Configurational stereoisomer of difethialone, composition and rodenticide bait comprising same, and method for controlling target rodent pests

Abstract

Disclosed is a laevorotatory enantiomer of the 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.

Claims

1. Laevorotatory enantiomer of 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-tetrahyd-ronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have the same absolute configuration, said laevorotatory enantiomer of said homo-stereoisomer being in isolated form.

2. Composition comprising the laevorotatory enantiomer of 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 with the exclusion of a racemic mixture of laevorotatory and dextrorotatory enantiomers of said homo-stereoisomer of difethialone and wherein the mass amount of the laevorotatory enantiomer of said homo-stereoisomer of difethialone is such that the ratio of this mass amount to the mass amount of difethialone is greater than 95%.

3. Rodenticidal bait comprising a composition at least one excipient that is edible for target rodent pests, and a composition comprising the laevorotatory enantiomer of 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, with the exclusion of a racemic mixture of laevorotatory and dextrorotatory enantiomers of said homo-stereoisomer of difethialone, wherein the mass amount of the laevorotatory enantiomer of said homo-stereoisomer of difethialone is such that the ratio of this mass amount to the mass amount of difethialone is greater than 95%, and wherein the mass amount of difethialone is such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is between 1 ppm and 5 ppm.

4. Bait according to claim 3, 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.

5. Process for controlling target rodent pests, in which there is spread an amount of rodenticidal bait comprising: at least one excipient that is edible for target rodent pests; and a laevorotatory enantiomer of the 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-tetrahyd-ronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have the same absolute configuration; with the exclusion of a racemic mixture of the laevorotatory and dextrorotatory enantiomers of said homo-stereoisomer of difethialone, and wherein the mass amount of the laevorotatory enantiomer of said homo-stereoisomer of difethialone is such that the ratio of this mass amount to the mass amount of difethialone is greater than 95%, and wherein the mass amount of difethialone is such that the ratio of this mass amount of difethialone to the mass amount of rodenticidal bait is between 1 ppm and 5 ppm.

6. Process according to claim 5, wherein the following are chosen in combination: the edible excipient; a proportion of laevorotatory enantiomer of said homo-stereoisomer of difethialone relative to said homo-stereoisomer of difethialone; a proportion of laevorotatory enantiomer of said homo-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.

7. Process according to claim 5, wherein the following are chosen in combination: the edible excipient; a proportion of laevorotatory enantiomer of said homo-stereoisomer of difethialone relative to said homo-stereoisomer of difethialone; a proportion of laevorotatory enantiomer of said homo-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.

8. Chromatographic process for obtaining a laevorotatory enantiomer of 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 of said homo-stereoisomer have the same absolute configuration, in which process: a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of cellulose tris(4-methylbenzoate) particles, said particles having a mean size of 3 pm 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 homo-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 homo-stereoisomer of difethialone is collected with a retention time t.sub.1 having a value such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.2, 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 homo-stereoisomer of difethialone, separately from a dextrorotatory enantiomer of said homo-stereoisomer of difethialone with a retention time t.sub.4 and separately from the laevorotatory and dextrorotatory enantiomers of a configurational stereoisomer of difethialone, named hetero-stereoisomer, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer have different absolute configurations, and of retention times t.sub.2 and t.sub.3; and then the liquid mobile phase of said fraction is removed so as to obtain said laevorotatory enantiomer of said homo-stereoisomer of difethialone.

Description

(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 is a chromatogram of an analysis by high-pressure liquid chromatography on a chiral column of difethialone (top) and of the laevorotatory enantiomer of said purified homo-stereoisomer of difethialone (bottom);

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

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

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

(6) FIG. 5 is a .sup.13C carbon NMR spectrum at 500 MHz of the laevorotatory enantiomer of said homo-stereoisomer of difethialone;

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

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

(9) FIG. 8 is a representation in graph form of the change over time of the hepatic concentration in rats (male and female) of the laevorotatory enantiomer of said homo-stereoisomer of difethialone (.circle-solid.) and of said homo-stereoisomer of difethialone (); and

(10) FIG. 9 is a representation in graph form of the change over time of the hepatic concentration in rats (male and female) of the laevorotatory enantiomer of said homo-stereoisomer of difethialone (.circle-solid.) and of the total difethialone ().

(11) A. Purification of the Laevorotatory Enantiomer of said Isolated Homo-Stereoisomer of Difethialone

(12) A.1. Identification of said Homo-Stereoisomer of Difethialone

(13) 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 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 as illustrated in FIG. 2.

(14) The hetero-stereoisomer of difethialone and the homo-stereoisomer of difethialone are distinguished by their proton NMR spectra. In the proton NMR spectrum acquired in CDCl.sub.3, the chemical shift of the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer of difethialone (FIG. 3) is between 5.2 ppm and 5.4 ppm.

(15) A.2. Separation of the Laevorotatory and Dextrorotatory Enantiomers of said Homo-Stereoisomer of Difethialone by High-Pressure Liquid Chromatography

(16) The inventors solved the complex problem, which was not solved to date, of the separation of the laevorotatory and dextrorotatory enantiomers of said homo-stereoisomer of difethialone from a commercial preparation of difethialone. They succeeded in performing an analytical separation of the enantiomers of said homo-stereoisomer of difethialone by high-pressure (high-performance) liquid chromatography on a LUX Cellulose-3 chiral column

(17) (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 water (B), with an A/B volume proportion 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.

(18) In a process for separating the enantiomers of said homo-stereoisomer of difethialone, it is possible to detect said enantiomers leaving the column by tandem mass spectrometry (MS/MS) in negative electrospray ionization mode (ESI: ElectroSpray Ionization). The temperature of the carrier gas is 350 C. and its flow rate is 8 L/minute. The pressure of the nebulizer 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 analysed. FIG. 1 represents the chromatograms of difethialone (top) and of the laevorotatory enantiomer of the isolated homo-stereoisomer of difethialone (bottom).

(19) Under these experimental conditions, the value of the retention time (t.sub.1) for the laevorotatory enantiomer of said homo-stereoisomer according to the invention is about 8.1 minutes as described in FIG. 1. By way of comparison, 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, such that the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer may be efficiently separated by high-pressure liquid chromatography on a chiral column.

(20) The value of the retention time (t.sub.3) for the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone is about 11.7 minutes and the value of the retention time (t.sub.2) for the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is about 9.4 minutes. Thus, under these experimental conditions, the order of elution of the difethialone enantiomers is such that t.sub.1<t.sub.2<t.sub.3<t.sub.4.

(21) 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 homo-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.

(22) B. Structural Characterization

(23) B.1. UV Spectroscopy

(24) The UV spectrum of the laevorotatory enantiomer of said homo-stereoisomer of difethialone dissolved in chloroform (CHCl.sub.3) shows absorbance peaks at 238.2 nm and 259.5 nm.

(25) B.2. Optical Rotation

(26) The inventors characterized the laevorotatory enantiomer of said homo-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 an optically active and dextrorotatory solution and compound, and deviation of the polarization plane of polarized light anticlockwise facing the polarized light beam characterizes an optically active and laevorotatory solution and compound.

(27) The optical rotation of a solution of laevorotatory enantiomer of said homo-stereoisomer of difethialone in chloroform (CHCl.sub.3) is measured at a concentration of 11.05 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 obtained on two series of 10 measurements is 1.635. The specific optical rotation at 25 C. [].sup.25 C..sub.589 nm for the laevorotatory enantiomer of said homo-stereoisomer of difethialone dissolved in chloroform, measured on the sodium D line (589 nm), is 14.8.

(28) B.3. Circular Dichroism

(29) The circular dichroism spectrum of the laevorotatory enantiomer of said isolated homo-stereoisomer of difethialone reflects the difference in absorbance (A=A.sub.L-A.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 homo-stereoisomer of difethialone in methanol (CH.sub.3OH) at a concentration of 0.81 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 homo-stereoisomer of difethialone measured under these conditions is shown in FIG. 7. The circular dichroism value is negative between the wavelengths of 220 nm and 300 nm.

(30) B.4. Nuclear Magnetic Resonance

(31) FIGS. 2 and 3 represent, respectively, a proton nuclear magnetic resonance spectrum (.sup.1H-NMR) at 300 MHz of the homo-stereoisomer of difethialone in CDCl.sub.3 (FIG. 2) and a proton nuclear magnetic resonance spectrum at 300 MHz in CDCl.sub.3 of the hetero-stereoisomer of difethialone in CDCl.sub.3 (FIG. 3). Said homo-stereoisomer of difethialone has (FIG. 2) 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. Said hetero-stereoisomer of difethialone has (FIG. 3) a multiplet whose chemical shift () is between 5.2 ppm and 5.4 ppm.

(32) FIG. 4 is a proton NMR spectrum at 500 MHz of the laevorotatory enantiomer of said homo-stereoisomer of difethialone in CDCl.sub.3. The proton NMR spectra of the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer of difethialone are indistinguishable from each other.

(33) FIG. 5 is a .sup.13C NMR spectrum of the laevorotatory enantiomer of said homo-stereoisomer of difethialone dissolved in CDCl.sub.3 at a concentration of 40 mg/mL, acquired on a Bruker 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 homo-stereoisomer of difethialone is not distinguished from the .sup.13C-NMR spectrum of the dextrorotatory enantiomer of said homo-stereoisomer of difethialone. Said homo-stereoisomer of difethialone has a characteristic signal between 34 ppm and 38 ppm which is distinctive for said hetero-stereoisomer of difethialone.

(34) FIG. 6 is a two-dimensional proton NMR (2D .sup.1H-NMR) spectrum obtained by correlation spectroscopy of the laevorotatory enantiomer of said homo-stereoisomer of difethialone dissolved in CDCl.sub.3 at a concentration of 40 mg/mL acquired on a Bruker 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 with the protons borne by carbon 2 of the 1,2,3,4-tetrahydronaphthalene group at 2.27 ppm.

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

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

(37) 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.

(38) C.2. Lipid Removal

(39) 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.

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

(41) 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 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.

(42) C.4. Analysis

(43) The acetonitrile solution containing difethialone is analysed by high-pressure liquid chromatography on a LUX Cellulose-3 chiral column (1502 mm, particle size of 3 m) (Phenomenex, Le Pecq, France) as described in point A2) above. The retention time value (t.sub.1) of the laevorotatory enantiomer of said homo-stereoisomer of difethialone according to the invention is between 7.8 minutes and 8.4 minutes (the maximum value of the peak corresponding to the dextrorotatory enantiomer of said homo-stereoisomer being about 8.1 minutes, as described in FIG. 1), depending on the operating conditions, especially depending on the column temperature conditions. The retention time value (t.sub.4) of the dextrorotatory enantiomer of said homo-stereoisomer is between 13.3 minutes and 15.1 minutes (the maximum value of the peak corresponding to the dextrorotatory enantiomer of said homo-stereoisomer being about 14.4 minutes), depending on the operating conditions, especially depending on the column temperature conditions. The retention time value (t.sub.3) of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone is between 11.2 minutes and 12.3 minutes (the maximum value of the peak corresponding to the dextrorotatory enantiomer of said hetero-stereoisomer being about 11.7 minutes), depending on the operating conditions, especially depending on the column temperature conditions. The retention time value (t.sub.2) of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is between 9.0 minutes and 9.8 minutes (the maximum value of the peak corresponding to the laevorotatory enantiomer of said hetero-stereoisomer being about 9.4 minutes), depending on the operating conditions, especially depending on the column temperature conditions. The composition of the sample is analysed by high-pressure liquid chromatography as described in point A2) above.

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

(45) 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 mole ratio of the amount of said homo-stereoisomer to the amount of said hetero-stereoisomer is 4/6. Each difethialone diastereoisomer is formed from a racemic mixture of the two enantiomers of the corresponding diastereoisomer.

(46) The tube-feeding solution is administered (on DO) so that the amount of difethialone ingested by each rat is about 3.4 mg per kilogram of rat.

(47) 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.

(48) 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 and the amount of each of the configurational stereoisomers 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 according to the invention; the dextrorotatory enantiomer of said hetero-stereoisomer; the laevorotatory enantiomer of said hetero-stereoisomer; present in the liver of the tube-fed rats.

(49) In FIGS. 8 and 9, the difethialone concentrations recorded (mean of the values measured on six rats and expressed in nanograms of enantiomer per gram of liver (ng/g) in the liver of the tube-fed rats) are given as a function of the time (in hours) after tube-feeding. In FIG. 8, the concentration of the laevorotatory enantiomer of said homo-stereoisomer according to the invention in the liver is represented by filled circles (.circle-solid.) on the left-hand scale and the concentration of said homo-stereoisomer of difethialone in the liver is represented by open circles () on the right-hand scale.

(50) The laevorotatory enantiomer of said homo-stereoisomer according to the invention has inexplicably high hepatic persistence relative to the persistence of the homo-stereoisomer of difethialone.

(51) In FIG. 9, the concentration of the laevorotatory enantiomer of said homo-stereoisomer according to the invention in the liver is represented by filled circles (.circle-solid.) on the left-hand scale and the total difethialone concentration in the liver is represented by open triangles () on the right-hand scale. The corresponding values are given in table 1 below.

(52) TABLE-US-00001 TABLE 1 Hepatic content, ng/g Time Total difethialone after Homo-stereoisomer Hetero-stereoisomer 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

(53) The laevorotatory enantiomer of said homo-stereoisomer according to the invention has high hepatic persistence relative to the persistence of difethialone.

(54) Rodenticidal Bait Comprising a Proportion of 13.7 ppm of Difethialone

(55) A pasty rodenticidal bait according to the invention is prepared by dispersing an amount of laevorotatory 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 13.7 ppm and the proportion of laevorotatory enantiomer of said homo-stereoisomer relative to the difethialone is 95.4% (13.1 mg of laevorotatory enantiomer of said homo-stereoisomer of difethialone per kilogram of bait). The bait also comprises a mass proportion of 3.3% of dextrorotatory enantiomer of said homo-stereoisomer of difethialone relative to the difethialone and a mass proportion of 1.4% of dextrorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone.

(56) On D0, ten coumaphen-sensitive Sprague-Dawley (SD) rats (five male rats and five female 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. The weight gain of each of the rats is also measured daily. 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.

(57) The mean amounts of bait consumed daily by a rat on D4, D5, D6 and D7 expressed in grams per day are given in table 2 below.

(58) TABLE-US-00002 TABLE 2 Bait consumed Mean Standard deviation D4 17.8 5.7 D5 16.0 4.7 D6 14.2 4.1 D7 9.4 3.8

(59) 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.

(60) The bait containing a low dose of 13.7 ppm of difethialone makes it possible to obtain a mortality rate of 100% while at the same time minimizing the risks of intoxication of animalsespecially birdswhich prey or carrion-feed on weakened target rodent pests that have consumed a rodenticidal bait according to the invention.

(61) 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.