STEREOISOMER OF FLOCOUMAFEN, COMPOSITION AND RODENTICIDE BAIT COMPRISING SAME, AND METHOD FOR CONTROLLING TARGET RODENT PESTS

Abstract

Disclosed is a configurational stereoisomer, referred to as enantiomer E.sub.2, of flocoumafen, the enantiomer E.sub.2 having, as determined by the chromatographic analysis of a flocoumafen composition including four configurational stereoisomers of flocoumafen, carried out under conditions described hereinafter, a retention time t.sub.2 with 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 the configurational stereoisomers of flocoumafen different from the enantiomer E.sub.2, the chromatographic analysis being carried out at a temperature of 23.5 C.

Claims

1-11. (canceled)

12. Configurational stereoisomer, named enantiomer E.sub.2, of flocoumafen, said enantiomer E.sub.2 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the conditions described below, 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 the configurational stereoisomers of flocoumafen different from said enantiomer E.sub.2, the chromatographic analysis being performed at a temperature of 23.5 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 ; using, as liquid mobile phase, a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute; by injection into the chromatography column of a volume of 1 L of flocoumafen composition at a concentration of 1 g of flocoumafen per millilitre of acetonitrile.

13. Composition comprising a configurational stereoisomer, named enantiomer E.sub.2, of flocoumafen, with the exclusion of a racemic mixture of said enantiomer E.sub.2 and of a configurational stereoisomer, named enantiomer E.sub.3, of flocoumafen; said enantiomer E.sub.2 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the conditions described below, a retention time t.sub.2; said enantiomer E.sub.3 having, by analysis of flocoumafen comprising four configurational stereoisomers of flocoumafen performed under these same conditions, a retention time t.sub.3; t.sub.2 and t.sub.3 being values such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1 and t.sub.4 representing the retention times of each of the configurational stereoisomers of flocoumafen different from said enantiomer E.sub.2 and from said enantiomer E.sub.3, the chromatographic analysis being performed at a temperature of 23.5 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 ; using, as liquid mobile phase, a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute; by injection into the chromatography column of a volume of 1 L of flocoumafen composition at a concentration of 1 g of flocoumafen per millilitre of acetonitrile.

14. Composition according to claim 13, wherein the amount of said enantiomer E.sub.2 is greater than the amount of said enantiomer E.sub.3 in the composition.

15. Composition according to claim 13, wherein the flocoumafen is predominantly in the form of said enantiomer E.sub.3 in the composition.

16. Composition according to claim 13, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 25%.

17. Composition according to claim 13, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 95%.

18. Rodenticidal bait comprising a composition according to claim 13 and at least one excipient that is edible for target rodent pests.

19. Bait according to claim 18, 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.

20. Bait according to claim 18, comprising a mass amount of flocoumafen such that the ratio of this mass amount of flocoumafen to the mass amount of rodenticidal bait is less than 200 ppm.

21. 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, a configurational stereoisomer, named enantiomer E.sub.2, of flocoumafen, with the exclusion of a racemic mixture of said enantiomer E.sub.2 and of a configurational stereoisomer, named enantiomer E.sub.3, of flocoumafen; said enantiomer E.sub.2 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under the conditions described below, a retention time t.sub.2; said enantiomer E.sub.3 having, by chromatographic analysis of a flocoumafen composition comprising four configurational stereoisomers of flocoumafen performed under these same conditions, a retention time t.sub.3; t.sub.2 and t.sub.3 being values such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.1 and t.sub.4 representing the retention times of each of the configurational stereoisomers of flocoumafen different from said enantiomer E.sub.2 and from said enantiomer E.sub.3, said analysis being performed at a temperature of 23.5 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, said particles having a mean size of 3 m and having a mean pore size of 1000 ; using, as liquid mobile phase, a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 92/8 and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute; by injection into the chromatography column of a volume of 1 L of flocoumafen composition at a concentration of 1 g of flocoumafen per millilitre of acetonitrile.

22. Chromatographic process for obtaining said enantiomer E.sub.2 according to claim 12, in which: a high-pressure liquid chromatography column of dimensions 1502 mm, and comprising a chiral stationary phase constituted of particles of tris(4-chloro-3-methylphenyl carbamate) cellulose, is chosen, said particles having a mean size of 3 m and having a mean pore size of 1000 ; a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an A/B volume ratio of 92/8 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 flocoumafen is performed at room temperature, during which: a liquid composition comprising said enantiomer E.sub.2 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 flocoumafen, and a fraction of the mobile phase comprising said enantiomer E.sub.2 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 flocoumafen different from said enantiomer E.sub.2, is collected separately from said enantiomer E.sub.3 of retention time t.sub.3; and the liquid mobile phase of said fraction is removed so as to obtain said enantiomer E.sub.2.

23. Composition according to claim 14, wherein the flocoumafen is predominantly in the form of said enantiomer E.sub.3 in the composition.

24. Composition according to claim 14, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 25%.

25. Composition according to claim 15, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 25%.

26. Composition according to claim 14, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 95%.

27. Composition according to claim 15, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 95%.

28. Composition according to claim 16, comprising an amount of said enantiomer E.sub.2 such that the ratio of this amount to the amount of flocoumafen in the composition is greater than 95%.

29. Rodenticidal bait comprising a composition according to claim 14 and at least one excipient that is edible for target rodent pests.

30. Rodenticidal bait comprising a composition according to claim 15 and at least one excipient that is edible for target rodent pests.

31. Rodenticidal bait comprising a composition according to claim 16 and at least one excipient that is edible for target rodent pests.

Description

[0148] 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:

[0149] FIG. 1 is a representative chromatogram of the separation of the configurational stereoisomers of flocoumafen on a chiral column;

[0150] FIG. 2 is a histogram representation of the persistence of the configurational stereoisomers of flocoumafen in rat liver.

[0151] Analysis of the Configurational Stereoisomers of Flocoumafen

[0152] The configurational stereoisomers of flocoumafen are separated by high-pressure liquid chromatography using a LUX Cellulose-4 chiral column (00F-4490-B0, Phenomenex, Le Pecq, France) as stationary phase and a mixture formed from acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with an AB volume ratio of 92/8, as mobile phase with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL/minute. 1 L of extract to be analysed at a concentration of 1 g/mL of acetonitrile is injected. Detection is performed by tandem mass spectrometry (MS/MS) in negative electrospray ionization (ESI) mode. 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 541.1.fwdarw.382.1 and m/z 541.1.fwdarw.161, corresponding to the flocoumafen signal, are analysed.

[0153] The retention time values for each of the flocoumafen enantiomers are, under the conditions described:

[0154] t.sub.1=4.5 min for enantiomer E.sub.1 of said diastereoisomer D.sub.1,4;

[0155] t.sub.2=6.2 min for said enantiomer E.sub.2 according to the invention;

[0156] t.sub.3=6.8 min for said enantiomer E.sub.3 of said diastereoisomer D.sub.2,3; and

[0157] t.sub.4=9.3 min for enantiomer E.sub.4 of said diastereoisomer D.sub.1,4.

[0158] The retention time values t.sub.1, t.sub.2, t.sub.3 and t.sub.4 are liable to vary, especially with the temperature of the chromatography column. However, under these chromatographic conditions, the order of elution of the flocoumafen enantiomers remains unchanged. As a guide, the retention time value (t.sub.1) of enantiomer E.sub.1 of said diastereoisomer D.sub.14 may range between 4.4 min and 4.6 min. The retention time value (t.sub.2) of enantiomer E.sub.2 of said diastereoisomer D.sub.2,3 may range between 5.9 min and 6.4 min. The retention time value (t.sub.3) of enantiomer E.sub.3 of said diastereoisomer D.sub.23 may range between 6.4 min and 6.9 min. The retention time value (t.sub.4) of said enantiomer E.sub.4 according to the invention may range between 8.9 min and 9.4 min.

[0159] Extraction of Flocoumafen from the Liver of Rats Tube-Fed with Flocoumafen

[0160] Homogenization of the Liver Sample

[0161] 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.

[0162] Lipid Removal

[0163] 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.

[0164] Solid-Phase Extraction (SPE) of Flocoumafen

[0165] 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 flocoumafen 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 flocoumafen 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 flocoumafen is filtered through a 0.2 m porosity filter.

[0166] Hepatic Persistence of the Configurational Stereoisomers of Flocoumafen in Rats

[0167] A solution comprising flocoumafen in a mixture of vegetable oil and 5% of DMSO so that the amount of flocoumafen ingested by each rat is about 2.3 mg per kilogram of rat is administered orally (per os) to 8-week-old male and female laboratory rats (Sprague-Dawley rats, Charles River, Saint-Germain-sur-l'Arbresle, France) weighing between 180 and 200 g. The tube-fed rats are treated daily by subcutaneous administration of a dose of vitamin K1 at a rate of 1 U per rat so as to keep the rats alive throughout the experiment. The ratio of the sum of the amounts of said enantiomer E.sub.2 and of said enantiomer E.sub.3 (diastereoisomer D.sub.2,3) to the amount of flocoumafen in the tube-feeding solution is 59% and the ratio of the sum of the amounts of said enantiomer E.sub.1 and of said enantiomer E.sub.4 (diastereoisomer D.sub.1,4) to the amount of flocoumafen in the tube-feeding solution is 41%.

[0168] At 1 day (D+1), 3 days (D+3) and 7 days (D+7) after tube-feeding, three male rats and three female rats anaesthetized beforehand with isoflurane are euthanized, the liver of the euthanized rats is removed and the amounts of each of the configurational stereoisomers of flocoumafen present in the liver of the tube-fed male and female rats are then extracted from the liver and assayed. The results obtained on the male rats are given in table 1 below and the results obtained on the female rats are given in table 2 below in which the values of the concentration of each of the configurational stereoisomers of flocoumafen in the liver are the mean of the values measured on three rats expressed in nanograms (ng) per gram of liver.

TABLE-US-00001 TABLE 1 (male rats) Hepatic concentration, ng/g D + 1 D + 3 D + 7 Enantiomer E.sub.1 196 47 0 Enantiomer E.sub.2 3904 1962 319 Enantiomer E.sub.3 3301 1862 647 Enantiomer E.sub.4 8143 9906 2623

TABLE-US-00002 TABLE 2 (female rats) Hepatic concentration, ng/g D + 1 D + 3 D + 7 Enantiomer E.sub.1 4585 2692 1196 Enantiomer E.sub.2 5396 2522 469 Enantiomer E.sub.3 5478 2734 1228 Enantiomer E.sub.4 10342 7765 5054

[0169] FIG. 2 represents the change in the percentage of the mass amount of each enantiomer (E.sub.1 and E.sub.4) of diastereoisomer D.sub.1,4 and of each enantiomer (E.sub.2 and E.sub.3) of diastereoisomer D.sub.2,3 relative to the amount of flocoumafen retained in the liver of the tube-fed rats (male and female) euthanized on D+1, D+3 and D+7. The percentage of said enantiomer E.sub.1 is represented by oblique-hatched columns, the percentage of said enantiomer E.sub.4 is represented by horizontally-hatched columns, the percentage of said enantiomer E.sub.2 is represented by black columns and the percentage of said enantiomer E.sub.3 is represented by white columns. The respective proportions of each of the enantiomers (E.sub.1 and E.sub.4) of diastereoisomer D.sub.1,4 and of the enantiomers (E.sub.2 and E.sub.3) of diastereoisomer D.sub.2,3 in the flocoumafen of the tube-feeding composition are represented in column X FIG. 2 and are 20.5% for enantiomer E.sub.1, 20.5% for enantiomer E.sub.4, 29.5% for enantiomer E.sub.2 of diastereoisomer D.sub.2,3 and 29.5% for enantiomer E.sub.3 of diastereoisomer D.sub.23.

[0170] The persistence of said enantiomer E.sub.2 in the liver of the male and female rats is lower than the persistence of enantiomer E.sub.4 of said diastereoisomer D.sub.1,4 and lower than the persistence of flocoumafen in the liver of the rats. Said enantiomer E.sub.2 of flocoumafen is thus able to be used as a rodenticidal substance which has reduced toxicity to the environment.

[0171] 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 rodenticidal bait and in the embodiments of the process.