N-heterocyclic compounds as nitrification inhibitors

Abstract

Use of an N-heterocyclic compound of general formula (a) or (b) as a nitrification inhibitor, in general formula (a) or (b): X1 is S or O, X2 is S or O, and at least one of X1 and X2 is S; r2 is H or C1-4 alkyl; r3 is H or a C1-4 alkyl group; r6 and R7 are hydrogen or together form a covalent carbon-carbon bond; in the general formula (a), R1 is H, C1-12 alkyl or-CH2-NR4R5, and R4 is hydrogen or C1-4 alkyl; r5 is a C1-12 hydrocarbon residue which may comprise 1-3 halogen atoms and / or 1-4 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, R4 and R5 may also form together with the nitrogen atom linking them a 5-or 6-membered saturated or unsaturated heterocyclic group which may optionally further contain one or two further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; in general formula (b), R1 is H or a C1-17 hydrocarbon, preferably H, or-CH2-R5, where R5 is H or a C1-16 hydrocarbon residue, the hydrocarbon may contain 1-3 halogen atoms and / or 1-6 heteroatoms, selected from nitrogen, oxygen and sulfur; preferably, X1 and X2 in the general formulae (a) and (b) are S. # imgabs0 #

Description

[0001] This application is a divisional application of the application with application date of September 12, 2021, application number 202180064751.8, and invention name “N-heterocyclic compounds used as nitrification inhibitors”. Technical Field

[0002] The present invention relates to the use of a specific N-heterocyclic compound as a nitrification inhibitor or nitrogen stabilizer, a fertilizer mixture containing the same, a method for preparing the same, and a soil fertilization method comprising applying the same. Background Art

[0003] In order to provide agricultural or horticultural plants with the nitrogen they require, fertilizers comprising ammonium compounds are frequently used.

[0004] With the invention of the Haber-Bosch process in 1908, fertilizers fueled the Green Revolution of the 1950s and 1960s and helped feed the exponentially growing world population. Since nitrogen (N) is one of the most important nutrients for plant growth, the industrial synthesis of ammonia (NH3) has pushed the limits of the Earth's ability to produce food crops. However, the nitrogen sources used for fertilizers (primarily ammonium (NH4 + ) and nitrate (NO3 - ) is not only used by plants but also leached into the environment or converted by microorganisms through the nitrogen cycle into inaccessible nitrogen forms. As a result, the average nitrogen fertilizer loses nearly 50%. In addition, N leaching can lead to eutrophication of groundwater and free water and cause toxic algal blooms, reduced recreational water value, or contamination of drinking water. Microbial conversion to the greenhouse gas N2O significantly exacerbates global warming. However, the global population is expected to reach 9.7 billion by 2050, and the Food and Agriculture Organization of the United Nations (FAO) expects fertilizer demand to increase by 50% or more. Therefore, mitigation strategies are very important to limit N emissions as much as possible. One way to do this is to inhibit nitrification, that is, to convert NH4 + or NH3 is converted to NO3 - .

[0005] With NO3 - Different, positively charged NH4 + It will bind to negatively charged soil particles and will hardly leach into the soil. + is the preferred nitrogen source and should be kept in the soil for plant uptake. However, NH4 + May be converted to NO3 through nitrification - Nitrification is a microbial process in the global nitrogen cycle, in which NH4 + NH3 in pH-dependent equilibrium is transported by soil microorganisms through nitrite (NO2 - ) is converted into NO3- Therefore, nitrification is the cause of NH4 + The core step of the nitrogen cycle and subsequent conversion to undesirable leachable or volatile nitrogen forms.

[0006] The first step of nitrification, NH3 oxidation, occurs in agricultural soils and substrates and is primarily carried out by ammonia-oxidizing bacteria (AOB). They first oxidize NH3 to hydroxylamine (NH2OH), which is catalyzed by ammonia-monooxygenase (AMO). Subsequently, the enzyme hydroxylamine oxidoreductase (HAO) catalyzes the second step: the oxidation of NH2OH to form nitrite (NO2 - ).

[0007] In the past, various organic and inorganic compounds have been identified as nitrification inhibitors, but only a few are currently commercially available, including dicyandiamide (DCD), nitrapyrin (2-chloro-6-(trichloromethyl)-pyridine), or 3,4-dimethylpyrazole phosphate (DMPP). However, the effectiveness of nitrification inhibitors can vary among different strains and genera of AOB. Therefore, expanding the range of applicable nitrification inhibitors is of great importance.

[0008] One problem with the use of pyrazole compounds as nitrification inhibitors is their high volatility. When fertilizer formulations containing pyrazole compounds are stored, the active ingredient is continuously lost due to evaporation. Therefore, pyrazole compounds must be formulated in a non-volatile form through appropriate measures.

[0009] EP-B-1120388 describes phosphoric acid addition salts of 3,4-dimethylpyrazole and 4-chloro-3-methylpyrazole as nitrification inhibitors. This salt form significantly reduces volatility.

[0010] WO 96 / 24566 relates to the use of low-volatility pyrazole derivatives with hydrophilic groups as nitrification inhibitors. For example, 2-(N-3-methylpyrazole)succinic acid (DMPSA) is proposed as a nitrification inhibitor. Suitable mineral fertilizers listed are ammonium-containing nitrates, sulfates, or phosphates.

[0011] WO 2011 / 032904 and WO 2013 / 121384 describe pyrazole derivatives as nitration inhibitors, one of which is DMPSA.

[0012] A. Saha et al. described the green synthesis of 5-substituted-1-3,4-thiadiazole-2-thiols as novel potent nitration inhibitors in J. Heterocyclic Chem., 47, 838 (2010). The 1,3,4-thiadiazole-2-thiols were 5-substituted with alkyl or aromatic groups. These compounds were screened for in vitro nitration inhibitory activity. Compounds 5-substituted with heptyl, 2-chlorophenyl, 2,4-dichlorophenyl, 2-methylphenyl, 3-methylphenyl, 3,4-dimethoxyphenyl, 2-hydroxyphenyl, and 4-hydroxy-3-methoxyphenyl groups were considered promising nitration inhibitors.

[0013] WO 2020 / 020765A1 discloses the use of substituted thiazolidine compounds as nitration inhibitors. These compounds are derived from 1,3-thiazolidine-2-thione, as shown in the table on page 71.

[0014] WO 2020 / 020777A1 discloses substituted 2-thiazolines as nitration inhibitors. These compounds are derived from 2-mercapto-2-thiazoline by substituting the hydrogen atoms of the ring structure or the thiol group.

[0015] CN 103524159 A discloses that oxazolidinthione may be present in fertilizers, among other ingredients, but does not mention the function of the compound.

[0016] There is a continuing need for new nitrification inhibitors with high nitrification inhibitory activity, low toxicity, and low volatility. Summary of the Invention

[0017] Therefore, an object of the present invention is to provide a novel nitrification inhibitor preferably having high nitrification inhibitory activity.

[0018] Another object of the present invention is to provide a fertilizer mixture containing the nitrification inhibitor, a preparation method thereof, and a soil fertilization method using the same.

[0019] These objects are achieved by the use of N-heterocyclic compounds of the general formula (a) or (b) as nitration inhibitors,

[0020]

[0021] It has the following definition:

[0022] X1 is S or O, X2 is S or O, and at least one of X1 and X2 is S;

[0023] R 2 H or C 1-4 alkyl;

[0024] R 3 H or C 1-4 alkyl;

[0025] R 6 and R 7 for hydrogen or together form a covalent carbon-carbon bond;

[0026] In the general formula (a), R 1 H, C 1-12 Alkyl or -CH2-NR 4 R 5 ,in:

[0027] R 4 is hydrogen or C 1-4 alkyl;

[0028] R 5 C 1-12 A hydrocarbon residue which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur, R 4 and R 5 They may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally contain one or two further heteroatoms selected from nitrogen, oxygen and sulfur;

[0029] In the general formula (b), R 1 H or C 1-17 Hydrocarbon, preferably H, or -CH2-R 5 , where R 5 H or C 1-16 a hydrocarbon residue which may contain one to three halogen atoms and / or one to six heteroatoms selected from nitrogen, oxygen and sulfur;

[0030] Preferably, in general formulae (a) and (b), X 1 and X 2 For S.

[0031] These objects are preferably achieved by the use of N-heterocyclic compounds of the general formula (I), (II), (III) or (IV) as nitrification inhibitors, preferably against AOB and (possibly) comammox,

[0032]

[0033] It has the following definition:

[0034] X 1 S or O, X 2 is S or O, and X 1 and X 2 At least one of them is S;

[0035] R 2 H or C 1-4 alkyl;

[0036] R 3 H or C 1-4 alkyl;

[0037] In the general formulas (I) and (III), R 1 H, C 1-12 Alkyl or -CH2-NR 4 R 5 ,in:

[0038] R 4 is hydrogen or C 1-4 alkyl;

[0039] R 5 C 1-12 A hydrocarbon residue which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur, R 4 and R 5 They may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally contain one or two further heteroatoms selected from nitrogen, oxygen and sulfur;

[0040] In the general formulas (II) and (IV), R 1 H or C 1-17 Hydrocarbon, preferably H, or -CH2-R 5 , where R 5 H or C 1-16 a hydrocarbon residue which may contain one to three halogen atoms and / or one to six heteroatoms selected from nitrogen, oxygen and sulfur;

[0041] Preferably, in the general formulae (II), (III) and (IV), X1 and X 2 For S.

[0042] In the following, reference is often made to N-heterocyclic compounds of the general formulae (I) to (IV). The compounds of the general formulae (a) and (b) described above can also be employed, and these references also apply to the general formulae (a) and (b). General formula (a) is a combination of general formulae (I) and (III). General formula (b) is a combination of general formulae (II) and (IV).

[0043] The N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) are used as nitrification inhibitors for solid or liquid fertilizers. In addition, they are used as nitrogen stabilizers in liquid fertilizers or manure. The fertilizers may be organic and / or inorganic and / or organomineral fertilizers.

[0044] This object is further achieved by the use of the N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) as additives or coating materials for fertilizers, preferably inorganic fertilizers, more preferably ammonium- and / or urea-containing nitrogen fertilizers.

[0045] This object is further achieved by the use of N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) for reducing nitrogen or carbon losses from inorganic and / or organic and / or organomineral fertilizers or nitrogen- or carbon-containing compounds or materials and from harvested litter and grazing land or during storage of liquid manure, and for reducing the ammonia load in animal stalls.

[0046] Furthermore, it is advantageous to use the N-heterocyclic nitrification inhibitors of the general formula (I) and / or (II) and / or (III) and / or (IV) in combination with further agrochemicals, which are preferably selected from:

[0047] at least one additional nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridinium chloride (2-Cyano ... ine), 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide azide), potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate carbamate) and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester (N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester), AOA inhibitors or comammox inhibitors;

[0048] - at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT);

[0049] at least one customary agrochemical adjuvant, preferably selected from aqueous and / or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, photoprotectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and / or vegetable oils and / or waxes, colorants and drift control agents;

[0050] and mixtures thereof.

[0051] When an additional nitrification inhibitor is used, the weight ratio of the N-heterocyclic compound of formula (I) and / or (II) and / or (III) and / or (IV) to the additional nitrification inhibitor is preferably 0.1 to 10:1, more preferably 0.2 to 5:1, most preferably 0.5 to 2:1.

[0052] Therefore, the nitrification inhibitors according to the present invention can be advantageously used or combined or mixed with other nitrification inhibitors, which preferably inhibit ammonia oxidizing bacteria (AOB) or archaea (AOA), such as ammonia-binding liquid inoculum (ABIL) or complete ammonia oxidation (comammox) bacteria.

[0053] Furthermore, specifically, if the inorganic fertilizer contains urea, the nitrification inhibitor may also be used together with, combined with, or mixed with a urease inhibitor, which is preferably selected from n-butylthiophosphoric triamide (NBTPT or NBPT) and / or n-propylthiophosphoric triamide (NPTPT or NPPT).

[0054] If the N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) of the present invention are combined with n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT), the weight ratio of the nitrification inhibitor to the urease inhibitor is preferably in the range of 0.1 to 10:1, more preferably in the range of 0.5 to 8:1, and most preferably in the range of 1 to 6:1.

[0055] Therefore, the present invention also relates to a mixture comprising: at least one N-heterocyclic compound of the general formula (I), (II), (III) or (IV),

[0056]

[0057] It has the following definition:

[0058] X 1 S or O, X 2 is S or O, and X 1 and X 2 At least one of them is S;

[0059] R 2 H or C 1-4 alkyl;

[0060] R 3 H or C 1-4 alkyl;

[0061] In the general formulas (I) and (III), R 1 H, C 1-12 Alkyl or -CH2-NR 4 R 5 ,in:

[0062] R 4 is hydrogen or C 1-4 alkyl;

[0063] R 5 C 1-12 A hydrocarbon residue which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur, R 4 and R 5They may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally contain one or two further heteroatoms selected from nitrogen, oxygen and sulfur;

[0064] In the general formulas (II) and (IV), R 1 H or C 1-17 Hydrocarbon, preferably H, or -CH2-R 5 , where R 5 H or C 1-16 a hydrocarbon residue which may contain one to three halogen atoms and / or one to six heteroatoms selected from nitrogen, oxygen and sulfur;

[0065] Furthermore, preferably, in the general formulae (II), (III) and (IV), X 1 and X 2 For S;

[0066] and, at least one additional agrochemical, preferably selected from:

[0067] at least one additional nitrification inhibitor, preferably selected from the group consisting of 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridinium chloride (2-Cyano ... ine), 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide azide), potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate carbamate) and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester (N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester), AOA inhibitors or comammox inhibitors;

[0068] - at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT);

[0069] at least one customary agrochemical adjuvant, preferably selected from aqueous and / or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, photoprotectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anticaking agents, mineral and / or vegetable oils and / or waxes, colorants and drift control agents;

[0070] and mixtures thereof.

[0071] Preference is given to combinations with at least one further nitrification inhibitor and / or urease inhibitor.

[0072] According to the present invention, it has been found that N-heterocyclic compounds of the general formula (I), (II), (III) and (IV) are novel potent nitrification inhibitors and target microorganisms, preferably ammonia oxidizing bacteria (AOB) and possibly also ammonia oxidizing archaea (AOA) and / or comammox bacteria, such as Candidatus Nitrospira kreftii. They can be used in combination with known nitrification inhibitors and / or urease inhibitors.

[0073] These new nitrification inhibitors are able to inhibit nitrification, particularly in soils.

[0074] Thiazolidine-thiols, particularly structural variants of these molecules in formula (I), (II), (III), and (IV), have been found to be nitrification inhibitors. These nitrification inhibitors specifically inhibit ammonia oxidation in ammonia-oxidizing bacteria, effectively reducing ammonium losses in agricultural soils. They are also copper chelators, thereby eliminating the effects of copper. Copper is essential for the activity of the AMO enzyme complex, which is essential for ammonia oxidation. Therefore, these compounds inhibit nitrification by chelating copper.

[0075] According to the invention, N-heterocyclic compounds of the general formula (a) and / or (b), preferably of the general formula (I) and / or (II) and / or (III) and / or (IV), are used as nitration inhibitors.

[0076]

[0077] It has the following definition:

[0078] X 1 S or O, X 2 is S or O, and X 1 and X 2 At least one of them is S;

[0079] R 2 H or C 1-4 alkyl;

[0080] R 3 H or C 1-4 alkyl;

[0081] In the general formulas (I) and (III), R 1 H, C 1-12 Alkyl or -CH2-NR 4 R 5 ,in:

[0082] R 4 is hydrogen or C 1-4 alkyl;

[0083] R 5 C 1-12 A hydrocarbon residue which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur, R 4 and R 5 They may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally contain one or two further heteroatoms selected from nitrogen, oxygen and sulfur;

[0084] In the general formulas (II) and (IV), R 1 H or C 1-17 Hydrocarbon, preferably H, or -CH2-R 5 , where R 5 H or C 1-16 a hydrocarbon residue which may contain one to three halogen atoms and / or one to six heteroatoms selected from nitrogen, oxygen and sulfur;

[0085] Preferably, the N-heterocyclic compound is combined with a fertilizer or manure, more preferably with a nitrogen (ammonium)-containing fertilizer, for example, a solid or liquid inorganic, organic and / or organomineral fertilizer. The heterocyclic compound is used, for example, as a nitrification inhibitor for solid fertilizers or as a nitrification inhibitor or nitrogen stabilizer in liquid organic, inorganic or organomineral fertilizers or manure.

[0086] The N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) are mostly known per se and can be synthesized according to standard techniques. Some of them are commercially available compounds and can be obtained from ENAMINE Ltd., UkrOrgSynthesis Ltd. or Vitas-M Laboratory, Ltd. or from Merck Millipore, Burlington, MA, USA or Merck KGaA.

[0087] In the general formulas (I), (II), (III) and (IV), X 1 and X 2 Preferably, it is sulfur (S). In this case, the N-heterocyclic compound is a thiazolidine-2-thiol / thione compound, or a compound that can be easily cleaved to form a thiazolidine-2-thiol compound that is tautomeric with a thiazolidine-2-thione compound. The simplest molecule of this type is X in the general formula (I) and (III). 1 and X 2 is S, and R 1 、R 2 and R 3 These compounds are shown in Examples 3 and 26 below.

[0088] If X 1 is oxygen (O) and X 2is sulfur (S), these compounds are oxazolidine-2-thiol / thione compounds or compounds that are easily cleaved to form oxazolidine-2-thiol compounds that are tautomers with oxazolidine-2-thione compounds.

[0089] When X in the general formula (I) 1 is sulfur (S) and X 2 When it is oxygen (O), the compound is a thiazolidine-2-one compound.

[0090] Preferably, in the general formulae (I), (II), (III) and (IV), X 2 is S, and X 1 is O or S; more preferably, X 1 and X 2 It’s S.

[0091] The compounds of general formula (II) are preferably thiazolidine-2-thioethers, which can be cleaved to form thiazolidine-2-thiols and tautomeric thiazolidine-2-thions.

[0092] So, for example, X 1 and X 2 The compounds of formula (I) and formula (II) where S is S - and formula (III) and (IV) in a similar manner - are technically related in that they are "capped" thiazolidine-thiol compounds or compounds containing a thiazolidine-thiol derived substructure. In both substructures, the residue R 1 can be cleaved to produce thiazolidine-thiol compounds.

[0093] The most preferred is X 1 and X 2 The compounds of formula (I), (II), (III) and (IV) are S.

[0094] These classes of compounds are effective nitrification inhibitors, preferably against AOB, and possibly also AOA and / or comammox bacteria.

[0095] In the compounds of formula (I), (II), (III) and (IV), some of the residues are hydrocarbon residues. Hydrocarbon residues consist of hydrogen and carbon atoms. They may be saturated, unsaturated or aromatic. In addition, they may contain heteroatoms as defined above. The hydrocarbon residues may be linear, branched, cyclic, or may contain at least one linear or branched residue and at least one cyclic residue in the same structure.

[0096] If more than one heteroatom is present in the structure, heteroatoms of the same type are not directly covalently bonded to each other. Preferably, heteroatoms are not directly covalently bonded to other heteroatoms, but they intersect with hydrocarbon residues, with the exception of sulfonamides and sulfonic acid groups. Thus, heteroatoms are preferably not adjacent.

[0097] Therefore, in R 5 In the embodiment, heteroatoms of the same chemical element are not adjacent. 5 In the present invention, the heteroatoms are not directly connected to each other.

[0098] In the general formulas (I) and (III), R 5 May contain at least one cyclic structure. 5 It comprises a 5- or 6-membered ring structure, which may be fused to a second 5- or 6-membered ring structure. In summary, the compounds of formula (I) and (III) contain additional ring structures as shown in formula (I) and (III).

[0099] The compounds of general formula (I) and (III) preferably contain two, three or four ring structures, which may be further condensed or fused, or may not be condensed or fused. These ring structures are preferably 5- or 6-membered rings. The ring structure shown in general formula (I) and (III) is one of the ring structures that form the compound of general formula (I).

[0100] In the general formulas (II) and (IV), R 5 It may contain at least one ring structure, preferably no ring structure, one or two ring structures, and most preferably one or two ring structures. 5 There are two ring structures in R, which may be condensed or fused, or not condensed or fused. These ring structures are preferably 5- or 6-membered rings. Therefore, preferably, the compounds of formula (II) and (IV) have R 5 The cyclic ring structure comprises a 5- or 6-membered ring structure, which may be fused to a second 5- or 6-membered ring structure.

[0101] The compounds of the general formulae (II) and (IV) preferably contain two or three ring structures, one of which is a ring structure represented by the general formulae (II) and (IV).

[0102] R of compounds (I), (II), (III) and (IV) 5 The ring structures in the may be carbocyclic or heterocyclic. In a condensed or fused structure, one or more ring structures may be heterocyclic.

[0103] A condensed or fused ring structure is one in which two ring structures share two chemical elements, preferably two carbon atoms, in their respective ring structures.

[0104] The ring structure shown in general formula (I), (II), (III) or (IV) cannot condense with another ring structure. Therefore, only when two ring structures are present in addition to the ring structure shown in general formula (I), (II), (III) or (IV) can these two additional ring structures be condensed. These additional ring structures can be saturated, unsaturated or aromatic. If two condensed ring structures are present, one ring structure can be aromatic and the other ring structure can be non-aromatic.

[0105] If the two additional ring structures are not condensed, they can be directly covalently linked to each other. Alternatively, they can be linked via a spacer that can contain carbon atoms, heteroatoms, or both. For example, the spacer can be C 1-6 An alkylene group, or a heteroatom selected from oxygen, sulfur and nitrogen, or the spacer may be, for example, an amido group -C(=O)-NH-. If the cyclic group is attached via a nitrogen atom, the nitrogen is preferably -NH- or -NR-, wherein R is C 1-4 Alkyl, more preferably R is methyl or ethyl.

[0106] Ring structures may also be described as structural elements that form part of the compounds of formula (I), (II), (III) and (IV).

[0107] Preferably, R 2 is hydrogen, methyl or ethyl. Preferably, R 3 is hydrogen, methyl or ethyl. Preferably, R 4 is hydrogen, methyl or ethyl.

[0108] Most preferably, R 2 and R 3 For hydrogen.

[0109] Most preferably, R 4 It is methyl or ethyl.

[0110] Preferred are compounds of the general formula (I), wherein, in the general formula (I), R5 It is C 3-10 A hydrocarbon residue which may contain one to two halogen atoms and / or one to three heteroatoms and contains at least one ring structure.

[0111] More preferred are compounds of formula (I), wherein, in formula (I), R 5 It is C 3-8 A hydrocarbon residue which may contain one to two halogen atoms and / or one to three heteroatoms and which contains a 5- or 6-membered ring structure which may be fused to a second 5- or 6-membered ring structure.

[0112] Preferred are compounds of the general formula (II), wherein, in the general formula (II), R 1 It is C 1-4 Alkyl or -CH2-R 5 , where R 5 It is C 3-14 A hydrocarbon residue which may contain one or two halogen atoms and / or two to six heteroatoms selected from nitrogen, oxygen and sulfur.

[0113] More preferred are compounds of formula (II), wherein, in formula (II), R 1 is methyl, ethyl or -CH2-R 5 , where R 5 It is C 5-12 A hydrocarbon residue which may contain one halogen atom and / or two to four heteroatoms selected from nitrogen, oxygen and sulfur. The halogen atom is preferably selected from F, Cl and Br, more preferably from F and Cl. 5 Halogen atoms may be included in combination with heteroatoms or as alternatives to heteroatoms.

[0114] In another embodiment, R in formula (I) 4 and R 5 Together with the nitrogen atom to which they are attached, they form a 5- or preferably a 6-membered saturated or unsaturated heterocyclic group. 4 and R 5 Together with the nitrogen atom to which they are attached, they form a 5- or preferably 6-membered saturated or unsaturated heterocyclic radical, which optionally may also contain one further heteroatom selected from nitrogen and oxygen, and which may be a C 4-16 The hydrocarbon residue is part of the 4-16 The hydrocarbon residue itself is connected to R 4 and R 5 In addition to the nitrogen atoms, the carbonyl group may contain (in total) 1 to 4 heteroatoms selected from nitrogen, oxygen, sulfur and fluorine.

[0115] More preferably, R 4 and R 5 Together with the nitrogen atoms that connect them, they can form saturated or unsaturated structural elements

[0116]

[0117] It can be C 4-15 The hydrocarbon residue is part of the 4-15 In addition to the hydrocarbon residues connected to R 4 and R 5 In addition to the nitrogen atoms, the alkylene group may contain from 1 to 4 heteroatoms selected from nitrogen, oxygen, sulfur and fluorine. Thus, the structure shown may or may not contain C=C or C=N bonds.

[0118] They are preferably C 4-13 hydrocarbon residues, more preferably C 4-10 Hydrocarbon residues, most preferably C 7-9 The hydrocarbon residue is a part of the hydrocarbon residue. 4 and R 5 In addition to the nitrogen atom, the carbonyl group may further contain 1 to 3 heteroatoms selected from nitrogen, oxygen, sulfur and fluorine, and more preferably may contain 1 or 2 heteroatoms selected from nitrogen, oxygen, sulfur and fluorine.

[0119] Saturated structural elements as shown above are preferred.

[0120] According to one embodiment of the present invention, the above structural element or ring structure may contain one or two additional bonds to carbon atoms on the structural element or ring structure. Therefore, the above structural element or ring structure may contain one or two substituents connected to the structural element or ring structure through carbon atoms. These one or two substituents together with the structural element or ring structure form a C 4-16 Hydrocarbon residue, preferably C 4-13 hydrocarbon residue, more preferably C 4-10 Hydrocarbon residues, especially C 7-9 Hydrocarbon residues.

[0121] Preferably, the structural elements shown above may include one or two 4 R 5 More preferably, in addition to the bond to the NR 4 R 5 The structural element contains no other substituents besides one or two bonds to other carbon atoms in the meta or para position to the nitrogen of the radical.

[0122] Preferably, R 4 and R 5 Both are connected to NR through carbon atoms 4 R 5 The N in .

[0123] In the examples, specific residues R are disclosed for general formula (I) and (II). 1 、R 2、R 3 、X 1 / X 2 、R 4 and R 5 Each of these residues can be used to define the compounds of formula (I) and (II) independently of the other residues. Thus, the compounds of formula (I) and (II) may contain the residue R 1 、R 2 、R 3 、R 4 、R 5 、X 1 and X 2 One or more of, as disclosed in each embodiment.

[0124] In the compounds of formula (III) and (IV), preferably R1 is hydrogen, so that the compounds of formula (III) and (IV) are tautomers of a single compound. Preferably, in the compounds of formula (III) and (IV), R 2 and R 3 is hydrogen, methyl or ethyl, more preferably hydrogen or methyl, especially hydrogen. Therefore, in the most preferred embodiment of the compounds of formula (III) and (IV), X 1 and X 2 is S, and R 1 、R 2 and R 3 For hydrogen.

[0125] The alkyl residue may be linear or branched.

[0126] The cyclic group in the residue is preferably a 5- or 6-membered ring, which can be saturated, unsaturated or aromatic. These rings can be pure hydrocarbons, for example, phenyl. They can also contain nitrogen, oxygen and / or sulphur atoms as heteroatoms, preferably nitrogen or sulphur atoms. Examples of these residues are shown in the following embodiments.

[0127] The compounds of the general formula (I), (II), (III) and (IV) are nitrification inhibitors which inhibit ammonia oxidizing bacteria (AOB) and possibly also ammonia oxidizing archaea (AOA), in particular Nitrosomonas europaea and / or Nitrosospira multiformis.

[0128] The nitrification inhibitor of the present invention is particularly noteworthy for its long-term effectiveness in inhibiting the nitrification of ammonium nitrogen in the soil.

[0129] Compounds that can form thiol tautomers are highly active and are preferred. For example, 3H-1,3-thiazole-2-thione tautomers conform to the general formulas (III) and (IV), respectively, where R 1 to R 3 is hydrogen, and X 1 and X 2 Sulfur. This includes compounds with a labile aminomethyl linker or H at the N of the 5-ring. Occupancy of the N in the 5-ring prevents possible thiol formation and also reduces nitrification inhibition. Similarly, the absence of a thiol group reduces activity. Side groups on the carbon atoms of the thiazole 5-ring have little or no effect on activity. In addition, the most important substructure, 1,3-thiazol-2-thione or 2-thiazoline-2-thiol derived from it, also exhibits strong nitrification inhibition in soil.

[0130] The relative amount (equilibrium concentration) of each tautomer can be altered by, for example, adjusting the pH of a solution of the compound to the desired value.

[0131] Furthermore, molecules containing a thiazolidine-thiol derived substructure were found to possess excellent nitration inhibition activity. These conform to the general formula (II) and are preferably thiazolidine-2-thioethers.

[0132] Nitrification inhibitors are described as those of formula (I), (II), (III), and (IV). One or more compounds of formula (I), (II), (III), or (IV) can be used as nitrification inhibitors and in fertilizers (discussed below). In addition, mixtures of one or more compounds of formula (I) with one or more compounds of formula (II) can be used, as well as mixtures of one or more compounds of at least two of formulas (I) to (IV). This is reflected by expressions (I) and / or (IV) and / or (III) and / or (IV).

[0133] According to one embodiment of the present invention, compounds No. 1-12 shown in the table on page 71 of WO 2020 / 020765 are excluded from the N-heterocyclic compounds to be used. In addition, compounds No. 1-11 disclosed in WO 2020 / 020777 are preferably excluded from the N-heterocyclic compounds.

[0134] Preferably, in the general formula (I), X 1 and X 2 is S and R 1 The compounds are excluding compounds that are H or CH3 or C(=O) hydrocarbon groups. In this case, hydrocarbon groups are C 1-20 A hydrocarbon residue which may contain 1 to 3 halogen atoms and / or 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur.

[0135] Preferably, in the compound of formula (I), R 1 Not hydrogen, C 1-6 Alkyl and preferably not hydrogen or C 1-12 Therefore, in the general formula (I), R 1 Preferably -CH2-NH4R 5 .

[0136] In the general formula (II), R 1 Preferably, it is not hydrogen, methyl, ethyl, propagyl, C(=O)NH(phenyl), CH2C(=O)(phenyl)H + Br - , 2-methyl-4-chloro-phenyl, C(=O)NH(CH2CH3).

[0137] CH2C(=O)OCH3 or C(=S)N(CH3)2.

[0138] Preferably, R in the general formula (I) 1 Not the one in WO 2020 / 020765 for R N Any of the disclosed residues, and R in the general formula (II) 1 Not the one in WO 2020 / 020777 for R S Any of the disclosed residues.

[0139] The above waiver of rights relates to X 1 and X 2 A sulfur compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 The copper chelating ability of the nitrification inhibitor of the present invention is shown by analyzing the absorption spectra with and without copper. DETAILED DESCRIPTION

[0141] According to one embodiment of the present invention, in contrast to CN 103524159 A, the fertilizer mixture according to the invention does not contain oxazolidinthione.

[0142] The nitrification inhibitors of the present invention are expected to have favorable toxicological properties, low vapor pressure, and good soil adsorption. Therefore, the nitrification inhibitors of the present invention are neither emitted to the atmosphere by sublimation to a significant extent nor readily leached by water. This results in, firstly, economic advantages, such as high profitability due to the long-term, sustained effects of the nitrification inhibitors, and environmental advantages, such as reduced air pollution (reduction of climate gases) and the burden on surface and groundwater.

[0143] Nitrification inhibitors can be applied to soil or substrates fertilized with inorganic, organic, or organo-mineral fertilizers. Typically, they are used in fertilizer mixtures comprising (preferably inorganic) fertilizers and N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV). Typically, the amount of N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) used is 10 to 10,000 ppm by weight, more preferably 100 to 10,000 ppm by weight, based on the (preferably inorganic) fertilizer without water. The amount applied is based on dry fertilizer.

[0144] The nitrification inhibitors of the present invention can be used in the form of a substance, solution, dispersion or emulsion. Therefore, the present invention also relates to solutions, dispersions or emulsions containing preferably 0.1 to 50% by weight, more preferably 0.5 to 30% by weight, and most preferably 1 to 20% by weight of the N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) of the present invention.

[0145] Preferably, according to the invention, inorganic fertilizers are used to form a fertilizer mixture containing compounds A and B:

[0146] A: Inorganic and / or organic and / or organo-mineral fertilizers; and

[0147] B: 10 to 10,000 ppm by weight, more preferably 100 to 10,000 ppm by weight, of an N-heterocyclic compound of the general formula (I) and / or (II) and / or (III) and / or (IV) as defined above, based on the fertilizer, preferably the inorganic fertilizer.

[0148] The water content of the mixture of compound A and fertilizer is generally not more than 1.5% by weight, preferably not more than 1.0% by weight, more preferably not more than 0.5% by weight, and most preferably not more than 0.3% by weight, and is therefore negligible in terms of the quantitative balance. Compounds A and B preferably constitute at least 95% by weight, more preferably at least 98% by weight, of the fertilizer mixture.

[0149] The nitrogen content of component A (excluding water) is generally at least 12% by weight, preferably at least 20% by weight, and more preferably at least 22% by weight. For example, the nitrogen content may be 25% to 29% by weight, in particular 26% to 28% by weight. The nitrogen content can be divided into fast-acting nitrate nitrogen and slow-acting ammonium nitrogen.

[0150] The inorganic fertilizer is preferably an ammonium-containing fertilizer and / or a urea-containing fertilizer, and more preferably an ammonium-containing fertilizer further containing urea.

[0151] Urea-containing fertilizers are further described in WO 2016 / 207210.

[0152] The fertilizer used according to the present invention can be of natural or synthetic origin and is applied to soil or plant tissue to provide one or more plant nutrients necessary for plant growth. The fertilizer used according to the present invention should at least provide nitrogen as a nutrient. Other nutrients are, for example, K and P. Multinutrient fertilizers or complex fertilizers provide two or more nutrients. Inorganic fertilizers do not include carbonaceous materials other than urea. Organic fertilizers are typically materials of plant or animal origin. Organomineral fertilizers (combinations of inorganic and organic fertilizers) can also be used.

[0153] The main nitrogen-based straight fertilizer is ammonia or its solution. Ammonium nitrate is also widely used. Urea is another popular nitrogen source, having the advantage of being solid and explosion-proof. Another nitrogen-based fertilizer is calcium ammonium nitrate.

[0154] The main straight phosphate fertilizers are superphosphates, including single superphosphate, phosphogypsum, and triple superphosphate. The main potassium-based straight fertilizer is muriate of potash (MOP).

[0155] The binary fertilizer is preferably NP or NK fertilizer, which may be monoammonium phosphate (MAP) and diammonium phosphate (DAP).

[0156] NPK fertilizer is a three-component fertilizer that provides nitrogen, phosphorus and potassium. NPK fertilizer can be produced by mixing the above single fertilizers in bulk or in each granule, such as In some cases, a chemical reaction occurs between two or more components.

[0157] In addition to the main components such as N, P and K, fertilizers can also contain micronutrients (trace elements). The main micronutrients are molybdenum, zinc, boron and copper. These elements are usually provided in the form of water-soluble salts.

[0158] Preferred fertilizers contain ammonium or urea. Examples of preferred ammonium-containing fertilizers are NPK fertilizers, calcium ammonium nitrate, ammonium sulfate nitrate, ammonium sulfate, and ammonium phosphate.

[0159] Further preferred ingredients of the fertilizer compositions are, for example, trace elements, other minerals, standardizers, binders.

[0160] Organic fertilizers can describe those of organic or biogenic origin, that is, fertilizers derived from living or formerly living organisms, such as animals, plants, or algae. Fertilizers of organic origin include animal waste, plant waste (e.g., from food processing or agriculture), compost, and treated sewage sludge (biosolids). Animal sources can be manures or products from slaughtered animals, such as blood meal, bone meal, feather meal, hides, hooves, and horns.

[0161] Soil amendments such as peat or coir, bark and sawdust may also be included.

[0162] Fertilizers may include, but are not limited to, ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, hampene (chelated iron), dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, potassium nitrate, potassium bicarbonate, monopotassium phosphate, magnesium nitrate, magnesium sulfate, potassium sulfate, potassium chloride, sodium nitrate, magnesian limestone, limestone), magnesium oxide, disodium dihydromolybdate, cobalt chloride hexahydrate, nickel chloride hexahydrate, indole butyric acid, L-tryptophan, urea, ureaformaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, iso-butylidene diurea, K2SO4-2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cake, fish fecesmanure, blood meal, rock phosphate, superphosphates, slag, bone meal, wood ash, biochar, algae, algae extract, struvite, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof. The micronutrient fertilizer material may include boric acid, a borate salt, boron frit, copper sulfate, copper frit, copper chelate, sodium tetraborate decahydrate, iron sulfate, iron oxide, iron ammonium sulfate, iron frit, iron chelate, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdic acid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate, or a combination thereof. In a specific embodiment, the fertilizer or fertilizer composition does not contain insoluble selenium, selenium minerals, soluble selenium, or salts thereof.

[0163] The treated (inorganic, organic or organo-mineral) fertilizer according to the invention is preferably in powder form, prill form or granule form.

[0164] In addition to the N-heterocyclic compounds of formula (I) and / or (II), formulations containing these compounds and agricultural adjuvants can also be used to include nitrification inhibitors in fertilizers. Agricultural adjuvants are, for example, solvents, dispersants, pH regulators, fillers, stability improvers, and surfactants.

[0165] The nitrification inhibitor may be included in a fertilizer mixture by mixing the nitrification inhibitor or a formulation containing it with a solid or liquid fertilizer or fertilizer formulation. Preferably, the fertilizer mixture is in solid form and the nitrification inhibitor is applied to the surface of the (inorganic, organic or organo-mineral) fertilizer.

[0166] During the production of the fertilizer mixtures according to the invention, the nitrification inhibitor or a preparation containing it can be introduced into the (inorganic, organic or organo-mineral) fertilizer and / or applied to the surface of inorganic fertilizers.

[0167] Fertilizer granules are impregnated or coated with a nitrification inhibitor, for example, by spraying a preparation (e.g., a solution or dispersion) of the nitrification inhibitor and subsequently drying. This process is known, for example, from DE-A-4128828. The impregnated granules can be sealed, for example, with paraffin wax (another proposal in the latter document), but this is generally unnecessary.

[0168] Granulating assistants that can be used in the preparation of solid fertilizer compositions may be lime, gypsum, silica or kaolinite.

[0169] An alternative is to add the nitrification inhibitor during the actual production of the fertilizer, for example to the slurry.

[0170] Generally speaking, nitrification inhibitors are usually applied to the soil in an amount of 100 g / ha to 10 kg / ha, preferably in a range of 300 g / ha to 5 kg / ha.

[0171] Delivery of nitrification inhibitors in liquid fertilizer formulations can be achieved, for example, by fertigation with or without excess water as described in DE-C-10230593.

[0172] The above fertilizer mixture may contain at least one additional nitrification inhibitor. Preferably, the at least one additional nitrification inhibitor inhibits ammonia oxidizing bacteria (AOB) and is preferably selected from the group consisting of: 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid, 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazolephosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine ), 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazol, 2-amino-4-chloro-6-methyl-pyrimidine, 2-mercapto-benzothiazole, 2-sulfanilamidothiazole, thiourea, sodium azide azide), potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbonbisulfide, ammonium thiosulfate, sodium tri-thiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate carbamate) and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester (N-(2,Nitrification inhibitors that inhibit ammonia-oxidizing archaea (AOA) can also be used together with the N-heterocyclic compounds of general formula (I).

[0173] When an additional nitrification inhibitor is used, the weight ratio of the N-heterocyclic compound of formula (I) and / or (II) and / or (III) and / or (IV) to the additional nitrification inhibitor is preferably 0.1 to 10:1, more preferably 0.2 to 5:1, most preferably 0.5 to 2:1.

[0174] Furthermore, the fertilizer mixture may contain at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT). Urease inhibitors are often added when the fertilizer contains urea. Urea nitrogen is released as ammonium by the action of urease, which can undergo nitrification. Therefore, combining a urease inhibitor with a nitrification inhibitor is advantageous.

[0175] If the N-heterocyclic compounds of the general formula (I) and / or (II) and / or (III) and / or (IV) according to the present invention are combined with n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT), the weight ratio of nitrification inhibitor to urease inhibitor is preferably 0.1 to 10:1, more preferably 0.5 to 8:1, most preferably 1 to 6:1.

[0176] It is known that thiophosphoric triamides are relatively easy to convert into the corresponding phosphoric triamides and thiophosphoric diamides and other metabolites. Since water cannot generally be completely eliminated, thiophosphoric triamides and the corresponding phosphoric triamides often exist in the form of mixtures with each other. Therefore, in this specification, the term "(thio)phosphoric triamide" refers not only to pure thiophosphoric triamides and phosphoric triamides, respectively, but also to mixtures thereof.

[0177] According to the invention, it is also possible to use mixtures of n-butylthiophosphoric triamide and n-propylthiophosphoric triamide, as described in EP-A-1 820 788.

[0178] The above fertilizer mixtures may contain other ingredients, such as coatings, for example inorganic or organic polyacids, which are described in US 6,139,596.

[0179] Furthermore, the coatings of powders, prills and granules can be formed from inorganic materials, for example, sulphur-based or mineral-based coatings, or from organic polymers. WO 2013 / 121384, page 23, line 37 to page 24, line 16, describes corresponding coatings.

[0180] As mentioned above, the agrochemical formulations comprising the compounds of the general formula (I) and / or (II) are used in an "effective amount". This means that they are used in an amount which allows to obtain the desired effect, which is a (synergistic) increase in plant health without causing any phytotoxic symptoms in the treated plants.

[0181] For use according to the invention, the agrochemical formulations comprising the compounds of formula (I) and / or (II) and / or (III) and / or (IV) can be converted into conventional formulations, for example, solutions, emulsions, suspensions, dusts, powders, pastes and granules. The form of use depends on the specific intended purpose; in each case, a fine and uniform distribution of the agrochemical formulation comprising the compounds of formula (I) and / or (II) and / or (III) and / or (IV) according to the invention should be ensured. The formulations are prepared in a manner known to those skilled in the art.

[0182] The above-mentioned agricultural chemical preparations may also be included in adjuvants commonly used in agricultural chemical preparations. The adjuvants used depend on specific application forms and active substances respectively. Examples of suitable adjuvants include solvents, solid carriers, dispersants or emulsifiers (for example, other solubilizers, protective colloids, surfactants and adhesives), organic and inorganic thickeners, bactericides, antifreezes, defoamers, (if appropriate) colorants and tackifiers or binders (for example, for seed treatment formulations).

[0183] Suitable solvents are: water; organic solvents, for example, medium- to high-boiling mineral oil fractions, such as kerosene or diesel oil, and also coal tar oils, and oils of vegetable or animal origin; aliphatic, cyclic and aromatic hydrocarbons, for example, toluene, xylene, paraffin, tetrahydronaphthalene, alkylated naphthalenes or derivatives thereof; alcohols, for example, methanol, ethanol, propanol, butanol and cyclohexanol, glycols; ketones, for example, cyclohexanone and gamma-butyrolactone; fatty acid dimethylamides; fatty acids and fatty acid esters and highly polar solvents, for example, amines, such as N-methylpyrrolidone.

[0184] Solid carriers are: mineral earths, for example, silicates, silica gel, talc, kaolin, limestone, lime, chalk, bole, loess, clays, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide; ground synthetic materials; fertilizers, for example, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea; and products of plant origin, for example, cereal meal, tree bark meal, wood meal and nutshell meal, cellulose powders; and other solid carriers.

[0185] Suitable surfactants (auxiliaries, wetting agents, tackifiers, dispersants or emulsifiers) are alkali metal, alkaline earth metal and ammonium salts of aromatic sulfonic acids, such as ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid, dibutylnaphthalenesulfonic acid, and fatty acids; alkylsulfonates; alkyl-arylsulfonates; alkylsulfates; lauryl ether sulfate; fatty alcohol sulfates and sulfated hexa-, hepta- and octadecanolates; sulfated fatty alcohol glycol ethers;Condensate of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde, polyoxy-ethyleneoctylphenyl ether, ethoxylated isooctylphenol, octylphenol, nonylphenol, alkylphenyl polyglycolethers, tributephenyl polyglycol ether, tristearyl-phenyl polyglycol ether, alkylarylpolyether alcohols, alcohol and fatty alcohol / ethylene oxide condensate, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycolether acetal, sorbitol esters esters), lignin-sulfite waste liquid and proteins, denatured proteins, polysaccharides (e.g., methylcellulose), hydrophobically modified starches, polyvinyl alcohols, polycarboxylates, polyalkoxylates, polyvinylamines, polyvinylpyrrolidone, and copolymers thereof. Examples of thickeners (i.e., compounds that impart improved flow properties to a formulation (i.e., high viscosity under static conditions and low viscosity during stirring)) are polysaccharides, and organic and inorganic clays, e.g., xanthan gum.

[0186] "Pesticide" means a substance used to prevent, destroy or control harmful organisms ("pests") or diseases, or to protect plants or plant products during production, storage and transportation.

[0187] The term includes, inter alia, herbicides, fungicides, insecticides, acaricides, nematicides, molluscicides, rodenticides, growth regulators, repellents, rodenticides and biocides, as well as plant protection products.

[0188] Plant protection agents are pesticides used to protect crops or desirable or target plants. They are primarily used in the agricultural sector, but are also used in forestry, horticulture, landscaping, and home gardens. They contain at least one active substance and have one of the following effects:

[0189] - Protecting plants or plant products from pests / diseases before and after harvest;

[0190] - substances that affect plant life processes (e.g. substances that affect plant growth, excluding nutrients);

[0191] - Preservation of plant products;

[0192] -Destroy or prevent the growth of unwanted plants or plant parts.

[0193] They may also contain other ingredients including safeners and synergists.

[0194] An active substance is any chemical, plant extract, pheromone or microorganism (including viruses) that has an effect on a "pest" or on a plant, plant part or plant product.

[0195] The most common form of pesticides used is plant protection products (PPPs).

[0196] The term "pesticide" is often used interchangeably with "plant protection agent", however, pesticide is a broader term that also covers non-plant / crop uses, for example, biocides.

[0197] Biocides may be added, such as herbicides, bactericides, molluscicides, algicides, phytotoxicants, fungicides, and mixtures thereof.

[0198] Bactericides may be added to preserve and stabilize the formulation. Examples of suitable bactericides are based on dichlorophene and benzylalcohol hemiformal (ICI's or Thor Chemie's RS and Rohm & Haas MK) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones ( M BS from Raytheon Chemical). Examples of suitable antifreeze agents are ethylene glycol, propylene glycol, urea and glycerol. Examples of defoaming agents are emulsified silicone oils (e.g. SRE, Wacker, Germany or Rhodia, France), long-chain alcohols, fatty acids, fatty acid salts, fluoroorganic compounds and agrochemical preparations containing compounds of general formula (I) or (II) thereof.

[0199] Suitable colorants are low water-soluble pigments and solvent-soluble (eg, water-soluble) dyes.

[0200] Examples of adhesion promoters (e.g., tackifiers or adhesives) include polyvinylpyrrolidons, polyvinylacetates, polyvinylalcohols, and cellulose ethers. Shin-Etsu, Japan).

[0201] Granules, for example, coated granules, impregnated granules and homogeneous granules, can be prepared by binding the active substance to a solid carrier. Examples of solid carriers are mineral earths, for example, silica gel, silicates, talc, kaolin, attaclay, limestone, lime, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials; fertilizers, for example, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and products of plant origin, for example, corn flour, bark flour, wood flour and nut shell flour, cellulose powder; and other solid carriers.

[0202] Anticaking agents such as oils and / or waxes may be added.

[0203] The above-mentioned agrochemical formulations usually contain (by weight) 0.01% to 95%, preferably 0.1% to 90%, most preferably 0.5% to 90% of active substance. The compounds of the agrochemical formulations containing compounds of general formula (I) are used with a purity of 90% to 100%, preferably 95% to 100% (according to their NMR spectra).

[0204] The compounds of the agrochemical preparations comprising the compounds of the general formula (I) or (II) or (III) or (IV) can be used as such or in the form of their agrochemical compositions, for example in the form of directly sprayable solutions, powders, suspensions, dispersions, emulsions, oil dispersions, pastes, dustable products, spreading materials or granules, which can be applied by spraying, atomizing, dusting, spreading, brushing, dipping or pouring. The form of application depends entirely on the intended purpose; it is intended to ensure the best possible distribution of the compound present in each case in the agrochemical preparation comprising the compound of the general formula (I) or (II) or (III) or (IV).

[0205] Aqueous application forms can be prepared from emulsion concentrates, pastes or wettable powders (sprayable powders, oil dispersions) by adding water. To prepare emulsions, pastes or oil dispersions, the substances, as such or dissolved in an oil or solvent, can be homogenized in water with the aid of a wetter, tackifier, dispersant or emulsifier. Alternatively, concentrates composed of active substance, wetter, tackifier, dispersant or emulsifier and, if appropriate, solvent or oil can be prepared, and such concentrates are suitable for dilution with water.

[0206] The concentration of the active substance in the ready-to-use formulations can vary within a relatively wide range. Typically, they represent 0.0001% to 10%, preferably 0.001% to 1%, by weight of the compound of the agrochemical formulation comprising the compound of formula (I) or (II) or (III) or (IV).

[0207] Agrochemical formulations comprising compounds of the general formula (I) can also be successfully used in the ultra-low-volume process (ULV), making it possible to apply compositions comprising more than 95% by weight of active substance, or even to apply the active substance without additives.

[0208] Various types of oils, wetters, adjuvants, herbicides, fungicides, other pesticides, or bactericides may be added to the active compounds, if appropriate just before use (tank mix). These agents may be mixed with the compounds of the agrochemical formulations comprising the compounds of the general formula (I) or (II) or (III) or (IV) in a weight ratio of 1:100 to 100:1, preferably 1:10 to 10:1.

[0209] The compositions of the present invention may also contain fertilizers (e.g., ammonium nitrate, urea, potassium carbonate (potash), and superphosphate (superphosphate)), phytotoxicants, plant growth regulators (plant growth improvers), and safeners. These may be used sequentially or in combination with the above-described compositions, or, if appropriate, added immediately prior to use (tank mix). For example, plants may be sprayed with the compositions of the present invention before or after treatment with fertilizers.

[0210] In agrochemical formulations comprising compounds of formula (I) or (II) or (III) or (IV), the weight ratio of the compounds generally depends on the nature of the compounds of the agrochemical formulation comprising compounds of formula (I) or (II) or (III) or (IV).

[0211] The compounds of the agrochemical formulations comprising the compounds of formula (I) or (II) or (III) or (IV) can be used individually or partially or completely mixed with one another to prepare the compositions of the present invention. They can also be packaged and further used as a combination composition, for example, a kit of parts.

[0212] The user typically applies the composition of the present invention from a pre-dosage device, a knapsack sprayer, a spray tank, or a spray plane. The agrochemical composition is prepared with water and / or a buffer to the desired application concentration, and, if appropriate, further adjuvants are added to obtain a ready-to-use spray liquid or the agrochemical composition of the present invention. Typically, 50 to 500 liters, preferably 50 to 400 liters, of this ready-to-use spray liquid are applied per hectare of agricultural area.

[0213] In a particular embodiment, the absolute amount of the active compound represented by the general formula (I) or (II) or (III) or (IV) is 1 mg / L to 100 mg / L, in particular 1 mg / L to 20 mg / L, in particular 1 mg / L to 25 mg / L, in particular 2 mg / L to 200 mg / L, in particular 2 mg / L to 100 mg / L, in particular 2 mg / L to 50 mg / L, in particular 2 mg / L to 25 mg / L, in particular 4 mg / L to 40 mg / L, in particular 4 mg / L to 20 mg / L, in particular 4 mg / L to 16 mg / L, in particular 4 mg / L to 12 mg / L.

[0214] According to one embodiment, the individual compounds of the agrochemical preparations comprising the compounds of the general formula (I) or (II) or (III) or (IV) formulated as compositions (or preparations) (e.g., parts of a kit of parts or parts of a mixture according to the invention) can be mixed by the user himself in a spray tank, if appropriate with the addition of further adjuvants (tank mix).

[0215] As used herein, "agrochemical" refers to any active substance that can be used in the agrochemical industry (which includes agricultural, horticultural, floriculture, and home and garden uses, but also includes products for non-crop related uses, such as use by public health / pest control professionals to control unwanted insects and rodents; household uses, such as household fungicides and insecticides, and agents for protecting plants or plant parts, crops, bulbs, tubers, fruits (e.g., from harmful organisms, diseases or pests), for controlling (preferably promoting or increasing) the growth of plants, and / or for promoting the yield of plants, crops or harvested plant parts (e.g., their fruits, flowers, seeds, etc.)).

[0216] As used herein, an "agrochemical composition" refers to a composition for agrochemical use as defined herein, comprising at least one active substance of a compound of formula (I), optionally with one or more additives that facilitate optimal dispersion, atomization, deposition, leaf wetting, distribution, retention and / or absorption of the agrochemical. As non-limiting examples, such additives are diluents, solvents, adjuvants, surfactants, wetting agents, spreading agents, oils, stickers, viscosity regulators (e.g., thickeners, penetrants), pH regulators (e.g., buffers, acidifiers), anti-settling agents, anti-freeze agents, photoprotectors, defoamers, biocides and / or drift control agents.

[0217] As used herein, "carrier" refers to any solid, semisolid, or liquid vehicle into or upon which an active substance can be suitably incorporated, contained, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached, or contained. Non-limiting examples of such vehicles include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, gels, weak ionic resin particles, liposomes, cochleate delivery vehicles, small granules, granulates, nano-tubes, bucky-balls, water droplets as part of a water-in-oil emulsion, oil droplets as part of an oil-in-water emulsion, organic materials (e.g., cork, wood, or other plant-derived materials (e.g., seed hulls, wood chips), chips, pulp, spheres, beads, sheets or any other suitable form), paper or cardboard, inorganic materials (such as talc, clay, microcrystalline cellulose, silica, alumina, silicates and zeolites), or even microbial cells (such as yeast cells) or suitable parts or fragments thereof.

[0218] As used herein, the terms "effective amount," "effective dose," and "effective amount" refer to the amount required to achieve the desired result or results. Further exemplary information regarding dosages, modes of application, and suitable ratios is provided below. Those skilled in the art will appreciate that such amounts can vary widely and depend on various factors, such as the cultivated plant being treated and climatic and soil conditions.

[0219]

[0046] The terms "determine," "measure," "assess," "monitor," and "measure" are used interchangeably herein and include both quantitative and qualitative measurements.

[0220] It should be understood that the above-mentioned agrochemical compositions are stable during storage and during use, which means that the integrity of the agrochemical composition is maintained under the conditions of storage and / or use of the agrochemical composition, which may include elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, ultraviolet radiation, the presence of harmful chemicals, etc. More preferably, the compounds of formula (I), (II), (III), (IV) as described herein remain stable in the agrochemical composition, which means that the integrity and activity of these compounds are maintained under the conditions of storage and / or use of the agrochemical composition, which may include elevated temperature, freeze-thaw cycles, changes in pH or ionic strength, ultraviolet radiation, the presence of harmful chemicals, etc. Most preferably, the compounds of formula (I), (II), (III), (IV) remain stable in the agrochemical composition during storage of the agrochemical composition for two years at ambient temperature or during storage of the agrochemical composition for two weeks at 54°C. Preferably, the agrochemical compositions of the present invention retain at least about 70% activity, more preferably at least about 70% to 80% activity, and most preferably about 80% to 90% activity or more. Examples of suitable carriers include, but are not limited to, alginates, gums, starches, p-cyclodextrins, cellulose, polyurea, polyurethane, polyesters, or clays.

[0221] The above-mentioned agrochemical compositions may be in the form of any type of formulation, preferably dusts, wettable powders, wettable granules, water dispersible granules, emulsions, emulsifiable concentrates, dusts, suspensions, suspension concentrates, suspoemulsions, capsule suspensions, aqueous dispersions, oil dispersions, aerosols, pastes, foams, slurries or flowable concentrates.

[0222] In yet another embodiment, the present invention provides use of the agrochemical composition of the present invention for enhancing abiotic stress tolerance in plants.

[0223] The agrochemical composition of the present invention can be applied to crops once or twice or more with an interval between each application. The agrochemical composition of the present invention can be applied to crops alone or mixed with other substances (preferably other agrochemical compositions) to crops; alternatively, the agrochemical composition of the present invention and other substances (preferably other agrochemical compositions) can be applied to the same crop separately at different times.

[0224] In another embodiment, the present invention provides a method for manufacturing ("producing" is an equivalent expression) an agrochemical composition of the present invention, which comprises forming a molecule of general formula (I) or (II) or (III) or (IV) as defined above, together with at least one commonly used agrochemical adjuvant. Suitable manufacturing methods are known in the art and include, but are not limited to, high or low shear mixing, wet or dry milling, drip-casting, encapsulating, emulsifying, coating, encrusting, pilling, extrusion granulation, fluidized bed granulation, co-extrusion, spray drying, spray chilling, atomization, addition polymerization or condensation polymerization, interfacial polymerization, in situ polymerization, coacervation, spray encapsulation, cooling melted dispersions, solvent evaporation, phase separation, solvent extraction, sol-gel polymerization, fluidized bed coating, pan coating, and the like. coating), melting, passive or active absorption or adsorption.

[0225] Commonly used agrochemical adjuvants are well known in the art and preferably include, but are not limited to, aqueous and / or organic solvents, pH regulators (such as buffers, acidifiers), surfactants, wetting agents, spreading agents, adhesion promoters (such as tackifiers, stickers), carriers, fillers, viscosity regulators (such as thickeners), emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, photoprotectants, antifreeze agents, biostimulants (including bacterial and / or fungal inoculants or microorganisms), biocides (preferably selected from herbicides, bactericides, phytotoxins, fungicides, insecticides and mixtures thereof), plant growth regulators, safeners, penetrants, anticaking agents, mineral oils and / or vegetable oils and / or waxes, colorants and drift control agents. agents) or any suitable combination thereof.

[0226] The insecticide may include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkylphthalate, a boric acid, a borate, a fluoride, a sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof.Herbicides used to remove unwanted vegetation may include chlorophenoxy compounds, nitrophenolic compounds, nitrocresolic compounds, dipyridyl compounds, acetamide, aliphatic acide, anilide, benzamide, benzoic acid, benzoic acid derivatives, anisic acid, anisic acid derivatives, benzonitrile, benzothiadiazinone dioxide, thiocarbamate, carbamate, phenylcarbamate, chloropyridinyl, cyclohexenone derivatives, dinitroaminobenzenederivatives, fluorodinitrotoluidine compounds, and the like. compound, isoxazolidinone, nicotinic acid, isopropylamine, an isopropylamine derivative, oxadiazolinone, phosphate, phthalate, picolinic acid compound, triazine, triazole, uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof.The fungicide may include substituted benzene, thiocarbamate, ethylene bis dithiocarbamate, thiophthalidamide, copper compound, organomercury compound, organotin compound, cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination thereof. The inoculant may include a Glomeraceae fungal inoculant, a Claroidoglomeraceae fungal inoculant, an Acaulosporaceae fungal inoculant, a Sacculospraceae fungal inoculant, an Entrophosporaceae fungal inoculant, a Pacidsproraceae fungal inoculant, a Diversisporaceae fungal inoculant, a Paraglomeraceae fungal inoculant, a Protosporaceae fungal inoculant, a fungal inoculants from the family Archaeosporaceae, fungal inoculants from the family Geosiphonaceae, fungal inoculants from the family Ambisporaceae, fungal inoculants from the family Scutellosproaceae, fungal inoculants from the family Dentiscultataceae, fungal inoculants from the family Racocetraceae, fungal inoculants from the phylum Basidiomycota, fungal inoculants from the phylum Ascomycota, fungal inoculants from the phylum Zygomycota, fungal inoculants from the genus Glomus, or a combination thereof.The bacterial inoculant may include a bacterial inoculant of the genus Rhizobium, a bacterial inoculant of the genus Bradyrhizobium, a bacterial inoculant of the genus Mesorhizobium, a bacterial inoculant of the genus Azorhizobium, a bacterial inoculant of the genus Allorhizobium, a bacterial inoculant of the genus Burkholderia, a bacterial inoculant of the genus Sinorhizobium, a bacterial inoculant of the genus Kluyvera, a bacterial inoculant of the genus Azotobacter, a bacterial inoculant of the genus Pseudomonas, or a bacterial inoculant of the genus S. inoculant, a bacterial inoculant of the genus Azosprillium, a bacterial inoculant of the genus Bacillus, a bacterial inoculant of the genus Streptomyces, a bacterial inoculant of the genus Paenibacillus, a bacterial inoculant of the genus Paracoccus, a bacterial inoculant of the genus Enterobacter, a bacterial inoculant of the genus Alcaligenes, a bacterial inoculant of the genus Mycobacterium, a bacterial inoculant of the genus Trichoderma, a bacterial inoculant of the genus Gliocladium, a bacterial inoculant of the genus Klebsiella, or a combination thereof.

[0227] In addition, the mixture may further comprise at least one microorganism selected from the group consisting of Bacillus subtilis strain 713, Bacillus amyloliquefaciens MBI 600, Bacillus pumillus QST2808, Pseudomonas fluorescens, Bradyrhizobium japonicum, Trichoderma vireus, Pseudomonas putida, Trichoderma harzianum Rifai strain T22, Penicillium bilaii, Mesorhizobium, Azospirillum, Azotobacter vinifera. vinelandii), Clostridium pasteurianum, and Glomus species.

[0228] The N-heterocyclic compounds of formula (I) and / or (II) and / or (III) and / or (IV) used according to the present invention can be used in combination with these adjuvants. The adjuvants used depend on the specific application form and the active substance and preferably include solvents, solid carriers, dispersants or emulsifiers, for example, solubilizers, protective colloids, surfactants and adhesion agents. In addition, organic and inorganic thickeners, bactericides, antifreeze agents, defoamers, (if appropriate) colorants and tackifiers or binders can be combined with nitrification inhibitors and used in the fertilizer mixture. Suitable adjuvants are discussed in WO 2013 / 121384, pages 25 to 26.

[0229] Further possible preferred ingredients are oils, wetters, adjuvants, biostimulants, herbicides, bactericides, other fungicides and / or pesticides. They are discussed, for example, in WO 2013 / 121384, pages 28 / 29.

[0230] The fertilizer mixture is preferably in solid form, including powders, prills, and granules. Additionally, a nitrification inhibitor in the form of a formulation, solution, or dispersion may be delivered separately or simultaneously with the fertilizer.

[0231] Furthermore, the nitrification inhibitors of the present invention can be used to reduce nitrogen losses in organic fertilizers and during harvesting of litter and grazing land or storage of liquid manure, and can be used to reduce the ammonia load in animal stalls.

[0232] For respective applications, reference may be made to US 6,139,596 and WO 2013 / 121384 as well as WO 2015 / 086823 and WO 2016 / 207210.

[0233] The present invention also relates to a method for fertilizing agricultural or horticultural soil, wherein a fertilizer mixture comprising the following compounds A and B is applied to the soil, or a fertilizer mixture comprising compounds A and B, respectively, is applied to the soil within a period of 0 to 5 hours, preferably 0 to 1 hour, more preferably approximately simultaneously:

[0234] A: Inorganic and / or organic and / or organo-mineral fertilizers; and

[0235] B: 10 to 10,000 ppm by weight of an N-heterocyclic compound of the general formula (I) and / or (II) and / or (III) and / or (IV) as defined above, based on the inorganic fertilizer.

[0236] The use of the nitrification inhibitors according to the invention and compositions containing them, while improving the utilization efficiency of nitrogen in ammonium- or urea-containing mineral fertilizers, organic fertilizers and organo-mineral fertilizers, has the effect of (in some cases significantly) increasing the yield and biomass of crop plants.

[0237] Likewise, during the actual storage of organic fertilizers (e.g., liquid manure), the nitrification inhibitors of the present invention can be added to such fertilizers to prevent nitrogen nutrient losses by slowing the conversion of various forms of nitrogen into gaseous forms (volatile nitrogen compounds), while also helping to reduce ammonia loads in animal stables. Furthermore, the nitrification inhibitors of the present invention or compositions containing the nitrification inhibitors can be used on agricultural stovers and grazed land to reduce gaseous nitrogen losses and prevent nitrate leaching.

[0238] In general, the N-heterocyclic compounds of the general formula (I) or (II) or (III) or (IV) can be used to reduce nitrogen or carbon losses from inorganic and / or organic and / or organomineral fertilizers or nitrogen- or carbon-containing compounds or materials and from harvested litter and grazing land or during storage of liquid manure, and can be used to reduce the ammonia load in animal stables.

[0239] Nitrogen losses are usually due to N2O and / or NO emissions or NO3 - Leaching. Nitrogen losses can occur from nitrogen-containing compounds or materials, such as roots, plants, fertilizers, animals, etc. Typically, nitrogen losses occur from nitrogen-containing mineral fertilizers or any organic nitrogen-containing substances. As mentioned above, the term "nitrogen losses" includes all forms of nitrogen or nitrogen compounds lost through emission or leaching. One example is greenhouse gas emissions, which can be reduced by using the N-heterocyclic compounds of the present invention.

[0240] The same applies to carbon losses from carbon-containing compounds or materials, such as carbonate-containing mineral fertilizers and carbon-containing organic matter, such as roots, plants, animals, and organic fertilizers. This carbon loss usually takes the form of carbon dioxide emissions, which are part of greenhouse gas emissions.

[0241] Thus, the N-heterocyclic compounds of the present invention can be used to prevent or reduce greenhouse gas emissions from nitrogen- or carbon-containing compounds or materials. These materials typically contain nitrogen and / or carbon in the form of covalent or ionically bound forms, for example, ammonium, proteins, nitrates, carbonates, carbohydrates, cellulose, or other organic carbon-containing compounds. Thus, the terms "carbon" and "nitrogen" can refer to elemental states or to compounds or materials containing carbon and / or nitrogen atoms.

[0242] The nitrogen-containing and / or carbon-containing compounds or materials may be present in fertilizers, soil or the environment. The most significant effect of the N-heterocyclic compounds of the present invention is to reduce greenhouse gas emissions from soil containing nitrogen-containing compounds or materials.

[0243] The most effective approach is to reduce greenhouse gas emissions from fertilized soils, which are often caused by downstream processes such as nitrification.

[0244] The plants treated according to the invention or the plants rooted in the soil treated according to the invention are preferably selected from agricultural plants, silvicultural plants, ornamental plants and horticultural plants, each in its natural or genetically modified form. Preference is given to treating non-transgenic agricultural plants.

[0245] Preferred agricultural plants are field crops selected from the group consisting of potatoes, sugar beets, wheat, barley, rye, oats, sorghum, rice, corn, cotton, rapeseed / oilseed rape, canola, soybeans, peas, field beans, sunflower, sugar cane, cucumbers, tomatoes, onions, leeks, lettuce, squashes; even more preferably, the plants are selected from the group consisting of wheat, barley, oats, rye, soybeans, corn, rapeseed / oilseed rape, cotton, sugar cane, rice and sorghum.

[0246] In a preferred embodiment of the present invention, the plants to be treated are selected from the group consisting of tomatoes, potatoes, wheat, barley, oats, rye, soybeans, corn, oilseed rape, canola, sunflower, cotton, sugarcane, sugar beet, rice, sorghum, pasture grass and grazed land.

[0247] In another preferred embodiment of the invention, the plants to be treated are selected from the group consisting of tomatoes, potatoes, wheat, barley, oats, rye, soybeans, corn, oilseed rape, canola, sunflower, cotton, sugarcane, sugarbeet, rice and sorghum.

[0248] In a particularly preferred embodiment of the invention, the plants to be treated are selected from the group consisting of tomatoes, wheat, barley, oats, rye, corn, oilseed rape, canola, sugarcane and rice.

[0249] In one embodiment, the plant treated according to the methods of the present invention is an agricultural plant. "Agricultural plant" refers to a plant that is harvested or grown on a commercial scale, either in part (e.g., seeds) or in whole, or is an important source of feed, food, fiber (e.g., cotton, linen), fuel (e.g., wood, bioethanol, biodiesel, biomass), or other compounds. Preferred agricultural plants are, for example, cereals, such as wheat, rye, barley, triticale, oats, sorghum or rice; beets, such as sugar beets or fodder beets; fruits, such as pomes, stone fruits or soft fruits, for example apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries or gooseberries; leguminous plants, such as lentils, peas, alfalfa or soybeans; oil plants, such as rapeseed (oilseed rape), canola, linseed, mustard, olives, sunflower, coconut, cocoa beans, castor oil plants, oil palm, groundnuts, nuts, or soybeans; cucurbits, such as squash, cucumbers, or melons; fiber plants, such as cotton, flax, or jute; citrus fruit, such as oranges, lemons, grapefruits, or mandarins; vegetables, such as spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, cucurbits, or paprika; lauraceous plants, such as avocados, cinnamon, or camphor; energy and raw material plants, such as corn, soybeans, rapeseed, canola, sugarcane, or oil palm. palm; tobacco; nuts; coffee; tea; bananas; vines (tablegrapes and grape juice grape vines); hops; turf; and natural rubber plants.

[0250] Pastures and grasslands consist of grasses or grass mixtures including, for example, bluegrass (Poa spp.), bentgrass (Agrostis spp.), ryegrasses (Lolium spp.), fescues (Festuca spp., hybrids and cultivars), zoysiagrass (Zoysia spp.), bermudagrass (Cynodon spp.), St. Augustine grass, bahiagrass (Paspalum), centipedegrass (Eremachloa), carpetgrass (Axonopus), buffalograss, and gramagrass. Grazing land may also consist of a mixture including the above grasses, for example, Ryegrass and Trifolium species (such as Trifolium umpratensis and Trifolium repens), Medicago species (such as Medicago sativa), Lotus species (such as Lotus corniculatus), and Melilotus species (such as Melilotus albus).

[0251] In one embodiment, the plant treated according to the method of the present invention is a horticultural plant. The term "horticultural plant" is understood to mean a plant commonly used in gardening, for example, a plant used in the cultivation of ornamental plants, herbs, vegetables and / or fruits. Examples of ornamental plants include turf, geranium, pelargonia, petunia, begonia and fuchsia. Examples of vegetables include potatoes, tomatoes, peppers, cucurbits, cucumbers, melons, watermelons, garlic, onions, carrots, cabbages, beans, peas and lettuce, more preferably from tomatoes, onions, peas and lettuce. Examples of fruits include apples, pears, cherries, strawberries, citrus, peaches, apricots and blueberries. In gardening, substrates typically replace (part of) soil.

[0252] In one embodiment, the plants treated according to the method of the invention are ornamental plants. "Ornamental plants" are plants commonly used in gardening (e.g., in parks, gardens, and on balconies), for example, turfgrass, geraniums, pelargoniums, petunias, begonias, and fuchsias.

[0253] In one embodiment, the plant processed according to the method of the present invention is a silvicultural plant. The term "silvicultural plant" is understood to mean trees, more specifically trees for reforestation or industrial plantations. Industrial plantations are generally used in the commercial production of forest products, and forest products are, for example, timber, paper pulp, paper, rubber trees, Christmas trees or saplings for gardening purposes. Examples of silvicultural plants include conifers, such as pine trees, particularly Pinus spec, fir and spruce, eucalyptus, tropical trees, such as teak, rubber trees, oil palm, salix, salix spec, poplar (cottonwood), particularly Populus spec, beech, particularly Fagus spec, birch, oil palm and oak.

[0254] The following definitions apply:

[0255] The term "plant" is understood to mean plants of economic value and / or artificially cultivated plants. They are preferably selected from agricultural plants, afforestation plants, ornamental plants and horticultural plants, each in its natural or transgenic form. The term "plant" as used herein includes all parts of a plant, for example, germinating seeds, emerging seedlings, herbaceous vegetation, and rooted woody plants, including all underground parts (e.g., roots) and above-ground parts.

[0256] The term "soil" is understood to mean a natural body composed of living (e.g., microorganisms such as bacteria and fungi, animals and plants) and non-living (e.g., mineral and organic matter such as organic compounds in varying degrees of decomposition, liquids and gases) matter present on the land surface, characterized by soil horizons that are distinguished from the initial material as a result of various physical, chemical, biological and anthropogenic processes.

[0257] The term "nitrification inhibitor" is to be understood as any chemical substance that slows down or delays the nitrification process that normally occurs in (fertilized) soil. Nitrification inhibitors slow the natural conversion of ammonium to nitrate and target microorganisms, preferably ammonia oxidizing bacteria (AOB), preferably by inhibiting the activity of bacteria, for example, Nitrosomonas spp. and / or Nitrosospira spp. They can also act on ammonia oxidizing archaea (AOA). Nitrification inhibitors are most often used in combination with fertilizers, preferably (ammonium) nitrogen-containing fertilizers, for example, solid or liquid inorganic, organic and / or organomineral fertilizers or manures.

[0258] The term "nitrification" is understood to mean the reaction of ammonia (NH3) or ammonium (NH4 + ) is biologically oxidized by oxygen to nitrite (NO2 - ), these nitrites are then oxidized by microorganisms into nitrates (NO3 - ). In addition to nitrate (NO3 - ) Nitrification also produces nitrous oxide. Nitrification is an important step in the nitrogen cycle in the soil.

[0259] The term "denitrification" is understood to mean the denitrification of nitrates (NO3 - ) and nitrite (NO2 - ) to gaseous nitrogen (usually N2 or N2O). This respiration process reduces oxidized forms of nitrogen in response to the oxidation of electron donors (e.g., organic matter). Preferred nitrogen electron acceptors, in order of decreasing thermodynamic favorability, include: nitrate (NO3 - ), nitrite (NO2 - ), nitric oxide (NO), and nitrous oxide (N2O). In the general nitrogen cycle, denitrification completes the cycle by returning N2 to the atmosphere. This process is primarily performed by heterotrophic bacteria (e.g., Paracoccus denitrifican and various pseudomonads), but autotrophic denitrifiers (e.g., Thiobacillus denitrificans) have also been identified. Denitrifying bacteria appear in all major phylogenetic groups. When faced with an oxygen shortage, many bacterial species are able to switch from using oxygen to using nitrates to support respiration in a process called denitrification, in which water-soluble nitrates are converted to gaseous products (including nitrous oxide) that are emitted to the atmosphere.

[0260] Nitrous oxide, commonly known as "happy gas" or "laughing gas," is a chemical compound with the chemical formula N2O. At room temperature, it is a colorless, non-flammable gas. Nitrous oxide is produced naturally in soil through the microbial processes of nitrification and denitrification. These natural emissions can be increased through various agricultural practices and activities, including: a) adding nitrogen directly to the soil through the use of mineral and organic fertilizers, b) growing nitrogen-fixing crops, and c) cultivating soils with high organic content.

[0261] The term "fertilizer" should be understood as a (chemical) compound used to promote the growth of plants and fruits. Fertilizers are usually applied through the soil (for absorption by plant roots) or foliar application (for absorption through leaves). The term "fertilizer" can be divided into two major categories: a) organic fertilizers (composed of decayed plant / animal matter) and b) inorganic fertilizers (composed of chemicals and minerals). Organic fertilizers include slurry, worm castings, peat, seaweed, sewage and guano. Artificial organic fertilizers include compost, blood meal, bone meal and seaweed extract. Further examples are enzymatically digested protein, fish meal and feather meal. Decomposed crop residues from previous years and manure are another source of fertilizer. In addition, naturally occurring minerals such as mine rock phosphate, sulfate of potash and limestone are also considered to be inorganic fertilizers. Inorganic fertilizers are typically manufactured by chemical processes (e.g., the Haber-Bosch process), but also from natural deposits that are chemically altered (e.g., concentrated triple superphosphate). Naturally occurring inorganic fertilizers include Chilean sodium nitrate, mine rock phosphate, and limestone. A third category, organo-mineral fertilizers, is a combination of inorganic and organic fertilizers.

[0262] The term "urea-containing fertilizer" (urea fertilizer) is defined as a synthetic fertilizer containing urea, but does not include any naturally occurring fertilizer containing urea (e.g., manure as an example of a naturally occurring fertilizer containing urea). Examples of fertilizers containing urea are urea ammonium nitrate (UAN), isobutylidene diurea (IBDU), crotonylidene diurea (CDU), and urea formaldehyde (UF). Urea is usually prepared as a granulated material or prills. Urea fertilizer can be produced by dripping liquid urea from a prilling tower while drying the product. Urea is also available as a liquid formulation that can be used for foliar fertilization (e.g., application to potatoes, wheat, vegetables, and soybeans) and liquid field fertilization. It is usually mixed with ammonium nitrate to form UAN containing 28% N.

[0263] The term "locus" (plant habitat) is to be understood as any type of environment, soil, area or material in which plants are growing or are intended to be grown. Particularly preferred according to the invention is soil.

[0264] The present invention is further described by the following examples, which show:

[0265] - the compounds of the general formulae (I) and (II) and (III) and (IV) are strong nitrification inhibitors;

[0266] -The new nitrification inhibitor is highly effective and specific;

[0267] - These new nitrification inhibitors act specifically on nitrifying bacteria, targeting the NH3 oxidation step in nitrifying bacteria;

[0268] - These new nitrification inhibitors reduce the depletion of ammonium in the soil.

[0269] Example

[0270] 1. Heterocyclic compounds of general formula (I) to (IV)

[0271] Different heterocyclic compounds of general formula (I) to (IV) were obtained from ENAMINE Ltd., UkrOrgSynthesis Ltd. or Vitas-M Laboratory, Ltd. The compounds are shown in the following Table 1. 2-Thiazoline-2-thiol was also available from Sigma-Aldrich.

[0272] The nitrification inhibition of compounds of formula (I), (II), and (III) / (IV) was determined in two screening assays using the nitrifying bacteria N. europaea and N. multiformis. In practice, ammonium and candidate nitrification inhibitors (100 μM) were added to dense bacterial cultures in multiwell plates and, after 24 hours of incubation, nitrite levels were measured. To normalize for differences between batches of cultures and multiwell plates, the relative nitrification activity of each well was calculated and normalized to negative and positive controls (see the "Materials and Methods" section below for details).

[0273] 2. Materials and Methods

[0274] 2.1 Nitrite determination

[0275] Nitrite (NO2) was determined using Griess reagent (Product No. G4410, Griess reagent (modified), Sigma-Aldrich). - ). An equal volume of sample or diluted sample was mixed with Griess reagent in a transparent flat-bottom multiwell plate and incubated in the dark at room temperature for 10 min. Spectrophotometry (EnVision, Perkin ) was used to determine the absorbance at 540 nm for calculating [NO2 - ].

[0276] 2.2 Ammonium determination

[0277] Ammonium (NH4) was determined by modified Berthelot's reagent protocol. + 8 μl of culture sample, 35 μl of reagent A (0.5 g NaOH and 8 ml NaClO (2.5%) in 92 ml MilliQ), and 33 μl of reagent B (1 g salicylic acid, 0.5 g NaOH, and 1.0237 g sodium nitroprusside dihydrate in 100 ml MilliQ) were successively added to a flat-bottom 96-well plate (Cat. No. 353072, After 30 min of incubation, the cells were analyzed by spectrophotometry (EnVision, PerkinElmer). ) was used to determine the absorbance at 635 nm for calculating [NH4 + ].

[0278] 2.3 Culture maintenance

[0279] 2.3.1 Nitrosomonas europaea

[0280] Nitrosomonas europaea growth medium was prepared by aseptically mixing 900 mL of stock solution 1 (27.75 mM (NH4)2SO4, 3.35 mM KH2PO4, 0.83 μM MgSO4, 0.22 μM CaCl2, 11 μM FeSO4, 18.33 μM EDTA, and 0.56 μM CuSO4) with 100 mL of stock solution 2 (400 mM KH2PO4 and 40 mM NaH2PO4, pH 8.0 (NaOH)) and 8 mL of stock solution 3 (5% anhydrous Na2CO3). All three stock solutions were pre-autoclaved. Nitrosomonas europaea (ATCC 25978) cells were grown on adhesive tape (Micropore TM Surgical Tape 1530-1, 3M TM ) in sealed sterile Erlenmeyer flasks at 28°C with shaking (±150 rpm) in the dark. Late logarithmic phase cultures ([NO2 - ] = 10-20 mM) were subcultured by centrifugation (4000 rpm, 15 min, 5°C), and the medium was refreshed by discarding the supernatant and resuspending the bacterial cell pellet in freshly prepared Nitrosomonas europaea growth medium.

[0281] 2.3.2 Nitrosospira multiformis

[0282] Nitrosospira multiformis (NCIMB 11849) cells were grown in autoclaved 181 medium for AOB (NCIMB Ltd) in Erlenmeyer flasks containing 1.78 mM (NH4)2SO4, 1.47 mM KH2PO4, 272 μM CaCl2 × 2H2O, 162 μM MgSO4 × 7H2O, 1 mL of stock solution 1 (1.8 mM FeSO4 × 7H2O and 1.49 mM NaEDTA), and 1 mL of stock solution 2 (0.5% phenol red, pH indicator) (pH 7.5-8). The pH was maintained by periodically adding sterile 5% Na2CO3. The flasks were sealed with tape (Micropore). TMSurgical Tape 1530-1,3M TM ) and cultured in the dark at 30°C with shaking (±150 rpm). Late logarithmic phase cultures ([NO2 - ] = ± 3 mM) were subcultured by centrifugation (4000 rpm, 15 min, 5°C), and the medium was renewed by discarding the supernatant and resuspending the bacterial cell pellet in pH-adjusted 181 medium.

[0283] Nitrification inhibition test

[0284] 2.4.1 Nitrosomonas europaea

[0285] To perform a high-throughput nitrification inhibition assay using N. europaea, late logarithmic phase cultures were subcultured two days before the assay. TM Conical Centrifuge Tubes, After centrifugation (4000 rpm, 15 min, 5°C), the culture was over-concentrated 5-fold with fresh growth medium. For each batch, all cultures were pooled into a sterile Schott bottle and then distributed through a dispenser (Multidrop) that was first rinsed with growth medium. TM Combi Reagent Dispenser, Thermo Scientific TM ) were distributed to 384-well plates (Cat.No.781086, Plate, Greiner Bio-One; 50 μl / well). Specifically for high-throughput screening of novel nitration inhibitors, 0.5 μl of 99.99% DMSO (negative control - final concentration 1%) was added to the two outer columns on the left side of the plate, and 0.5 μl of 10 mM DMP (3,4-dimethylpyrazole, positive control - final concentration 100 μM) was manually added to the two outer columns on the right side of the plate. Finally, a Tecan robot (Freedom 0.5 μl of a candidate nitrification inhibitor (5 mM stock solution in 99.99% DMSO - final concentration 50 μM) was added to the central well using a pin tool on a Tecan. Between additions, the pin was rinsed with 99.5% DMSO, MilliQ water, and 100% ethanol and air-dried. All plates were individually wrapped in Parafilm. Stacks of four plates were placed on top of a 96-well plate containing 100 μl of MilliQ per well, covered with aluminum foil, and shaken at 150 rpm at 28°C. After 24 h, NO2 was assessed. - To this end, the sample was first diluted 200-fold by pipetting 1.5 μl of the sample into the middle plate (Cat. No. 353077, 96-well Clear Round Bottom Microplate, ) in 300 μl of fresh growth medium, then 15 μl of diluted sample was mixed with 15 μl of Griess reagent in the wells of a transparent flat-bottom 384-well plate (Cat. No. X7001, Low Profile Microplate, Molecular Devices) and the color was measured spectrophotometrically at 540 nm (EnVision, Perkin ) determination.

[0286] 2.4.2 Nitrosospira multiformis

[0287] The late log phase ([NO2 - ]≥3mM) and in the past 24h [NO2 - ] 500 mL of culture grown in a 1-L Erlenmeyer flask with an increase of ≥ 500 μM was used for high-throughput nitrification inhibition assay. First, the nitrification inhibition assay was performed by adding 50 mL of culture medium to a 50 mL centrifuge tube (Cat. No. 430829, CentriStar TM Conical Centrifuge Tubes, ) were centrifuged (4000 rpm, 15 min, 5 ° C), and the culture was over-concentrated 5 times in fresh 181 medium. In each batch, all cultures were pooled into a sterile Schott bottle (Schottbottle), which was then dispensed using a dispenser (Multidrop) that was first rinsed with 181 medium. TM Combi Reagent Dispenser, Thermo Scientific TM ) were distributed to 384-well plates (Cat.No.781086, Plate, Greiner Bio-One; 50 μl / well). 0.5 μl of 99.99% DMSO (negative control - final concentration 1%) was added to the two outer columns on the left side of the plate, and 0.5 μl of 10 mM DMP (3,4-dimethylpyrazole, positive control - final concentration 100 μM) was manually added to the two outer columns on the right side of the plate. Finally, a Tecan robot (Freedom 0.5 μl of a candidate nitrification inhibitor (5 mM stock solution in 99.99% DMSO - final concentration 50 μM) was added to the central well using a pin tool on a Tecan. Between additions, the pin was rinsed with 99.5% DMSO, MilliQ water, and 100% ethanol and air-dried. All plates were individually wrapped in Parafilm. A stack of four plates was placed on top of a 96-well plate containing 100 μl of MilliQ per well, covered with aluminum foil, and shaken at 30°C (±150 rpm). After 24 h, NO2 was assessed. - To this end, first, the sample was diluted 100-fold by pipetting 3 μl of the sample into the middle plate (Cat. No. 353077, 96-well Clear Round Bottom Microplate, ) in 300 μl of fresh growth medium, then 15 μl of diluted sample was mixed with 15 μl of Griess reagent in the wells of a transparent flat-bottom 384-well plate (Cat. No. X7001, Low Profile Microplate, Molecular Devices) and the color was measured spectrophotometrically at 540 nm (EnVision, Perkin ) determination.

[0288] 2.4.3 Quantification of nitrification inhibition

[0289] To assess the efficacy of each compound (in terms of inhibiting nitration) and enable comparisons between culture plates and culture batches, we calculated relative nitration. More specifically, all nitrite results were normalized to a negative control containing no compound and a positive control containing a benchmark (100 μM DMP) using Equation 1. This normalization was performed in each multiwell plate, which contained 32 positive controls and 32 negative controls.

[0290]

[0291] As a result, a compound that allows complete nitration (no nitration inhibition) shows a relative nitration of 1 (or 100%). A compound that shows the same nitration inhibition as the positive control shows a nitration inhibition of 0.

[0292] The IC50 value, the concentration that inhibits nitration by 50%, was calculated by fitting a logistic curve to the 8-point dose response data, using doses between 2 and 100 μM with a step size of 1.75. If a concentration of 2 μM already inhibits nitration, an IC50 value cannot be calculated. In this case, the IC50 value represents the dose that most closely resembles a 50% inhibitory effect.

[0293] Cell growth inhibition assay

[0294] The structures studied had no toxic / non-specific effects (at least in the assays tested), no effect on the AOA system (ABIL, from Avecom and Nitrososphaera viennensis) and no effect on microtox.

[0295] 2.6 Nitrosomonas europaea ammonia and hydroxylamine oxidation test

[0296] To determine whether compounds specifically inhibit NH3 or NH2OH oxidation, N. europaea cells were provided with NH3 or hydroxylamine (NH2OH) as the N source. - Two-day-old late logarithmic phase cultures at concentrations between 10 and 20 mM were washed three times in fresh growth medium (without N) and finally overconcentrated 4-fold by centrifugation (4000 rpm, 15 min, 4°C). TM Combi Reagent Dispenser, Thermo Scientific TM ) a transparent flat-bottom 96-well plate (Cat. No. 353072, 96Well Clear Flat Bottom Microplate, ) were filled with 150 μl of culture per well. A Tecan robot (Freedom Tecan) were added to the multiwell plate in triplicate (final concentration 50 μM). Thiourea (positive control for inhibition of NH3 oxidation - final concentration 100 μM) and phenylhydrazine hydrochloride (positive control for inhibition of NH2OH oxidation - final concentration 1 mM) were added to the two outer columns. Each plate was run in duplicate and 500 μM (NH4)2SO4 (final [NH4 +]=1mM) or 1mM NH2OH. All plates were individually wrapped in sealing film. A stack of 4 plates was placed on top of a 96-well plate containing 100μl MilliQ in each well, covered with aluminum foil, and shaken at 28°C (±150rpm). In order to prevent the reading of NO2 - The secondary effect is only measured after 30 minutes. - To this end, first, the samples were diluted 2-fold by pipetting 15 μl into a 96-well PCR microplate (Cat. Ref. PCR-96-FS-C, 96-well PCR Microplate, ) in 15 μl of fresh growth medium, then 15 μl of diluted sample was mixed with 15 μl of Griess reagent in a transparent flat-bottom 384-well plate (Cat. No. X7001, Low Profile Microplate, Molecular Devices) and the color was measured spectrophotometrically at 540 nm (EnVision, Perkin ) determination.

[0297] 2.7 Test in soil

[0298] Top soil samples (0 to 10 cm) were collected from different fields in Belgium (Merelbeke and Moorslede). Field vegetation was removed, and samples were collected from different plots. All soil samples were pooled and sieved (2.8 mm mesh size) to remove large debris and homogenize the soil. The soil was stored at 5°C in plastic containers covered with Saran foil to prevent changes in the microbial community composition and maintain the original soil moisture content.

[0299] Soil moisture content was determined by drying 20 g of soil in an oven at 60°C for more than 48 h and measuring the weight before and after drying. Based on the soil moisture content (±20%), compound solutions were prepared such that the addition of 200 μl of compound and 200 μl of NH4Cl solution would result in a final compound concentration of 50 μM and a final NH4Cl solution of 50 μM. + The concentration was 10mM. For each treatment, 5 small pots were filled with 20g of soil. Next, the soil was first treated with the corresponding compound solution and then with the NH4Cl solution. Each tray included a positive control (50μM DMP) and a negative control (DMSO). The pots were incubated at 21°C (light from 6 am to 10 pm) for 7 days. Demineralized water was added to the soil every 2 to 3 days to a soil weight of 20g. Finally, each sample of 20g of soil was dissolved in 100ml of 1M KCl and shaken for 1h, and then, by Filter with filter paper. The filtrate is used to determine pH and NH4+ and NO3 - concentration.

[0300] 2.8. Copper binding test

[0301] DMSO solutions containing compounds at a final concentration of 2.5 mM or 10 mM and CuSO4 at a final concentration of 5 mM were compared with pure compounds or CuSO4 solutions at the same concentrations. 100 μl of the solution was added to a 96-well plate, and the full absorbance spectrum was measured using a SpectraMex 2550 plate reader (Molecular devices).

[0302] Two agricultural soils from fields in Morse Lade and St. Lawrence were used. 10 g of soil was placed in screw-top vials. The vials were sealed with Parafilm and pre-cultured for 5 days in the dark at 21 ° C. After pre-culture, the vials were ventilated with a fan. 100 μL of 1 mM compound solution (10% DMSO) was added, followed by 100 μL of 200 mM NH4Cl. The vials were airtightly closed and 5 mL of synthetic air was added to the headspace using a plastic syringe, and then the 5 mL headspace was transferred to an emptied 3 mL exetainer. The vials were incubated in the dark at 21 ° C, and background NO concentration was recorded. At each sampling time point, 5 mL of synthetic air was added to the headspace again using a plastic syringe, and then the 5 mL headspace was transferred to an emptied 3 mL exetainer again. The vials were ventilated and closed again, and new gas samples were transferred to the exetainer as before. Determination of N2O, CO2, and CH4 in the texetainer by gas chromatography. Direct determination of NO concentration.

[0303] 3. Results

[0304] The nitrification inhibition of compounds of formula (I) and (II) was determined in two screening assays using the nitrifying bacteria N. europaea and N. multiformis. In practice, ammonium and candidate nitrification inhibitors (100 μM) were added to high-concentration bacterial cultures in multiwell plates. After 24 hours of incubation, nitrite levels were measured. To normalize for differences between culture batches and multiwell plates, relative nitrification was calculated for each well and normalized to negative and positive controls (see the "Materials and Methods" section above for details). Thus, a relative nitrification of 100% or 0% indicates no nitrification inhibition or nitrification inhibition at the level of the positive control (100 μM 3,4-dimethylpyrazole (DMP)), respectively.

[0305] 3.1 Results in Table 1

[0306] The results shown in Table 1 below are based on the use of the nitrifying bacteria Nitrosomonas europaea and Nitrosospira multiformis. N-heterocyclic compounds of the general formulae (I), (II), (III) and (IV) inhibited nitrification in at least one screening system. The inhibitory activity of this class of molecules was demonstrated against a number of structures with different sub-groups. Eight different doses and four biological replicates were used. The results in Table 1 show that all (tested) substances significantly inhibited nitrification in the test system, and that some inhibitors showed strong inhibition at very low doses (some inhibitors showed complete inhibition at 2 μM).

[0307] For reference, 3,4-dimethylpyrazole was used in Comparative Example C1. IC50 values ​​represent the estimated concentration that causes 50% inhibition of nitration. These values ​​were predicted by fitting a logistic curve to the 8-point dose-response data or based on the lowest tested dose that produced greater than 50% inhibition.

[0308] IC50 represents the concentration at which 50% inhibition occurs. Therefore, lower values ​​indicate stronger inhibition of nitrification. All tested nitrification inhibitors showed a stronger effect on Nitrosospira multiformis.

[0309] As is evident from Table 1, all (tested) molecules were able to inhibit nitrification. Therefore, the presence of a substructure of formula (I), (II), (III) or (IV) is necessary and appears to be sufficient to achieve nitrification inhibition. Different side groups on the aminomethyl group bonded via the nitrogen atom in formulas (I) and (III) or on the thioether group in formulas (II) and (IV) have little influence on nitrification inhibition. It is also possible to add molecular structures to the R2 and R3 groups of the structures of formulae (I) and (II), but particularly complex substructures may weaken the nitrification inhibition capacity. Nevertheless, all molecules of the substructures of formulae (I) and (II) and (III) and (IV) inhibited nitrification to some extent by at least one of the two tested nitrifying bacteria.

[0310] Not all molecules shown in Table 1 were tested.

[0311] All tested molecules are strong nitrification inhibitors, acting specifically on nitrifying bacteria. NA stands for Not Analyzed.

[0312] Table 1

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] In particular, the molecule of Example 1, 3-[[(6-chloroimidazo[2,1-b][1,3]thiazol-5-yl)methyl-methylamino]methyl]-1,3-thiazolidine-2-thione, strongly inhibited nitrification by both test bacteria. Two similar molecules, 3-[[1,3-benzothiazol-2-ylmethyl(methyl)amino]methyl]-1,3-oxazolidine-2-thione of Example 5 and 3-[[(2-chloro-6-fluorophenyl)methyl-methylamino]methyl]-1,3-oxazolidine-2-thione of Example 6, showed strong nitrification inhibition against these two ammonia oxidizing bacteria.

[0322] The three structures of particularly preferred embodiments 1, 5 and 6 are shown below:

[0323]

[0324] 3.2 Ammonia and hydroxylamine oxidation test

[0325] In order to gain a deeper understanding of the target metabolic pathways and to distinguish compounds that specifically affect NH3 oxidation or affect other pathways, three representative new nitrification inhibitors (Examples 1, 5 and 6) were tested in the "Ammonia and Hydroxylamine Oxidation Assay", in which NO2 generated from NH3 - With NO2 produced by NH2OH- For comparison. Since NH3 is in NO2 - Comparison of the inhibition of nitrification by these novel compounds against NH3 (see "1." in the diagram below) and NH2OH (see "2." in the diagram below) indicates which part of the pathway is inhibited.

[0326] No nitrification inhibitors:

[0327]

[0328] Nonspecific or general metabolic inhibitors:

[0329] NH3→X

[0330] NH2OH→X

[0331] Ammonia oxidation specific inhibitors:

[0332] NH3→X

[0333]

[0334] Ammonia oxidation is a crucial step in the metabolism of nitrifying bacteria. Therefore, inhibition of ammonia oxidation by nitrification inhibitors indirectly affects all other enzymatic steps, including the second step, although less strongly than the first. Indeed, even in a 30-min assay, DMP, known to target ammonia oxidation, reduced NH₂OH oxidation, but to a lesser extent than its effect on ammonia oxidation.

[0335] Table 2 below shows that all three novel nitrification inhibitors significantly inhibited ammonia oxidation but had little effect on hydroxylamine oxidation. The limited effect on hydroxylamine oxidation is likely indirect, as inhibition of ammonia oxidation affects the entire metabolism. Indeed, DMP, known to target the first step, also had limited effect on the second step in the experiment (Table 2). Therefore, the novel nitrification inhibitors specifically target ammonia oxidation (the first step of nitrification), further confirming their specific action on AOB and their general lack of toxicity.

[0336] Table 2

[0337]

[0338] 3.3 Experiments in agricultural soils

[0339] Finally, the effects of a representative panel of novel nitrification inhibitors were tested in soil.

[0340] Nitrification inhibitors were used in nitrification inhibition experiments using agricultural soil. In practice, ammonium with or without the novel nitrification inhibitor was added to soil from a field in Merelbeke (Belgium). Ammonium levels were measured at the beginning and end of the experiment (after one week of incubation). The percentage nitrification inhibition was calculated by comparing the decrease in ammonium levels with the nitrification inhibitor with the decrease in ammonium levels without the inhibitor.

[0341] Ammonium was added to soil with high nitrification activity and treated with a nitrification inhibitor (final concentration of 50 μM). Table 3 shows that after one week of treatment, the thiazolidine compound of Example 1, in particular, strongly inhibited ammonium consumption. Thioether-forming thiazolidine compounds (Examples 9 and 10) and the oxazolidine compound of Example 5 also showed significant inhibitory effects. This confirms that the novel nitrification inhibitor effectively inhibits ammonia oxidation in soil communities.

[0342] Table 3

[0343]

[0344]

[0345] The presence of a thiazolidine-thiol substructure has been found to be associated with nitration inhibition. Evaluation of different structural variants indicates that structures containing thiazolidine-thiol and oxazolidine-thiol are preferred nitration inhibitors. Preferred compounds have substructures that are bound by an amino-methyl group in combination with more complex structures. If R1 in the general formula (I) is hydrogen, such molecules can also form thiol tautomers. Preferred are compounds containing a thiazolidine-thiol substructure, an oxazolidine-thiol substructure, or a thiazolidine-thioether substructure.

[0346] A group of examples were tested under the same conditions (Moorslede soil, 3 days of incubation) at a concentration of 50 μM. The results are shown in Table 4.

[0347] Table 4

[0348]

[0349] Another set of examples was also tested in Moorslede soil at different concentrations and samples were taken after 5 days. The results are shown in Table 5. Tables 3-7 show that different variations within the claimed formula effectively inhibit nitrification in soil.

[0350] Table 5

[0351]

[0352] 3.4 Nitrification inhibition test using 3H-1,3-thiazole-2-thione

[0353] According to Sections 1., 2.4.1 to 2.4.3 above, the compound of Example 24, 3H-1,3-thiazole-2-thione, was used in a full dose-response assay in two ammonia-oxidizing bacteria, Nitrosomonas europaea and Nitrosospira multiformis.

[0354] Table 6

[0355]

[0356]

[0357] 3H-1,3-thiazole-2-thione (the tautomeric form is 3H-1,3-thiazole-2-thiol), 2-(methylsulfanyl)-1,3-thiazole, and 4-methyl-1,3-thiazole-2-thiol were tested at concentrations ranging from 2 to 100 μM. The results are shown in Table 5 below. The results at 100 μM are also included in Table 1.

[0358] 3H-1,3-Thiazol-2-thione has a potent effect on Nitrosospira multiformis (only 25% nitration after 2 μM application), but still almost completely inhibits nitration by Nitrosomonas europaea at higher concentrations.

[0359] 3.5 Experiments in agricultural soils

[0360] The effect of 3H-1,3-thiazole-2-thione was tested in soil according to the test described in Section 3.3 above. The results are summarized in Table 7 below.

[0361] Table 7

[0362]

[0363] 3H-1,3-thiazole-2-thione effectively inhibits nitrification in soil.

[0364] In the data presented, the extent of inhibition appears to be limited, but the experiments were intentionally performed at low concentrations (20 μM) in order to compare the efficacy between different inhibitors.

[0365] 3.6 Control of greenhouse gas emissions

[0366] To further confirm the inhibitory effect of the new nitrification inhibitor on nitrogen loss, gaseous emissions from soils fertilized with ammonium and treated with 1,3-thiazolidine-2-thione were captured. NO, N₂O, CO₂, and CH₄ were measured. Table 7 shows the cumulative values ​​over the 99-hour incubation period, demonstrating that NO and N₂O, in particular, as well as CO₂, were significantly reduced after application of the new nitrification inhibitor, particularly in two different agricultural soils. No significant effect on CH₄ was detected.

[0367] Table 8

[0368]

[0369]

[0370] 4. New nitrification inhibitors are copper chelators

[0371] The new nitrification inhibitor forms a complex with copper.

[0372] Since many known nitrification inhibitors are copper chelators, the copper chelation ability of the novel inhibitors was tested by analyzing their absorption spectra in the presence and absence of Cu. If Cu forms a complex with the nitrification inhibitor, the absorption spectrum would be expected to shift. In fact, the absorption spectra of the novel inhibitors shifted significantly upon binding to Cu, indicating that they form a complex with Cu and further confirming their role as nitrification inhibitors.

Claims

1. An N-heterocyclic compound for use as a nitrification inhibitor, wherein the N-heterocyclic compound has the general formula (a): It has the following definition: X1 is 0, X2 is S; R2 is H or C1-4 alkyl; R3 is H or C1-4 alkyl; R6 and R7 are hydrogen or together form a covalent carbon-carbon bond; R1 is H, C1-12 alkyl or -CH2-NR4R5, wherein: R4 is hydrogen or C1-4 alkyl; R5 is a C1-12 hydrocarbon residue, which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur. R4 and R5 may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic group, which may optionally also contain one or two additional heteroatoms selected from nitrogen, oxygen and sulfur.

2. The N-heterocyclic compound for use according to claim 1, wherein The N-heterocyclic compound of general formula (a) is an N-heterocyclic compound of general formula (I), It has the following definition: X1 is O, X2 is S; R2 is H or C1-4 alkyl; R3 is H or C1-4 alkyl; R1 is H, C1-12 alkyl or -CH2-NR4R5, wherein: R4 is hydrogen or C1-4 alkyl; R5 is a C1-12 hydrocarbon residue, which may contain one to three halogen atoms and / or one to four heteroatoms selected from nitrogen, oxygen and sulfur. R4 and R5 may also form, together with the nitrogen atom to which they are attached, a 5- or 6-membered saturated or unsaturated heterocyclic group, which may optionally also contain one or two additional heteroatoms selected from nitrogen, oxygen and sulfur.

3. The N-heterocyclic compound for use according to claim 1 or 2, wherein R2, R3 and R4 are independently hydrogen, methyl or ethyl.

4. The N-heterocyclic compound for use according to claim 2 or 3, wherein In the general formula (I), R5 is a C3-10 hydrocarbon residue, which may contain 1-2 halogen atoms and / or 1-3 heteroatoms and contain at least one cyclic structure, wherein preferably R5 in the general formula (I) is a C3-8 hydrocarbon residue, which may contain 1-2 halogen atoms and / or 1-3 heteroatoms and contain a 5- or 6-membered ring structure that may be fused to a second 5- or 6-membered ring structure.

5. The N-heterocyclic compound for use according to any one of claims 1 to 3, wherein the N-heterocyclic compound is Or wherein the N-heterocyclic compound is 4,4-dimethyloxazolidine-2-thione.

6. An N-heterocyclic compound as defined in any of the preceding claims for use as an additive or coating material for inorganic and / or organic and / or organomineral fertilizers, preferably inorganic fertilizers, more preferably nitrogen fertilizers containing ammonium and / or urea.

7. The N-heterocyclic compound for use according to any one of the preceding claims, wherein The N-heterocyclic compound is delivered in the form of a formulation, solution or dispersion, either alone or simultaneously with a fertilizer, or incorporated into or applied to a fertilizer.

8. Use of an N-heterocyclic compound as defined in any of the preceding claims for reducing nitrogen or carbon losses from inorganic and / or organic and / or organomineral fertilizers or nitrogen- or carbon-containing compounds or materials and from harvested litter and grazing land or during storage of liquid manure, and for reducing the ammonia load in animal stables.

9. The N-heterocyclic compound for use according to any one of the preceding claims, wherein The N-heterocyclic compound is used together with at least one additional agrochemical, preferably selected from: At least one additional nitrification inhibitor, preferably selected from 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, 2-amino-4-chloro-6-methyl-pyrimidine, 2 -mercaptobenzothiazole, sulfathiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon disulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranylmethylcarbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester; at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT); at least one commonly used agrochemical adjuvant, preferably selected from aqueous and / or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, chelating agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, light protectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anti-caking agents, mineral oils and / or vegetable oils and / or waxes, colorants and anti-drift agents; and mixtures thereof.

10. A mixture comprising at least one N-heterocyclic compound as defined in any one of the preceding claims and at least one further agrochemical, said at least one further agrochemical being preferably selected from: At least one additional nitrification inhibitor, which is preferably selected from 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, 2-amino-4-chloro-6-methyl-pyrimidine, 2 -mercaptobenzothiazole, sulfathiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon disulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranylmethylcarbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester; at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT); at least one commonly used agrochemical adjuvant, preferably selected from aqueous and / or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, chelating agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, light protectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anti-caking agents, mineral oils and / or vegetable oils and / or waxes, colorants and anti-drift agents; and mixtures thereof.

11. A fertilizer mixture comprising: A: Inorganic and / or organic and / or organo-mineral fertilizers; and B: 10 to 10 000 ppm by weight of at least one N-heterocyclic compound as defined in any one of claims 1 to 4, based on the inorganic fertilizer.

12. The fertilizer mixture according to claim 11, wherein The fertilizer mixture is in solid form, and the N-heterocyclic compound of formula (I) is applied to the surface of the fertilizer, which is preferably an inorganic fertilizer.

13. The fertilizer mixture according to claim 11 or 12, wherein The fertilizer mixture contains at least one additional agrochemical, which is preferably selected from: At least one additional nitrification inhibitor, which is preferably selected from 2-(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD), 1H-1,2,4-triazole, 3-methylpyrazole (3-MP), 2-chloro-6-(trichloromethyl)-pyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, 2-amino-4-chloro-6-methyl-pyrimidine, 2 -mercaptobenzothiazole, sulfathiazole, thiourea, sodium azide, potassium azide, 1-hydroxypyrazole, 2-methylpyrazole-1-carboxamide, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, 2,4-diamino-6-trichloromethyl-5-triazine, carbon disulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranylmethylcarbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester; at least one urease inhibitor, preferably selected from n-butylthiophosphoric triamide (NBTPT) and / or n-propylthiophosphoric triamide (NPTPT); at least one commonly used agrochemical adjuvant, preferably selected from aqueous and / or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, chelating agents, anti-settling agents, coalescing agents, rheology modifiers, defoamers, light protectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anti-caking agents, mineral oils and / or vegetable oils and / or waxes, colorants and anti-drift agents; and mixtures thereof.

14. A method for producing a fertilizer mixture according to any one of claims 11 to 13, said method being carried out by introducing the N-heterocyclic compound into the fertilizer and / or applying the N-heterocyclic compound to the surface of the fertilizer.

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