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

Abstract

Disclosed is a configurational stereoisomer, named enantiomer E.sub.1, of bromadiolone having, by chromatographic analysis of bromadiolone including four configurational stereoisomers of bromadiolone performed under the conditions described below, 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 being the retention times of the configurational stereoisomers of bromadiolone different from the enantiomer E.sub.1, the analysis being performed at a temperature of 27.3 C.

Claims

1. Configurational stereoisomer, named enantiomer E.sub.1, of bromadiolone having, by chromatographic analysis of a bromadiolone composition comprising four configurational stereoisomers of bromadiolone performed under the conditions described below, 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 being the retention times of the configurational stereoisomers of bromadiolone different from said enantiomer E.sub.1, said analysis being performed at a temperature of 27.3 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and containing a chiral stationary phase constituted of particles of cellulose tris(3,5-dimethylphenyl carbamate), 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 80/20 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 bromadiolone composition at a concentration of 1 g of bromadiolone per millilitre of acetonitrile.

2. Composition comprising a configurational stereoisomer, named enantiomer E.sub.1, of bromadiolone, with the exclusion of a racemic mixture of said enantiomer E.sub.1 and of another configurational stereoisomer, named enantiomer E.sub.2, of bromadiolone different from said enantiomer E.sub.1; said enantiomer E.sub.1 having, by chromatographic analysis of a bromadiolone composition comprising four configurational stereoisomers of bromadiolone performed under the conditions described below, a retention time t.sub.1; said enantiomer E.sub.2 having, by chromatographic analysis of a bromadiolone composition comprising four configurational stereoisomers of bromadiolone performed under these same conditions, a retention time t.sub.2; t.sub.1 and t.sub.2 being values such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.3 and t.sub.4 representing the retention times of configurational stereoisomers of bromadiolone different from said enantiomer E.sub.1 and from said enantiomer E.sub.2, said analysis being performed at a temperature of 27.3 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and containing a chiral stationary phase constituted of particles of cellulose tris(3,5-dimethylphenyl carbamate) particles, 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 80/20 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 bromadiolone composition at a concentration of 1 g of bromadiolone per millilitre of acetonitrile.

3. Composition according to claim 2, wherein the amount of said enantiomer E.sub.1 is greater than the amount of said enantiomer E.sub.2 in the composition.

4. Composition according to claim 2, wherein the bromadiolone is predominantly in the form of said enantiomer E.sub.1 in the composition.

5. Composition according to claim 2, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 25%.

6. Composition according to claim 2, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

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 bromadiolone such that the ratio of this mass amount of bromadiolone to the mass amount of rodenticidal bait is less than 50 ppm.

10. 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 configurational stereoisomer, named enantiomer E.sub.1, of bromadiolone, with the exclusion of a racemic mixture of said enantiomer E.sub.1 and of another configurational stereoisomer, named enantiomer E.sub.2, of bromadiolone different from said enantiomer E.sub.1; said enantiomer E.sub.1 having, by chromatographic analysis of a bromadiolone composition comprising four configurational stereoisomers of bromadiolone performed under the conditions described below, a retention time t.sub.1; said enantiomer E.sub.2 having, by chromatographic analysis of a bromadiolone composition comprising four configurational stereoisomers of bromadiolone performed under these same conditions, a retention time t.sub.2; t.sub.1 and t.sub.2 being values such that t.sub.1<t.sub.2<t.sub.3<t.sub.4; t.sub.3 and t.sub.4 representing the retention times of configurational stereoisomers of bromadiolone different from said enantiomer E.sub.1 and from said enantiomer E.sub.2, said analysis being performed at a temperature of 27.3 C. and under the following conditions: on a high-pressure liquid chromatography column of dimensions 1502 mm, and containing a chiral stationary phase constituted of particles of cellulose tris(3,5-dimethylphenyl carbamate), 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 80/20 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 bromadiolone composition at a concentration of 1 g of bromadiolone per millilitre of acetonitrile.

11. Chromatographic process for obtaining said enantiomer E.sub.1 according to claim 1, in which: a high-pressure liquid chromatography column of dimensions 1502 mm, and containing a chiral stationary phase constituted of cellulose tris(3,5-dimethylphenyl carbamate) particles, 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 80/20, is chosen as liquid mobile phase; the separation is performed at room temperature; a liquid composition containing said enantiomer E.sub.1 dissolved in acetonitrile is introduced into the top of the chromatography column; and then the liquid mobile phase is passed at a flow rate of 0.25 mL/min through the chromatography column after the liquid composition, and a fraction of the mobile phase comprising said enantiomer E.sub.1 is collected at the column outlet, separately from the configurational stereoisomers of bromadiolone different from said enantiomer E.sub.1, and 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 being the retention times of each of the configurational stereoisomers of bromadiolone different from said enantiomer E.sub.1; and the liquid mobile phase of said fraction is removed so as to obtain said enantiomer E.sub.1.

12. Composition according to claim 3, wherein the bromadiolone is predominantly in the form of said enantiomer E.sub.1 in the composition.

13. Composition according to claim 3, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 25%.

14. Composition according to claim 4, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 25%.

15. Composition according to claim 3, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

16. Composition according to claim 4, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

17. Composition according to claim 5, comprising an amount of said enantiomer E.sub.1 such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

18. Rodenticidal bait comprising a composition according to claim 3 and at least one excipient that is edible for target rodent pests.

19. Rodenticidal bait comprising a composition according to claim 4 and at least one excipient that is edible for target rodent pests.

20. Rodenticidal bait comprising a composition according to claim 5 and at least one excipient that is edible for target rodent pests.

Description

[0146] 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:

[0147] FIG. 1 represents a chromatographic analysis of a mixture of the configurational stereoisomers of bromadiolone on a chiral column and of each configurational stereoisomer of bromadiolone on the same chiral column;

[0148] FIG. 2 is a histogram graph representation of the change in the hepatic concentration of each of the configurational stereoisomers of bromadiolone in rats tube-fed with bromadiolone;

[0149] FIG. 3 is a histogram representation of the coagulation time (Quick time) of rats tube-fed with each of the configurational stereoisomers of bromadiolone; and

[0150] FIG. 4 is a histogram graph representation of the hepatic concentration of each configurational stereoisomer of bromadiolone in rats tube-fed with a single configurational stereoisomer of bromadiolone.

[0151] Separation of the Configurational Stereoisomers of Bromadiolone

[0152] Separation of the configurational stereoisomers of bromadiolone is performed by high-pressure liquid chromatography on a LUX Cellulose-1 chiral column (Phenomenex, Le Pecq, France) containing a chiral stationary phase constituted of particles of cellulose tris(3,5-dimethylphenyl carbamate), 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 80/20 and with a flow rate of 0.25 mL/minute in the chromatography column, is used as mobile phase. The concentration of the composition to be analysed is 1 g of bromadiolone per millilitre of acetonitrile and the volume injected onto the column is 1 L. 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 525.1.fwdarw.250.1 and m/z 525.1.fwdarw.181, corresponding to the bromadiolone signal, are detected. An example of a chromatogram is presented in FIG. 1.

[0153] The retention time values for each of the configurational stereoisomers of bromadiolone are:

[0154] for enantiomer E.sub.1 of said diastereoisomer D.sub.1,2, t.sub.1=4.3 min;

[0155] for enantiomer E.sub.2 of said diastereoisomer D.sub.1,2, t.sub.2=5.0 min;

[0156] for enantiomer E.sub.3 of said diastereoisomer D.sub.3,4, t.sub.3=6.3 min; and

[0157] for enantiomer E.sub.4 of said diastereoisomer D.sub.3,4, t.sub.4=8.8 min.

[0158] The retention time values t.sub.1, t.sub.2, t.sub.3 and t.sub.4 are liable to vary, according to the chromatographic conditions. However, in any case, under these conditions (choice of these mobile and stationary phases), the order of elution of the configurational stereoisomers of bromadiolone remains unchanged. As a guide, the retention time values for each of the configurational stereoisomers of bromadiolone may vary with a variation of a few degrees Celsius of the temperature of the chromatography column. For said enantiomer E.sub.1, the retention time (t.sub.1) may range between 4.1 min and 4.4 min. For said enantiomer E.sub.2, the retention time (t.sub.2) may range between 4.8 min and 5.2 min. For said enantiomer E.sub.3, the retention time (t.sub.3) may range between 6.0 min and 6.6 min. For said enantiomer E.sub.4, the retention time (t.sub.4) may range between 8.3 min and 9.0 min.

[0159] Extraction of Bromadiolone from the Liver of Rats Treated with Bromadiolone

[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 Bromadiolone

[0165] 1 mL of dichloromethane (CH.sub.2Ce.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 bromadiolone 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.2Ce.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 bromadiolone 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 bromadiolone is filtered through a 0.2 m filter.

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

[0167] A mixture of diastereoisomers (diastereoisomer D.sub.1,2 and diastereoisomer D.sub.3,4) of bromadiolone dissolved in a mixture of vegetable oil and 5% of DMSO is administered by tube feeding (per os) to 8-week-old male and female Sprague-Dawley SD rats weighing about 200 g. The molar proportion of said diastereoisomer D.sub.1,2 is 56% and the molar proportion of said diastereoisomer D.sub.3,4 is 44%.

[0168] Tube-feeding solution is administered (on D0) so that the amount of bromadiolone ingested by each rat is about 13 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.

[0169] On D1 (D+1), D3 (D+3) and D7 (D+7) after tube feeding, four rats anaesthetized beforehand with isoflurane are euthanized, the liver of the euthanized rats is removed, the bromadiolone is then extracted from the liver and the amount of each of the configurational stereoisomers of bromadiolone is assayed by high-pressure liquid chromatography analysis on a chiral column as described above. The area under the peaks of the chromatogram obtained is measured and each configurational stereoisomer of bromadiolone is quantified by comparison with a calibration curve.

[0170] The results are shown in FIG. 2. Enantiomer E.sub.1 is represented by white columns, enantiomer E.sub.2 is represented by black columns, enantiomer E.sub.3 is represented by oblique-hatched columns and enantiomer E.sub.4 is represented by horizontally-hatched columns. Enantiomer E.sub.1 has the longest persistence in rat liver of the four enantiomers E.sub.1, E.sub.2, E.sub.3 and E.sub.4. This property makes enantiomer E.sub.1 the preferential candidate for preparing a rodenticidal bait with a low dose of rodenticidal agent, in particular for its use in a multi-feeding process.

[0171] Anticoagulant Power of Each Configurational Stereoisomer of Bromadiolone

[0172] The following are administered to male laboratory rats (OFA, Sprague-Dawley 200 g) by tube feeding on D0:

[0173] a composition comprising enantiomer E.sub.1 in a proportion of 1.5 mg of said enantiomer E.sub.1 per kilogram of rat; or

[0174] a composition comprising enantiomer E.sub.2 in a proportion of 1.5 mg of said enantiomer E.sub.2 per kilogram of rat; or

[0175] a composition comprising enantiomer E.sub.3 in a proportion of 1.5 mg of said enantiomer E.sub.3 per kilogram of rat; or

[0176] a composition comprising enantiomer E.sub.4 in a proportion of 1.5 mg of said enantiomer E.sub.4 per kilogram of rat.

[0177] On these rats, blood is collected on D1, D3, D7 and D14 and measurement of the coagulation time (Quick time, expressed in seconds) of the plasma is performed using the Neoplastine CI Determination of Prothrombin Time kit (Diagnostica Stago, Asnibre, France) by measurement on a Thrombotimer Option 2 Plus machine (Behnk Electronik, Norderstedt, Germany).

[0178] The results obtained are given in FIG. 3, in which:

[0179] the white histogram bars correspond to the coagulation time of the rats treated with said enantiomer E.sub.1 according to the invention;

[0180] the black histogram bars correspond to the coagulation time of the rats treated with said enantiomer E.sub.2;

[0181] the oblique-hatched histogram bars correspond to the coagulation time of the rats treated with said enantiomer E.sub.3; and

[0182] the horizontally-hatched histogram bars correspond to the coagulation time of the rats treated with said enantiomer E.sub.4.

[0183] As a guide, the normal value of the coagulation time for untreated rats is about 10 to 20 seconds.

[0184] Ingestion of a dose of 1.5 mg per kilogram of rat of said enantiomer E.sub.1 according to the invention is sufficient to maintain an anticoagulant effect up to D7.

[0185] Hepatic Persistence of Each Configurational Stereoisomer of Bromadiolone

[0186] The liver of the rats treated above is removed for the purposes of comparative analysis of the anticoagulant power of the configurational stereoisomers of bromadiolone, the bromadiolone is then extracted from the liver and the amount of each of the configurational stereoisomers of bromadiolone is assayed by high-pressure liquid chromatography analysis on a chiral column as described above. The area under the peaks of the chromatogram obtained is measured and each configurational stereoisomer of bromadiolone is quantified by comparison with a calibration curve. Enantiomers E.sub.1, E.sub.2, E.sub.3 and E.sub.4 of bromadiolone present in the rat liver are assayed. The hepatic persistence of each enantiomer is expressed in micrograms (g) of enantiomer per gram (g) of rat liver.

[0187] The results are shown in FIG. 4, in which:

[0188] the white histogram bar corresponds to the persistence of said enantiomer E.sub.1 according to the invention in the liver of rats treated with said enantiomer E.sub.1;

[0189] the black histogram bar corresponds to the persistence of said enantiomer E.sub.2 in the liver of rats treated with said enantiomer E.sub.2;

[0190] the oblique-hatched histogram bar corresponds to the persistence of enantiomer E.sub.3 of said diastereoisomer D.sub.3,4 of bromadiolone in the liver of rats treated with said enantiomer E.sub.3; and

[0191] the horizontally-hatched histogram bar corresponds to the persistence of enantiomer E.sub.4 of said diastereoisomer D.sub.3,4 of bromadiolone in the liver of rats treated with said enantiomer E.sub.4.

[0192] Said enantiomer E.sub.1 according to the invention has the highest hepatic persistence among the bromadiolone enantiomers. This property makes enantiomer E.sub.1 the preferential candidate for preparing a rodenticidal bait comprising a low proportion of rodenticidal substance, in particular for its use in a multi-feeding process.

[0193] 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.