EP3386964A1

EP3386964A1 - Stereoisomer of bromadiolone, composition and rodenticide bait comprising same, and method for controlling target rodent pests

Info

Publication number: EP3386964A1
Application number: EP16809716.0A
Authority: EP
Inventors: Hervé CARUEL; Etienne Benoit; Isabelle FOUREL; Virginie LATTARD
Original assignee: Liphatech SA; Institut Enseignement Superieur et Recherche en Alimentation Sante Animale Sciences Agronomiques et Environnement; LiphaTech Inc
Current assignee: Liphatech SA; Institut Enseignement Superieur et Recherche en Alimentation Sante Animale Sciences Agronomiques et Environnement; Novozymes BioAg Inc
Priority date: 2015-12-11
Filing date: 2016-12-06
Publication date: 2018-10-17
Also published as: WO2017097754A1; US20180362490A1; FR3045045A1; US10703736B2; FR3045045B1

Abstract

The invention relates to a configurational stereoisomer, referred to as enantiomer E1, of bromadiolone, having, as determined by the chromatographic analysis of bromadiolone comprising four configurational stereoisomers of bromadiolone, carried out under conditions described hereinafter, a retention time t1 with a value such that t1 < t2 < t3 < t4; t2, t3 and t4 representing the retention times of the configurational stereoisomers of bromadiolone different from said enantiomer E1, said analysis being carried out at a temperature of 27.3°C.

Description

BROMADIOLONE STEREO-ISOMER, COMPOSITION AND RODONTICIDE APPAT COMPRISING SAME, AND METHOD OF CONTROLLING HARMFUL TARGET RODENTS

The present invention relates to a bromadiolone configuration stereoisomer, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a method for controlling deleterious target rodents. The invention thus relates to the technical field of the fight against the development of rodent populations of harmful rodents.

It is known to use poisons in the form of rodenticide baits against harmful target rodents. It is known from EP 2,090,164 that bromadiolone is a second generation anticoagulant acting at a single dose. A bait comprising bromadiolone is also known, the proportion of bromadiolone in the bait being 50 ppm.

Such bait is likely to be consumed by animals other than harmful target rodents when made available to harmful target rodents. It can be consumed directly (primary consumption) by pets or pets. It can also be accidentally consumed by humans. Such consumption may produce poisoning in these pets, pets, or humans that can be lethal.

In addition, a fraction of the bromadiolone from these rodenticide baits can be ingested (secondary consumption) by animals - particularly by weakened target rodent predators - birds that have consumed such rodenticide bait or by dead rodenticide scavenging animals. have consumed such rodenticide bait. This secondary consumption is likely to eventually lead to the death of these predatory animals or scavengers that may be animals - especially birds - belonging to protected species.

The invention therefore aims to overcome these disadvantages by proposing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a stereoisomer of configuration of bromadiolone and a method of controlling harmful target rodents which are effective in controlling target rodent pest populations and which also limit the risk of poisoning non-target animals - especially domestic animals, animals or humans - accidentally consuming such rodenticide bait.

The invention also aims to overcome these drawbacks by proposing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a method for controlling harmful target rodents which are effective to control target rodent pest populations and also to minimize the risk of secondary poisoning of domestic animals (pets or farm animals) or wild animals-for example, foxes or rodent predators-harmful rodent predators weakened having consumed rodenticide bait or dead target poisonous rodent scavengers.

The invention also aims at providing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a method for controlling harmful target rodents whose implementation is in accordance with the rules of good practice - especially with respect to the protection of birds, and especially raptors.

The invention also aims at providing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a method for controlling harmful target rodents which are respectful of the environment, human health and non-target animals-especially birds, especially raptors-.

The invention also aims at providing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a A method of controlling harmful rodent rodents which makes it possible to use a low dose of bromadiolone to control a target rodent population.

The invention also aims at providing a configuration stereoisomer of bromadiolone, a composition and a rodenticide bait comprising such a bromadiolone configuration stereoisomer and a method for controlling harmful target rodents which are capable of being used to control target rodents that are resistant to known baits for the control of target rodents.

The invention therefore aims to propose an alternative to known rodenticide baits.

To this end, the invention relates to a configuration stereoisomer, referred to as the enantiomer E ₁ , of bromadiolone having, by chromatographic analysis, a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under the conditions described above. after, a retention time ti of value such that ti <t ₂ <t ₃ <t ₄ ; t ₂ , t ₃ and t ₄ being the retention times of bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ , said analysis being carried out at the temperature of 27.3 ° C and under the following conditions:

on a high-pressure liquid chromatography column of dimensions 150 × 2 mm, and containing a chiral stationary phase consisting of tris (3,5-dimethylphenylcarbamate) cellulose particles, said particles having an average size of 3 μπι and exhibiting an average pore size of 1000 Å;

by using, as liquid mobile phase, a mixture formed of acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with a volume ratio A / B of 80/20; and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL / min;

by injection into the chromatography column of a volume of 1 of bromadiolone composition at a concentration of 1 μg of bromadiolone per milliliter of acetonitrile. Throughout the text:

the term "bromadiolone" designates the compound 3- [3- (4'-bromo [1,1'-biphenyl] -4-yl) -3-hydroxy-1-phenylpropyl] -4-hydroxy-2H-1- benzopyran-2-one or 3- [3- [4- (4-bromophenyl) phenyl] -3-hydroxy-1-phenylpropyl] -2-hydroxychromen-4-one of formula (I) below:

in which are represented the numbers of carbons 1 and 3 of the 3-hydroxy-1-phenylpropyl group;

"Stereoisomers" means isomers of the same semi-developed formula, but whose relative position of atoms differs in space.

The term "configuration stereo-isomers" denotes stereoisomers whose conversion of these configuration stereoisomers into one another requires a rupture / reformation of an interatomic covalent bond. Thus, the term "configuration stereoisomers" refers to stereoisomers that are not conformational isomers (or "rotamers"), whose conversion from one to the other of the conformational isomers is accompanied only by a rotation of a part of the molecule along the axis of a bond σ (sigma) formed by axial overlap of orbitals);

the term "quantity" means a molar amount, a mass quantity or a volume quantity. The proportions are therefore proportions of a molar amount relative to a molar amount, of a mass quantity relative to a mass quantity, or of a volume quantity referred to a volume quantity;

- the term "substantially" indicates, in the usual way, that a structural or functional characteristic should not be taken as marking an abrupt discontinuity, which would have no physical meaning, but covers not only that structure or function, but also slight variations of this structure or function which produce, in the technical context considered, an effect of the same nature, if not of the same degree;

the terms "high pressure liquid chromatography" or "high performance liquid chromatography" ("HPLC") refer to "HPLC" or "High Performance Liquid Chromatography" chromatography, and;

the expression "retention time" designates the duration, measured at the peak of the chromatogram peak, during which a compound is retained on a chromatography column.

The invention therefore relates to said enantiomer E ₁ in the isolated state and which has the property of being elutable, under the chromatography conditions described above, the first with respect to the four configuration stereoisomers of bromadiolone.

The inventors have observed that the analysis of bromadiolone by high pressure liquid chromatography under the conditions described above reveals four signals or peaks corresponding to four compounds of the same chemical formula developed and corresponding to the formula (I) of bromadiolone . Two of the detected signals correspond to one of the diastereoisomers of bromadiolone, the other two signals corresponding to the other diastereoisomer of bromadiolone. One of the two diastereoisomers of bromadiolone is a bromadiolone configuration stereoisomer in which carbons 1 and 3 of the 3-hydroxy-1-phenylpropyl group are of identical absolute configurations. The other of the two diastereoisomers of bromadiolone is a bromadiolone configuration stereoisomer in which carbons 1 and 3 of the 3-hydroxy-1-phenylpropyl group are of distinct absolute configurations, said absolute configurations being determined according to the sequential rules. of priority and the nomenclature of Cahn, Ingold and Prelog (CIP).

They determined, by the analysis of bromadiolone preparations comprising varying proportions of the two diastereoisomers of bromadiolone, that the two compounds corresponding to the signal exhibiting a retention time of a value of the order of 4.3 min and the signal having a retention time (t ₂ ) of a value of the order of 5.0 min correspond to the two enantiomers of one of the two diastereoisomers of bromadiolone and that the two compounds corresponding to the signal having a retention time (t ₃ ) of a value of the order of 6.3 min and the signal having a retention time (t ₄ ) of a value of the order of 8.8 min correspond to the two enantiomers of the other of the two diastereoisomers of bromadiolone.

Due to slight variations in the chromatographic conditions-in particular temperature variations of the chromatography column-said enantiomer E ₁ may have a retention time (t 1) of between 4, 1 min and 4.4 min. retention time (ti) of said enantiomer Ei being less than the retention time (t ₂ ) of another configuration stereo-isomer, said enantiomer E ₂ , of a diastereoisomer, said diastereoisomer D _{1 2} of bromadiolone, the time Retention (t ₂ ) may have a value between 4.8 min and 5.2 min.

For the same reasons, said enantiomer E ₃ may have a retention time (t ₃ ) of between 6.0 min and 6.6 min, the retention time (t ₃ ) of said E ₃ enantiomer being less than retention time of another configuration stereo-isomer, referred to as the enantiomer E ₄ , of a diastereoisomer, referred to as diastereoisomer D _{3 4} , of the bromadiolone which is eluted, under the chromatography conditions described above, in fourth position by relative to the four configuration stereoisomers of bromadiolone and with a retention time t of between 8.3 min and 9.0 min.

The values of the retention times t ₁₅ t ₂ , t ₃ and t ₄ are therefore likely to vary according to variations in the chromatographic conditions and in particular with the temperature of the chromatography column. However, in these chromatographic conditions (choice of these mobile and stationary phases), the order of elution of configuration stereoisomers of bromadiolone remains unchanged.

Said enantiomer Ei according to the invention is thus obtained in a form separate from the enantiomers of said diastereoisomer D _{3 4} and under a separated form of said enantiomer E _{2 of} said diastereoisomer D _{1 2} . It is possible to realize such a separation by high pressure liquid chromatography on a chiral column LUX ^® cellulose-one (00F-4458-B0, Phenomenex ^®, Le Pecq, France). If necessary, it is possible to successively carry out several steps of high-pressure liquid chromatography on a chiral column in order to obtain the amount of said enantiomer E ₁ sought and the desired level of purity. It is also possible to carry out such a separation by high-pressure liquid chromatography on a preparative chiral column, that is to say on a chromatography column of larger dimensions - in particular with an internal diameter greater than 2 mm - and in which the stationary phase has a particle size greater than 3 μπι. This purified enantiomer E ₁ is obtained and isolated from other bromadiolone configuration stereoisomers by removal of the mobile phase from the fraction collected and containing said enantiomer E ₁ .

It has not previously been known to be able to separate the configuration stereoisomers of bromadiolone and the enantiomers of said diastereoisomer D _{1; 2} . Under these experimental conditions, the enantiomers (E ₁ and E ₂ ) of said diastereoisomer D _{1 2} are separated and purified by high pressure liquid chromatography.

In these chromatographic conditions, the enantiomers of said diastereoisomer D _{3 4} are also separated and purified. One of the enantiomers, said enantiomer E ₃ , of said diastereoisomer D _{3 4} is eluted, under the chromatography conditions described above, the third with respect to the four configuration stereoisomers of bromadiolone. Under the conditions described above, said enantiomer E ₃ has a retention time (t ₃ ) of between 6.0 min and 6.4 min. The other enantiomer, said enantiomer E ₄ , of said diastereoisomer D _{3 4} is eluted, under the chromatography conditions described above, the fourth relative to the four configuration stereoisomers of bromadiolone. Under the conditions described above, said enantiomer E ₄ has a retention time (t ₄ ) of a value between 8.3 min and 9.0 min, so that the enantiomers E ₃ and E _{4 of} said diastereoisomer D _{3 4} are separated and purified by high pressure liquid chromatography on a chiral column under these conditions.

The invention therefore also relates to a chromatographic method for separating bromadiolone configuration stereoisomers-in particular the enantiomers E ₁ and E _{2 of} said diastereoisomer D _{1 2} and the enantiomers E ₃ and E _{4 of} said diastereoisomer D _{3 4} - bromadiolone . The invention thus relates to such a chromatographic process for obtaining said enantiomer E ₁ according to the invention, in which:

a high-pressure liquid chromatography column of dimensions 150 × 2 mm, and containing a chiral stationary phase consisting of cellulose particles tris (3,5-dimethylphenylcarbamate), is chosen, said particles being of average size of 3 μπι and having an average pore size of 1000 Å;

a mixture of acetonitrile (A) and water comprising 0.1% by volume of formic acid (B) is chosen as a liquid mobile phase, with a volume ratio A / B of 80/20; ;

the separation is carried out at room temperature;

a liquid composition containing said enantiomer Ei in solution in acetonitrile is introduced at the top of the chromatography column, and then;

the liquid mobile phase is passed at a flow rate of 0.25 ml / min into the chromatography column and following the liquid composition, and a fraction of the mobile phase comprising the said enantiomer E ₁ is collected at the column outlet; , separately bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ , and with a retention time ti of value such that ti <t ₂ <t ₃ <t; t ₂ , t ₃ and t being the retention times of each of the bromadiolone configuration stereoisomers distinct from said enantiomer E ₁₅ and;

the liquid mobile phase of said fraction is removed so as to obtain said enantiomer Ei. The invention also relates to a configuration stereoisomer - in particular said enantiomer E of bromadiolone obtained by a process according to the invention.

The invention also relates to a composition comprising said E ₁₅ enantiomer excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ . The invention also relates to a composition comprising said enantiomer Ei wherein the amount of said enantiomer Ei in the composition is different from the amount of said enantiomer E ₂ in the composition.

The invention therefore relates to a composition comprising a configuration stereoisomer, said enantiomer E ₁ , of bromadiolone, excluding a racemic mixture of said enantiomer Ei and another configuration stereoisomer, said enantiomer E ₂ , bromadiolone distinct from said enantiomer Ei;

said enantiomer E ₁ having, by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under the conditions described below, a retention time ti;

said enantiomer E ₂ having, by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under these same conditions, a retention time t ₂ ;

t ₁ and t ₂ being of values such that t i <t ₂ <t ₃ <t ₄ ; t ₃ and t ₄ represent retention times of bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ and said enantiomer E ₂ , said analysis being carried out at a temperature of 27.3 ° C:

on a high-pressure liquid chromatography column of dimensions 150 × 2 mm, and containing a chiral stationary phase consisting of tris (3,5-dimethylphenylcarbamate) cellulose particles, said particles having an average size of 3 μπι and exhibiting an average pore size of by using, as liquid mobile phase, a mixture formed of acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with a volume ratio A / B of 80/20; and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL / min;

by injection into the chromatography column of a volume of

1 μl of bromadiolone composition at a concentration of 1 μg of bromadiolone per milliliter of acetonitrile.

The invention therefore relates to such a composition comprising said enantiomer E _15, excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ , that is to say excluding a composition in which said enantiomer E ₁ and said enantiomer E ₂ are in equimolar and non-optically active mixture. In a composition according to the invention, said enantiomer E ₁ and said enantiomer E ₂ are therefore not in equimolecular and racemic mixtures. The said enantiomer E ₁ and the said enantiomer E _{2 are} quantified by chromatographic analysis under the conditions evoked and by performing a detection of configuration stereoisomers by absorption photometry or by absorption spectrometry, by adjusting the concentration of bromadiolone in the solution. of bromadiolone and the volume injected according to the sensitivity of the detector and by measuring the value of the area under the peak corresponding to each enantiomer E ₁ and E ₂ . It is also possible to assay said enantiomer E ₁ and said enantiomer E ₂ of any composition comprising bromadiolone by carrying out a mass spectrometric detection at the outlet of the separating column.

Advantageously and according to the invention, the amount of said enantiomer E ₁ is greater than the amount of said enantiomer E ₂ in the composition. Advantageously, said diastereoisomer D _{1 2} of bromadiolone is predominantly in the form of said enantiomer E ₁ in the composition. Advantageously, a composition according to the invention comprises diastereoisomer D _{1 2} of bromadiolone predominantly in the form of said enantiomer E 1.

Throughout the text, the expression "said diastereoisomer Ό _{1 2} is mainly in the form of said enantiomer E ₁ "means that the ratio of the quantity (mass, molar or volume) of said enantiomer Ei to the quantity (mass, molar or corresponding volume) of said diastereoisomer D _{1 2} (in all its forms enantiomers) is greater than 50%.

Thus, in particular in a composition according to the invention:

the ratio of the amount of said enantiomer Ei to the sum of the amounts of each enantiomer (Ei and E ₂ ) of said diastereoisomer D _{1 2} is greater than 0.5 (greater than 50%);

the ratio of the concentration of said enantiomer Ei to the sum of the concentrations of each enantiomer (Ei and E ₂ ) of said diastereoisomer D _{1 2} is greater than 0.5 (greater than 50%), and;

the proportion of said enantiomer Ei in the composition is greater than the proportion of said enantiomer E ₂ in the composition.

Advantageously and according to the invention, the composition comprises an amount of said enantiomer Ei such that the ratio of this amount to the sum of the amount of said enantiomer E ₁ and the amount of said enantiomer E ₂ in the composition is greater than 50%, in particular greater than 60%, in particular greater than 70%, more preferably greater than 80%, preferably greater than 90%, more preferably greater than 95%, particularly preferably greater than 98%, still more preferably greater than 99% or more preferably 100% order.

In a particular embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer E ₁ such that the ratio of this quantity to the sum of the amount of said enantiomer E ₁ and the amount of said enantiomer E ₂ in the composition is greater than 75%, preferably between 85% and 100%, more preferably between 90% and 98%.

In another embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer Ei such that the ratio of this quantity to the sum of the quantity of said enantiomer E ₁ and the amount of said enantiomer E ₂ in the composition is between 98% and 100%.

The composition may also comprise an amount of said enantiomer E ₂ such that the ratio of this quantity to the sum of the amount of said enantiomer E ₁ and the amount of said enantiomer E ₂ is less than 50, especially less than 25, preferably between 0%. and 25, especially less than 10.

Advantageously and according to the invention, the bromadiolone is predominantly in the form of said enantiomer Ei in the composition. The composition therefore comprises an amount of said enantiomer E ₁ such that the ratio of this amount to the amount of bromadiolone in the composition is greater than the ratio of the amount of said enantiomer E ₂ to the amount of bromadiolone in the composition and greater than the ratio the amount of each enantiomer (enantiomer E ₃ and enantiomer E ₄ ) of said diastereoisomer D _{3 4} on the amount of bromadiolone in the composition.

Thus, in particular, in a composition according to the invention:

the ratio of the amount of said enantiomer E ₁ to the amount of bromadiolone is greater than 0.25 (greater than 25%);

the ratio of the amount of said enantiomer E ₁ to the sum of the quantities of the enantiomers E ₃ and E _{4 of} said diastereoisomer D _{3 4} and the enantiomers E ₁ and E _{2 of} said diastereoisomer D _{1 2} is greater than 0.25 (greater than 25% );

the ratio of the concentration of said enantiomer Ei in the composition to the concentration of bromadiolone in the composition is greater than 0.25 (greater than 25%);

the proportion of said enantiomer Ei in the composition is greater than the proportion of each of the enantiomers E ₃ and E _{4 of} said diastereoisomer D _{3 4} in the composition and greater than the proportion of said enantiomer E ₂ in the composition.

Advantageously and according to the invention, the composition comprises an amount of said enantiomer E ₁ such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 25%, especially greater than 50%, in particular greater than 70%, more particularly greater than 80%, of preferably greater than 90%, particularly preferably greater than 95%, more preferably greater than 98%, still more preferably greater than 99% or of the order of 100%.

In a particular embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer Ei such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 70%, preferably between 80% and 100%, more preferably between 90% and 100%.

In another embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer Ei such that the ratio of this amount to the amount of bromadiolone in the composition is between 95% and 99%.

In another particular embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer Ei such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

In another embodiment, advantageously and according to the invention, the composition comprises an amount of said enantiomer E ₁ such that the ratio of this amount to the amount of bromadiolone in the composition is between 98% and 100% including -.

In another particularly advantageous embodiment according to the invention, the composition comprises an amount of said enantiomer E ₁ such that the ratio of this amount to the amount of bromadiolone in the composition is substantially of the order of 100%.

A composition according to the invention may be substantially free of said enantiomer E ₂ , that is to say that said enantiomer E _{2 of} said diastereoisomer D _{1 2} may be present in the composition but only at the state of traces. A composition according to the invention may also be substantially free of said diastereoisomer D _{3 4,} i.e. that said diastereoisomer D _{3 4} may be present in the composition but only in trace amounts.

Advantageously and according to the invention, the composition is in the liquid state and comprises a liquid solvent for bromadiolone. It may be a bromadiolone solution in a bromadiolone solvent, excluding a racemic mixture of said enantiomer Ei and said enantiomer E ₂ . It may be a solution of bromadiolone in a bromadiolone solvent and wherein said diastereoisomer D _{1 2} is predominantly in the form of said enantiomer Ε

It may also be a solid comprising bromadiolone, excluding a racemic mixture of said enantiomer Ei and said enantiomer E ₂ . It may also be a solid comprising bromadiolone and wherein said diastereoisomer D _{1 2} is predominantly in the form of said enantiomer. It may also be a solid comprising bromadiolone and in which bromadiolone is predominantly in the form of said enantiomer Ε

The invention therefore also relates to a composition comprising said enantiomer Ei according to the invention, excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ , said composition being optically active. However, it is not excluded that the composition according to the invention, comprising said enantiomer E ₁ , excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ , is itself optically inactive.

The invention also relates to the use of a composition according to the invention for the preparation of a rodenticide bait for harmful target rodents.

The invention also relates to a rodenticide bait comprising a composition according to the invention, and at least one edible excipient for harmful target rodents. A rodenticide bait according to the invention comprises:

at least one edible carrier for harmful target rodents, and

a configuration stereo-isomer, referred to as the enantiomer E ₁ , of bromadiolone, excluding a racemic mixture of said enantiomer Ei and another configuration stereoisomer, referred to as the enantiomer E ₂ , of the bromadiolone distinct from said enantiomer Ei;

t ₁ and t ₂ being of values such that t i <t ₂ <t ₃ <t ₄ ; t ₃ and t represent retention times of bromadiolone configuration stereoisomers distinct from said enantiomer Ei and said enantiomer E ₂ , said analysis being carried out at a temperature of 27.3 ° C and under the following conditions:

by injection into the chromatography column of a volume of 1 μl of bromadiolone composition at a concentration of 1 μg of bromadiolone per milliliter of acetonitrile. Advantageously, a rodenticide bait according to the invention comprises an edible excipient for harmful rodent targets and said enantiomer E ₁ , excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ .

The inventors have observed that the two diastereoisomers

(Diastereoisomer D _{1 2} and diastereoisomer D _{3 4} ) of bromadiolone do not have the same hepatic persistence in harmful target rodents and that said diastereoisomer D _{1 2} is in fact the diastereoisomer of bromadiolone which is the most remanent in the liver target rodents, including harmful target rodents. They observed that said diastereoisomer D _{1 2} of a rodenticide bait, when ingested by a harmful target rodent, disappears from the target harmful rodent liver that has consumed such a rodenticide bait according to the invention less rapidly than said diastereoisomer D _{3 4} .

The inventors who have succeeded in separating the enantiomers of said diastereoisomer D _{1 2} and in isolating said enantiomer E ₁ have also discovered that said enantiomer E ₁ has:

an anticoagulant activity higher than the anticoagulant activity of said enantiomer E ₂ and also higher than the anticoagulant activities of said two enantiomers of said diastereoisomer D ₃ , and;

- Remanence in the liver of harmful target rodents greater than the remanence in the liver of rodent harmful targets of said enantiomer E ₂ and greater than the remanence in the liver of harmful target rodents of each enantiomer of said diastereoisomer D ₃ .

Thus, said enantiomer E ₁ according to the invention allows the production of rodenticide baits with a high efficiency for a reduced amount of bromadiolone ingested.

Such rodenticide baits comprising said Ei enantiomer as a novel rodenticide agent are likely to be used to control target rodent-resistant rodent resistant populations of known rodenticide treatments. Advantageously, a rodenticide bait according to the invention comprises a mass quantity of bromadiolone such that the ratio (mass proportion) of this mass quantity of bromadiolone to the mass quantity of the rodenticide bait is less than 50 ppm-that is, say less than 50 mg bromadiolone per kilogram of rodenticide bait. Advantageously, the mass proportion of bromadiolone relative to the rodenticide bait is greater than 1 ppm. Advantageously, the mass proportion of bromadiolone relative to the amount of rodenticide bait is between 1 ppm and 50 ppm (1 mg to 50 mg of bromadiolone per kilogram of rodenticide bait), especially between 1 ppm and 30 ppm ( 1 mg to 30 mg bromadiolone per kilogram of rodenticide bait), preferably between 1 ppm and 20 ppm (1 mg to 20 mg bromadiolone per kilogram of rodenticide bait), more preferably between 1 ppm and 10 ppm (1 mg to 10 mg bromadiolone per kilogram of rodenticide bait).

Advantageously, a rodenticide bait according to the invention comprises a quantity of said enantiomer Ei such that the ratio of this quantity to the amount of bromadiolone in the bait is greater than 70%, more particularly greater than 80%, preferably greater than 90%. %, particularly preferably greater than 95%, more preferably greater than 98%, still more preferably greater than 99% or of the order of 100%, and bromadiolone in a mass proportion of less than 25 ppm relative to the rodenticide bait .

Advantageously and according to the invention, the edible excipient for harmful rodent targets is chosen to allow consumption of the bait by harmful target rodents. Advantageously and according to the invention, each edible excipient is non-lethal for harmful target rodents. The edible carrier is not a rodenticide in itself.

Advantageously and according to the invention, the edible excipient comprises at least one food selected from the group consisting of cereal seeds-notably dehulled cereal seeds-cereal seed mills, cereal seed flours, flakes of seeds cereal bran, cereal bran and non-cereal seeds, eg alfalfa seeds - in particular in the form of husks, in the form of milling, in the form of flour, in the form of flakes or in the form of cereals. The edible carrier may include any carrier that may be consumed by harmful target rodents.

Advantageously, the edible carrier comprises at least one food selected from the group consisting of plant foods and foods of animal origin. Advantageously, the edible carrier comprises at least one food chosen to stimulate the appetite of harmful target rodents. In particular, this food is selected from the group consisting of seeds of one or more cereals, seeds of one or more grains, seeds of one or more grains, seed flakes of one or more cereals, the sound of one or more cereals, and the grain meal of one or more cereals. For example, cereals are selected from the group consisting of oats, wheat, barley, corn, soybeans and rice.

Advantageously, the food is selected from the group consisting of sweet foods. For example, these may be foods comprising at least one sugar selected from the group consisting of sucrose, lactose, fructose and glucose. It may be a sugar syrup-for example, a sugar syrup obtained by hydrolysis of the starch-or a sugar syrup obtained by hydrolysis of sucrose (invert sugar syrup), or beet sugar syrup, or maple syrup or sugar cane syrup, or syrup obtained from a plant of the genus stevia.

Advantageously, the food is chosen from the group consisting of flakes and coconut albumen flour (copra). Advantageously, the food is selected from the group consisting of nuts, hazelnuts and almonds-shredded and / or powdered.

Advantageously, the food is chosen from the group consisting of vegetable fats, vegetable oils (for example rapeseed oil, soy fat, sunflower oil, cocoa butter, peanut oil, butter peanuts, corn oil, palm oil), animal fats and animal oils (butter, lard, fish oil).

Advantageously, the food is selected from the group consisting of proteins of plant origin and proteins of animal origin. By way of example, mention may be made, for example, of powdered milk, particularly powdered skimmed milk, eggs, in particular powdered eggs, protein hydrolysates of animal origin and protein hydrolysates of origin. vegetable.

Advantageously and according to the invention, the rodenticide bait is chosen from the group consisting of solid baits comprising bromadiolone and a solid edible excipient. Advantageously, the rodenticide bait may be a solid in the divided state, for example in the form of pellets or granules. Advantageously, the rodenticide bait may also be a solid in the form of a block or paste that can be consumed by the target harmful rodents or a solid material that may be eaten by the target harmful rodents. Advantageously, the solid rodenticide bait according to the invention can be in the form of a rigid block, a semi-rigid block, a foam, a powder or a gel.

Advantageously, the rodenticide bait in the form of a powder, in the form of a foam or in the form of a gel is adapted to be able to soil the fur of the target rodent (s) harmful (s) ) and to be ingested by him (them) during his (their) grooming.

It may be a solid rodenticide bait comprising bromadiolone, excluding a racemic mixture of said enantiomer Ei and said enantiomer E ₂ . It may also be a solid rodenticide bait comprising bromadiolone and wherein the amount of said enantiomer E ₁ is greater than the amount of said E ₂ enantiomer. It may also be a solid rodenticide bait comprising bromadiolone in which the bromadiolone is predominantly in the form of said enantiomer Ei.

Advantageously and according to the invention, the rodenticide bait is chosen from the group consisting of liquid baits comprising bromadiolone and a liquid edible excipient. The rodenticide bait is then a drink for harmful target rodents. It may be a bromadiolone solution in a bromadiolone solvent, excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ . It may also be a bromadiolone solution in a bromadiolone solvent and in which the amount of said enantiomer Ei is greater than the amount of said E ₂ enantiomer. It may also be a bromadiolone solution in a bromadiolone solvent and in which the bromadiolone is predominantly in the form of said enantiomer E ₁ . It can also be a suspension of bromadiolone in the solid state in a liquid medium. It may also be a bromadiolone emulsion in a liquid medium.

The invention therefore also relates to a rodenticide bait in which bromadiolone is optically active. However, it is not excluded that the bromadiolone of the rodenticide bait according to the invention is optically inactive.

Advantageously, the rodenticide bait comprises at least one dye. Such a dye makes it possible in particular to give said rodenticide bait a color easily detectable and identifiable by a person handling the rodenticide bait.

Advantageously, the rodenticide bait comprises at least one preservative able to ensure its conservation during storage. Advantageously, the rodenticide bait includes at least one compound bittering agent denatonium benzoate type, also known as the "Bitrex ^®" and to reduce the risk of accidental consumption by non-target organisms.

Advantageously, in a particular variant, the composition and the rodenticide bait according to the invention comprise exclusively bromadiolone - excluding a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ - as a rodenticide substance. In particular, the composition and the rodenticide bait according to the invention are free of any other anticoagulant substance for rodenticide use distinct from bromadiolone. However, in this variant according to the invention, the composition and the bait rodenticide may include any anti-harmful substance other than a rodenticide, such as an insecticidal and / or acaricidal substance.

Advantageously, in another particular variant, the composition and the rodenticide bait according to the invention comprise bromadiolone excluding a racemic mixture of said enantiomer Ei and said enantiomer E ₂ and at least one other substance distinct from bromadiolone as a rodenticide. This other rodenticide substance distinct from bromadiolone may be another anticoagulant, particularly anti-vitamin K or non-anticonagulant, or another non-anticoagulant rodenticide.

The invention also relates to a method for controlling harmful target rodents in which a quantity of rodenticide bait comprising is dispersed comprising:

at least one edible carrier for harmful target rodents, and

a configuration stereo-isomer, referred to as the E ₁₅ enantiomer of bromadiolone, excluding a racemic mixture of said enantiomer E ₁ and another configuration stereoisomer, referred to as the E ₂ enantiomer, of the bromadiolone distinct from said enantiomer Ei;

t ₁ and t ₂ being of values such that t i <t ₂ <t ₃ <t ₄ ; t ₃ and t represent retention times of bromadiolone configuration stereoisomers distinct from said enantiomer Ei and said enantiomer E ₂ , said analysis being carried out at a temperature of 27.3 ° C and under the following conditions: ^■ on a liquid chromatography column at high pressure of 150 x 2 mm dimensions and containing a chiral stationary phase consisting of cellulose particles tris (3,5-dimethylphenylcarbamate), said particles being of an average size of 3 μπι and having an average pore size of 1000 Å;

^By using, as the liquid mobile phase, a mixture of acetonitrile (A) and water comprising 0.1% by volume of formic acid (B), with a volume ratio A / B of 80/20; and with a flow rate of the liquid mobile phase in the chromatography column of 0.25 mL / min;

By injection into the chromatography column of a volume of 1 μl of bromadiolone composition at a concentration of 1 μg of bromadiolone per milliliter of acetonitrile.

The invention also relates to a method for controlling harmful target rodents in which a quantity of rodenticide bait according to the invention is dispersed, said quantity of bait being sufficient to be rodenticide.

Advantageously and according to the invention, a quantity of rodenticidal bait comprising said enantiomer E ₁ is dispersed, with the exclusion of a racemic mixture of said enantiomer E ₁ and said enantiomer E ₂ . A quantity of rodenticide bait is dispersed, said rodenticide bait comprising a reduced amount of bromadiolone and being selected to limit secondary intoxications of non-target species consuming poisoned rodents. Advantageously, the amount of said enantiomer Ei in the rodenticide bait may be greater than the amount of said E ₂ enantiomer in the rodenticide bait. In such a process, advantageously and according to the invention, the bromadiolone may be predominantly in the form of said enantiomer Ei.

Advantageously and alternatively according to the invention, one chooses in combination:

- the edible excipient;

an amount of said enantiomer E i distinct from the quantity of said enantiomer E ₂ in the rodenticide bait; a proportion of said enantiomer E ₁ with respect to bromadiolone, and

- a mass proportion of bromadiolone in the rodenticide bait, and;

- a quantity of disseminated rodenticide bait;

such that harmful target rodents consume a quantity of bromadiolone sufficient to be lethal to said harmful target rodents consuming said bait during a single 24-hour period.

A rodenticide bait according to this variant of the invention is a deadly bait in a single catch or "one-shot" in English. Advantageously and according to this variant of the invention, the mass proportion of bromadiolone in the bait is less than 50 ppm, especially between 5 ppm and 25 ppm.

Advantageously and in another variant according to the invention, one chooses in combination:

- the edible excipient;

an amount of said enantiomer E i distinct from the quantity of said enantiomer E ₂ in the rodenticide bait;

a proportion of said enantiomer E ₁ with respect to bromadiolone, and

- a mass proportion of bromadiolone in the rodenticide bait, and; - a quantity of disseminated rodenticide bait;

such that harmful target rodents consume a quantity of bromadiolone;

o non-lethal for harmful target rodents, i.e., generally non-lethal to harmful target rodents, consuming said bait for a period of 24 consecutive hours, and;

o sufficient to be lethal to harmful target rodents consuming said bait for several 24-hour periods.

Advantageously and according to the invention, the periods of 24 hours are consecutive.

This other variant of the invention therefore also aims at a method for controlling harmful rodent rodents in which it spreads a quantity of lethal rodenticide bait for harmful target rodents consuming this rodenticide and non-lethal rodent bait for rodents or non-target animals accidentally consuming this rodenticide bait. This is called a "multi-dose" or "multi-feeding" control method. In such a method according to the invention, the consumption of rodenticide bait by a target rodent harmful for a period of 24 hours is insufficient to cause the death of said rodent, while repeated consumption of rodenticide bait for at least two days resulting in the death of the target rodent.

This other variant of the invention therefore relates to a method for controlling a population of harmful target rodents in which harmful rodent rodents are provided with a quantity of rodenticide bait capable of being ingested by the target harmful rodents, said quantity of rodenticide. rodenticide bait being sufficient to kill harmful target rodents consuming said rodenticide bait for several days.

Advantageously, in this other variant of a process according to the invention, the quantity of disseminated rodenticide bait, the mass proportion of bromadiolone relative to the rodenticide bait and the proportion of said enantiomer Ei with respect to the proportion of said enantiomer E ₂ so that the consumption of the rodenticide bait is lethal to harmful target rodents consuming bait daily for at least 2 periods of 24 hours, especially 3 to 7 periods, said periods being consecutive.

Advantageously, in this other variant of a process according to the invention, the proportion of said enantiomer E ₁ being greater than 95% - in particular of the order of 100% - relative to bromadiolone, the proportion by mass of bromadiolone relative to the rodenticide bait may be between 1 ppm and 25 ppm, especially between 1 ppm and 15 ppm, in particular of the order of 10 ppm.

In a method according to the invention, harmful target rodents are provided with a quantity of rodenticide bait sufficient to daily satisfying the appetite of harmful target rodents, said rodenticide bait comprising predominantly said enantiomer Ei with respect to said diastereoisomer D _{1 2} and with respect to bromadiolone.

In a method according to the invention, the amount of disseminated rodenticide bait, the amount of said enantiomer Ei with respect to said enantiomer E ₂ and with respect to bromadiolone and the mass proportion of bromadiolone relative to the rodenticide bait of to allow the consumption of rodenticide bait for several days by harmful target rodents, while limiting:

- the risk of primary intoxication of mammals and non-target birds likely to consume only occasionally and accidentally such rodenticide bait;

the risks of secondary intoxication, for example predators of rodents targets, likely to consume rodents-dead or alive targets-ingested a quantity of said bait.

The invention also relates to a configuration stereoisomer of bromadiolone, a process for obtaining this configuration stereoisomer, a composition comprising this configuration stereoisomer, a rodenticide bait and a method for controlling rodents. pests characterized in combination by all or some of the characteristics mentioned above or hereafter.

Other objects, features and advantages of the invention will appear on reading the following description and examples given solely by way of non-limiting example and which refer to the appended figures, in which:

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

FIG. 2 is a graphical representation in histogram of the evolution of the hepatic concentration of each of the bromadiolone configuration stereoisomers in rats gavaged with bromadiolone; FIG. 3 is a histogram representation of the coagulation time (quick time) of gaved rats with each of the configuration stereoisomers of bromadiolone, and;

FIG. 4 is a graphical representation in histogram of the hepatic concentration of each bromadiolone configuration stereoisomer in rats gavaged with a single bromadiolone configuration stereoisomer.

Separation of configuration stereoisomers from bromadiolone

One carries out a separation bromadiolone the configuration of the stereoisomers by high pressure liquid chromatography on a chiral column LUX ^® cellulose-1 (Phenomenex, Le Pecq, France) containing a chiral stationary phase consisting of cellulose particles tris (3, 5-dimethylphenylcarbamate), said particles having an average size of 3 μπι and having an average pore size of 1000 A. As a mobile phase, a mixture of acetonitrile (A) and water is used. comprising 0.1% by volume of formic acid (B), with a volume ratio A / B of 80/20 and with a flow rate of 0.25 ml / min in the chromatography column. The concentration of the composition to be analyzed is 1 μg bromadiolone per milliliter of acetonitrile and the volume injected onto the column is 1 μl ·. Detection is performed by tandem mass spectrometry (MS / MS) in Electrospray Ionization (ESI) mode. The nebulizer gas temperature is 350 ° C and its flow rate is 8 L / min. The nebulizer gas pressure is raised to 2700 hPa. In particular, the MRM (Multiple Reaction Monitoring) transitions m / z 525.1 → 250.1 and m / z 525.1 → 181 corresponding to the bromadiolone signal are detected. An example of a chromatogram is presented in FIG.

The values of the retention times of each of the configuration stereoisomers of bromadiolone are:

for the enantiomer E _{1 of} said diastereoisomer D _{1; 2} , ti = 4.3 min;

for the enantiomer E _{2 of} said diastereoisomer D _{1 2} , t ₂ = 5.0 min; for the enantiomer E _{3 of} said diastereoisomer D _{3 4} , t ₃ = 6.3 min, and;

for the enantiomer E _{4 of} said diastereoisomer D ₃ , t = 8.8 min.

The retention time values t ₁₅ t ₂ , t ₃ and t ₄ are likely to vary according to the chromatographic conditions. However, in any case, under these conditions (choice of these stationary and mobile phases), the order of elution of the configuration stereoisomers of bromadiolone remains unchanged. As an indication, the values of the retention times of each of the bromadiolone configuration stereoisomers may vary with a variation of a few degrees Celsius in the temperature of the chromatography column. For said enantiomer E ₁ , the retention time ¾) can vary between 4.1 min and 4.4 min. For said enantiomer E ₂ , the retention time (t ₂ ) can vary between 4.8 min and 5.2 min. For said enantiomer E ₃ , the retention time (t ₃ ) can vary between 6.0 min and 6 min. , 6 min. For said enantiomer E ₄ , the retention time (t) can vary between 8.3 min and 9.0 min.

Bromadiolone extraction from livers of bromadiolone-treated rats

Homogenization of the liver sample

0.525 g (± 0.025 g) of rat liver are weighed accurately and placed in a 50 ml polypropylene tube. 10 mL of acetone was added and the suspension is subjected to homogenization using a homogenizer / disperser ^® Ultra-Turrax for a period of about 30 sec. The rod of the homogenizer / disperser 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 rate of 3000 rpm (rotation per minute). The supernatant is collected and decanted into a test tube. The sample is evaporated under a stream of nitrogen (N ₂ ) at a temperature of 40 ° C to form a dry extract.

Elimination of lipids

In the tube containing the dry extract, 1 mL of acetonitrile is added so as to solubilize the dry extract. The acetonitrile solution is washed twice in succession with 1 mL of hexane. The extract cleared lipids are dried under a flow of nitrogen (N ₂ ) at the temperature of 40 ° C, then taken up with 0.5 mL of methanol and solubilized by vortexing. 0.5 ml of ultrapure water (Milli-Q) is then added. The sample is homogenized under a vortex.

Extraction of solid phase bromadiolone ("SPE,

Solid Phase Extraction ")

1 ml of dichloromethane (CH ₂ C ₂ ), then 1 ml of methanol (CH ₃ OH) and then 1 ml of ultrapure water (Milli-Q) are passed through an Oasis HLB lcc cartridge (WAT094225, Waters). . The lipid-free liver extract (lmL MeOH / H ₂ O Milli-Q) containing the bromadiolone is then placed at the top of the previously conditioned cartridge. The liver extract enters the cartridge by gravity in contact with the solid phase of the cartridge. 1 mL of washing solution consisting of methanol (CH ₃ OH) and ultrapure water (H ₂ O) in a volume proportion of 90/10 is deposited at the top of the cartridge. The cartridge is dried by vacuum suction connected at the bottom of the cartridge. 1 mL of elution solution consisting of dichloromethane (CH ₂ Cl ₂ ) and of methanol (CH ₃ OH) in a volume proportion of 90/10 is then deposited at the top of the cartridge and an eluate comprising bromadiolone is collected at the bottom of the cartridge. . The eluate solvent was evaporated under a stream of nitrogen (N ₂ ) at a temperature of 40 ° C. The sample is taken up in 0.5 ml of acetonitrile (NC-CH ₃ ) and the bromadiolone-containing acetonitrile solution is filtered through a 0.2 μm filter.

Hepatic remission of bromadiolone configuration stereoisomers in rats

Gavage ("per os") is administered to SD rats

"Sprague Dawley" males and females, aged 8 weeks and weighing about 200 g, a mixture of diastereoisomers (diastereoisomer D _{1 2} and diastereoisomer D _{3 4} ) bromadiolone in solution in a mixture of vegetable oil and from DMSO to 5. The molar proportion of said diastereoisomer D _{1 2} is 56% and the molar proportion of said diastereoisomer D _{3 4} is 44%. The gavage solution is administered (on day 0) so that the amount of bromadiolone ingested by each rat is of the order of 13 mg per kilogram of rat. To avoid hemorrhage, fed rats are treated daily by subcutaneous administration of a dose of vitamin K1 (as an antidote to bleeding) at the rate of 0.1U per 200g of rat's weight.

On D1 (D + 1), D3 (D + 3) and D7 (D + 7) after gavage, four rats previously anesthetized with isoflurane were euthanized, the liver was removed from the euthanized rats, and bromadiolone was extracted from the liver. and the amount of each of the bromadiolone configuration stereoisomers 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 bromadiolone configuration stereoisomer is quantified by comparison with a calibration curve.

The results are shown in FIG. 2. The enantiomer E ₁ is represented by white columns, the E ₂ enantiomer is represented by black columns, the E ₃ enantiomer is represented by oblique hatched columns and the enantiomer E ₄ is represented by horizontal hatched columns. The enantiomer Ei has the highest persistence in the liver of the rats of the four enantiomers E ₁ , E ₂ , E ₃ and E ₄ . This property makes the enantiomer Ei the preferred candidate for the production of a low-dose rodenticide bait rodenticide - especially for its use in a multi-feeding process.

Anticoagulant potency of each configuration stereoisomer of bromadiolone

On day 0 gavage was administered to male laboratory rats (OFA, Sprague Dawley 200 g), that is:

a composition comprising enantiomer Ei in a proportion of 1.5 mg of said enantiomer E ₁ per kilogram of rat, ie;

a composition comprising enantiomer E ₂ at the rate of 1.5 mg of said enantiomer E ₂ per kilogram of rat, ie; a composition comprising enantiomer E ₃ in a proportion of 1.5 mg of said enantiomer E ₃ per kilogram of rat, that is;

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

On these rats, blood is taken at J1, J3, J7 and J14 and a measurement of the coagulation time (Quick time, expressed in seconds) of the plasma is carried out using the "Neoplastine CI Determination of Prothrombin Time" kit (Diagnostica Stago, Asnière, France) by measurement on a Thrombotimer device option 2 plus (Behnk Electronik, Norderstedt, Germany).

The results obtained are given in FIG. 3 in which:

the white histogram bars correspond to the coagulation time of the rats treated with the said enantiomer E 1 according to the invention;

the black histogram bars correspond to the coagulation time of the rats treated with the said enantiomer E ₂ ;

the obliquely hatched histogram bars correspond to the coagulation time of the rats treated with said E ₃ enantiomer, and;

the horizontal hatched histogram bars correspond to the coagulation time of the rats treated with the said enantiomer E ₄ .

As an indication, the normal value of the coagulation time of untreated rats is of the order of 10 to 20 seconds.

Ingesting a dose of 1.5 mg per kilogram of rat of said enantiomer E ₁ according to the invention is sufficient to maintain an anticoagulant effect until J7.

Hepatic remission of each bromadiolone configuration stereoisomer

The liver from the above-treated rats is taken for comparative analysis of the anticoagulant properties of the bromadiolone configuration stereoisomers, bromadiolone is removed from the liver and the amount of each of the stereoisomers of the configuration is assayed. bromadiolone 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 bromadiolone configuration stereoisomer is quantified by comparison with a calibration curve. The E ₁₅ E ₂ , E ₃ and E ₄ enantiomers of bromadiolone present in the liver of rats are dosed. Hepatic persistence of each enantiomer is expressed in microgram (g) of enantiomer per gram (g) of rat liver.

The results are represented in FIG. 4 in which:

the white histogram bar corresponds to the remanence of said enantiomer E ₁ according to the invention in the liver of rats treated with said enantiomer E ₁ ;

the black histogram bar corresponds to the remanence of said enantiomer E ₂ in the liver of rats treated with said enantiomer E ₂ ;

the oblique hatched histogram bar corresponds to the remanence of the enantiomer E _{3 of} said diastereoisomer D _{3 4} of bromadiolone in the liver of rats treated with said enantiomer E ₃ , and;

the horizontally hatched histogram bar corresponds to the remanence of the enantiomer E _{4 of} said diastereoisomer D _{3 4} of bromadiolone in the liver of rats treated with said enantiomer E ₄ .

Said enantiomer E ₁ according to the invention has the highest hepatic persistence among the enantiomers of bromadiolone. This property makes Γ enantiomer E ₁ the preferred candidate for producing a rodenticide bait comprising a small proportion of rodenticide substance - especially for its use in a multi-feeding process.

It goes without saying that the invention can be the subject of many variants and applications. In particular, a composition, a rodenticide bait and a method for controlling harmful target rodents are subject to infinity of variants both in the formulation of the bait and in the methods of implementation of the method.

Claims

1 / - Stereo-isomer configuration, said enantiomer E ₁₅ of bromadiolone exhibiting by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under the conditions described below, a retention time ti of value such that ti <t ₂ <t ₃ <t ₄ ;

t ₂ , t ₃ and t ₄ being the retention times of bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ , said analysis being carried out at the temperature of 27.3 ° C and under the following conditions:

21 - A composition comprising a stereoisomer configuration, said enantiomer S _l5 bromadiolone, excluding a racemic mixture of said enantiomer and E ₁ of another stereoisomer configuration, said enantiomer E _2, of the bromadiolone distinct from said enantiomer Ei;

said enantiomer E ₁ having, by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under the conditions described below, a retention time ti; said enantiomer E ₂ having, by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of bromadiolone configuration carried out under these same conditions, a retention time t ₂ ;

t ₁ and t ₂ being of values such that t i <t ₂ <t ₃ <t ₄ ;

t ₃ and t ₄ represent retention times of bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ and said enantiomer E ₂ , said analysis being carried out at the temperature of 27.3 ° C and under the following conditions:

3 / - Composition according to claim 2, characterized in that the amount of said enantiomer Ei is greater than the amount of said enantiomer E ₂ in the composition.

4 / - Composition according to one of claims 2 or 3, characterized in that the bromadiolone is predominantly in the form of said enantiomer E ₁ in the composition.

5 / - Composition according to one of claims 2 to 4, characterized in that it comprises an amount of said enantiomer E ₁ such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 25%. 6 / - Composition according to one of claims 2 to 5, characterized in that it comprises an amount of said enantiomer Ei such that the ratio of this amount to the amount of bromadiolone in the composition is greater than 95%.

11 - Rodenticide bait comprising a composition according to one of claims 2 to 6, and at least one edible excipient for harmful target rodents.

8 / - Bait according to claim 7, characterized in that the edible carrier comprises at least one food selected from the group consisting of cereal seeds, cereal grain mills, cereal seed meal, seed flakes cereal, cereal bran and non-cereal seeds.

91 - Bait according to one of claims 7 or 8, characterized in that it comprises a mass quantity of bromadiolone such that the ratio of this mass quantity of bromadiolone on the mass quantity of the rodenticide bait is less than 50 ppm.

10 / - A method of controlling harmful target rodents, wherein a quantity of rodenticide bait is dispersed comprising:

at least one edible carrier for harmful target rodents, and

a configuration stereo-isomer, referred to as the E ₁₅ enantiomer of bromadiolone, excluding a racemic mixture of said enantiomer E ₁ and another configuration stereoisomer, referred to as the E ₂ enantiomer, of the bromadiolone distinct from said enantiomer E ₁ ;

said enantiomer E ₂ having, by chromatographic analysis of a bromadiolone composition comprising four stereoisomers of configuration of bromadiolone carried out under these same conditions, a retention time t ₂ ;

t ₁ and t ₂ being of values such that t i <t ₂ <t ₃ <t ₄ ;

^■ on a liquid chromatography column at high pressure of 150 x 2 mm dimensions and containing a chiral stationary phase consisting of cellulose particles tris (3,5-dimethylphenylcarbamate), said particles being of an average size of 3 μπι and having an average pore size of 1000 Å;

^■ by injection into the chromatography column with a volume of 1 .mu.l · bromadiolone composition at a concentration of 1 mcg bromadiolone per milliliter of acetonitrile.

11 / - Chromatographic process for obtaining said enantiomer Ei according to Claim 1, in which:

the separation is carried out at room temperature; a liquid composition containing said enantiomer Ei in solution in acetonitrile is introduced at the top of the chromatography column, and then;

the liquid mobile phase is passed at a flow rate of 0.25 ml / min into the chromatography column and following the liquid composition, and a fraction of the mobile phase comprising the said enantiomer E ₁ is collected at the column outlet; separately from bromadiolone configuration stereoisomers distinct from said enantiomer _E15 and with a retention time ti of value such that ti <t ₂ <t ₃ <t ₄ ;

t ₂ , t ₃ and t ₄ being the retention times of each of the bromadiolone configuration stereoisomers distinct from said enantiomer E ₁ , and;

the liquid mobile phase of said fraction is removed so as to obtain said enantiomer Ei.