EP3386972A1

EP3386972A1 - Configurational stereoisomer of difethialone, composition and rodenticide bait comprising same, and method for controlling target rodent pests

Info

Publication number: EP3386972A1
Application number: EP16809710.3A
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: FR3045051A1; US20180362494A1; BR112018011659A2; US10501434B2; FR3045051B1; WO2017097747A1

Abstract

The invention relates to a levorotatory enantiomer of the configurational stereoisomer of difethialone, referred to as a hetero-stereoisomer, of formula 3-(4'-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which the carbon atoms 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have different absolute configurations.

Description

DIFETHIALONE CONFIGURATION STEREO-ISOMER, COMPOSITION AND RODONTICIDE BAIT COMPRISING SAME, AND METHOD FOR CONTROLLING HARMFUL TARGET RODENTS

A configuration stereoisomer of difethialone, a composition and a rodenticide bait comprising such a configuration stereo-isomer and a method for controlling deleterious target rodents. The invention thus relates to the technical field of the fight against target rodent pest populations.

It is known to use rodenticide baits as a poison for harmful target rodents. It is known from EP 2,090,164 that difethialone is a second-generation anticoagulant acting at a single dose. US 2005/181003 discloses a rodenticide bait in gel form comprising difethialone with a mass proportion of 25 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 difethialone of these rodenticide baits may be ingested (secondary consumption) by animals - in particular by weaker rodent-killing predatory birds that have consumed such a rodenticide bait or by dead rodent-killing 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 difethialone, a composition and a rodenticide bait comprising such a configuration stereo-isomer and a a method of controlling harmful target rodents which are at the same time effective in controlling target rodent pest populations and which also limit the risk of poisoning non-target animals - including domestic animals, pets or humans - accidentally consuming such a rodenticide bait and the risk of poisoning domestic animals (pets or farm animals) or wildlife - for example foxes or birds - predators of rodents weakened harmful target having consumed rodenticide bait or dead target rodent scavengers poisoned by consumption of such rodenticide bait.

The invention also aims at providing a configuration stereoisomer of difethialone, a composition and a rodenticide bait comprising such a 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, especially raptors.

The invention also aims at providing a configuration stereoisomer of difethialone, a composition and a rodenticide bait comprising such a configurational stereoisomer and a method for controlling harmful target rodents that do not require, to control a population harmful rodent rodents, use massive doses of a rodenticide and that are environmentally friendly, human health and non-target animals - especially birds-.

The invention also aims to propose a configuration stereoisomer of difethialone, a composition and a rodenticide bait comprising such a configuration stereoisomer and a method for controlling harmful target rodents that are likely to be used to control against harmful target rodents that have become 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 laevorotatory enantiomer of a configuration stereoisomer of the hetero-stereoisomeric difethialone of the formula 3- (4'-bromobiphenyl-4-yl) -1- (4- hydroxythiocoumarin-3-yl) -1,2,3,4-tetrahydronaphthalene, wherein the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of distinct absolute configurations.

Throughout the text:

the term "difethialone" refers to the compound 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) -1,2,3,4-tetrahydronaphthalene or 3- [3- [4] - (4-bromophenyl) phenyl] -1-tetralinyl] -2-hydroxy-4-thiochromenone or 3- [3- (4'-bromo [1,1'-biphenyl] -4-yl) -1,2 3,4-tetrahydro-1-naphthalenyl] -4-hydroxy-2H-1-benzothiopyran-2-one of formula I) below:

in which the numbers of carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are presented;

the term "stereoisomers" refers to isomers of the same semi-expanded formula, but whose relative position of atoms differs in space. The term "configuration stereo-isomers" refers to stereoisomers whose conversion of these configuration stereoisomers into one another requires a break / reformation of an interatomic covalent bond. Thus, the term "configuration stereo-isomers" refers to stereoisomers which are not conformational isomers (or "rotamers", whose conversion of the 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 "hetero-stereoisomer" of difethialone refers to the configuration stereoisomer of the difethialone of formula 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) - 1, 2,3,4- tetrahydronaphthalene, wherein the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene moiety of said hetero-stereoisomer are of distinct absolute configurations (1R, 3S and 1S, 3R), said absolute configurations being determined according to the sequential rules priority and nomenclature of Cahn, Ingold and Prelog (CIP);

the term "homo-stereoisomer" of difethialone refers to the configuration stereoisomer of the difethialone of formula 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) - 1,2,3,4-tetrahydronaphthalene, wherein carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene moiety of said homo-stereoisomer are of the same absolute configuration (1R, 3R and 1S, 3S);

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 relates to the levorotatory enantiomer of said hetero-stereoisomer of difethialone. It concerns the laevorotatory enantiomer of said hetero-stereoisomer of the difethialone in the isolated state and in particular separated from the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone and of the levorotatory and dextrorotatory enantiomers of a configuration stereoisomer of difethialone, said homo-stereo-isomer, in which the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of the same absolute configuration.

The inventors have discovered that it is possible to separate the levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer from difethialone and the laevorotatory and dextrorotatory enantiomers of said homo-isomer of difethialone by high pressure liquid chromatography in isocratic and under particular conditions using a chromatography column comprising a chiral stationary phase. It was not known at the time of the invention to be able to separate the levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer from difethialone and said homo-stereoisomer of difethialone.

The inventors have succeeded in achieving this separation using a LUX ^® Cellulose-3 HPLC column (phenomenex, Le Pecq, France) of dimension 150 x 2 mm and comprising a chiral stationary phase consisting of porous cellulose particles tris (4-methylbenzoate) , a particle size of 3 μπι and a porosity of 1000 A. The inventors used, as mobile phase, an eluent obtained by mixing acetonitrile (A) and water comprising formic acid in proportion by volume of 0.1% in water (B) with a volume ratio A / B of 80/20. The flow rate of the mobile phase in the column is maintained at a value of 0.25 ml / min and the separation is carried out at a temperature of 23.2 ° C. The composition to be analyzed is at a concentration of 1 μg of difethialone per milliliter in acetonitrile and the volume of composition injected on the column is 1 μl ·. Detection can be performed by tandem mass spectrometry (MS / MS). Detection can also be performed photometrically or spectrophotometrically by adjusting the difethialone concentration and injection volume for optimal detection and by measuring the area under the peak of each enantiomer. Under these experimental conditions, the value of the retention time (t ₂ ) of the laevorotatory enantiomer of said hetero-stereoisomer of the difethialone according to the invention may vary according to the operating conditions -particularly according to the temperature conditions of the column- and be between 9.0 min and 9.5 min. The value of the retention time (t ₃ ) of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone may vary according to the operating conditions - in particular according to the temperature conditions of the column - and be between 11.3 min and 11.8 min, so that the dextrorotatory and laevorotatory enantiomers of said heterois stereoisomer can be separated by high pressure liquid chromatography on this chiral column.

The value of the retention time (ti) of the laevorotatory enantiomer of said ho-stereo-isomer may vary according to the operating conditions - in particular according to the temperature conditions of the column - and be between 7.8 min and 8.2 min. The value of the retention time (t ₄ ) of the dextrorotatory enantiomer of said homo-stereoisomer according to the invention may vary according to the operating conditions - in particular according to the temperature conditions of the column - and be between 14.0 min. and 14.4 min. so that the dextrorotatory and laevorotatory enantiomers of said homo-isomer can be separated by high pressure liquid chromatography on a chiral column.

Thus, under these analytical conditions, the order of elution of the configuration stereoisomers of the difethialone is such that t <t ₂ <t ₃ <t. The values of the retention times t ₁₅ t ₂ , t ₃ and t ₄ are likely to vary, in particular with the temperature of the chromatography column. However, in these chromatographic conditions, the order of elution of the configuration stereoisomers of the difethialone remains unchanged.

The invention thus relates to the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the isolated state and having the property of being elutable, under the chromatography conditions described above, the second of the four stereoisomers. configuration of difethialone.

The laevorotatory enantiomer of said hetero-stereoisomer of difethialone isolated in pure form according to the invention, dissolved in methanol at a concentration of 0.94 g / L and placed in a spectrophotometer quartz tank, has a circular dichroism spectrum produced at 25 ° C of negative circular dichroism values between 225 nm and 250 nm.

The laevorotatory enantiomer of said hetero-stereoisomer of difethialone isolated in the pure state according to the invention, in solution in chloroform (CHCt ₃ ) has a specific rotational power [a] ^C 589 _nm measured at 25 ° C. and on the sodium D line (589 nm) of a value of -13 °.

The laevorotatory enantiomer of said hetero-stereoisomer of difethialone isolated in the pure state according to the invention has in magnetic resonance spectroscopy (1H-NMR) of the 500 MHz proton in CDCt ₃ a multiplet exhibiting a chemical shift (δ ) of the order of 5.3 ppm corresponding to the proton carried by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer of difethialone.

These hetero-stereoisomers are distinguished from difethialone and said homo-stereoisomer of difethialone by their proton NMR spectra. On a proton NMR spectrum produced in CDCt ₃ , the chemical shift of the proton carried by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer of difethialone is between 4.9 ppm and 5 , 1 ppm.

The invention also relates to a composition comprising the levorotatory enantiomer of said hetero-stereoisomer of the difethialone according to the invention, excluding a racemic mixture of levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone.

The invention therefore also relates to a composition comprising a levorotatory enantiomer of a configuration stereoisomer of the hetero-stereoisomeric difethialone of the formula 3- (4'-bromobiphenyl-4-yl) -1 (4). -hydroxythiocoumarin-3-yl) -1,2,3,4-tetrahydronaphthalene, in which the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of distinct absolute configurations, with the exception of a mixture racemic laevorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone, that is to say excluding a composition in which the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is in racemic mixture (equimolecular) with the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone.

The invention thus relates to such a composition in which the levorotatory enantiomer of said hetero-stereoisomer of difethialone and the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone are in unequal (distinct) amounts.

Advantageously and according to the invention, said hetero-stereoisomer is predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone. Advantageously, a composition according to the invention comprises said hetero-stereoisomer of difethialone predominantly in the form of levorotatory enantiomer. The invention therefore relates to a composition wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer.

In the following, the expression "said hetero-stereoisomer is predominantly in the form of a laevorotatory enantiomer" means that the quantity (mass, molar or volume) of laevorotatory enantiomer of said hetero-stereoisomer of difethialone is predominant. greater than 50% in the totality of said hetero-stereoisomer of the difethialone present in the composition (in all of its dextrorotatory and laevorotatory enantiomeric forms).

Advantageously and according to the invention, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of said hetero-stereoisomer of difethialone is greater than 50% in particular greater than 60%, in particular greater than 70%, more particularly greater than 80%, preferably greater than 90%, more preferably greater than 95%, particularly preferably greater than 98%, even more preferably greater than 99% or of the order of 100% -. Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of said hetero-stereoisomer of difethialone is greater than 75%, of preferably between 85% and 100, more preferably between 90% and 98%. Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of said hetero-stereoisomer of difethialone is between 98% and 100.

The invention therefore relates to a composition wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer.

In particular, in a composition according to the invention: the ratio of the amount of laevorotatory enantiomer of said hetero-stereoisomer of difethialone to the sum of the amounts of each of the enantiomers (levorotatory and dextrorotatory) of said heterois stereoisomer difethialone is greater than 0.5 (greater than 50%);

the ratio of the laevorotatory enantiomer concentration of said hetero-stereoisomer of difethialone to the sum of the concentrations of each of the enantiomers (levorotatory and dextrorotatory) of said hetero-stereoisomer of difethialone is greater than 0.5 (greater than at 50), and;

the proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the composition is greater than the proportion of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone. In a composition according to the invention, the proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the composition is more than 50% relative to said hetero-stereoisomer of difethialone.

Advantageously, the composition may also comprise an amount of dextrorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of said hetero-stereoisomer of difethialone in the composition is less than 50%. in particular less than 25, preferably between 0% and 25, in particular less than 10.

Advantageously and according to the invention, the difethialone is predominantly in the form of a laevorotatory enantiomer of said heterois stereoisomer of the difethialone. The composition comprises the levorotatory enantiomer of said hetero-stereoisomer of difethialone with a proportion greater than each of the proportions of each of the other enantiomers of the difethialone relative to the difethialone in the composition.

Advantageously, in a composition according to the invention:

the ratio of the amount of laevorotatory enantiomer of said hetero-stereoisomer of difethialone to the sum of the amounts of each of the enantiomers (levorotatory and dextrorotatory) of said hetero-stereoisomer of difethialone and the amount of said homo-stereo; isomer of difethialone is greater than 0.25 (greater than 25%);

the ratio of the laevorotatory enantiomer concentration of said hetero-stereoisomer of difethialone to the sum of the concentrations of each of the enantiomers (levorotatory and dextrorotatory) of said hetero-stereoisomer of difethialone and the concentration of said homo-stereo; isomer of difethialone is greater than 0.25 (greater than 25%), and;

the proportion of laevorotatory enantiomer of said hetero-stereoisomer of the difethialone in the composition is greater than the proportion of each of the enantiomers of said homo-stereoisomer of difethialone and of the other enantiomer of said hetero-stereoisomer of difethialone. In a composition according to the invention, the proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the composition is more than 25% relative to the total difethialone.

Advantageously and according to the invention, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is greater than 25%, especially greater than 50%, in particular 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% -. A composition according to the invention therefore comprises difethialone predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is greater than 70, preferably between 80% and 100, more preferably between 90% and 100% -. Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is between 95% and 99%. Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is between 98% and 100%. Advantageously, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is substantially of the order of 100%.

Advantageously and according to the invention, the composition comprises an amount of the levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the (total) amount of difethialone is greater than 97% relative to difethialone.

A composition according to the invention may be substantially free of dextrorotatory enantiomer of said hetero-stereoisomer of difethialone, that is to say that the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone may be present in the composition but only in the form of traces. The composition may also be substantially free of a stereoisomer, called a ho-stereo-isomer, of the configuration of the difethialone in which the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of the same absolute configuration. i.e., said homo-isomer of difethialone 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 of difethialone. It could be a solution of difethialone in a solvent of difethialone, excluding a racemic mixture of said levogyre and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone. It may also be a solution comprising difethialone in a solvent of difethialone and wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer. It may also be a solution comprising difethialone in a solvent of difethialone and in which the difethialone is predominantly in the form of laevorotatory enantiomer of said hetero-stereoisomer of difethialone. It can also be a suspension or an emulsion of difethialone in a liquid medium.

Advantageously and according to the invention, the composition is in the solid state. It may also be a solid comprising difethialone, excluding a racemic mixture of dextrorotatory and levorotatory enantiomers of said hetero-stereoisomer of difethialone. It may also be a solid comprising difethialone and wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer. It may also be a solid comprising difethialone and in which the difethialone is predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

The invention therefore also relates to a composition comprising difethialone, the difethialone of the composition being optically active. However, it is not excluded that the difethialone of the composition according to the invention is 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 excipient for harmful target rodents,

the laevorotatory enantiomer of the hetero-stereoisomeric configuration stereoisomer of the formula 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) - 1,2,3,4-tetrahydronaphthalene, in which the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of distinct absolute configurations, excluding a racemic mixture of laevorotatory and dextrorotatory enantiomers; hetero-stereoisomer of difethialone. In a rodenticide bait according to the invention, the amount of laevorotatory enantiomer of said hetero-stereoisomer of difethialone is distinct from the amount of dextrorotatory enantiomer of said hetero-stereoisomer of difethialone.

The inventors who succeeded in separating the dextrorotatory enantiomer of said hetero-stereoisomer from difethialone and the laevorotatory enantiomer of said hetero-stereoisomer of difethialone and obtaining the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the isolated state, have also observed, in a totally surprising manner, that the laevorotatory enantiomer of said hetero-stereoisomer of difethialone and the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone do not exhibit the same hepatic persistence in harmful target rodents and that the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is in fact the enantiomer of said hetero-stereoisomer of difethialone which is of lower hepatic persistence in target rodents, especially in target rodents harmful.

Even more unexpectedly, they have observed that, although said hetero-stereoisomer of difethialone has a higher hepatic persistence than said ho-stereo-isomer of difethialone, the laevorotatory enantiomer of said hetero-stereoisomer difethialone has in fact a low hepatic remanence, and in any case lower than that of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone, lower than that of said hetero-stereoisomer of difethialone and lower than that of difethialone itself. They observed that the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, when ingested by a harmful target rodent, is less persistent and disappears from the target harmful rodent's liver having consumed a bait according to the invention more rapidly. that the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone disappears. The amount of laevorotatory enantiomer of said hetero-stereoisomer of the residual difethialone in the body of a poisoned rodent decreases more rapidly than the amount of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone. The dead or alive harmful target rodent having previously ingested the levorotatory enantiomer of said hetero-stereoisomer of difethialone is less toxic to non-rodent mammals and to birds consuming the target harmful rodent-dead or alive - and in particular vis-à-vis predators (including non-rodent mammals and birds) who preferentially consume the viscera of their prey and in particular their liver.

The inventors have also observed that the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, although having a lower hepatic persistence in harmful target rodents, makes it possible, in a completely surprising manner, to effectively fight against rodents. harmful targets.

The laevorotatory enantiomer of said hetero-stereoisomer of difethialone is a prime candidate for controlling target rodent pest populations that have become resistant to known rodenticide treatments.

Advantageously and according to the invention, the rodenticide bait may comprise an edible excipient for harmful target rodents and said hetero-stereoisomer of difethialone predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone. Advantageously and according to the invention, the rodenticide bait may comprise an edible excipient for harmful target rodents and difethialone predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone. Advantageously and according to the invention, the rodenticide bait comprises a mass quantity of difethialone such that the ratio (mass proportion) of this mass quantity of difethialone to the mass quantity of rodenticide bait is less than 200 ppm-that is less than 200 mg of difethialone per kilogram of bait. Advantageously, the mass proportion of the difethialone in the rodenticide bait is between 1 ppm and 100 ppm (1 mg to 100 mg of difethialone per kilogram of rodenticide bait), in particular between 5 ppm and 100 ppm (5 mg to 100 ppm). mg of difethialone per kilogram of rodenticide bait), preferably of between 5 ppm and 50 ppm (5 mg to 50 mg of difethialone per kilogram of rodenticide bait), more preferably between 10 ppm and 50 ppm (10 mg to 50 ppm). mg of difethialone per kilogram of rodenticide bait), more preferably between 15 ppm and 50 ppm (15 mg to 50 mg of difethialone per kilogram of rodenticide bait), for example of the order of 15 ppm (15 mg of difethialone per kilogram of 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 cereal seeds, 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 of ses-. 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 food 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 selected from the group consisting of vegetable fats, vegetable oils (for example rapeseed oil, soy fat, sunflower oil, cocoa butter, peanut oil, peanut butter, 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 difethialone and a solid edible excipient. Advantageously, the rodenticide bait is a solid in the divided state, for example in the form of pellets or granules. Advantageously, the rodenticide bait can 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 can 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 difethialone, excluding a racemic mixture of dextrorotatory and levorotatory enantiomers of said homo-stereoisomer of difethialone. It may also be a solid rodenticide bait comprising difethialone and wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer. It may also be a solid rodenticide bait comprising difethialone in which the difethialone is predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

Advantageously and according to the invention, the rodenticide bait is chosen from the group consisting of liquid baits comprising difethialone and a liquid edible excipient. The rodenticide bait is then a drink for harmful target rodents. It may be a solution of difethialone in a solvent of difethialone, excluding a racemic mixture of dextrorotatory and levorotatory enantiomers of said hetero-stereoisomer of difethialone. It may also be a solution of difethialone in a solvent of difethialone and wherein said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer. It can also be a solution of difethialone in a solvent of difethialone and in which the difethialone is predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone. It can also be a suspension or an emulsion of difethialone in a liquid medium.

The invention also relates to a rodenticide bait in which the difethialone is optically active. However, it is not excluded that the difethialone 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 bittering type compound denatonium benzoate, also known as the "Bitrex ^®" designed 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 difethialone-in which the hetero-stereoisomer is not a racemic mixture-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.

However, in this variant according to the invention, the composition and rodenticide bait may include any antinuisible substance other than a rodenticide, such as an insecticide and / or acaricide.

Advantageously, in another particular variant, the composition and the rodenticide bait according to the invention comprise difethialone, excluding a racemic mixture of levogyre and dextrorotatory enantiomers of said heterois stereoisomer and at least one other substance. separate difethialone as a rodenticide. This other rodenticide substance, distinct from difethialone, 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

the laevorotatory enantiomer of the hetero-stereoisomeric configuration stereoisomer of the formula 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) - 1,2,3,4-tetrahydronaphthalene, wherein carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene moiety are of distinct absolute configurations;

excluding a racemic mixture of levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone, i.e. excluding a mixture in which the laevorotatory enantiomer of said hetero- the stereoisomer of difethialone and the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone are in equal amounts.

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.

Thus, a quantity of rodenticide bait comprising said hetero-stereoisomer of difethialone is mainly disseminated in the form of a laevorotatory enantiomer, with decreased hepatic persistence in the target rodent but a sufficient rodenticidal efficacy. The method according to the invention thus makes it possible to limit the secondary poisoning of non-rodent mammals and of birds capable of feeding with dead or poisoned live rodents, but comprising a reduced amount, and in particular a non-lethal quantity, of difethialone. . The process according to the invention It also makes it possible to limit such secondary poisoning of non-rodent mammals and birds that may preferentially consume the viscera, in particular the liver, of said dead or poisoned rodents.

Advantageously and according to the invention, said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomer.

Advantageously and according to the invention, the difethialone is predominantly in the form of a laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

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

- the edible excipient;

a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to said hetero-stereoisomer of difethialone;

a proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to difethialone;

- a mass proportion of difethialone compared to the rodenticide bait, and;

- a quantity of disseminated bait;

so that harmful target rodents consume a quantity of difethialone 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 difethialone in the rodenticide bait is between 2 ppm and 200 ppm, in particular between 5 ppm and 100 ppm, preferably between 10 ppm and 50 ppm, more preferably between 15 ppm and 50 ppm.

Advantageously and in another variant according to the invention, one chooses in combination: - the edible excipient;

- a mass proportion of difethialone compared to the rodenticide bait, and;

- a quantity of disseminated bait;

so that harmful target rodents consume a quantity of difethialone;

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, said periods being consecutive.

This other variant of the invention therefore also relates to a method for controlling harmful target rodents in which a quantity of lethal rodenticide bait is dispersed for the rodent harmful rodent consuming this rodenticide and non-lethal rodent bait for rodents or non-rodent animals durably. targets 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 generally insufficient to cause the death of said rodent, while a repeated consumption of rodenticide bait for at least two consecutive days can lead to the death of the rodent harmful target.

The invention therefore relates to a method for controlling a target rodent nuisance population in which rodenticide rodent bait capable of being ingested by the target harmful rodent is made available to harmful target rodents, said quantity of rodenticide bait being sufficient to kill harmful target rodents consuming the rodenticide bait for several days.

Advantageously, the quantity of disseminated rodenticide bait, the mass proportion of difethialone relative to the rodenticide bait and the proportion of levorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone are adapted so that the consumption of the rodenticide bait is lethal to harmful target rodents consuming bait daily for at least 2 24-hour periods, including 3 to 7 periods, the said periods being consecutive.

Advantageously, in this other variant of a process according to the invention, the proportion of laevorotatory enantiomer of said hetero-stereoisomer of difethialone being greater than 95%, in particular of the order of 100%, relative to the difethialone. , the mass proportion of difethialone relative to the rodenticide bait is between 5 ppm and 50 ppm, especially between 10 ppm and 20 ppm, for example of the order of 15 ppm. In a method according to the invention, harmful target rodents are provided with a quantity of rodenticidal bait sufficient to daily satisfy the appetite of the target harmful rodents, said rodenticidal bait comprising a major proportion of levorotatory enantiomer of said hetero-steroid. isomer of difethialone.

In a process according to the invention, the amount of disseminated rodenticide bait, the proportion of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone relative to the difethialone and the mass proportion of difethialone relative to the rodenticide bait to allow rodenticide bait consumption 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 target rodents, likely to consume rodents-dead or alive-targets having ingested a quantity of said bait.

The invention also relates to a chromatographic process for obtaining a laevorotatory enantiomer of a configuration stereoisomer of the hetero-stereoisomer difethialone of formula 3- (4'-bromobiphenyl-4-yl) - 1- (4-hydroxythio coumarin-3-yl) -1,2,3,4-tetrahydronaphthalene, wherein carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene moiety of said heteroisomer are distinct absolutes, a process in which:

a column for high pressure liquid chromatography of dimensions 150 × 2 mm, and comprising a chiral stationary phase consisting of tris (4-methylbenzoate) cellulose particles, said particles having an average size of 3 μπι and presenting a 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 liquid mobile phase, 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 / min;

a separation of the configuration stereoisomers of the difethialone at room temperature during which;

a liquid composition comprising said laevorotatory enantiomer of said hetero-stereoisomer of difethialone is introduced at the top of the chromatography column, and then;

the liquid composition is entrained with the mobile phase in the column for chromatography under conditions suitable for separating the configuration stereoisomers from the difethialone, and;

collecting a fraction of the mobile phase comprising said laevorotatory enantiomer of said hetero-stereoisomer of difethialone with a retention time t ₂ of value such that t i <t ₂ <t ₃ <t ₄ ; t _l5 1 ₃ and t ₄ represent the retention times of each of the configuration stereoisomers of the difethialone distinct from the laevorotatory enantiomer of said hetero-stereoisomer of the difethialone, separately from a dextrorotatory enantiomer of said hetero-stereoisomer of the retention time difethialone t ₃ and separately from the levorotatory and dextrorotatory enantiomers of a difethialone configuration stereoisomer, said homo-isomer, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer are of the same absolute configuration, and of retention time t ₁ and t ₄ , then;

the liquid mobile phase of said fraction is removed so as to obtain said laevorotatory enantiomer of said hetero-stereoisomer of difethialone.

The invention also relates to said laevorotatory enantiomer of said hetero-stereoisomer of difethialone obtained by a process according to the invention.

The invention also relates to a configuration stereoisomer of difethialone, a process for obtaining such a configuration stereo-isomer, a composition and a rodenticide bait comprising such a configuration stereo-isomer and a method of controlling harmful target rodents, 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 chromatogram for the separation of the enantiomers of difethialone (top) and a chromatogram for the analysis of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone (bottom) by high pressure liquid chromatography on a chiral column. ;

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

FIG. 3 is a 300 MHz proton NMR spectrum of said homo-isomer of difethialone;

FIG. 4 is a 500 MHz proton NMR spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone according to the invention; FIG. 5 is a 500 MHz proton NMR correlation spectroscopy analysis (1H-NMR) of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone;

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

FIG. 7 is a circular dichroism spectrum of the levorotatory enantiomer of said hetero-stereoisomer of difethialone;

FIG. 8 is a graphical representation of the temporal evolution of the average concentration measured in the liver of rats (5 male rats and 5 female rats) of said ho-stereo-isomer (·) and of said hetero-stereoisomer ( O), and;

FIG. 9 is a comparative graphical representation of the temporal evolution of the concentration in the liver of rats of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone (·), of said hetero-stereoisomer (■) of difethialone and total difethialone (Δ).

A. Purification of the levorotatory enantiomer of said hetero-stereoisomer of difethialone

A. L. Identification of said hetero-stereoisomer of difethialone and said ho-stereo-isomer of difethialone

The hetero-stereoisomer of difethialone is identified by proton magnetic resonance spectroscopy (1H NMR). Said hetero-stereoisomer of the difethialone in solution in CDCt ₃ has a signal with a chemical shift (δ) of the order of 5.3 ppm and corresponding to the proton carried by the carbon 1 of the 1,2,3 group, 4-tetrahydronaphthalene of difethialone as illustrated in FIG. 2. This hetero-isomer is distinguished from the difethialone of said homo-isomer of difethialone, the latter having in solution in CDCt ₃ a multiplet with a chemical shift (δ) between 4.9 ppm and 5.1 ppm and corresponding to the proton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of difethialone (FIG. 3). A.2. Separation of the levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer from difethialone by chiral column high pressure liquid chromatography

The inventors have solved the complex and unsolved problem to date of the enantiomeric separation of difethialone and in particular the levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone. They succeeded in separating the enantiomers of said hetero-stereoisomer from difethialone and preparing the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone by high performance liquid chromatography (high performance) on a chiral column LUX ^® Cellulose-3 (Phenomenex, Le Pecq, France) dimension 150 x 2 mm and comprising a chiral stationary phase consisting of porous particles of cellulose tris (4-methylbenzoate), a particle size of 3 μπι and a porosity of 1000 A. On uses, as a mobile phase, an eluent consisting of a mixture of acetonitrile (A) and water comprising formic acid in a volume proportion of 0.1% in water (B) with a volume ratio A / B 80/20. The flow rate of the mobile phase in the column is 0.25 ml / min and the separation is carried out at a temperature of 23.2 ° C. The solution containing the sample to be analyzed is at a concentration of 1 μg of difethialone per milliliter in acetonitrile and is filtered on a regenerated cellulose membrane with a cut-off of 0.2 μιη. The volume of solution containing the sample to be analyzed injected on the column is 1 μL ·.

In a process for separating the enantiomers of said hetero-stereoisomer from difethialone, it is possible to detect said enantiomers at the outlet of the high-pressure liquid chromatography column by tandem mass spectrometry (MS / MS) in negative ionization mode. by electrospray (ESI, "ElectroSpray Ionization"). 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 537.1 → 151.0 and m / z 537.1 → 78.99 corresponding to the signals of the difethialone are detected. Figure 1 shows the chromatograms of difethialone (top) and laevorotatory enantiomer of the hetero-stereoisomer of difethialone (bottom) isolated.

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

Under these same conditions, the value of the retention time

(t ₄ ) of said dextrorotatory enantiomer of said homo-stereoisomer according to the invention is of the order of 14.4 min and the value of the retention time () of the levorotatory enantiomer of said homo-stereo-isomer is order of 8.1 min so that the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer can also be separated by high pressure liquid chromatography on a chiral column. Thus, under these conditions, the order of elution of the enantiomers of the difethialone is such that ti <t ₂ <t ₃ <t ₄ .

It is possible to carry out under these experimental conditions (stationary phase, mobile phase, temperature) a preparative separation of the levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone by using a similar stationary phase and with a particle size greater than 3 μπι, and a chromatographic column of larger dimensions, in particular with a diameter of 20 mm.

B. Structural characterization

B.l. UV spectroscopy

The UV spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in solution in chloroform (CHCt ₃ ) has an absorbance peak centered at 238.2 nm and an absorbance peak centered at 259.5 nm. B.2. Rotating power

The inventors have characterized the levorotatory enantiomer of said hetero-stereoisomer of the difethialone in the isolated state by its rotatory power (also called optical activity or circular birefringence), that is to say by its property to deflect the plane. polarization of a polarized light. A deflection of the plane of polarization of the polarized light in the clockwise direction of rotation by facing the polarized light beam characterizes a dextrorotatory solution, and a deflection of the plane of polarization of the polarized light in the anticlockwise direction of rotation facing the Polarized light beam characterizes a solution and a levorotatory compound.

The rotatory power of a laevorotatory enantiomer solution of said hetero-stereoisomer of difethialone in chloroform (CHCt ₃ ) is measured at a concentration of 6.95 g / L. The rotary power of this solution is measured by means of a P 2000 digital polarimeter (JASCO, Bouguenais, France) operating with excitation light with a wavelength of 589 nm. The average rotary power obtained over two series of ten measurements is -0.904 °. The specific rotation power at 25 ° C [α] ^{25 C} 589 _nm of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in solution in chloroform, measured on the sodium D-line (589 nm) is -13 °.

B.3. Circular Dichroism

The circular dichroism spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of the isolated difethialone expresses the difference in absorbance (AA = A _G -A _D ) of the two left circular polarization waves (PCG) of intensity A _G and right circular polarization (PCD) of intensity A _D. It makes it possible to distinguish the dextrorotatory and laevorotatory enantiomers of said homo-stereoisomer from difethialone. This difference in absorbance of the two circular polarization waves is measured in a J-815 circular dichroism spectrometer (JASCO, Bouguenais, France). 2 mL of laevorotatory enantiomer solution of said hetero-stereoisomer of difethialone in methanol (CH ₃ OH) at a concentration of 0.94 mg / mL are prepared. The solution is transferred to a quartz tank for spectrophotometer. At 25 ° C the spectrum of Circular dichroism of the solution between 200 nm and 400 nm. The circular dichroism spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of the difethialone measured under these conditions is shown in FIG. 7. The circular dichroism value is negative between the wavelengths between 225 nm and 250 nm.

B.4. Nuclear Magnetic Resonance

The 300 MHz proton nuclear magnetic resonance spectrum (1 H NMR) of the hetero-stereoisomer of difethialone in CDC ₃ (FIG. 2) shows a multiplet whose chemical shift (δ) is of the order 5.3 ppm. The nuclear magnetic resonance spectrum of the 300 MHz proton Γ ho mo-stereoisomer of difethialone in CDCC ₃ (FIG. 3) shows a multiplet whose chemical shift (δ) is between 4.9 ppm and 5.1 ppm corresponding to carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer of difethialone. The proton NMR spectra of the dextrorotatory and laevorotatory enantiomers of said hetero-stereoisomer of difethialone are indistinguishable from each other.

FIG. 4 is a 1H-NMR spectrum of the proton of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in solution at a concentration of 40 mg / ml in CDCC ₃ having a multiplet whose chemical shift (δ ) is of the order of 5.3 ppm characteristic of said hetero-stereoisomer of difethialone.

FIG. 5 is a two-dimensional (1H-NMR) two-dimensional proton NMR spectrum obtained by correlation spectroscopy of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in solution at a concentration of 40 mg / mL in CDCC ₃ performed on a Bruker AVANCE III HD (500 MHz) spectrometer equipped with a PRODIGY motorized multi-core direct cryoprobe. It makes it possible to identify the coupling of the proton carried by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer of difethialone (5.3 ppm) with the protons carried by carbon 2 of group 1 2,3,4-tetrahydronaphthalene at 2.0 ppm and 2.4 ppm. FIG. 6 is a ¹³ C NMR spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in solution in CDCt ₃ at a concentration of 40 mg / ml produced on a Bruker AVANCE III HD (500 MHz) spectrometer equipped of a PRODIGY motorized multi-core direct cryoprobe. It allows an identification of the 31 carbon atoms of the difethialone. The ¹³ C-NMR spectrum of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is indistinguishable from the ¹³ C-NMR spectrum of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone. However, said hetero-stereoisomer of difethialone has a signal with a chemical shift of the order of 38.2 ppm and a signal with a chemical shift of the order of 40.3 ppm, characteristic and distinctive of said homo- stereoisomer of difethialone.

C. Extraction of difethialone from difethialone-treated rat liver for analysis of different enantiomers of difethialone

Cl. 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.

C.2. 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 lipid-freed extract is dried under a flow of nitrogen (N ₂ ) at a temperature of 40 ° C., then taken up with 0.5 ml of methanol and solubilized by vortexing. We add then 0.5 mL of ultra-pure water (Milli-Q). The sample is homogenized under a vortex.

C.3. Extraction of Solid Phase Extraction (SPE)

1 ml of dichloromethane (CH ₂ C ₂ ) is passed, then

1 mL of methanol (CH ₃ OH), then 1 mL of ultrapure water (Milli-Q) on an Oasis HLB lcc cartridge (WAT094225, Waters). The lipid-free liver extract (lmL MeOH / H ₂ O Milli-Q) and containing the difethialone at the top of the previously conditioned cartridge is then deposited. 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 proportion by volume 90/10 is then deposited at the top of the cartridge and an eluate comprising difethialone 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 (N≡CCH ₃ ) and the acetonitrile solution containing the difethialone is filtered through a 0.2 μιη filter.

C.4. Analysis

The acetonitrile solution containing difethialone is analyzed by high pressure liquid chromatography on a chiral column (150 x 2 mm, particle size 3 μιη) LUX® Cellulose-3 (phenomenex, Pecq, France) as described in point A2) above.

D. Persistence of conferring stereoisomers of difethialone in rat liver

Gents ("per os") are administered to male and female coumafen-sensitive rats (Rattus norvegicus), a solution of a mixture of said ho-stereo-isomer and said hetero-stereoisomer of difethialone in a mixture. of vegetable oil and 5% DMSO. The proportion molar of said homo-stereoisomer is 40% and the molar proportion of said hetero-stereoisomer is 60%. Each configuration stereoisomer of the difethialone is formed from a racemic mixture of the two levogyre and dextrorotatory enantiomers of said corresponding configuration stereo-isomer. The solution comprising 40% of said homo-isomer and 60% of said hetero-stereoisomer is administered (on D0) so that the amount of difethialone ingested by each rat is of the order of 3.4 mg per kilogram of rat. To avoid hemorrhage, fed rats are treated daily by subcutaneous administration of a dose of vitamin K1 (as antidote to bleeding) at a rate of 0.1U per 200g of rat's weight.

At 4 hours (H + 4), 9 hours (H + 9), 24 hours (H + 24), 120 hours (H + 120), 168 hours (H + 168) and 216 hours (H + 216) after the 6 rats (3 male rats and 3 female rats) anesthetized with isoflurane were first euthanized, the liver was taken from the euthanized rats and then the difethialone was extracted from the liver and the amount of each stereoisomeric configuration was determined. difethialone by analysis by high pressure liquid chromatography on a chiral column according to the method described above, measuring the area under the peaks of the chromatogram obtained and quantifying each enantiomer by comparison with a calibration curve. We dose:

the dextrorotatory enantiomer of said homo-stereoisomer;

the laevorotatory enantiomer of said homo-stereoisomer;

the dextrorotatory enantiomer of said heterois stereoisomer;

the laevorotatory enantiomer of said hetero-stereoisomer;

present in the liver of gaved rats.

The results of the analysis of the content (expressed in enantiomeric nanogram per gram of liver (ng / g)) of each enantiomer of said hetero-stereoisomer of difethialone and said homo-stereoisomer of difethialone in the liver of the rats as a function of time (in hours) after the gavage of the rats are given in Table 1 below. Hepatic content, ng / g

Delay after

Total difethialone

gavage

Hetero-stereo-isomer hours Homo-stereo-isomer

DFN-Hetero-dextro DFN-hetero-lévo

4 10258.5 10589.5 5380.5

9 11833.5 12155.5 5869.5

24 5647 8102 3613.5

48 4594.5 7974.5 2804

120 2279.5 5431.5 1211

168 1007.5 3011 392.5

216 1301.5 4030.5 545

Table 1

The results of the analysis of the content of said homo-isomer of the difethialone and of said hetero-stereoisomer of the difethialone are deduced from Table 1 and represented in FIG. 8. Said hetero-stereoisomer (O) has a hepatic remanence greater than the value of the hepatic remanence of said ho mo-stereoisomer (·). However, the inventors have also observed that the laevorotatory enantiomer of said hetero-stereoisomer (said hetero-stereoisomer being the stereoisomer of the most remanent difethialone) has in fact a low hepatic remanence conferring on it surprising properties for the protection of the environment and wild animals - for example, foxes or target predatory rodent birds that have consumed rodenticide bait or scavengers from poisoned target rodent carcasses.

FIG. 9 depicts the evolution over time of the content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone in the liver represented by solid circles (·), of the content of said hetero-stereoisomer in the liver represented by solid squares (■) and total difethialone content in the liver represented (right scale) by open triangles (Δ). The average content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, representing of the order of 30% of the total starting difethialone ingested by the rats, decreases rapidly in the liver of the sacrificed rats. This decay is faster than the decrease of the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone (not shown in FIG. 9), faster than the decrease of said hetero-stereoisomer of difethialone and faster than the decay of total difethialone. The laevorotatory enantiomer of said hetero-stereoisomer of difethialone has a significantly lower hepatic persistence relative to the dextrorotatory enantiomer of said hetero-stereoisomer of difethialone, but also with respect to the laevorotatory enantiomer of said ho stereoisomer (less persistent stereoisomer) of difethialone in male rats.

E. Rodenticide bait with a proportion of 11.85 ppm difethialone

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

On D0, ten male-female Sprague Dawley rats (SD, 5 male and female rats) were placed in individual cages with a reference diet free of rodenticide. On D3, each rat is weighed and then 50 g of rodenticide bait as described above are made available to each rat. This supply of 50 g of rodenticide bait is renewed daily. The bait consumed by the rats is supplemented with 50 g of bait on D4, D5 and D6. As of J7, residual rodenticide baits are discarded and a rodenticide free diet is provided to all rats. The rats are monitored for 3 weeks. The average amounts of bait consumed daily by a rat on day 4, day 5, day 6 and day 7 expressed in grams per day are given in table 2 below.

Table 2

It should be noted that no rat consumed a daily amount of bait less than 1 g / day. The average daily bait consumption relative to the average mass of the rats over the period from D4 to D7 is 64.7 g of bait per kilogram of rat. 9 out of 10 rats die between D9 and D12. The bait mortality is 90% at D12.

The hepatic content of the laevorotatory enantiomer of said hetero-stereoisomer of difethialone is measured in each of the rats that died between D9 and D10 by analysis by high pressure liquid chromatography on a chiral column. The values, expressed in micrograms of laevorotatory enantiomer of said heterois stereoisomer ("DFN-hetero-levo") per gram of liver are given in Table 3 below.

Table 3

The bait dosed at 11.81 ppm of laevorotatory enantiomer of said hetero-stereoisomer of difethialone makes it possible to obtain a mortality rate of the order of 90% by minimizing the risks of secondary intoxication of animals. -in particular, bird-predators or scavengers of weakened target rodents that have consumed a rodenticide bait. 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 / - Laevorotatory enantiomer of the hetero-stereoisomeric configuration stereoisomer of the formula 3- (4'-bromobiphenyl-4-yl) -1- (4-hydroxythiocoumarin-3-yl) -1 , 2,3,4-Tetrahydronaphthalene, wherein the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group are of distinct absolute configurations.

21. A composition comprising a laevorotatory enantiomer according to claim 1, excluding a racemic mixture of levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone.

3 / - Composition according to claim 2, characterized in that said hetero-stereoisomer is predominantly in the form of levorotatory enantiomer.

4 / - Composition according to one of claims 2 or 3, characterized in that the diféthialone is predominantly in the form of levorotatory enantiomer of said hetero-stereoisomer of difethialone.

5 / - Composition according to one of claims 2 to 4, characterized in that it comprises an amount of Γ laevorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of the difethialone is greater than 25%.

6 / - Composition according to one of claims 2 to 5, characterized in that it comprises a quantity of Γ levorotatory enantiomer of said hetero-stereoisomer of difethialone such that the ratio of this amount to the amount of the difethialone is greater than 97% in the composition.

Il - Rodenticide bait comprising a composition according to one of claims 2 to 6, and at least one edible carrier for harmful rodent targets.

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 difethialone such that the ratio of this mass quantity of difethialone on the mass quantity of rodenticide bait is less than 200 ppm.

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

at least one edible carrier for harmful target rodents, and

excluding a racemic mixture of enantiomerically levorotatory and dextrorotatory enantiomers of said hetero-stereoisomer of difethialone.

11 / - Method according to claim 10, characterized in that said hetero-stereoisomer of difethialone is predominantly in the form of levorotatory enantiomers.

12 / - Method according to one of claims 10 or 11, characterized in that the difethialone is predominantly in the form of levorotatory enantiomer of said hetero-stereoisomer of difethialone.

13 / - Method according to one of claims 10 to 12, characterized in that chosen in combination:

- the edible excipient;

- a mass proportion of difethialone compared to the rodenticide bait, and;

- a quantity of disseminated bait; so that harmful target rodents consume a quantity of difethialone sufficient to be lethal to said harmful target rodents consuming said bait during a single 24-hour period.

14 / - Method according to one of claims 10 to 12, characterized in that in combination:

- the edible excipient;

- a mass proportion of difethialone compared to the rodenticide bait, and;

- a quantity of disseminated bait;

so that harmful target rodents consume a quantity of difethialone;

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

15 / - Chromatographic process for obtaining a laevorotatory enantiomer of a configuration stereoisomer of difethialone, called hetero-stereoisomer, of formula 3- (4'-bromobiphenyl-4-yl) -1- ( 4-hydroxythio coumarin-3-yl) -1,2,3,4-tetrahydronaphthalene, wherein the carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer are of distinct absolute configurations, process in which

a column for high pressure liquid chromatography of dimensions 150 × 2 mm, and comprising a chiral stationary phase consisting of tris (4-methylbenzoate) cellulose particles, said particles being of average size of 3 μπι and having an average pore size of 1000 Å;

collecting a fraction of the mobile phase comprising said laevorotatory enantiomer of said hetero-stereoisomer of difethialone with a retention time t ₂ of value such that t i <t ₂ <t ₃ <t ₄ ; t _l5 1 ₃ and t ₄ representing the retention times of each of the difethialone configuration stereoisomers distinct from the laevorotatory enantiomer of said hetero-stereoisomer of difethialone, separately from a dextrorotatory enantiomer of said hetero-stereoisomeric difethialone isomer of t ₃ retention time and separately from the levorotatory and dextrorotatory enantiomers of a difethialone configuration stereoisomer, referred to as homo-stereoisomer, in which carbons 1 and 3 of the 1,2,3 group , 4-tetrahydronaphthalene of said homo-stereoisomer are of the same absolute configuration, and of retention time ^ and t ₄ , then;