viruses such as Papillomavirus and Epstein-Barr virus. classification. It thus includes, genus, species, cultivars, vari The present invention also extends to methods for produc 15 eties, variants and other minor taxonomic groups which lack ing resistance in plants to pests including, but not limited to, a consistent nomenclature. insects, arachnids, helminths, molluscs, protozoa and viruses In order that the invention may be readily understood and by crossing a plant expressing a B-dione compound according put into practical effect, particular preferred embodiments to the invention with pest Susceptible lines. Crossing a B-di will now be described by way of the following non-limiting one-producing plant into a pest Susceptible background examples. would produce a resistant plant with a high level of pest resistance. Plants that could be made pest resistant include, EXAMPLES but are not limited to, dicotyledonous plants, especially trees and more especially members of the Myrtaceae family. For Example 1 example E. cloeziana commonly known as Gympie Mess 25 mate is one of the many Eucalyptus species grown for hard wood production. However the oil present in this chemotype B-Triketone-Containing Oils Obtained from Australian does not contain B-diones and hence an intra species cross Myrtaceae Species with the unique North Queensland tasmanone chemotype Australia has an extensive number of volatile oils from would introduce this phenotypic trait. Such a process would 30 species of the Myrtaceae that are rich in a diversity of struc be readily applicable to other Eucalyptus species of commer turally related constituents known as B-triketones. These oils cial interest. Interspecific crossing within the Myrtaceae fam often show not only a high yield of oil, but also a high degree ily is well established to those skilled in the art and inclusion of biosynthetic selectivity that produces ?-triketones in a high of B-dione as an additional trait into formal breeding pro proportion of the oil composition. The major constituents of grams is acknowledged. 35 the published Myrtaceae essential oils (Hellyer, 1968; Boland Suitable 3-dione-producing plants may be selected from and Brophy, 1990, 1993: Brophy, et al., 1995; Bignall et al., the families Alliaceae, Apiaceae, Asteraceae, Cannabinaceae, 1997: Southwell and Brophy 2000) are listed in Table 1 and Lamiaceae, Pteridaceae, Myrtaceae, Myoporaceae, Pro their structures are included in FIG. 1. teaceae, Rutaceae and Zingiberaceae. Preferably, the volatile oil-bearing plant is selected from genera of the Myrtaceae 40 TABLE 1 family including, but not limited to, Angophora, Austromyr tus, Backhousia, Baeckea, Callistemon, Corymbia, Dar 3-Triketone Profiles of Australian Essential Oils winia, Eucalyptus, Kunzea, Leptospermum, Melaleuca, Syzy Species Yield B-Triketone (%) Distribn gium and Xanthostemon. Preferred B-dione-producing plants are Leptospermum morrisonii, Eucalyptus bensonii, Euca 45 Backhousia anglisiifolia 2.5 Angustione (85) QLD Backhousia anglisiifolia 2.5 Dehyroangustione (80) QLD lyptus megacornuta, Eucalyptus pilularis, Eucalyptus cor Eucalyptus Cloeziana 3.0 Tasmanone (95) QLD nuta, Eucalyptus baxteri, Eucalyptus macrorhyncha, Euca Eucalyptus Suberea 1.4 Tasmanone (94) WA lyptus cloeziana, Melaleuca Cajuputi, Eucalyptus jensenii, Eucalyptus lateritica 0.9 Tasmanone (37), WA Backhousia angustifolia and Leptospermum scoparium. A lateriticone (14) Eucalyptus Canfieldii Tasmanone (40) NSW particularly preferred B-dione-producing plant is Eucalyptus 50 Leptospermain scoparium 0.4 Leptospermone (19), NSW, NZ Cloeziana. Flavesone (8), As used herein, the term “plant includes reference to Isoleptospermone (5) whole plants, plant organs (e.g., leaves, stems, roots, etc.), Eucalyptus grandis 0.6 Leptospermone (20), NSW Flavesone (13), seeds and plant cells and progeny of same. Plant cell, as used Isoleptospermone (3) herein includes, without limitation, seeds Suspension cul 55 Eucalyptus agglomerata Agglomerone (40) NSW tures, embryos, meristematic regions, callus tissue, leaves, Eucalyptus mckieana Agglomerone (60) NSW roots, shoots, gametophytes, sporophytes, pollen, and Eucalyptus bensonii 2.5 Agglomerone (72) Eucalyptus insularis 1 Agglomerone (19) WA microspores. The class of plants which can be used in the Eucalyptusiensenii 0.3 Jensenone (70) NT methods of the invention is generally as broad as the class of Eucalyptus paptiana 0.7 Papuanone (40) Nth Aus higher plants amenable to transformation techniques, includ 60 Leptospermain morrisonii 1.8 Grandiflorone (58) NSW ing both monocotyledonous and dicotyledonous plants. Melaletica Caiepitti Platyphyllol Nth Aus Xanthostenon Xanthostemone Thus, the present invention contemplates conventional oppositifolius plant breeding methods to transfer the genetic material asso ciated with B-dione production via crossing and backcross ing. Such methods will comprise the steps of: (1) sexually 65 The B-triketones obtained from selected Myrtaceous vola crossing the B-dione-producing plant with a plant from a pest tile oils were shown to have significant insecticidal and/or Susceptible taxon; (2) recovering reproductive material from acaricidal activity. US 7,820,209 B2 27 28 Example 2 TABLE 3-continued Insecticidal Activite Initial insecticidal screening against two important arthro B-Triketone Profiles of Selected Oils pod species, two-spotted mite (Tetranychus urticae) and dia mondback moth (Plutella xylostella) lavae highlighted three Plant Source Principle Component (%) oils on the basis of efficacy, oil yield, oil profile and ease of recollection (Table 2). Where feasible, the LDs and LDs Backhousia angustifolia - IV Angustinone (28%) values were determined. 10 Eucalyptus cloeziana Tasmanone (84-96%) Melaleuca cajuputisubsp platyphylla Platyphyllol (64-71%) TABLE 2 Percentage Mortality of Three Efficacious Oils As determined by gas chromatography
Two Spotted Mite Diamond Back Moth 15 % Mortality % Mortality The most promising oils were derived from B. angustifolia, Species (0.5%/1.0%) (0.5%/1.0%) M. Cajuputi Subsp platyphylla and E. cloeziana and these Backhousia anglisiifolia 98.98 100,100 were then subjected to additional chemical fractionation. The Backhousia anglisiifolia 100,100 100,100 lower insecticidal activity observed in the recollected B. Eucalyptus cloeziana 100,100 70,100 angustifolia (IV) was in part attributed to the lower levels of B-triketone. The level of B-triketone was elevated by the Insecticidal tests using the oil from fresh plant recollec removal of the more Volatile monoterpenes using vacuum tions and steam distillations varied occasionally and, in this distillation. regard, it is believed that improving storage conditions One of the most efficacious oils was from E. cloeziana, a including temperature, light and exposure to air and inclusion 25 tasmanone (84-96%) rich oil with additional terpenes and of a dessicant can enhance the stability of the active fraction B-triketones (FIG. 2, Table 4), which displayed consistent of such oils. activity at every stage of processing and formulation. This oil E. cloeziana oil continued to show high potency against both insect tests. This oil exhibited an LDs between 0.04 was, therefore, fractionated using column chromatography 0.20% (depending on formulation and treatment) against 30 on silica gel with a hexane-diethyl ether gradient and a final two-spotted mite. An LDs of 0.10% was observed against 1 methanol elution. The profiles of the fractions are sum instar lavae of diamond back moth and this rose to 0.78% marised in Table 5. when tested against 3" instar lavae. In additional preliminary investigations with greenhouse thrips (Heliothrips haemor TABLE 4 rhoidalis), a 0.1% concentration of E. cloeziana oil induced 35 100% mortality. Chemical Profile of E. cloeziana Oil Additional work was carried out on the E. Cloeziana oil to Peak No Compound Composition (%) explore the contribution the various components make to the 1 a-pinene O5-19 overall efficacy of this oil. Fractions 1, 3, 4, and 5 outlined in 2 B-pinene 1.5-5.7 Example 3 were screened against two-spotted mite. Fractions 40 3 Limonene O.1-0.6 1 and 5 showed no significant insecticidal effect. Fractions 3 4 a-terpineol O.7-2.O 5 Globulol O.O1-05 and 4, comprising 98% and 99% tasmanone were active and 6 Agglomerone O.O1-0.6 showed little difference to the activity of the whole oil. 7 Tasmanone 84-96 This Suggests that not only is tasmanone the principle 8 Lateriticone O.2-0.7 component in the oil, but it is also the principle bioactive 45 9 Isolateriticone O3-12 constituent. It is also reasonable to assume that as the activity of E. cloeziana oil has been demonstrated against a number of different arthropod species, namely a mite (T. urticae), a caterpillar (P xylostella) and a thrips (H. haemorrhoidalis), TABLE 5 its insecticidal activity is broad in nature. 50 Fractions Cut From E. cloeziana Oil Example 3 Fraction No Solvent System Composition Amount F1 Hexane Hydrocarbons 73 mg Chemistry F2 Hex:Et2O (9:1) 80% Tasmanone 4 mg 55 F3 Hex:Et2O (1:1) 98% Tasmanone 3.66 g Chemical analysis (GC-MS) of the steam-distilled oils F4 Hex:Et2O (9:1) +99% Tasmanone 694 mg from the collected plants in this work are summarised in Table F5 MeOH Terpene alcohols 64 mg 3.
TABLE 3 "H NMR data (FIG.3) were recorded on F4 and confirmed 60 the structure of tasmanone. Moreover the compound exists in 3-Triketone Profiles of Selected Oils solution (CDC1) in tautomeric forms (FIG. 4) in the ratio Plant Source Principle Component (%) 2:0:1 (A:B:C). Another efficacious oils was from M. Cajuputi Subsp platy Backhousia anglisiifolia - I Dehydroangustione 85% Backhousia anglisiifolia - II Dehydroangustione (80%) as phylla, a platyphyllol (64-71%) rich oil (Table 6), which also Backhousia anglisiifolia - III Angustinone (65%) displayed consistent activity at every stage of processing and formulation. US 7,820,209 B2 29 30 eye closure, slow breathing, piloerection and reduced motor TABLE 6 activity, and had returned to normal by 24 hours after dosing. The animals in the 50 mg/kg group did not show any signs of Typical Chemical Profile of M. Caiupui subsp platyphylla Oil toxicity during the seven day experimental period. Peak No Compound Composition (%) There were no other clinical abnormalities in any animal 1 a-pinene Tr-O.8 throughout the seven (7) day observation period. The stomach 2 1,8-cineole Tr-O.7 3 B-caryophyllene O6-32 of one animal in the 250 mg/kg group (5F) had a single ulcer. 4 Humulene O6-13 There were no other gross abnormalities in the major organs 5 Spathulenol 4.0-9.0 10 6 Caryophyllene oxide Tr-36 of any animal at autopsy. 6 Platyphyllol 64-71 7 MW 234 - unknown 4.3 Based on the results obtained from this study 50, 100 and 200 mg/kg can be selected for a main study as the maximum As determined by gas chromatography non-toxic dose, intermediate dose and high dose, respec 15 tively. Example 4 Full Study The acute oral toxicity of E. cloeziana oil was investigated Phytotoxicity in thirty (30) Sprague Dawley Specific Pathogen Fee rats (15 Initial investigations using leaves and leaf discs of several males and 15 females) at doses of 50, 100 and 200 mg/kg. plant species including French bean (Phaseolus vulgaris) These doses were chosen following a dose range finding in lemon (Citrus limon) Orange (Citrus sinensis) and Cabbage the above study. The experimental procedure was based on (Brassica oleracea) indicated that phytotoxicity did not occur OECD guidelines for the testing of chemicals, No. 420. for most oils below 0.5%. More detailed investigations using 25 Soft intact leaves of young greenhouse-grown French bean The acute NOAEL of E. cloeziana oil was determined to be Subsequently showed E. cloeziana oil applied as a spray 50 mg/kg, and the MTD was 200 mg/kg under the conditions caused some phytotoxicity at concentrations of 0.5% and of this study. above. Acute Dermal 30 Example 5 The acute dermal toxicity of E. cloeziana oil was investi gated in ten (10) Sprague Dawley Specific Pathogen Free rats (5 males and 5 females) at a dose of 2000 mg/kg. A prelimi Toxicity of E. cloeziana Oil nary study (SIGHTING) indicated no signs of toxicity at this Bacterial Reverse Mutation Assay 35 dose. The experimental procedure was based on OECD This study investigated the potential of E. cloeziana oil to guidelines for the testing of chemicals, No. 420. induce reverse mutations at the histidine locus in the genome of one strain of Salmonella typhimurium TA100 in the pres No clinical abnormalities, skin irritations or body weight ence and absence of a metabolic activation system (mamma losses were observed in any animal throughout the fourteen lian microsomal enzymes, S9 mix). The test sample was 40 (14) day observation period. No deaths occurred. No abnor dissolved in dimethylsulfoxide (DMSO). In this assay, an E. malities were seen in the major organs at necropsy. The rat cloeziana oil test sample did not induce an appropriate-fold acute dermal LD50 of E. cloeziana oil was determined to be increase (a 2-fold increase for TA100) in the mean revertants greater than 2000 mg/kg under the conditions of this study. per plate in the tester strain TA100 over the mean revertants 45 Skin Irritancy/Corrosion per plate of the appropriate vehicle control. Accordingly, the test sample was considered to be non-mutagenic under the The potential of a test sample of E. cloeziana oil to provoke conditions of the assay. skin irritation/corrosion reactions was investigated using a primary dermal irritation/corrosion test in three (3) New Acute Oral 50 Zealand White albino rabbits (OECD Guidelines for the Test Sighting ing of Chemicals, No. 404). The results obtained from this The acute oral toxicity of E. cloeziana oil was investigated study indicated that E. cloeziana oil is a non-irritant accord in ten (10) Sprague Dawley Specific Pathogen Free female ing to the National Occupational Health and Safety Commis rats (groups of 2) at doses of 500, 250, 125 and 50 mg/kg. The 55 sion (NOHSC) “Approved Criteria for Classifying Hazard experimental procedure was based on OECD guidelines for ous Substances NOHSC: 1008(1999). the testing of chemicals, No. 420. Clinical signs of toxicity occurred between one (1) and Example 6 twenty-four (24) hours after dosing. Both animals in the 500 mg/kg group exhibited Subdued behaviour, partial eye clo 60 Isolation and Purification of B-Triketones from Australian Sure, slow breathing, reduced motor activity, ataxia followed Myrtaceae Species by death within 24 hours. The animals in the 250 mg/kg group Plant collections were commissioned at the Mt. Annan exhibited subdued behaviour, partial eye closure, slow Botanic Garden, Sydney, Australia, and the Darwin Botanic breathing, social isolation, and reduced motor activity, and 65 Garden, Northern Territory, Australia to provide potential had returned to normal by 24 hours after dosing. The animals Sources of a range of B-triketone isolation. The plants inves in the 125 mg/kg group exhibited subdued behaviour, partial tigated for B-triketone exploration are listed in Table 6. US 7,820,209 B2 31 32
TABLE 6 Plants Sourced for B-Triketone Exploration B-triketone # Source Plant Plant Location Accession if anticipated 1 Leptospermum morrisonii Mt. Annan, NSW 873247 grandiflorone 2 Eucalyptus bensonii Mt. Annan, NSW 88.1045 agglomerone 3 Eucalyptus megacornuta Mt. Annan, NSW 831078 jensenone" 4 Eucalyptus pilularis Mt. Annan, NSW 873166 torquatone 5 Eucalyptus cornutta Mt. Annan, NSW 852618 jensenone 6 Eucalyptus baxteri Mt. Annan, NSW 860999 agglomerone taSmalOne 7 Eucalyptus macrorhyncha Mt. Annan, NSW 860896 conglomerone 8 Eucalyptus cloeziana Lappa, QLD PF 2513 taSmalOne 9. Melaleuca Caiutputti SSp Bensbach, WP, PNG KW16-19 platyphyllol platyphyla 10 Eucalyptusiensenii NT Botanic Gardens, RK114- jensenone Darwin, NT LUS 11 Backhousia anglisiifolia Wilgavale, Texas, PF1712 dehydroangustione QLD 12 Backhousia anglisiifolia Didcott Creek, QLD PF 1708 angustione 13 Leptospermin Scoparitin NA commercial oil NA flavesone, sample isoleptospermone, leptospermone 14 Eucalyptus conjuncta Mt Annan 854097 conglomerone "Oil contains other B-triketone constituents
All plants, apart from 9 and 13, were steam distilled to yield The chemical structures and identities of the f-triketones various quantities of essential oil. All oils were analysed by isolated were confirmed by GCMS and Nuclear Magnetic Gas Chromatography Mass Spectrometry (GCMS) to deter Resonance (NMR) analysis. mine the presence and abundance of B-triketones. Based on 30 this information, particular oils were targeted for isolation Example 7 and purification off-triketones using wet chemistry and pre oarative HPLC techniques. Efficacy of E. cloeziana Oil on Target Organisms The B-triketones listed in Table 7 were isolated in quanti Target Organisms ties adequate for insecticidal screening. A minimum of 50 mg 35 of each compound was required. Isoleptospermone and lep Two spotted mite (TSM) Tetranychus urticae Koch Aca tospermone were difficult to separate due to their structural rina; Tetranychidae were collected from a mass culture similarity as were angustione and dehydroangustione. Con maintained at the University of Western Sydney's Hawkes sequently, mixtures of these compounds, where one isomer bury Campus in Richmond, NSW, Australia. They were 40 reared on potted French beans (Phaseolus vulgaris L. Fa was significantly more abundant, were provided for insecti bales: Fabaceae in a glasshouse maintained at 25+5° C. cial screening as this will still allow for differentiation in 65+5% RH and 14 h D:L photoperiod. Only young females observed activity. were selected for bioassay. Adult parthenogenetic female greenhouse thrips (GHT), TABLE 7 45 Heliothrips haemorrhoidalis Bouché (Thysanoptera: Thripi 3-Triketones Isolated for Insecticidal Screening dae) of similar age were obtained from a colony reared on orange fruits and maintained in an insectary at UWS Hawkes Amount buryry under conditions of 25+3° C., 65% RH and 16 h D:L RESample % Screeningsent for photoperiod B-triketones Plant source by GCMS (mg) 50 Young nymphs of citrus. aphids Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae) were collected from grandiflorone Leptospermum morrisonii 100.00 99.5 lemon seedlings grown underglasshouse conditions at UWS Jensenone Eucalyptusiensenii 100.00 67.9 Hawkesb dehydro- Backhousia anglisiifolia 95.02 165.02 awkesbury. angustione PF1712 Workers of the termite Nasutitermes walkeri Hill (Isoptera: angustione Backhousia anglisiifolia 66.30 107.1 5.5 Termitidae) were collected from a laboratory culture at UWS PF1708 iR0. Richmond which was initiated from soil, termitaria and wood angustione) on which termites were feeding were field collected at Rich agglomerone Eucalyptus bensonii 99.28 109.2 mond NSW, and maintained in a darkened container under flavesone Leptospermum scoparium 99.32 101.8 conditions of 25+2° C., 35-68% RH. Termites were fed on isoleptospermone Leptospermain scoparium (c. 113.4 60 wood collected from near the original nest. Moistened soil lepto- 0. from the nest together with paper towel were placed on top of spermone) the nest and made it possible to maintain this culture for leptospermone (E.g. Scoparitim 95.33 77.0 several months in the laboratory at UWS Hawkesbury. taSmanone Eucalyptus Cloeziana 99.92 110.1 platyphyllol Melaletica Cajuputti Subsp. 99.62 255.6 Workers of the whitefooted house ant Technomyrmex albi platyphylia 65 pes (F. Smith) (Hymenoptera: Formicidae) were field col lected at UWS Hawkesbury by baiting in an empty glass jar containing Sugar granules. US 7,820,209 B2 33 34 Pupae of housefly, Musca domestica L (Diptera: Mus caused only 35.5-6.9 mortality. The lowest concentration of cidae) and different stages of American Cockroach, Periplan E. cloeziana oil extract tested in this combination (i.e. 0.01%) eta americana L (Blattodea: Blattidae) were initially supplied resulted in 100% mortality in TSM, which is significantly by C.E.R.I.T and maintained in laboratory culture at UWS lower than that reported earlier in this document to produce Hawkesbury. 100% mortality with E. cloeziana oil extract alone (>0.06%). Tomato russet mites (TRM) Aculops lycopersici (Massee) (Acarina: Eriophyidae) were collected from infested tomato Greenhouse Thrips plants near Riverstone, NSW. The same experimental procedure used for TSM was Mixed sex adults of the mosquito Culex quinquefaciatus repeated for GHT except that lemon leaf discs were used (Diptera: Culicidae) were supplied by C.E.R.I.T. held at the in 10 instead of French beans. The required number of adult thrips the Centre for Horticulture & Plant Science, University of for each treatment was transferred with a fine brush to the Western Sydney, Richmond NSW and treated one day after underside of a lemon disc of the same diameter (6 cm) as the arrival. base of a petridish. The lemon disc was mounted on agar with Workers of the honeybee Apis mellifera (Hymenoptera: its adaxial side uppermost. Immediately after treatment the Apidae) were collected from several field hives maintained at 15 petridish was covered with perforated plastic wrap. Mortality the apiary in the Centre for Horticulture & Plant Sciences, was assessed 24 h after treatment. University of Western Sydney, Richmond NSW Tomato Russet Mite Adults of ash-white-flies Aleaurocanthus woglumi (Ho moptera: Aleyrodidae) were field collected from an ornamen The same experimental procedure for TSM was repeated tal pear (Prunus sp) in the Centre for Horticulture & Plant except that tomato leaf discs were used instead of French Sciences, University of Western Sydney, Richmond NSW. bean. Drug Store beetle Sitodrepa panicea (Coleoptera: Anobi Brown Citrus Aphid idae) were reared on curry powder under laboratory condi Lemon leaf discs 2.5 cm diam. were cut from tender young tions of 25.1° C. and 65.5% RH. leaves and mounted on moistened absorbent cotton wool in Mixed age groups of Snails Helix apersa (Mollusca: Gas 25 90 mm petri dish with their adaxial surface uppermost. Each tropoda) were collected from infested plants in the Centre for petri dish contained four leaf discs. Uniform early instar Horticulture & Plant Science, University of Western Sydney, nymphs were then transferred with a fine brush to the leaf Richmond NSW. discs (each containing 8-10 nymphs). A Potter tower was used Bioassays to apply 5 mL aliquots to each petri dish. A control (Solvent 30 and Surfactant only) was also included in the assessment. TSM Mortality was assessed 24 h after treatment. From an E. cloeziana oil extract (containing 85% tas manone), 1.1765 g was dissolved in 5 mL ethyl alcohol and Termites distilled water containing 200 ppm Triton X-100TM was Twenty uniform termite workers were transferred to 90 added to prepare a 1% stock solution. From this homogenised 35 mm petridishes lined with the same diameter moistened filter stock solution, further serial dilutions of 0.0125, 0.025, 0.05, paper (Whatman No 2). A preliminary trial was carried out 0.10, and 0.12% were prepared by mixing the required using a Potter tower to apply 5 mL aliquots of each concen amount of stock solution in distilled water and Triton X-100 tration. Using this method, all termite workers died 4 h after solution. Each treatment was conducted on 60-80 TSM, treatment in all concentrations, including the lowest concen which were evenly distributed on four French bean leaf discs 40 tration of 0.015%. There was no mortality recorded in the (25 mm diam) contained in 90 mm diam. petridishes. The leaf blank control treatment, and all workers remained alive and discs were placed with their underside uppermost on moist active for >48 h after treatment. Identical results were absorbent cotton wool covered with muslin netting. Water obtained from these investigations, whether the petri dishes was added to the dishes daily to prevent desiccation of the leaf were covered or uncovered after application of the E. cloezi discs. Five mL aliquots (unless otherwise stated) were 45 ana oil extract. applied to each petri dish with a Potter precision spray tower Subsequent investigations further assessed efficacy of E. as described by Herron et al (1995). The average mass of cloeziana oil extract by releasing termite workers on fresh solution applied to each dish was calculated to be 3.95 dried residues. This was carried out by uniformly distributing mg/cm. Mortality was recorded 24 h after treatment. Death one mL of each concentration over the entire Surface area of was recognised by the absence of movement when the test 50 a 90 mm diam filter paper. When the paper was air dry, 20 organisms were mechanically stimulated. Data were analy termite workers were placed in each petri dish. One hundred sedusing SPSS for WindowsTMVersion 7. Probitanalysis was percent mortality was recorded in all four replicates even at carried out for dose-mortality data and heterogeneity of the lowest concentration applied (0.015% w/v=150 ppm). regressions was determined by the Pearson chi-squared char This suggests that the plant extract is a highly toxic contact acteristic. 55 poison to termites. In addition, TSM was treated with E. Cloeziana oil extract in combination with paraffin oil. In particular, E. cloeziana oil Ants extract at levels of 0.1, 0.2,0.3, 0.4 and 0.5 g were weighed The same experimental procedure for termites was out and each was made up to a weight of 10.0 g with a repeated for ants, with the required number of worker ants for formulated paraffin oil (BioPest(R), which was then sonicated 60 each treatment transferred with a fine brush to the filter paper for 10 min. A 1.0% V/v of each blend was prepared by mixing containing a fresh dried residue of the E. cloeziana oil extract. 1.0 mL with distilled water in a 100.0 mL volumetric flask. Immediately after release of the ants the petri dish was cov TSM were transferred to the petri dish following the same ered with perforated plastic wrap, which enabled any excess standard method. Five-mLaliquots were applied to each petri vapours to escape while retaining the ants. Mortality was dish and mortality was recorded 24 h after treatment. All 65 assessed 4h after treatment. In all 4 replicates 100% mortality blends of E. cloeziana oil extract with paraffin oil produced was obtained at concentrations as low as 0.0075% w/v ai 100.0% mortality, compared with Biopest(R) alone which when applied at a rate of 1.0 mL to a 90 mm diam. filter paper. US 7,820,209 B2 35 36 Houseflies Honey Bees One percent E. cloeziana oil extract was prepared using Ten worker honeybees, anaesthetised with carbon dioxide, pure acetone as a diluent. From this solution, further serial were transferred to 90 mm diam. petri dishes lined with dilutions were prepared by adding the required amount in moistened filter paper. Five mLaliquots were applied using a acetone. Five mL aliquots of each concentration of E. cloezi ana oil extract were dispensed into 500 mL kilner jars. The Potter Spray Tower while the bees were still anaesthetised. kilner jars were immediately rotated until dryness to coat the Yellow sponges Soaked in 7% Sugar Solution were then placed inner surface uniformly with the E. cloeziana oil residue. inside petri dishes for bee sustenance, and the bees after After complete dryness, 30-50 pupae were transferred to a 10 treatment were placed in these dishes. The lids that were series of clean uncovered petri dishes (45 mm diam.), one of perforated and covered with muslin netting were placed on which was placed inside each jar. The jar mouth was then the dishes, and mortality was assessed 24 hours after treat covered with nylon netting supported by a rubber band. All ment. adulthouseflies started to emerge from pupae after 48 h and most emerged within a 3 h period. Flies were fed 5% sugar 15 Ash White Flies Solution soaked in absorbent cotton wool. Jars were main For each treatment, a 2.5 cm diam. leaf disc was mounted tained in an incubator at 29°C. on a moistened Whatman #2 filter paper lining the bottom of Mortality was assessed at the end of the third day (i.e., petri dishes. Adult white flies were anaesthetised using car approx. 72h) after application of the E. cloeziana oil residues bon dioxide and 30-50 adults were transferred onto leaf discs. in the kilner jar and the placement of pupae inside the jars. Serial dilutions of 0.00546, 0.0220, 0.0894 & 0.1788% con (This comprised 48 h for pupae to emerge and 24 h exposure centration of E. cloeziana oil extract were prepared using to E. cloeziana oil residues which were now 48 hold). Flies distilled water containing 200 ppm Triton X 100. Three rep were observed to die within a few hours after emergence, whereas in the control they remained alive for >48 h after 25 licates were treated for each concentration. Five-mLaliquots emergence. The total number of adulthouseflies that emerged were applied to each petri dish using a Potter Spray Tower. in each kilner jar was counted and their mortality was After treatment, petridishes were left to dry and then covered recorded. with muslin netting. Mortality assessment was assessed 24h American Cockroaches 30 after white flies had been transferred to petri dishes. Tests were conducted on 10-20 three months old nymphs Drug Store Beetle (mean individual mass 0.2-0.3 g) and replicated three times. One mL of each concentration of E. cloeziana oil extract in One mL of 1.0% E. cloeziana oil extract in acetone was pure acetone was uniformly dispensed on 90 mm diam. What uniformly distributed on 90 mm diam. Whatman No 2 filter paper. A control treatment was also carried out using 1 mL 35 man No 2 filter paper. The latter was left for 1 h to air dry acetone only (i.e., minus E. cloeziana oil extract). After com before being used to line the lid of a 90 mm diam. petri dish. plete dryness of the filter paper, the required number of cock Between 10-15 mixed adults were then transferred into the roaches was transferred inside the kilner jars, and were fed petri dish, which was sealed with ParafilmTM. Mortality dry dog food. Kilner jar necks were covered with muslin 40 assessment was carried out 24 h after sealing the petri dish. netting supported by rubber bands. Mortality was assessed 24 h after releasing the cockroaches, and death was recognised Snails by the absence of movement when the test animals were mechanically stimulated. Two methods were used to assess the efficacy of E. cloe 45 Ziana oil extract against Snails. In the first method different Adult Mosquitoes age groups of adult Snails were dipped directly into a solution A 0.3576 g of 85% ai of E. cloeziana oil extract was dissolve in pure acetone as a diluent to give approximately of 0.5% concentration of E. cloeziana oil extract in distilled 0.304% concentration stock solution. From this solution, fur water containing 200 ppm Triton X 10. Snails were dipped for ther serial dilutions 0.152, 0.076, 0.0043 and 0.00215 were 50 10 seconds and thereafter immediately filtered in a sieve. The prepared by adding the required amount in acetone. Aliquots treated snails were divided in three 500 mL kilner jars con (2.5 mL) of each concentration of E. Cloeziana oil extract taining French bean leaves as a food source. The control was were dispensed into 500 mL kilner jars with total internal carried out using 0% E. cloeziana oil extract. Mortality was surface area as 286.53 cm. The kilnerjars were immediately 55 assessed 24 h after treatment. rotated to coat the inner surface uniformly with the E. cloe Allowing the Snails to crawl on a E. cloeziana oil extract Ziana oil extract residue, until dry. Once completely dry, contaminated Surface carried out the second method. Two mL 10-25 mixed sex adult mosquitos were released into each kilner jar by allowing the mosquitoes to fly from a darkened of 0.08% concentration of E. cloeziana oil extract in pure cage into the naturally lit jars. The jar mouth was Subse 60 acetone were dispensed in 500 mL kilner jars which were quently placed on 110 mm diam. filter paper onto which had rotated to uniformly cover all inner surfaces until dry. Six been placed a yellow sponge soaked in 7.0% Sugar Solution. differentage groups of snails were put into each jar along with Treated jars were kept under laboratory temperature and plant material for food and covered with muslin netting held humidity conditions, (viz. 24+1° C. and 65+5% RH respec 65 by a rubber band. Three replicates of each treatment (0.0 & tively). Mortality was assessed 24 h after releasing the mos 0.08% concentration) were carried out. Mortality was quitoes in the jars. assessed 24 h after releasing the Snails inside the jars. US 7,820,209 B2 37 38 The above bioassay results are summarised in Table 8.
TABLE 8 Summary of E. Cloeziana Oil Extract Efficacy Against Various Target Pests Method of Target Pest LDso (95% CL) LDos (95% CL) application Remarks TSM O.O7 O.14 Potter spray Knockdown effect (0.06-0.08) (0.12–0.16) ower; Aliquot observed 2 h after applied 2.5 mL treatinent TSM <0.01 E. Cloeziana oil 100% mortality at extract + paraffin O.O1% oil TRM O.O2 O.04 Potter spray tower GHT O.10 O.12S Potter spray tower Aphid O.08 O.30 Potter spray tower American 157.27 g/cm E. cloeziana oil cockroach residues on filter nymph paper Ant <0.0075 E. Cloeziana oil 100% mortality at residues on filter 0.0075% when 1 mL paper applied as residue on filter paper Termite <0.0075 E. Cloeziana oil 100% mortality at residues on filter 0.0075% when 1 mL paper applied as residue on filter paper DBM 1 O.09 O.2O 5 mL Potter spray instar larva OWe Housefly 69.8 g/cm 130.87 g/cm E. cloeziana oil adult residues on kilner iar wall Adult O.OO387 O.OO694 Residues on Apparently very mosquitoes (0.00328-0.00451) (0.00596-0.01051) kilner jar wall rapid knock down. Ash white O.O1773 O.O.O3728 Potter spray Assessed 4h after fly (0.01179-0.03446) (0.02657-0.08337) tower, 2.5 mL treatinent aliquot Drug store O.O3S48 0.37275 Self Mortality assessed beetle contaminating by 24 h after releasing walking on filter adults. paper Honeybees O.12 O4O Potter spray tower Knockdown effect Aliquot applied 5 mL observed 2 h after treatinent Snails --O.O84 E. Cloeziana oil Three mL acetone residues on kilner solution for each of jar wall 3 replicates. Snails crawled on the wall on dried residues. Snails 100% Dipping method Three replicates mortality in 0.5%
Potted Plant Investigations revealed that the mean percent mortality in E. cloeziana oil The efficacy of E. cloeziana oil extract against TSM was extract treatment and control were 92.19+6.39 and further assessed under greenhouse conditions. French bean 0.25+0.46, respectively. 50 In Summary, E. cloeziana oil extract was efficacious plants were grown in 15 cm diam. plastic pots in an insecti against all pests tested namely TSM, TRM, GHT, aphids, cide-free glasshouse maintained at 27°C., RH 65% and natu termites, houseflies, American cockroaches, whitefooted ants ral light. Plants were used when they reached the two true-leaf adult mosquitoes, ash whitefly, drug store beetle and Snails. It stage (i.e., before trifoliate leaves appeared). One plant was was also toxic to honey bees. maintained in each pot (i.e., two leaves/pot). Twenty gravid 55 TSM females were then transferred with a fine brush to the Example 8 upper surface of each leaf. Mites were left to settle for 4 h before treatment, during which time they usually settled on E. cloeziana Oil Extract as a Fumigant the lower leaf Surfaces. E. cloeziana oil extract at a concen An investigation was undertaken to determine the fumiga 60 tion action of E. cloeziana oil extract against arthropods. The tration of 0.07% as well as a blank control were prepared test organisms used were termites, Nasutitermes walkeri. A using the same procedures described above for the laboratory Whatman No 2 filter paper (90 mm diam.) was immersed in bioassays. A 400 mL hand sprayer was used to apply the distilled water five seconds and left to drain excess water pesticide evenly to all aerial surfaces of the plants, to run-off. before placing it on the bottom of a 90 mm diam. petridish, to Each treatment was replicated four times. The mortality was 65 provide moisture for termite workers during the experimental assessed 24 h after treatment. The results were recorded as period. A 1.0% E. cloeziana oil solution was prepared in pure mean percent mortality with standard deviation. These results acetone. One mL was uniformly spread on a second filter US 7,820,209 B2 39 40 paper, which was allowed to air dry on a sheet of aluminium effects at all concentrations tested. Convulsion and lack of foil. It was then placed under the lid of the petri dishes movement were consistently observed and 60.0% mortality containing the moist filter paper on their base. Twenty uni form worker termites were transferred to the moist base of was recorded 72 h after application in the 0.4% treatment. each test petri dish, which was subsequently covered with its 5 lid containing the treated filterpaper, and the dishes were then Examples 11 sealed with ParafilmTM. A similar series of control treatments was also prepared, using acetone only, for comparison. Five replicates were used in each treatment. The termites did not move to the dry top surface and 10 remained on the water-moistened filter paper lining the base Ready-to-use miticide spray - I of the petri dishes throughout the experimental period. Mor tality was assessed 5 h after termite release. The results revealed that E. cloeziana oil extract has highly Ingredient Parts significant fumigant effects on termites. One hundred percent 15 mortality was recorded in all replicates of the E. cloeziana oil E. Cloeziana extract O.1 treatment whereas no mortality occurred in any of the control Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.O (acetone only) replicates. polyoxyethylenesorbitan, organosilicate Example 9 2O Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 5 Tannic acid 1.O Efficacy of Purified B-Triketones Against TSM Carrier e.g. water 92.9 The efficacy of purified B-triketones against TSM was investigated using the TSMbioassay described in Example 8. All B-triketones tested demonstrated a high level of activity 25 against TSM (see Table 9).
TABLE 9
Efficacy of B-Triketones Against TSM
Sample wt No. (mg) Chemical Name LDso and 95% CL LDos and 95% CL
1 109.2 Agglomerone 0.15 (0.11-021) 0.33 (0.22-1.06) 2 99.5 Grandiflorone O.04 O.13 3 165.0 Dehydroangustione 0.36 (0.33-0.41) 0.69 (0.61-0.81) 4 107.1 Angustione 0.22 (0.21-0.24) 0.35 (0.31-0.40) 5 67.9 Jensenone No direct mortality occurred in any concentration tested (0.05-0.4%) within 24 h. However, all treated TSM were unable to move normally and continued to convulse until they commenced to die 72 h after treatment. There was no recovery. 6 110.1 Tasmanone 0.055 O.1SO 7 101.8 Flavesone 0.020 (0.006-0.043) 0.0876 (0.076-0.114) 8 77.0 Leptospermone O.O37 O.169 9 113.4 Isoleptospermone 0.043 (0.027-0.057) 0.071 (0.058–0.109) 10 141.1 Platyphyllol O.O70 O.23
Example 10 Examples 12 55 Efficacy of Purified B-Triketones Against GHT The efficacy of purified B-triketones against GHT was investigated using the GHT bioassay described in Example 8. 60 Concentrated natural emulsifiable concentrate spray (4.4%) - I All B-triketones tested demonstrated a high level of activity Ingredient Parts against GHT (see Table 9). E. Cloeziana extract 4.4 Pyrethrins 7.4 All B-triketones exceptjensenone caused 100%O mortality Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 14.7 on GHT at a concentration of 0.3% when 5 mL aliquots were polyoxyethylenesorbitan, organosilicate applied with a Potter spray tower. The latter B-triketone did 65 Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.5 not cause direct mortality within 24h, but caused behavioural US 7,820,209 B2 41 42 Example 12 Example 16
Molluscidal dust Concentrated emulsifiable concentrate spray (44%) Ingredient Parts Ingredient Parts E. Cloeziana extract 2 10 Anti-caking agent (e.g. silica gel) 2 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 3 E. Cloeziana extract 22.0 polyoxyethylenesorbitan, organosilicate Platyphyllol (natural or synthetic) 22.0 Inert carrier (talc, kaolin, diatomaceous earth) 93 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 34.0 polyoxyethylenesorbitan, organosilicate 15 Example 17 Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 22
Example 13 2O Aerosol insecticidal and acaricidal Spray Ingredient Parts E. Cloeziana extract 1.O Piperonyl butoxide O.9 25 Propellent hydrocarbon 98.1 Natural ready to use insecticide spray
Ingredient Parts Example 18
E. Cloeziana extract O.3 30 Lavender oil 1.O Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.0 polyoxyethylenesorbitan, organosilicate RepellentD Solvent e.g. ethyl alcohol, isopropyl alcohol etc 40 Ingredient Parts 35 Carrier: Water 57.7 E. Cloeziana extract 19.5 Citronella oil 29.1 Phthalic acid dibutyl ester 29.1 N-octyl bicycloheptenedicarboxamide 22.3 Example 14 40 Examples 19
Oil-based natural spray concentrate 45 Ingredient Parts Ready-to-use miticide spray - II E. Cloeziana extract 1O.O Petroleum oil 89.0 Ingredient Parts Ellispinylylyethanol. 1. Melaletica Caieptiti extract O.2 polyoxyetny , Org 50 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.O polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol, isopropyl alcohol etc S.O Tannic acid 1.O Example 15 Carrier e.g. water 92.8
55 Examples 20 Concentrated emulsifiable concentrate spray (10%
Ingredient Parts 60 E. Cloeziana extract 1O.O Concentrated natural emulsifiable concentrate Spray (4.4%) - II Permethrin 1O.O Piperonyl butoxide 28.0 Ingredient Parts Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 31.0 polyoxyethylenesorbitan, organosilicate Melaletica Caieptiti extract 8.8 Solvent e.g. ethyl alcohol, isopropyl alcohol etc 21 65 Pyrethrins 7.4 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 14.7 US 7,820,209 B2 43 44 Example 25 -continued Intra-Specific Crosses for Imparting Pest Resistance to a Pest Concentrated natural emulsifiable concentrate spray (4.4%) - II Susceptible Plant Ingredient Parts Controlled and wild pollination within Eucalyptus and polyoxyethylenesorbitan, organosilicate other important commercial Myrtaceae are thoroughly Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 69.1 addressed in the CRC for Sustainable Production Forestry Symposium on Hybrid Breeding and Genetics Controlled Pollination of Eucalypts on 12th Apr., 2000 and published as 10 the proceedings of that symposium. Genetic Pollution from Examples 21 Farm Forestry (Potts et al., 2001) deals more specifically with intra and inter species crosses occurring within the Myrta CCaC.
15 Using the protocols described in the above publications, an Ready-to-use miticide spray - III intra-specific Eucalyptus cross breeding technique will employ the following protocol. This begins with the selection Ingredient Parts of Supreme individuals as parent stock. Within a selected 99% Tasmanone O.09 parent stock having Superior pest resistant characteristics Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.O (e.g., E. cloeziana) male and female trees are identified for polyoxyethylenesorbitan, organosilicate cross-pollination experiments. Pollen is harvested from the Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 5 male trees and either stored or directly transferred to the Tannic acid 1.O female trees if flowering is synchronous. Emasculation is Carrier e.g. water 92.91 undertaken to preclude extraneous pollination occurring and flowers are often bagged as a further precaution. Seed set and 25 Subsequent embryo development proceeds over the ensuing Examples 22 12-24 months and F1 seeds are collected at full maturation. Seeds are then germinated to produce seedlings that are Subjected to detailed analysis to assess the transfer of traits from parent to progeny. If the F1 progeny show a desirable 30 mix of phenotypic traits these progeny can be used to vegeta Concentrated natural emulsifiable concentrate spray (4.4%) - III tively propagate the new variety. If the F1 progeny show some Ingredient Parts improvement in the selected phenotypic traits, but further improvement is required selected F1 progeny can be back 99% Tasmanone 4.0 Pyrethrins 7.4 35 crossed either within the F1 progeny or with one of the parent Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 14.7 trees to produce an F2 progeny using the methodology out polyoxyethylenesorbitan, organosilicate lined above. This iterative process can be continued ad infini Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.9 tum until the desired characteristics are achieved. Example 26 Examples 23 40 Inter-Specific Crosses for Imparting Pest Resistance to a Pest Susceptible Plant Ready-to-use miticide spray - IV Inter-species hybridisation in the wild, which is a common 45 phenomenon within the Subgenera of the Myrtaceae, have Ingredient Parts been recorded at almost 40% in the Eucalypts, 33% in Ango phora 19% in Corymbia and 19% in Symphomyrtus. Hybridi 99% Platyphyllol O.09 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.O sation between the major Subgenera may also occur (Potts et polyoxyethylenesorbitan, organosilicate al., 2001). For example, Eucalyptus camaldulensis displays Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 5 50 14 natural hybrid crosses including E. CamaldulensisXE. Tannic acid 1.O robusta, E. CamaldulensisXE. alba, E. CamaldulensisXE. cla Carrier e.g. water 92.91 docalyx, E. CamaldulensisXE. bigalaterita, E. Camaldulen sisXE. tereticornis, E. camaldulensisXE. blakelvi, E. cam aldulensisXE. dwyeri, E. CamaldulensisXE. rudis, E. Examples 24 55 camaldulensisXE. ovata, E. CamaldulensisXE. bridgesiana, E. CamaldulensisXE. viminalis, E. CamaldulensisXE. largiflo rens, E. camaldulensisXE. meliodora, E. CamaldulensisXE. leucoxylon, 15 manipulated hybrids including E. Camaldu Concentrated natural emulsifiable concentrate spray (4.4%) - IV lensisXE. diversicolor, E. camaldulensisXE. grandis, E. cam 60 aldulensisXE. botryoides, E. camaldulensisXE. cladocalyx, Ingredient Parts E. CamaldulensisXE. tereticornis, E. camaldulensisXE. 99% Platyphyllol 4.0 blakelyi, E. camaldulensisXE. urophylla, E. camaldulensisx Pyrethrins 7.4 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 14.7 E. macarthurii, E. camaldulensisXE. exerta, E. Camaldulen polyoxyethylenesorbitan, organosilicate sisXE. maidenii, E. camaldulensisXE viminalis, E. Camaldu Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.9 65 lensisXE. globulus, E. camaldulensisXE. gunnii, E. camaldulensisXE. laevOpinea, E. CamaldulensisXE. fasti gata. In another example Eucalyptus globulus displays 15 US 7,820,209 B2 45 46 natural hybrids including E. globulusXE. barberi, E. globu Southern Australian Species of the Genus Eucalyptus (Se lusXE. brookeriana, E. globulusXE. ovata, E. globulusXE. ries I). Part XIV. Subgenus Monocalyptus. Flav Frag. Jour kitsoniana, E. globulusXE.goniocalyx, E.globulusXE.. norto nal, 12, 177-183. nii, E. globulusXE. Cypellocarpa, E. globulusXE. pseudoglo Boland, D. and J. Brophy (1993). Essential Oils of the Euca bulus, E. globulusXE. bicostata, E.globulusXE...johnstonii, E. lyptus and Related Genera: Search for Chemical Trends. globulusXE. viminalis, E. globulusXE. Cordata, E. globulusX Bioactive Volatile Compounds from Plants. R. Teranishi, E. rubida, E. globulusXE. urnigera, E. globulusXE. perrini R. G. Buttery and H. Sugisawa. Washington D.C., Ameri ana and 13 Successful manipulated hybrids including E. can Chemical Society. 525: 72-87. globulusXE. urophylla, E. globulusXE. grandis, E. globulusX Brophy, J.J. and D. J. Boland (1990). “Leaf Essential Oil of E. robusta, E. globulusXE. pellita, E. globulusXE. longifolia, 10 Two Chemotypes of Eucalyptus cloeziana F.Muell.” Jour E. globulusXE. loxophloeba, E. globulusXE. Camaldulensis, nal of Essential Oil Research 2(March/April): 87-90. E. globulusXE. dunnii, E. globulusXE. Initens, E. globulusXE. Brophy, J. J. R. J. Goldsack, et al. (1995). “Leaf Oils of the maidenii, E.globulusXE. bicostata, E.globulusXE. viminalis, Genus Backhousia (Myrtaceae).” Journal of Essential Oil E. globulusXE. gunnii. In another example Eucalyptus gran Research 7(May/June): 237-254. dis displays 4 natural hybrids including E. grandisXE. uro 15 CRC for Sustainable Production Forestry. Symposium on phylla, E. grandisXE. robusta E. grandisXE. pellita E. gran Hybrid Breeding and Genetics Controlled Pollination of disxE. terreticornis and 14 manipulated hybrids including E. Eucalypts, Noosa Australia, 12th Apr. 2000 grandisXE. urophylla E. grandisXE. botryoides E. grandisx Hellyer, R. (1968). “The Occurrence of B-Triketones in the E. pellita E. grandisXE. alba E. grandisXE. terreticornis E. Steam-Volatile Oils of some Myrtaceous Australian grandisXE. Camaldulensis E. grandisXE. dunnii E. initensXE. Plants.” Aust. J. Chem. 21 (11): 2825-2828. maidenii E. grandisXE. globulus E. grandisXE. gunnii E. Herron GA, Beatie GAC, Parkes R A & Barchia I. 1995. grandisXE. pulverulenta E. grandisXE. leucoxylon, E. gran Potter spray tower bioassay of selected citrus pests to disxE. resinifera. In another example the Corymbia henryi/ petroleum spray oil. Journal of Australian Entomological variegata/maculata/citriiodora complex displays 14 natural Society 34: 225-263. hybrids including C. citriiodorax.C. catenaria, C. citriodorax 25 Potts, BM, Barbour, R C and Hingston, A H, 2001. Genetic C. variegata, C. citriodorax.C. maculata, C. maculataxC. Pollution from Farm Forestry. Rural Industries Research gummifiea, C. maculataxC. intermedia, C. maculataxC. cit and Development Corporation Publication (No. 01/114). riodora, C. maculataxC. variegata, C. variegataxC. blox Southwell, I. A. and J. J. Brophy (2000). “Essential oil iso Someri, C. variegataxC. Watsoniana, C. variegataxC. citri lates from the Australian Flora. Part 3.” Journal of Essen Odora, C. variegataxC. maculata, C. HenryixC. torelliana, 30 tial Oil Research 12: 267-278. C. henryixC. variegata and one manipulated hybrid C. torel What is claimed is: lianaxc. In one further example Eucalyptus cloeziana dis 1. A method for controlling insects, arachnids, molluscs, plays only 1 natural hybrid E. cloezianaxE. acnenoides. protozoa and helminths, said method comprising exposing The disclosure of every patent, patent application, and the insects, arachnids, molluscs, protozoa and helminths to a publication cited herein is hereby incorporated herein by 35 pest-controlling effective amount of the compound tas reference in its entirety. manone (1-isobutroyl-4-methoxy-3, 5,5-trimethylcyclohex The citation of any reference herein should not be con 3-en-2,6-dione). Strued as an admission that such reference is available as 2. The method of claim 1, wherein the compound is obtain “Prior Art” to the instant application able from a volatile oil-bearing organism. Throughout the specification the aim has been to describe 40 3. The method of claim 2, wherein the volatile oil-bearing the preferred embodiments of the invention without limiting organism is selected from Volatile oil-bearing plants. the invention to any one embodiment or specific collection of 4. The method of claim 2, wherein the volatile oil-bearing features. Those of skill in the art will therefore appreciate that, organism is selected from genera of the Myrtaceae family. in light of the instant disclosure, various modifications and 5. The method of claim 2, wherein the volatile oil-bearing changes can be made in the particular embodiments exempli 45 organism belongs to the genus Eucalyptus. fied without departing from the scope of the present inven 6. The method of claim 1, wherein the pest that is con tion. All Such modifications and changes are intended to be trolled is selected from insects, arachnids and molluscs. included within the scope of the appended claims. All figures, 7. The method of claim 1, wherein the helminth is a nema tables, and appendices, as well as publications, patents, and tode. patent applications, cited herein are hereby incorporated by 50 8. The method of claim 1, wherein the compound is used in reference in their entirety for all purposes. the form of a pest-controlling composition which comprises I. BIBLIOGRAPHY from about 0.00005% to about 90% by weight of said com pound. Bignall, C.M., Dunlop, P. J., Brophy, J.J. and Fookes, C.J. R. (1997). Volatile Leaf Oils of some South-western and UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,820,209 B2 Page 1 of 2 APPLICATIONNO. : 12/134035 DATED : October 26, 2010 INVENTOR(S) : Robert Neil Spooner-Hart and Albert Habib Basta It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
Title Page 1, Item (56) in column 2, (item 56) at line 12, Under Other Publications, change “manuku to -manuka--. Title Page, Item (56) Ref. Cited, Insert the Following: --AHMED, S.M., et al., “Vapor toxicity and repellency of some essential oils to insect pests, STN CAPLUS, Vol. 15, No. 105, 1988, No. 105 XP002089702 (Abstract). BLOOR, S.J., “Antiviral Phloroglucinois from New Zealand Kunzee species, J Nat Prod, Vol. 55, 1992, pp. 43-47. DUKE, S.O., et al., “Natural products as sources of herbicides: current status and status and future trends, Weed Research, Vol. 40, 2000, pp. 99-111. GHISALBERTI, E.L., “Bioactive Acylphloroglucinol derivatives from Eucalyptus species, Phytochemistry, Vol. 41, pp. 7-22. KERAMARIS, K.E., et al., “Egg laying suppression in Drosophila melanogaster (Diptera:Drosophilidae) and Dacus (Bactrocera) oleae (Diptera: Tephritidae) by phloroglucinol, a peroxidase inhibitor, Bull Entomological Res, Vol. 86, 1996, pp. 369-375. PORTER, N.G., et al., “Chemical, physical and antimicrobial properties of essential oils of Leptosppemum scoparium and Kunzea ericoldes”, Phytochemistry, Vol. 50, 1998, pp. 407-415.--. In column 3 at line 60, Change “thiazaoyl, to --thiazoly 1.--. In column 11 at line 62-63, Change “thioexpoxides to --thioepoxides--. In column 12 at line 12, Change "purpoSS: to -purposes:--. In column 15 at line 55, Change “Lawassons to --Lawessons--. In column 20 at line 59, Change “sorbitane to --Sorbitan--. In column 20 at line 59, Change “sorbitane to --Sorbitan--. In column 27 at line 2 (approx.), Change “Activite to --Activity--. Signed and Sealed this Thirteenth Day of December, 2011
David J. Kappos Director of the United States Patent and Trademark Office CERTIFICATE OF CORRECTION (continued) Page 2 of 2 U.S. Pat. No. 7.820,209 B2 In column 27 at line 7, Change “lavae to -larvae--. In column 27 at line 26, Change “dessicant to --desiccant--. In column 27 at line 32, Change “lavae to --larvae--. In column 27 at line 33, Change “lavae. to --larvae.--. In column 31 (Table 6) at line 12, Change “platyphyla to -platyphylla--. In column 31 (Table 6) at line 17, Change “Didcott to --Didcot--. In column 31 at line 32-33, Change “preoarative' to --preparative--. In column 31 at line 40-41, Change “insecticial to -insecticidal--. In column 32 at line 36-37, Change “Acarina; to --Acarina:--. In column 32 at line 49, After “photoperiod’ insert ----. In column 32 at line 51, Change "(Kirkaldy) to --(Kirkcaldy)--. In column 33 at line 9, Change "quinquefaciatus to --quinquefasciatus--. In column 33 at line 17, After “NSW’’ insert ----. In column 33 at line 18, Change “Aleaurocanthus to --Aleurocanthus--. In column 35 at line 58, Change “mosquitos to -mosquitoes--. In column 42 at line 6, Change “Molluscidal to --Molluscicidal--. In column 44 at line 47, Change "Symphomyrtus. to --Symphyomyrtus.--. In column 44 at line 58, Change “leucoxylon, to --leucoxylon;--. In column 45 at line 39, After “application insert ----.