Configurational stereoisomer of difethialone, composition and rodenticidal bait comprising same, and process for controlling target rodent pests

Abstract

Disclosed is to a laevorotatory enantiomer of the 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.

Claims

1. Laevorotatory enantiomer of 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, said laevorotatory enantiomer of said hereto-stereoisomer of difethialone being in isolated form.

2. Composition comprising the laevorotatory enantiomer of the configurational stereoisomer of difethialone, named hetero-stereoisomer, the formula of which is 3-(4 -bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-y;)-1,2,3,4-tetrahydronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have different absolute configurations, with the exclusion of a racemic mixture of laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone.

3. Composition according to claim 2, wherein said hetero-stereoisomer is predominantly in laevorotatory enantiomer form.

4. Composition according to claim 2, wherein the difethialone is predominantly in the laevorotatory enantiomer form of said hetero-stereoisomer of difethialone.

5. Composition according to claim 2, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 25%.

6. Composition according to claim 2, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 97% in the composition.

7. Rodenticidal bait comprising a composition according to claim 2 and at least one excipient that is edible for target rodent pests.

8. Bait according to claim 7, 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.

9. Bait according to claim 7, 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.

10. Process for controlling target rodent pests, in which there is spread an amount of bait comprising: at least one excipient that is edible for target rodent pests; and the laevorotatory enantiomer of the 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; with the exclusion of a racemic mixture of the laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone.

11. Process according to claim 10, wherein said hetero-stereoisomer of difethialone is predominantly in laevorotatory enantiomer form.

12. Process according to claim 10, wherein the difethialone is predominantly in the laevorotatory enantiomer form of said hetero-stereoisomer of difethialone.

13. Process according to claim 10, wherein the following are chosen in combination: the edible excipient; a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to said hetero-stereoisomer of difethialone; a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone; a mass proportion of difethialone relative to the rodenticidal bait; and an amount of spread bait; so that target rodent pests consume an amount of difethialone that is sufficient to be lethal to said target rodent pests which consume said bait in the course of a single period of 24 consecutive hours.

14. Process according to claim 10, wherein the following are chosen in combination: the edible excipient; a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to said hetero-stereoisomer of difethialone; a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone; a mass proportion of difethialone relative to the rodenticidal bait; and an amount of spread bait; so that target rodent pests consume an amount of difethialone: which is non-lethal to target rodent pests which consume said bait over a period of 24 consecutive hours; and which is sufficient to be lethal to target rodent pests which consume said bait over several 24-hour periods, said periods being consecutive.

15. Chromatographic process for obtaining a laevorotatory enantiomer of 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 of said hetero-stereoisomer have different absolute configurations, in which process: a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-methylbenzoate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 , is chosen; a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 80/20 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute, is chosen as liquid mobile phase; separation of the configurational stereoisomers of difethialone is performed at room temperature, during which: a liquid composition comprising said laevorotatory enantiomer of said hetero-stereoisomer of difethialone is introduced into the top of the chromatography column; and then the liquid composition is entrained with the mobile phase in the chromatography column under conditions suitable for separating the configurational stereoisomers of difethialone; and a fraction of the mobile phase comprising said laevorotatory enantiomer of said hetero-stereoisomer of difethialone is collected with a retention time t.sub.2 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1, t.sub.3 and t.sub.4 representing the retention times of each of the configurational stereoisomers of difethialone different from the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, separately from a dextrorotatory enantiomer of said hetero-stereoisomer of difethialone with a retention time t.sub.3 and separately from the laevorotatory and dextrorotatory enantiomers of a configurational stereoisomer of difethialone, named homo-stereoisomer, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer have the same absolute configuration, and of retention times t.sub.1 and t.sub.4; and then the liquid mobile phase of said fraction is removed so as to obtain said laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

16. Composition according to claim 3, wherein the difethialone is predominantly in the laevorotatory enantiomer form of said hetero-stereoisomer of difethialone.

17. Composition according to claim 3, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 25%.

18. Composition according to claim 4, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 25%.

19. Composition according to claim 3, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 97% in the composition.

20. Composition according to claim 4, comprising an amount of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of difethialone is greater than 97% in the composition.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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:

(2) FIG. 1 represents a chromatogram of separation of the enantiomers of difethialone (top) and a chromatogram of analysis of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone (bottom) by high-pressure liquid chromatography on a chiral column;

(3) FIG. 2 is a proton NMR spectrum at 300 MHz of said hetero-stereoisomer of difethialone;

(4) FIG. 3 is a proton NMR spectrum at 300 MHz of said homo-stereoisomer of difethialone;

(5) FIG. 4 is a proton NMR spectrum at 500 MHz of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone according to the invention;

(6) FIG. 5 is an analysis by proton NMR (.sup.1H-NMR) correlation spectroscopy at 500 MHz of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone;

(7) FIG. 6 is a .sup.13C carbon NMR spectrum at 500 MHz of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone;

(8) FIG. 7 is a circular dichroism spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone;

(9) FIG. 8 is a representation in graph form of the change over time of the mean concentration measured in the liver of rats (5 male rats and 5 female rats) of said homo-stereoisomer (.circle-solid.) and of said hetero-stereoisomer (); and

(10) FIG. 9 is a comparative representation in graph form of the change over time of the concentration in the liver of rats of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone (.circle-solid.), of said hetero-stereoisomer of difethialone (.square-solid.) and of the total difethialone ().

(11) A. Purification of the Laevorotatory Enantiomer of Said Hetero-stereoisomer of Difethialone

(12) A. 1. Identification of Said Hetero-stereoisomer of Difethialone and of Said Homo-stereoisomer of Difethialone

(13) Said hetero-stereoisomer of difethialone is identified by proton magnetic resonance (.sup.1H-NMR) spectroscopy. Said hetero-stereoisomer of difethialone dissolved in CDCl.sub.3 has a signal with a chemical shift () of about 5.3 ppm and corresponding to the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of difethialone as illustrated in FIG. 2. Said hetero-stereoisomer of difethialone is distinguished from said homo-stereoisomer of difethialone, the latter, dissolved in CDCl.sub.3, having a multiplet at 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 (FIG. 3).

(14) A.2. Separation of the Laevorotatory and Dextrorotatory Enantiomers of Said Hetero-Stereoisomer of Difethialone by High-Pressure Liquid Chromatography on a Chiral Column

(15) The inventors solved the complex and hitherto unresolved problem of separating the enantiomers of difethialone and in particular the laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone. They succeeded in separating the enantiomers of said hetero-stereoisomer of difethialone and in preparing the dextrorotatory enantiomer of said hetero-stereoisomer 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 tris(4-methylbenzoate) cellulose, with a particle size of 3 m and a porosity of 1000 . The mobile phase used is 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 A/B 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 in acetonitrile and is filtered through a regenerated cellulose membrane with a cut-off threshold of 0.2 m. The volume of solution containing the sample to be analysed injected onto the column is 1 L.

(16) In a process for separating the enantiomers of said hetero-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.78.99, corresponding to the difethialone signals, are detected. FIG. 1 represents the chromatograms of difethialone (top) and of the laevorotatory enantiomer of the isolated hetero-stereoisomer of difethialone (bottom).

(17) Under these conditions, the value of the retention time (t.sub.2) for the laevorotatory enantiomer of said hetero-stereoisomer of difethialone according to the invention is about 9.4 minutes as described in FIG. 1, and the value of the retention time (t.sub.3) for the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone is about 11.7 minutes, such that the dextrorotatory and laevorotatory enantiomers of said hetero-stereoisomer may be separated by high-pressure liquid chromatography on a chiral column.

(18) Under these same conditions, the value of the retention time (t.sub.4) for said dextrorotatory enantiomer of said homo-stereoisomer according to the invention is about 14.4 minutes, and the value of the retention time (t.sub.1) for the laevorotatory enantiomer of said homo-stereoisomer is about 8.1 minutes, such that the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer may also be separated by high-pressure liquid chromatography on a chiral column. Thus, under these conditions, the order of elution of the enantiomers of difethialone is such that t.sub.1<t.sub.2<t.sub.3<t.sub.4.

(19) It is possible under these experimental conditions (stationary phase, mobile phase, temperature) to perform a preparative separation of the laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone by using a similar stationary phase with a particle size of greater than 3 m, and a chromatography column of larger dimensions, especially a diameter of 20 mm.

(20) B. Structural Characterization

(21) B.1. UV Spectroscopy

(22) The UV spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone dissolved in chloroform (CHCl.sub.3) shows an absorbance peak centred at 238.2 nm and an absorbance peak centred at 259.5 nm.

(23) B.2. Optical Rotation

(24) The inventors characterized the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in isolated form by means of its optical rotation (also known as the optical activity or circular birefringence), i.e. its 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.

(25) The optical rotation of a solution of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in chloroform (CHCl.sub.3) is measured at a concentration of 6.95 g/L. 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 a obtained on two series of ten measurements is 0.904. The specific optical rotation at 25 C. [].sup.25 C..sub.589nm for the laevorotatory enantiomer of said hetero-stereoisomer of difethialone dissolved in chloroform, measured on the sodium D line (589 nm), is 13.

(26) B.3. Circular Dichroism

(27) The circular dichroism spectrum of the isolated laevorotatory enantiomer 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 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 a solution of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in methanol (CH.sub.3OH) at a concentration of 0.94 mg/mL are prepared. The solution is transferred into a quartz spectrophotometer cuvette. The circular dichroism spectrum of the solution is measured at 25 C. between 200 nm and 400 nm. The circular dichroism spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone measured under these conditions is shown in FIG. 7. The circular dichroism value is negative between the wavelengths of between 225 nm and 250 nm.

(28) B.4. Nuclear Magnetic Resonance

(29) The proton nuclear magnetic resonance (.sup.1H-NMR) spectrum at 300 MHz of the hetero-stereoisomer of difethialone in CDCl.sub.3 (FIG. 2) has a multiplet whose chemical shift () is about 5.3 ppm. The proton nuclear magnetic resonance spectrum at 300 MHz of the homo-stereoisomer of difethialone in CDCl.sub.3 (FIG. 3) has a multiplet whose chemical shift () is between 4.9 ppm and 5.1 ppm, corresponding to carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer of difethialone. The proton NMR spectra of the dextrorotatory and laevorotatory enantiomers of said hetero-stereoisomer of difethialone are indistinguishable from each other.

(30) FIG. 4 is a proton NMR (.sup.1H-NMR) spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone dissolved at a concentration of 40 mg/mL in CDCl.sub.3, having a multiplet whose chemical shift () is about 5.3 ppm, characteristic of said hetero-stereoisomer of difethialone.

(31) FIG. 5 is a two-dimensional proton NMR (2D .sup.1H-NMR) spectrum obtained by correlation spectroscopy of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone dissolved at a concentration of 40 mg/mL in CDCl.sub.3, acquired on a Brker Avance III HD spectrometer (500 MHz) equipped with a Prodigy motorized multi-core direct cryoprobe. It allows identification of the coupling of the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer of difethialone (5.3 ppm) with the protons borne by carbon 2 of the 1,2,3,4-tetrahydronaphthalene group at 2.0 and 2.4 ppm.

(32) FIG. 6 is a .sup.13C NMR spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone dissolved in CDCl.sub.3 at a concentration of 40 mg/mL, acquired on a Brker Avance III HD spectrometer (500 MHz) equipped with a Prodigy motorized multi-core direct cryoprobe. It allows identification of the 31 carbon atoms of difethialone. The .sup.13C-NMR spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is not distinguished from the .sup.13C-NMR spectrum of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone. However, said hetero-stereoisomer of difethialone has a signal at a chemical shift of about 38.2 ppm and a signal at a chemical shift of about 40.3 ppm, which are characteristic of and distinctive for said homo-stereoisomer of difethialone.

(33) C. Extraction Of Difethialone from the Liver of Rats Treated with Difethialone for the Purpose of Analysis of the Various Enantiomers of Difethialone

(34) C.1. Homogenization of the Liver Sample

(35) 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.

(36) C.2. Lipid Removal

(37) 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.

(38) C.3. Solid-phase Extraction (SPE) of Difethialone

(39) 1 mL of dichloromethane (CH.sub.2Cl.sub.2), then 1 mL of methanol (CH.sub.3OH), 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 MeOH/Milli-Q H.sub.2O) containing difethialone is then loaded onto the top of the preconditioned cartridge. The liver extract penetrates through the cartridge by gravity on contact with the solid phase of the cartridge. 1 mL of washing solution formed from methanol (CH.sub.3OH) and ultra-pure water (H.sub.2O) 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 (CH.sub.2Cl.sub.2) and methanol (CH.sub.3OH) 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 (NCCH.sub.3) and the acetonitrile solution containing difethialone is filtered through a 0.2 m filter.

(40) C.4. Analysis

(41) The acetonitrile solution containing difethialone is analysed by high-pressure liquid chromatography on a LUX Cellulose-3 chiral column (Phenomenex, Le Pecq, France) (1502 mm, particle size of 3 m) as described in point A2) above.

(42) D. Persistence of the Configurational Stereoisomers of Difethialone in Rat Liver

(43) A solution of a mixture of said homo-stereoisomer and of said hetero-stereoisomer of difethialone in a mixture of vegetable oil and 5% DMSO is administered by tube-feeding (per os) to male and female coumaphen-sensitive rats (Rattus norvegicus). The molar proportion of said homo-stereoisomer is 40% and the molar proportion of said hetero-stereoisomer is 60%. Each configurational stereoisomer of difethialone is formed from a racemic mixture of the two laevorotatory and dextrorotatory enantiomers of said corresponding configurational stereoisomer. The solution comprising 40% of said homo-stereoisomer and 60% of said hetero-stereoisomer is administered (on D0) so that the amount of difethialone ingested by each rat is about 3.4 mg per kilogram of rat. To avoid haemorrhage, the tube-fed rats are 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.

(44) 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, six rats (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 and the amount of each configurational stereoisomer of difethialone is assayed via analysis by high-pressure liquid chromatography on a chiral column according to the process described above, the area under the peaks in the chromatogram obtained is measured and each enantiomer is quantified by comparison with a calibration curve. The following are assayed: the dextrorotatory enantiomer of said homo-stereoisomer; the laevorotatory enantiomer of said homo-stereoisomer; the dextrorotatory enantiomer of said hetero-stereoisomer; the laevorotatory enantiomer of said hetero-stereoisomer; present in the liver of the tube-fed rats.

(45) The results of the analysis of the content (expressed as nanograms of enantiomer per gram of liver (ng/g)) of each enantiomer of said hetero-stereoisomer of difethialone and of said homo-stereoisomer of difethialone in the liver of the rats as a function of the time (in hours) after the tube-feeding of the rats are given in table 1 below.

(46) TABLE-US-00001 TABLE 1 Hepatic content, ng/g Total difethialone Time after Hetero-stereoisomer tube-feeding, DFN-Hetero- DFN-Hetero- hours Homo-stereoisomer dextro laevo 4 10258.5 10589.5 5380.5 9 11833.5 12155.5 5869.5 24 5647 8102 3613.5 48 4594.5 7974.5 2804 120 2279.5 5431.5 1211 168 1007.5 3011 392.5 216 1301.5 4030.5 545

(47) The results of the analysis of the content of said homo-stereoisomer of difethialone and of said hetero-stereoisomer of difethialone are deduced from table 1 and shown in FIG. 8. Said hetero-stereoisomer () has hepatic persistence of a higher value than the value of the hepatic persistence of said homo-stereoisomer (.circle-solid.). Now, the inventors also observed that the laevorotatory enantiomer of said hetero-stereoisomer (said hetero-stereoisomer being the most persistent stereoisomer of difethialone) in fact has low hepatic persistence, giving it surprising properties for protection of the environment and of wild animalsfor example foxes or birdswhich prey on target rodent pests which have consumed the rodenticidal bait or which carrion-feed on the dead bodies of poisoned target rodent pests.

(48) FIG. 9 describes the change over time of the content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the liver, represented by filled circles (.circle-solid.), of the content of said hetero-stereoisomer in the liver, represented by filled squares (.square-solid.) and of the content of total difethialone in the liver, represented (right-hand scale) by empty triangles ().

(49) The mean content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, representing about 30% of the total starting difethialone ingested by the rats, decreases rapidly in the liver of the sacrificed rats. This decrease is faster than the decrease of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone (not shown in FIG. 9), faster than the decrease of said hetero-stereoisomer of difethialone and faster than the decrease of the total difethialone. The laevorotatory enantiomer of said hetero-stereoisomer of difethialone has significantly lower hepatic persistence than that of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone, but also than that of the laevorotatory enantiomer of said homo-stereoisomer (less persistent stereoisomer) of difethialone in male rats.

(50) E. Rodenticidal Bait Comprising a Proportion of 11.85 Ppm of Difethialone

(51) A pasty rodenticidal bait according to the invention is prepared by dispersing an amount of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in an edible excipient comprising vegetable fat and cereal flour. The measured proportion of difethialone relative to the bait is 11.8 ppm (11.81 mg of laevorotatory enantiomer of said hetero-stereoisomer of difethialone per kilogram of bait). The proportion of laevorotatory enantiomer of said hetero-stereoisomer relative to the difethialone is 99.7%.

(52) 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.

(53) 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 2 below.

(54) TABLE-US-00002 TABLE 2 Bait consumed, g Mean Standard deviation D4 13.2 3.6 D5 12.7 4.6 D6 14.7 3.5 D7 12.1 2.4

(55) It should be noted that no rat consumed a daily amount of bait of less than 1 g/day. The mean daily consumption of bait relative to the mean mass of the rats over the period from D4 to D7 is 64.7 g of bait per kilogram of rat. Nine out of ten rats die between D9 and D12. The mortality of the bait is 90% on D12.

(56) The hepatic content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in each of the rats which die between D9 and D10 is measured by high-pressure liquid chromatography analysis on a chiral column. The values, expressed as micrograms of laevorotatory enantiomer of said hetero-stereoisomer (DFN-Hetero-laevo) per gram of liver, are given in table 3 below.

(57) TABLE-US-00003 TABLE 3 DFN-Hetero-laevo, Hepatic content, g/g g/g Total difethialone, g/g Mean 11.2 11.3 Standard deviation 4.8 0.48

(58) The bait containing an 11.81 ppm dose of laevorotatory enantiomer of said hetero-stereoisomer of difethialone makes it possible to obtain a mortality rate of about 90% 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.

(59) 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.