COMPOSITION AND RODENTICIDE BAIT COMPRISING DIFETHIALONE AND METHOD FOR CONTROLLING TARGET RODENT PESTS

Abstract

Disclosed is to a composition including difethialone, containing a configurational stereoisomer of difethialone, referred to as a hetero-stereoisomer, of formula 3-(4-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which the carbon atoms 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have different absolute configurations, the composition including an amount of a dextrorotatory enantiomer of the hetero-stereoisomer such that the ratio of this amount to the amount of difethialone in the composition is less than 10%; the amount of the dextrorotatory enantiomer of the hetero-stereoisomer of difethialone in the composition being different from the amount of levorotatory enantiomer of the hetero-stereoisomer of difethialone in the composition.

Claims

1-13. (canceled)

14. Rodenticidal bait comprising difethialone and at least one excipient that is edible for target rodent pests, comprising a configurational stereoisomer of difethialone, named hetero-stereoisomer, the formula of which is 3-(4-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have different absolute configurations, the rodenticidal bait comprising an amount of dextrorotatory enantiomer of said hetero-stereoisomer such that the ratio of this amount to the amount of difethialone in the rodenticidal bait is less than 10%; the amount of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone in the rodenticidal bait being different from the amount of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the rodenticidal bait.

15. Rodenticidal bait according to claim 14, further comprising a configurational stereoisomer of difethialone, named homo-stereoisomer, the formula of which is 3-(4-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have the same absolute configuration, the rodenticidal bait comprising an amount of a laevorotatory enantiomer of said homo-stereoisomer of difethialone such that the ratio of the amount of laevorotatory enantiomer of said homo-stereoisomer of difethialone to the total amount of difethialone in the rodenticidal bait is less than 10%.

16. Rodenticidal bait according to claim 14, further comprising an amount of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of the amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the rodenticidal bait to the total amount of difethialone in the rodenticidal bait is less than 10%.

17. Composition according to claim 14, further comprising an amount of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone such that the ratio of this amount to the total amount of difethialone in the composition is between 10% and 100%.

18. Rodenticidal bait according to claim 14, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

19. Rodenticidal bait according to claim 14, further comprising a mass amount of difethialone such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is less than 200 ppm.

20. Rodenticidal bait according to claim 15, further comprising an amount of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of the amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the rodenticidal bait to the total amount of difethialone in the rodenticidal bait is less than 10%.

21. Composition according to claim 15, further comprising an amount of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone such that the ratio of this amount to the total amount of difethialone in the composition is between 10% and 100%.

22. Composition according to claim 16, further comprising an amount of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone such that the ratio of this amount to the total amount of difethialone in the composition is between 10% and 100%.

23. Rodenticidal bait according to claim 15, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

24. Rodenticidal bait according to claim 16, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

25. Rodenticidal bait according to claim 17, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

26. Rodenticidal bait according to claim 15, further comprising a mass amount of difethialone such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is less than 200 ppm.

27. Rodenticidal bait according to claim 16, further comprising a mass amount of difethialone such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is less than 200 ppm.

28. Rodenticidal bait according to claim 17, further comprising a mass amount of difethialone such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is less than 200 ppm.

29. Rodenticidal bait according to claim 18, further comprising a mass amount of difethialone such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is less than 200 ppm.

30. Composition according to claim 20, further comprising an amount of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone such that the ratio of this amount to the total amount of difethialone in the composition is between 10% and 100%.

31. Rodenticidal bait according to claim 20, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

32. Rodenticidal bait according to claim 21, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

33. Rodenticidal bait according to claim 22, wherein the edible excipient comprises at least one food chosen from the group formed from cereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes, cereal bran and non-cereal seeds.

Description

[0130] Other aims, characteristics and advantages of the invention will emerge on reading the following description and the examples, which are given for purely non-limiting purposes and which refer to the attached figures, in which:

[0131] FIG. 1 relates to the dextrorotatory enantiomer of said hetero-stereoisomer of difethialoneFIG. a1) represents an analysis by high-pressure liquid chromatography on a chiral column of the configurational stereoisomers of difethialone (top) and of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone (bottom)FIG. b1) represents a circular dichroism spectrum of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialoneFIG. c1) is the proton NMR spectrum at 500 MHz of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone;

[0132] FIG. 2 relates to the laevorotatory enantiomer of said hetero-stereoisomer of difethialoneFIG. a2) represents an analysis by high-pressure liquid chromatography on a chiral column of the configurational stereoisomers of difethialone (top) and of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone (bottom)FIG. b2) represents a circular dichroism spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialoneFIG. c2) is the proton NMR spectrum at 500 MHz of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone;

[0133] FIG. 3 relates to the dextrorotatory enantiomer of said homo-stereoisomer of difethialoneFIG. a3) represents an analysis by high-pressure liquid chromatography on a chiral column of the configurational stereoisomers of difethialone (top) and of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone (bottom)FIG. b3) represents a circular dichroism spectrum of the dextrorotatory enantiomer of said homo-stereoisomer of difethialoneFIG. c3) is the proton NMR spectrum at 500 MHz of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone; and

[0134] FIG. 4 relates to the laevorotatory enantiomer of said homo-stereoisomer of difethialoneFIG. a4) represents an analysis by high-pressure liquid chromatography on a chiral column of the configurational stereoisomers of difethialone (top) and of the laevorotatory enantiomer of said homo-stereoisomer of difethialone (bottom)FIG. b4) represents a circular dichroism spectrum of the laevorotatory enantiomer of said homo-stereoisomer of difethialoneFIG. c4) is the proton NMR spectrum at 500 MHz of the laevorotatory enantiomer of said homo-stereoisomer of difethialone.

A. PURIFICATION OF THE CONFIGURATIONAL STEREOISOMERS OF DIFETHIALONE

A.1. Identification of said homo-stereoisomer of difethialone and of said hetero-stereoisomer of difethialone

[0135] The homo-stereoisomer of difethialone is identified by proton magnetic resonance (.sup.1H-NMR) spectroscopy. The homo-stereoisomer of difethialone dissolved in CDCl.sub.3 has a multiplet with a chemical shift () of between 4.9 ppm and 5.1 ppm and corresponding to the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of difethialone as illustrated in FIGS. c3) and c4).

[0136] The hetero-stereoisomer of difethialone is identified by its proton NMR spectrum acquired in CDCl.sub.3, in which the chemical shift of the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer of difethialone is about 5.3 ppm.

A.2. Separation of the Laevorotatory and Dextrorotatory Enantiomers of Said Homo-Stereoisomer and of Said Hetero-Stereoisomer of Difethialone by High-Pressure Liquid Chromatography

[0137] The inventors solved the complex and hitherto unresolved problem of separating the laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone and of the laevorotatory and dextrorotatory enantiomers of said homo-stereoisomer of difethialone from a composition of difethialone that is predominantly in the form of said hetero-stereoisomer. They succeeded in separating the configurational stereoisomers of difethialone by high-pressure (high-performance) liquid chromatography on a LUX Cellulose-3 chiral column (Phenomenex, Le Pecq, France) of dimensions 1502 mm and comprising a chiral stationary phase constituted of porous particles of cellulose tris(4-methylbenzoate), with a particle size of 3 m and a porosity of 1000 and using, as mobile phase, an eluent formed from a mixture of acetonitrile (A) and water comprising formic acid in a volume proportion of 0.1% in the water (B), with an AB volume ratio of 80/20. The flow rate of the mobile phase in the column is 0.25 mL/minute and the separation is performed at a temperature of 23.2 C. The solution containing the sample to be analysed is at a concentration of 1 g of difethialone per millilitre of acetonitrile and is filtered through a regenerated cellulose membrane with a cut-off threshold of 0.2 m. The volume injected onto the column is 1 L.

[0138] In a process for separating the enantiomers of said homo-stereoisomer of difethialone, it is possible to detect said enantiomers leaving the high-pressure liquid chromatography column by tandem mass spectrometry (MS/MS) in negative electrospray ionization mode (ESI: ElectroSpray Ionization). The temperature of the nebulizer gas is 350 C. and its flow rate is 8 L/minute. The pressure of the nebulizer gas is brought to 2700 hPa. In particular, the MRM (Multiple Reaction Monitoring) transitions m/z 537.1.fwdarw.151.0 and m/z 537.1.fwdarw.78.9, corresponding to the difethialone signals, are detected.

[0139] FIGS. a1), a2), a3) and a4) illustrate the separation of the configurational stereoisomers of difethialone.

[0140] Under these experimental conditions: [0141] the value of the retention time (t.sub.1) for the laevorotatory enantiomer of said homo-stereoisomer is about 8.1 minutes as represented in FIG. a4); [0142] the value of the retention time (t.sub.2) for the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is about 9.4 minutes as represented in FIG. a2); [0143] the value of the retention time (t.sub.3) for the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone is about 11.7 minutes as represented in FIG. a1); [0144] the value of the retention time (t.sub.4) for the dextrorotatory enantiomer of said homo-stereoisomer according to the invention is about 14.4 minutes as represented in FIG. a3);
such that the configurational stereoisomers of difethialone can be separated by high-pressure liquid chromatography on a chiral column. Thus, under these experimental conditions, the order of elution of the configurational stereoisomers of difethialone is such that t.sub.1<t.sub.2<t.sub.3<t.sub.4.

[0145] The inventors also observed that it is possible to perform preparative purification of each enantiomer of said homo-stereoisomer and of said hetero-stereoisomer of difethialone by chromatography on a preparative column of larger dimensions, especially a diameter of 20 mm, comprising a chiral stationary phase constituted of porous particles of cellulose tris(4-methylbenzoate) with a particle size of greater than 3 m and under conditions similar to those described for the analytical chromatography.

[0146] It is possible to measure the amount of each configurational stereoisomer of difethialone by evaluating the area under the peak of the chromatogram corresponding to each configurational stereoisomer and, where appropriate, by comparing this area measurement with the areas obtained by analysis of samples comprising known amounts of configurational stereoisomers of difethialone.

B. STRUCTURAL CHARACTERIZATION

B.1. UV Spectroscopy

[0147] The UV spectrum of the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer and of said hetero-stereoisomer of difethialone dissolved in chloroform shows absorbance peaks centred at 238.2 nm and 259.5 nm.

B.2. Optical Rotation

[0148] The inventors characterized the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer and of said hetero-stereoisomer of difethialone in isolated form by means of their optical rotation (also known as the optical activity or circular birefringence), i.e. their ability to deviate the polarization plane of polarized light. Deviation of the polarization plane of polarized light clockwise facing the polarized light beam characterizes a dextrorotatory solution, and deviation of the polarization plane of polarized light anticlockwise facing the polarized light beam characterizes a laevorotatory solution and compound.

[0149] The optical rotation of a solution of a configurational stereoisomer of difethialone in chloroform is measured. The optical rotation of this solution is measured by means of a P 2000 digital polarimeter (JASCO, Bouguenais, France) operating with excitatory light with a wavelength of 589 nm. The mean optical rotation obtained on two series of ten different measurements and the specific optical rotation at 25 C. [].sup.25 C..sub.589 of each configurational stereoisomer of difethialone dissolved in chloroform, measured on the sodium D line (589 nm), are calculated. The results are given in table 1 below.

TABLE-US-00001 TABLE 1 Concentration, g/L [].sup.25C.sub.589 nm Dextrorotatory 11.6 2.308 +19.9 homo-stereoisomer Laevorotatory 11.05 1.635 14.8 homo-stereoisomer Dextrorotatory 6.3 +0.573 +9.1 hetero-stereoisomer Laevorotatory 6.95 0.904 13 hetero-stereoisomer

B.3. Circular Dichroism

[0150] The circular dichroism spectrum of each isolated dextrorotatory and laevorotatory enantiomer of said homo-stereoisomer and of said hetero-stereoisomer of difethialone reflects the difference in absorbance (A=A.sub.LA.sub.R) of the two waves of left circular polarization (LCP) of intensity A.sub.L and of right circular polarization (RCP) of intensity A.sub.R. This makes it possible to distinguish the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer and of said hetero-stereoisomer of difethialone. This difference in absorbance of the two circularly polarized waves is measured in a J-815 circular dichroism spectrometer (JASCO, Bouguenais, France). 2 mL of solution of each configurational stereoisomer of difethialone in methanol at the concentration given in table 2 are prepared. The solution is transferred into a quartz spectrophotometer cuvette. The circular dichroism spectrum of the solution is measured at 25 C. between 163 nm and 900 nm.

TABLE-US-00002 TABLE 2 Circular dichroism Concentration between mg/mL FIG. 220 nm and 300 nm Dextrorotatory 0.96 3b) positive homo-stereoisomer Laevorotatory 0.81 4b) negative homo-stereoisomer Dextrorotatory 0.65 1b) positive hetero-stereoisomer Laevorotatory 0.94 1c) negative hetero-stereoisomer

C. EXTRACTION OF DIFETHIALONE FROM THE LIVER OF RATS TREATED WITH DIFETHIALONE FOR THE PURPOSE OF ANALYSIS OF THE CONFIGURATIONAL STEREOISOMERS OF DIFETHIALONE

C.1. Homogenization of the Liver Sample

[0151] About 0.525 g (0.025 g) of rat liver is weighed out accurately and placed in a 50 mL polypropylene tube. 10 mL of acetone are added and the suspension is homogenized using an Ultra-Turrax homogenizer/disperser for a time of about 30 seconds. The homogenizer/disperser shaft is rinsed with hot water and then twice with 20 mL of acetone in a polypropylene tube. The homogenate is centrifuged for 5 minutes at a centrifugation speed of 3000 rpm (revolutions per minute). The supernatant is collected and transferred into a test tube. The sample is subjected to evaporation under a stream of nitrogen (N.sub.2) at a temperature of 40 C. so as to form a dry extract.

C.2. Lipid Removal

[0152] 1 mL of acetonitrile is added to the tube containing the dry extract so as to dissolve it. The acetonitrile solution is washed twice successively with 1 mL of hexane. The lipid-free extract is dried under a stream of nitrogen (N.sub.2) at a temperature of 40 C. and is then taken up in 0.5 mL of methanol and dissolved by vortex stirring. 0.5 mL of ultra-pure (Milli-Q) water is then added. The sample is vortex-homogenized.

C.3. Solid-Phase Extraction (SPE) of Difethialone

[0153] 1 mL of dichloromethane, then 1 mL of methanol, then 1 mL of ultra-pure (Milli-Q) water are passed through an Oasis HLB 1 cc cartridge (WAT094225, Waters). The lipid-free liver extract (1 mL of MeOH/Milli-Q H.sub.2O) containing difethialone is then loaded onto the top of the cartridge so that the liver extract penetrates through the cartridge by gravity on contact with the solid phase. 1 mL of washing solution formed from methanol and ultra-pure water in a 90/10 volume proportion is then loaded onto the top of the cartridge. The cartridge is dried by suction under vacuum connected to the bottom of the cartridge. 1 mL of eluting solution formed from dichloromethane and methanol in a 90/10 volume proportion is then loaded onto the top of the cartridge and an eluate comprising difethialone is collected at the bottom of the cartridge. The solvent of the eluate is evaporated off under a stream of nitrogen (N.sub.2) at a temperature of 40 C. The sample is taken up in 0.5 mL of acetonitrile and the acetonitrile solution containing difethialone is filtered through a 0.2 m filter.

[0154] The composition is analysed by high-pressure liquid chromatography on a chiral column as described in point A2) above.

D. Study of the Hepatic Persistence of the Configurational Stereoisomers of Difethialone in Rats

[0155] A solution of a mixture of homo-stereoisomer (DFN-Homo-SI) and of hetero-stereoisomer (DFN-Hetero-SI) of difethialone in a mixture of vegetable oil and 5% DMSO is administered by tube-feeding (per os) to 8-week-old coumaphen-sensitive rats (male and female Rattus norvegicus) weighing about 200 g. The molar proportion of homo-stereoisomer is 40% and the molar proportion of hetero-stereoisomer is 60%. Each configurational stereoisomer of difethialone is formed from a racemic mixture of the two enantiomers of said corresponding configurational stereoisomer.

[0156] On D0, a tube-feeding solution is administered to each rat so that the amount of difethialone ingested by each rat is about 3.4 mg per kilogram of rat. To avoid haemorrhage, the rats are also treated daily by subcutaneous administration of a dose of vitamin K1 (as haemorrhage antidote) at a rate of 0.1 U per 200 g of live rat weight.

[0157] At 4 hours (H+4), 9 hours (H+9), 24 hours (H+24), 120 hours (H+120), 168 hours (H+168) and 216 hours (H+216) after tube-feeding, three male rats and three female rats anaesthetized beforehand with isoflurane are euthanized, the liver of the euthanized rats is removed, the difethialone is then extracted from the liver, the amount of each of the configurational stereoisomers of difethialone is assayed, the area under the peaks in the chromatogram obtained is measured and each configurational stereoisomer is quantified by comparison with a calibration curve. The following are assayed: [0158] the dextrorotatory enantiomer of said homo-stereoisomer (DFN-Homo-dextro); [0159] the laevorotatory enantiomer of said homo-stereoisomer (DFN-Homo-laevo); [0160] the dextrorotatory enantiomer of said hetero-stereoisomer (DFN-Hetero-dextro); [0161] the laevorotatory enantiomer of said hetero-stereoisomer (DFN-Hetero-laevo);
present in the liver of the tube-fed rats.

[0162] The results are given in table 3 below, in which each value is the mean of the contents measured on six rats (three male rats and three female rats) and expressed as nanograms of enantiomer per gram of liver (ng/g)).

TABLE-US-00003 TABLE 3 Hepatic content, ng/g Time Total difethialone after DFN-Homo-SI DFN-Hetero-SI tube-feeding, DFN-Homo- DFN-Homo- DFN-Hetero- DFN-Hetero- hours dextro laevo dextro laevo 4 4566 5692.5 10589.5 5380.5 9 4692.5 7141 12155.5 5869.5 24 1243.5 4403.5 8102 3613.5 48 720.5 3874 7974.5 2804 120 192.5 2087 5431.5 1211 168 129 878.5 3011 392.5 216 77.5 1224 4030.5 545

[0163] The dextrorotatory enantiomer of said hetero-stereoisomer of difethialone is the most persistent in the liver of target rodent pests.

[0164] Rodenticidal Bit Comprising a Mass Proportion of 14.7 ppm of Difethialone

[0165] A pasty rodenticidal bait according to the invention is prepared by dispersing an amount of dextrorotatory enantiomer of said homo-stereoisomer of difethialone in an edible excipient comprising vegetable fat and cereal flour. The measured proportion of difethialone relative to the bait is 14.7 ppm (14.7 mg of difethialone per kilogram of bait) and the proportion of dextrorotatory enantiomer of said homo-stereoisomer relative to the difethialone is 99.7%. The bait also comprises a mass proportion of 0.3% of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone. The rodenticidal bait according to the invention is substantially free of dextrorotatory enantiomer of said hetero-stereoisomer of difethialone.

[0166] On D0, ten coumaphen-sensitive Sprague-Dawley rats (five male and five female SD rats) are placed in individual cages with a rodenticide-free reference feed. On D3, each rat is weighed, and 50 g of rodenticidal bait as described above are then provided to each rat. This provision of 50 g of rodenticidal bait is renewed daily. The bait consumed by the rats is made up to 50 g of bait on D4, D5 and D6. Starting from D7, the residual rodenticidal baits are removed and rodenticide-free feed is provided to all the rats. The rats are monitored for 3 weeks.

[0167] The mean amounts of bait consumed daily by a rat at D4, D5, D6 and D7 expressed in grams per day are given in table 4 below.

TABLE-US-00004 TABLE 4 Bait consumed, g Mean Standard deviation D4 17.8 5.7 D5 16.0 4.7 D6 14.2 4.1 D7 9.4 3.8

[0168] It should be noted that no rat consumed a daily amount of bait of less than 1 g/day. All the rats (100%) die between D9 and D10. The mortality is 100% on D10.

[0169] The bait containing a 14.7 ppm dose of difethialone (including 99.7% of dextrorotatory enantiomer of said homo-stereoisomer of difethialone) makes it possible to obtain a mortality rate of 100% while minimizing the risks of secondary intoxication of animalsespecially birdswhich prey or carrion-feed on weakened target rodent pests that have consumed a rodenticidal bait.

[0170] It goes without saying that the invention may be the subject of numerous implementation variants and applications. In particular, a composition, a rodenticidal bait and a process for controlling target rodent pests are subject to an infinite number of variants both in the formulation of the bait and in the embodiments of the process.