BE1029769B1 - Use of pyrazolo[3,4-b]pyridine compounds as a nitrification inhibitor - Google Patents

Abstract

The use of pyrazolo[3,4-b]pyridine compounds as a nitrification inhibitor.

Description

Use of pyrazolo[3,4-b]pyridine compounds as a nitrification inhibitor

Description

The present invention relates to the use of a pyrazolo[3,4-b]pyridine compound as a nitrification inhibitor or as a nitrogen stabilizer, to fertilizer mixtures containing it, to a process for its preparation, and to the method of soil fertilization which includes its application.

Fertilizers containing ammonium compounds are often used to provide plants in agriculture or horticulture with the nitrogen they need.

Ammonium compounds are microbially converted into nitrate in the soil within a relatively short period of time (nitrification). However, nitrate can be leached from the soil. The washed-out fraction is no longer available for plant nutrition, which is why rapid nitrification is undesirable. To use the fertilizer more efficiently, nitrification inhibitors are therefore added to the fertilizer. A well-known group of nitrification inhibitors are pyrazole compounds.

A problem with the use of pyrazole compounds as nitrification inhibitors is their high volatility. Therefore, during the storage of fertilizer preparations containing pyrazole compounds, a continuous loss of active substance occurs as a result of evaporation. Therefore, the pyrazole compounds must be formulated in a non-volatile form by appropriate measures.

EP-B-1 120 388 describes addition salts in phosphoric acid of 3,4-dimethylpyrazole and 4-chloro-3-methylpyrazole for use as nitrification inhibitors. Due to the salt form it is possible to reduce the volatility considerably.

WO 96/24566 relates to the use of low volatility pyrazole derivatives with hydrophilic groups as nitrification inhibitors. As an example, 2-(N-3-methylpyrazole)succinic acid (DMPSA) is proposed as a nitrification inhibitor. Ammonium-containing nitrates, sulfates or phosphates are mentioned as suitable mineral fertilizers.

WO 2011/032904 and WO 2013/121384 describe pyrazole derivatives as nitrification inhibitors, of which DMPSA is one.

US 2018/0016199 A1 describes pyrazole compounds as nitrification inhibitors. The examples refer to compounds of a formula | where R3 and R4 are one

2 BE2022/5162 may form a carbocyclic residue. The definition in claim 1 also allows R3 and

R4 forms a heterocyclic ring, but no specific structural examples are given for this very broad definition. The compounds contain a C3-6 alkynyl group attached to one pyrazole nitrogen atom.

US 2020/0062755 A1 discloses pesticidally active heterocyclic derivatives with sulfur-containing substituents containing an N=S structural element. Specific compounds contain a pyrazolopyridine group in which the pyridine nitrogen is far from the pyrazolone nitrogen, see paragraphs [0151] and [0165] and Table Q.

WO 2010/100475 A1 mentions hydroxamic acid derivatives as gram-negative antibacterial agents. Example 145 B and Example 293 B show compounds containing a pyrazolo[3,4-b]pyridine-4-carboxamide group in which the nitrogen of the carboxamide contains a hydrogen atom and a hydroxyl group.

PCT/EP2021/075026 to the applicant describes the use of certain pyrazolo[3,4-b]pyridine-4-carboxamides as nitrification inhibitors.

The known nitrification inhibitors act on the nitrifying ammonia-oxidizing bacteria (AOB). But ammonia-oxidizing archaea (AOA) also promote the nitrification of ammonia. One of these is the ammonia-oxidizing archaeon

Nitrososphaera viennensis.

J. Zhao et al. in Soil Biology and Biochemistry 141 (2020) 107673 describe the use of simvastatin as a specific AOA inhibitor in pure culture and in soil.

Simvastatin is a substituted hexahydronaphthalene dimethyl butanuate.

It would be desirable to use (further) nitrification inhibitors that act on ammonia-oxidizing archaea (AOA) and/or comammox bacteria and that can preferably improve the nitrification inhibition when combined with an AOB inhibitor.

The object of the present invention is to provide new nitrification inhibitors. Another object of the invention is the development of a nitrification inhibitor that acts on microorganisms, in particular on ammonia-oxidizing archaea (AOA).

Another object of the invention is the development of a fertilizer mixture containing such a nitrification inhibitor, a method for its preparation and a method for fertilizing the soil using this mixture.

3 BE2022/5162

The objectives are achieved through the use of a pyrazolo[3,4-b]pyridine of the general formula (A)

RA

? /

RS ) u“ xt ad ER

R1 R3

R2 (A) with the following definitions:

R1 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, preferably an amide or carboxylic acid,

R2 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, also consisting of R1 and R2, together with the nitrogen atom connecting them, may form a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally also contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, R2 is preferably hydrogen,

R3 hydrogen, or C1-12 hydrocarbon residue which may contain one heteroatom selected from the group consisting of nitrogen, oxygen and sulphur, where R3 is preferably hydrogen

R4 hydrogen, or C1-8 hydrocarbon residue, preferably hydrogen

R5 hydrogen or C1-4 alkyl, preferably hydrogen.

4 BE2022/5162 as a nitrification inhibitor, preferably acting on an inhibitory AOA and/or comammox.

In one embodiment, R1 is hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide.

In one embodiment, R2 is hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide.

In one embodiment, R1 or R2 are hydrogen or methyl.

In one embodiment, R1 and R2 are hydrogen or methyl.

In one embodiment, R3 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and preferably hydrogen, methyl, ethyl or iso-propyl.

In one embodiment, R4 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and preferably hydrogen, methyl, ethyl or iso-propyl.

In one embodiment, R5 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and preferably hydrogen, methyl, ethyl or iso-propyl.

Accordingly, in a preferred embodiment, the compound of formula (A) has the following side chains:

R1 hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide,

R2 is hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide, where at least R1 or R2 is hydrogen or methyl,

R3 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and is preferably hydrogen, methyl, ethyl or isopropyl,

R4 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and which is preferably hydrogen, methyl, ethyl or isopropyl, and

R5 hydrogen or a C1 -C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and is preferably hydrogen, methyl, ethyl or isopropyl. In one aspect, the pyrazolo[3,4-b]pyridine of the general formula (A) is not a pyrazolo[3,4-b]pyridine-4-carboxamide of the general formula (1) as described in patent claim 1 and the patent claims referring to patent claim 1 of international patent application PCT/EP2021/075026 to the applicant.

In one aspect, the pyrazolo[3,4-b]pyridine of the general formula (A) is not one of the exemplary compounds 1 to 165 of Table 1 of International Patent Application

PCT/EP2021/075026 to the applicant.

Another object is the use of any of the pyrazolo[3,4-b]pyridine compounds of formulas (1) to (XX),

HAC CH,

HN N SNS

N | A \ A N A

H.C©

CHs

H,C \ O NH

N N CH, 1

N

“ \ A CH,

N N

H,C

OOH H,C CH;

CH:

6 BE2022/5162 #4

N N CH: H.C / “ | \n CH

N N 3 \ N / A

Lo a

Hd NS Na” " O H.C °

CH,

HO o H;C \ N CH,

SCT

A | \IN \A CH;

N N

H,C I CHs HNO

CH,

CH3 CH;

ACH, ACH,

N N

/CC| CH, /CC| CH;

N N

\ n A 0 NH» oO NH»

7 BE2022/5162

CH

H3C CH3 CH

Ç A CH; /pt| CH, 3N

N Dr CH,\N %>

CH

H3C

A vt CH \ ; NCH3

NOTE ° AT à A = | 5 \ > O

H,C

H,C 3 HN ° NP ch,

CH

A H3C CH3

N N CH;

AU N N

Ni\| Ç X CC | CH3

D À NEN o

H3COH

OH

8 BE2022/5162

CH;

CH3

N

N N

N

/ACCH/| CF

N | \ ° À _ N x © x © _ HN ei ch (XVII) (XVIII)

H3C CF HC CH, \N \N / 7 CHs / CC CH,

N \ | N

N Ad

H,C H3C (XVIV) (XX) as a nitrification inhibitor.

In another aspect, the pyrazolo[3,4-b]pyridine compound is one of the compounds in Table 1 below.

In another aspect, the pyrazolo[3,4-b]pyridine compound specifically of formula (I) to (VIII) inhibits microorganisms selected from the group of ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB), or combinations thereof.

In another aspect, the pyrazolo[3,4-b]pyridine compound is a pyrazolo[3,4-b]pyridine compound of formula (1) and wherein the pyrazolo[3,4-b]pyridine-4-carboxamide of formula (I ) has an inhibitory effect on ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB).

9 BE2022/5162

Another aspect is that the pyrazolo[3,4-b]pyridine compound of formulas (II) to (XX) has an inhibitory effect on ammonia-oxidizing archaea (AOA).

In another aspect, the pyrazolo[3,4-b]pyridine compound especially of formulas (Il) to (VIII), even more preferably of formula (VIII), is administered in combination or admixture with a compound selected from the group consisting of 2-(3,4-dimethyl-pyrazole-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazole phosphate (DMPP).

The compounds of general formula (A) act as nitrification inhibitors.

In addition, they also act as a nitrogen stabilizer in liquid fertilizers or manure.

Fertilizers can be organic and/or inorganic and/or organomineral fertilizers.

The object is further achieved by the use of pyrazolo[3,4-b]pyridine as an additive or coating material for, preferably inorganic, fertilizers, preferably ammonium and/or urea-containing nitrogenous fertilizers.

The objective is further achieved by the use of pyrazolo[3,4-b]pyridine for reducing the nitrogen or carbon losses in inorganic and/or organic and/or organomineral fertilizers and also on crop waste and feedlots or during the storage of liquid manure, and for reducing the ammonia load in animal stables.

It may further be useful to use the nitrification inhibitor of pyrazolo[3,4-b]pyridine in combination with or mixed with an additional agrochemical, preferably selected from the group consisting of - at least one other 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-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-mercapto-benzothiazole, 2-sulfanilamidothiazole, 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 bisulfide, ammonium thiosulfate, sodium trithiocarbonate, 2,3-dinydro-2,2-dimethyl-7-benzofuranol methylcarbamate and N-(2, 6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, comammox inhibitors,

10 BE2022/5162 - at least one urease inhibitor, preferably selected from N-butylthiophosphoric acid triamide (NBTPT) and/or N-n-propylthiophosphoric acid triamide (NPPTT), - at least one common agrochemical adjuvant, preferably selected from the group consisting of aqueous and/or organic solvents, pH adjusters, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, sequestering agents anti-zinc agents, coalescing agents, rheology modifiers, anti-foaming agents, photoprotectants, antifreeze agents, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrating agents agents, anti-caking agents, mineral and/or vegetable oils and/or waxes, dyes and antifouling agents, and mixtures thereof.

When the additional nitrification inhibitor is used, the weight ratio between pyrazolo[3,4-b]pyridine and the additional nitrification inhibitor is preferably 0.1 to 10 : 1, preferably 0.2 to 5 : 1, and most preferably 0 .5 to 2:1.

Therefore, the nitrification inhibitor of the present invention can advantageously be used together with or in combination with nitrification inhibitors that preferably inhibit ammonia oxidizing bacteria (AOB) and/or comammox bacteria.

In addition, archaea are not or only slightly targeted by most (AOB) nitrification inhibitors. When the new archaea-targeted nitrification inhibitors were used together with other nitrification inhibitors, nitrification in farmland was synergistically reduced. This indicates, on the one hand, that archaea make a significant contribution to nitrification in agricultural soil, and, on the other hand, more importantly, that the new type of nitrification inhibitor can be used to further reduce nitrogen losses, including reduction of greenhouse gas emissions.

Analysis of DMP-treated soil for the abundance of ammonia-oxidizing archaea showed that, as expected, DMP had no inhibitory effect on the presence of archaea. In contrast, DMP had a positive effect on the abundance of ammonia-oxidizing archaea in some cases. Thus, while DMP inhibits bacterial nitrification, it may enhance archaeal nitrification, probably because

DMP blocks bacteria that compete for the same substrate as the archaea. This also explains the synergistic effect. Combining the two types of inhibitors is possible

11 BE2022/5162 lead to a further reduction of nitrogen losses, and thus of the N demand and the adverse effect of N fertilizers on the environment and climate.

Furthermore, especially when the inorganic fertilizer contains urea, the nitrification inhibitor can also be used together with or in combination with a urease inhibitor, preferably selected from N-n-butylthiophosphoric triamide (NBTPT or

NBPT) and/or N-n-propylthiophosphoric triamide (NPPTT or NPPT).

When the carboxamide compound of general formula (A) of the present invention is combined with N-n-butylthiophosphorus triamide (NBTPT) and/or N-n-propylthiophosphorus triamide (NPPTT), the weight ratio between nitrification inhibitor(s) and urease inhibitor is preferably between 0.1 and 10 : 1, preferably between 0.5 and 8 : 1, and preferably between 1 and 6 : 1.

Therefore, the invention also relates to a mixture containing at least one pyrazolo[3,4-b]pyridine of the general formula (A)

N°

Î; /x

RS” zi

Te”Ki>

R1 R3

R2 (A) with the following definitions:

R1 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, preferably an amide or carboxylic acid,

12 BE2022/5162

R2 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, and it is also possible that R1 and R2, together with the nitrogen atom connecting them, a 5- or 6-membered saturated or unsaturated heterocyclic radical, which may optionally also contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, R2 is preferably hydrogen,

R3 hydrogen or C1-12 hydrocarbon residue which may contain one heteroatom selected from the group of nitrogen, oxygen and sulphur, wherein R3 is preferably hydrogen;

R4: hydrogen or C1-8 hydrocarbon residue, preferably hydrogen

R5 hydrogen or C1-4 alkyl, preferably hydrogen, and at least one additional agrochemical agent, preferably selected from the group consisting of - at least one other 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-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 - mercapto-benzothiazole, 2-sulfanilamidothiazole, 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-dinydro-2,2-dimethyl-7-benzofuranol methylcarbamate and N-(2,6-dimethylphenyl)-N-( methoxyacetyl)-alanine methyl ester, comammox inhibitors, - at least one urease inhibitor, preferably selected from N-butylthiophosphoric acid triamide (NBTPT) and/or N-n-propylthiophosphoric acid triamide (NPPTT), - at least one common agrochemical adjuvant, preferably selected from the group consisting from aqueous and/or organic solvents, pH regulators, surfactants, wetting agents, spreading agents, adhesion promoters, carriers, fillers, viscosity regulators, emulsifiers, dispersants, sequestering agents, anti-zinc agents, coalescing agents,

13 BE2022/5162 rheology modifiers, defoamers, photoprotectants, antifreezes, biostimulants, pesticides, biocides, plant growth regulators, safeners, penetrants, anti-caking agents, mineral and/or vegetable oils and/or waxes, dyes and anti-drift agents, and mixtures thereof.

Preferably, the usual agrochemical aid is not an aqueous and/or organic solvent, mineral and/or vegetable oil or wax.

A combination with at least one other nitrification inhibitor and/or urease inhibitor is preferred.

According to the present invention, it has been found that chemical structures containing pyrazolo[3,4-b]pyridine are strong new nitrification inhibitors and, unlike the usual nitrification inhibitors, are directed against ammonia-oxidizing archaea (AOA) and preferably also against comammox bacteria , but usually not against bacteria (AOB). Therefore, they can be combined with another nitrification inhibitor targeting bacteria (AOB) and/or comammox bacteria for a better nitrification-inhibiting effect.

The new type of nitrification inhibitors is able to inhibit nitrification, especially in soil, and when combined with a bacterial nitrification inhibitor, i.e. a nitrification inhibitor acting on ammonia oxidizing bacteria (AOB) and possibly comammox bacteria inhibitors, it increases the efficiency of nitrification inhibition to above the level possible with the currently used inhibitors.

In one aspect, a pyrazolo[3,4-b]pyridine of the general formula (A)

14 BE2022/5162

HA

/ / \

RS © Wed Sfee Nd

R1 R3

R2 (A) with the following definitions:

R1 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulphur, preferably an amide or carboxylic acid,

R2 hydrogen or C1-12 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur, where R1 and

R2, together with the nitrogen atom connecting them, may also form a five- or six-membered saturated or unsaturated heterocyclic radical, which may optionally also contain one or two other heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur , R2 is preferably hydrogen,

R3 hydrogen or C1-12 hydrocarbon residue which may contain one heteroatom selected from the group of nitrogen, oxygen and sulphur, wherein R3 is preferably hydrogen;

R4: hydrogen or C1-8 hydrocarbon residue, preferably hydrogen

R5-hydrogen or C1-4-alkyl, preferably hydrogen, is used as a nitrification inhibitor on solid fertilizers, preferably in combination with a fertilizer, preferably an (ammonium) nitrogen-containing fertilizer, e.g. solid or

15 BE2022/5162 liquid inorganic, organic and/or organomineral fertilizer, or manure. For example, the heterocyclic compound is used as a nitrification inhibitor or is used as a nitrogen inhibitor or as a nitrogen stabilizer in liquid organic and/or inorganic or organomineral fertilizers or manure.

The pyrazolo[3,4-b]pyridine compounds are mostly known per se and can be synthesized by standard techniques. They are partly commercially available compounds and can be involved in Enamine (SIA Enamine,

Vestienas iela 2 B, LV-1035 Riga, Latvia - https://enamine.net/).

Preferably, the compounds of general formula (A) are free of alkynyl or allenyl groups or structural elements. Preferably, the compounds of general formula (A) do not contain C3-6 alkynyl groups or C3-6 allenyl groups, especially those groups depicted as R1 in formula | of claim 1 of US 2018/0016199 A1.

Furthermore, the compounds of the general formula (A) are preferably free of the structural element N=S. More preferably, the compounds of the general formula (A) are free of the structural element C(=O)-N=S, in particular the corresponding structural element depicted in formula 1 in claim 1 of US 2020/0062755 A1.

The compounds of general formula (A) are canceled or substituted pyrazole compounds.

Specifically, R4 in general formula (A) is not an alkynyl or allenyl group and does not contain an alkynyl or allenyl group, more specifically a C3-6 alkynyl or C3-6 allenyl group, e.g. as described in US 2018/0016199 A1 in formula | as group R1.

In the compounds of the general formula (A), some of the residues are hydrocarbon residues. Hydrocarbon residues are composed of hydrogen and carbon atoms. They can be saturated, unsaturated or aromatic. In addition, they may contain the heteroatoms mentioned above. The hydrocarbon residues may be linear, branched or cyclic, or may contain a linear or branched residue and a cyclic residue in the same structure.

If more than one heteroatom is present in the structure, heteroatoms of the same type are not directly covalently linked. Preferably, the heteroatoms are not directly covalently linked to other heteroatoms, but

16 BE2022/5162 they cut through the hydrocarbon residue. Therefore, the heteroatoms are preferably non-adjacent.

Preferably R1 and/or R5 are independently hydrogen, methyl or ethyl, preferably hydrogen or methyl.

Preferably R2 is a C2-10 hydrocarbon residue, containing one or two heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur, and preferably a C3-8 hydrocarbon residue, which may contain one or two heteroatoms selected from the group nitrogen, oxygen and sulfur.

Preferably R2 contains as heteroatoms one or two oxygen atoms, or a nitrogen atom, or a nitrogen atom and an oxygen atom, or a nitrogen atom and a sulfur atom.

Preferably R2 is a saturated linear or branched hydrocarbon residue, a linear hydrocarbon residue containing a cyclic heteroalkyl residue or an aromatic or heteroaromatic residue, wherein the hydrocarbon residue may contain one or two heteroatoms selected from the group consisting of oxygen and nitrogen, or wherein R2 is an unsaturated heterocyclic residue is.

Preferably R3 is a C3-7 hydrocarbon residue which may contain one oxygen atom, preferably a branched or cyclic hydrocarbon residue which may contain one oxygen atom.

Preferably R4 is C1-8 alkyl, preferably C1-6 alkyl, more preferably C1-4 alkyl. Most preferably R4 is selected from methyl, ethyl, isopropyl and tert. -butyl.

Alkyl residues can be linear or branched.

Cyclic groups in the residues are preferably 5 or 6 membered rings which may be saturated, unsaturated or aromatic. The rings may be purely hydrocarbon, e.g. phenyl groups. They may also contain nitrogen or oxygen as a heteroatom, preferably one nitrogen or oxygen atom. Examples of these residues are mentioned in the examples below.

The compounds of general formula (A) are nitrification inhibitors that inhibit the ammonia-oxidizing archaea (AOA), including the ammonia-oxidizing archaeon Nitrososphaera viennensis, Ammonia Binding Liquid Inoculum (ABIL) and preferably also comammox bacteria (complete ammonia oxidation) such as Candidatus

Nitrospira kreftii.

17 BE2022/5162

The nitrification inhibitor of the present invention is particularly notable for effectively inhibiting the nitrification of ammonium nitrogen in the soil for a long period of time.

The nitrification inhibitor of the present invention is expected to have favorable toxicological properties, low vapor pressure and good soil sorbability. As a result, the nitrification inhibitor is not expelled into the atmosphere by sublimation to any significant extent, nor is it easily washed out by water. This primarily results in economic benefits, such as high profitability given the longer-lasting effect of the nitrification inhibitor, and environmental benefits, such as a reduction in the pollution of the air (climate gas reduction) and of surface and ground water.

The nitrification inhibitors can be applied to soils or substrates fertilized with an inorganic or organic or organomineral fertilizer. Usually they are applied in a fertilizer mixture containing a (preferably inorganic) fertilizer and the pyrazolo[3,4-b]pyridine compound. Usually, the pyrazolo[3,4-b]pyridine is used in an amount of 10 to 10,000 ppm by weight, preferably 100 to 10,000 ppm by weight, based on the (preferably inorganic) fertilizer without water. The amount used is based on the dry fertilizer.

The nitrification inhibitor of the present invention can be used in dust, solution, dispersion or emulsion. Therefore, the invention also relates to a solution, dispersion or emulsion containing the pyrazolo[3,4-b]pyridine of the present invention, preferably in an amount of 0.1 to 50 wt®%, preferably 0.5 to 30 wt%, and preferably 1 to 20 wt%.

Preferably, according to the present invention, a fertilizer mixture is formed which contains compounds A and B

A. an inorganic and/or organic and/or organomineral fertilizer and

B. 10 to 10000% by weight, preferably 100 to 10000% by weight, based on the, preferably inorganic, fertilizer, of the pyrazolo[3,4-b]pyridine compound as defined above.

The water fraction in component A and in the fertilizer mixture is often not more than 1.5 wt%, preferably not more than 1.0 wt%, preferably not more than 0.5 wt%, preferably not more than 0.3 wt%, and is therefore negligible in the balance of the

18 BE2022/5162 quantities. Components A and B preferably form at least 95% by weight, preferably at least 98% by weight of the fertilizer mixture.

The nitrogen content of compound A (without water) is often at least 12 wt%, preferably at least 20 wt%, preferably at least 22 wt%. The nitrogen content may be, for example, 25 to 29 wt%, in particular 26 to 28 wt%. The nitrogen content can be divided between fast-acting nitrate nitrogen and slow-acting ammonium nitrogen.

The inorganic fertilizers are preferably ammonium and/or urea-containing fertilizers, preferably ammonium-containing fertilizers which may additionally contain urea.

Fertilizers containing urea are further described in WO 2016/207210.

The fertilizers used in the present invention may be of natural or synthetic origin and are applied to the soil or plant tissues to provide one or more plant nutrients essential to plant growth. The fertilizers used according to the present invention must provide at least 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 contain carbonaceous substances, except urea. Organic fertilizers are usually plant or animal derived material. Organomineral fertilizers (combinations of inorganic and organic fertilizers) can also be used.

The main nitrogen-based straight fertilizer is ammonia or its solutions. Ammonia nitrate is also widely used. Urea is another popular source of nitrogen, with the advantage of being solid and non-explosive. Another nitrogen-based fertilizer is calcium ammonium nitrate.

The main single phosphate fertilizers are the superphosphates, including single superphosphate, phosphogypsum and triple superphosphate. The most important potassium-containing straight fertilizer is potassium muriate (MOP).

The binary fertilizers are preferably NP or NK fertilizers, which can consist of monoammonium phosphate (MAP) and diammonium phosphate (DAP).

NPK fertilizers are three-component fertilizers that provide nitrogen, phosphorus and potassium. NPK fertilizers can be produced by mixing the

19 BE2022/5162 the above straight fertilizers in bulk or in granules per piece, as in

Nitrophoska®. In some cases, chemical reactions can occur between the two or more components.

In addition to the main components, such as N, P and K, micronutrients (trace elements) may be present in the fertilizers. The most important micronutrients are molybdenum, zinc, boron and copper. These elements are usually supplied as water-soluble salts.

The preferred fertilizers contain ammonium or urea. Examples of preferred ammonium-containing fertilizers are NPK fertilizers, potassium ammonium nitrate, ammonium sulfate nitrate, ammonium sulfate and ammonium phosphate.

Other preferred ingredients of the fertilizer compositions are, for example, trace elements, other minerals, standardizers and binding agents.

Organic fertilizers are fertilizers of organic or biological origin, i.e. fertilizers derived from living or formerly living materials, such as animals, plants or algae. Fertilizers of biological origin include animal waste, vegetable waste, for example from food processing or agriculture, compost and treated sewage sludge (biosolids). Animal sources can be manure, but also products from animal slaughter, such as blood meal, bone meal, feather meal, hides, hooves and horns.

Soil conditioners, such as peat or coir, bark and sawdust, can also be added.

Fertilizers may include: 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, calcite limestone, calcium oxide, hampene (chelated iron), dolomite limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, potassium nitrate, potassium bicarbonate, monopotassium phosphate, magnesium nitrate, magnesium sulfate, potassium sulfate, potassium chloride, sodium nitrates, magnesian limestone, magnesia, disodium dihydromolybdate, cobalt chloride hexahydrate, nickel chloride hexahydrate, indolebutyric acid, L-tryptophan,

20 BE2022/5162 urea, urea-formaldehydes, urea-ammonium nitrate, urea with sulfur coating, urea with polymer coating, isobutylidene diurea, K2504-2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulphur, marl, ground oyster shells, fish meal, oil cake, fish manure, blood meal, rock phosphate, superphosphates, slag, bone meal, wood ash, biochar, algae, algae extracts, struvite, manure, bat guano, peat dust, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion or a combination thereof. The micronutrient fertilizer material can be boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron sfrit, an iron chelate, a manganese sulfate, a manganese sulfate, an iron oxide, an iron oxide, an iron chelate or a combination thereof, manganese sulfate, manganese oxide, manganese chelate, manganese chloride, manganese frit, sodium molybdate, molybdiacid, zinc sulfate, zinc oxide, zinc carbonate, zinc frit, zinc phosphate, zinc chelate or a combination thereof. In a particular embodiment, said fertilizer or fertilizer composition does not contain insoluble selenium, selenium mineral, soluble selenium or salts thereof.

The treated (inorganic, organic or organomineral) fertilizers according to the invention are preferably present in powder form, in prill form or in granular form.

In addition to the heterocyclic carboxamide compound of formula (I), formulations containing the compound and agronomic aids can be used to incorporate the nitrification inhibitor into the fertilizer. Agronomic aids are, for example, solvents, dispersants, pH regulators, fillers, stability improvers, surfactants.

The nitrification inhibitor can be incorporated into the fertilizer mixture by mixing the mixture or 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 organineral) fertilizer.

In a process for the production of the fertilizer mixture of the present invention, the nitrification inhibitor or the formulation containing it can be incorporated into the (inorganic, organic or organomineral) fertilizer and/or applied to the surface of the (inorganic, organic or organomineral) fertilizer.

21 BE2022/5162

Fertilizer granules are impregnated or coated with the nitrification inhibitor, for example by spraying them with a formulation such as a solution or a dispersion of the nitrification inhibitor and then drying them. The method is known, for example, from DE-

A-41 28 828. Sealing the impregnated granules with, for example, paraffin wax, an additional proposal in the latter document, is possible, but generally not necessary.

Granulating auxiliaries that can be used for the preparation of solid fertilizer compositions can be lime, gypsum, silica or kaolinite.

An alternative is the addition of the nitrification inhibitor during the actual production of the fertilizer, for example in the slurry.

As a rule, nitrification inhibitors are applied to the soil in amounts of 100 g/ha to 10 kg/ha. Preferably, the amount is between 200 g/ha and 5 kg/ha.

The application of the nitrification inhibitor in liquid fertilizer formulations can be effected, for example, by fertigation with or without excess water, as described in DE-C-102 30 593.

The fertilizer mixture may contain at least one additional nitrification inhibitor. This at least one further nitrification inhibitor preferably inhibits the 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-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-mercapto-benzothiazole, 2-sulfanilamidothiazole, 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 tricarbonate, 2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate and N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester.

When the additional nitrification inhibitor is used, the weight ratio between pyrazolo[3,4-b]pyridine and the additional nitrification inhibitor is preferably 0.1 to 10 : 1, preferably 0.2 to 5 : 1, and most preferably 0 .5 to 2:1.

22 BE2022/5162

Furthermore, the fertilizer mixture may contain at least one urease inhibitor, which is preferably selected from N-n-butylthiophosphoric acid triamide (NBTPT or NBPT) and/or N-n-propylthiophosphoric acid triamide (NPPTT or NPPT). A urease inhibitor is usually added when the fertilizer contains urea. Nitrogen from urea is released in the form of ammonium by the action of urease, and the ammonium can undergo nitrification.

Therefore, it may be advantageous to combine a urease inhibitor with the nitrification inhibitor.

When the pyrazolo[3,4-b]pyridine of the present invention is combined with

N-n-butylthiophosphorus triamide (NBTPT) and/or N-n-propylthiophosphorus triamide (NPPTT), the weight ratio between nitrification inhibitor and urease inhibitor is preferably between 0.1 and 10:1, preferably between 0.5 and 8:1, and preferably between 1 and 6:1.

Thiophosphorus triamides are known to be converted relatively readily to the corresponding phosphorus triamides and thiophosphorus diamides, as well as to other metabolites. Since moisture cannot generally be completely excluded, thiophosphorus-containing triamides and the corresponding phosphorus-containing triamides are often present in a mixture with each other. In this specification, the term "(thio)phosphoric acid triamide" therefore refers not only to the pure thiophosphoric acid triamides or phosphoric acid triamides, respectively, but also to mixtures thereof.

Mixtures of N-n-butylthiophosphoric triamide and N-(n-)propylthiophosphoric triamide can also be used according to the present invention, as described in EP-A-1 820 788.

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

Furthermore, coatings of powders, prills and granules can be formed from inorganic material, such as sulfur or mineral based coatings, or with an organic polymer.

Respective coatings are described in WO 2013/121384 at page 23, line 37 to page 24, line 16.

As mentioned above, the agrochemical formulations containing the compounds of formula (|) are used in "effective amounts". This means that they are used in an amount that makes it possible to obtain the desired effect, namely a (synergistic) improvement in the health of a plant, but which does not give rise to phytotoxic symptoms on the treated plant.

23 BE2022/5162

For use in accordance with the present invention, the agrochemical formulations containing the compounds of formula (|) may 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 any case, it should ensure a fine and uniform distribution of the agrochemical formulations containing the compounds of formula (1) according to the present invention. The formulations are prepared in a manner known to those skilled in the art.

The agrochemical formulations may also contain excipients commonly used in agrochemical formulations. Which excipients are used depends on the specific form of application and the active substance, respectively. Examples of suitable excipients are solvents, solid carriers, dispersants or emulsifiers (such as further solubilizers, protective colloids, surfactants and adhesives), organic and inorganic thickeners, bactericides, antifreezes, antifoams, optionally colorants and tackifiers or binders (for example for formulations for seed treatment).

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

Solid carriers are mineral earth metals such as silicates, silica gels, talc, kaolin, limestone, chalk, boulder clay, loess, clay, dolomite, diatomaceous earth, calcium sulphate, magnesium sulphate, magnesium oxide, ground synthetic substances, fertilizers, such as e.g. ammonium sulphate, ammonium phosphate, ammonium nitrate, urea and products of vegetable origin, such as cereal flours, bark flours, wood flours and nut shell flours, cellulose powders and other solid carriers.

Suitable surfactants (auxiliaries, wetting agents, tackifiers, dispersants or emulsifiers) are alkali metal, alkaline earth metal and

24 BE2022/5162 ammonium salts of aromatic sulfonic acids, such as lignin sulfonic acid, phenol sulfonic acid, naphthalene sulfonic acid, dibutylnaphthalene sulfonic acid and fatty acids van naftaleensulfonzuur met fenol en formaldehyde polyoxy-ethyleen-octylfenylether, geëthoxyleerd isooctylfenol, octylfenol, nonylfenol, alkylfenylpolyglycolethers, tributylfenylpolyglycolether, tristearylfenylpolyglycolether, alkylarylpolyetheralcoholen, alcohol- en = vetalcohol/ethyleenoxidecondensaten, geëthoxyleerde ricinusolie, polyoxyethyleenalkylethers, geëthoxyleerd polyoxypropyleen, lauylalcoholpolyglycoletheracetaal, sorbitolesters, lignine-sulfietafvalvloeistof and proteins, denatured proteins, polysaccharides (e.g., methylcellulose), hydrophobically modified starches, polyvinyl alcohols, polycarboxylates, polyalkoxylates, polyvinylamines, polyvinylpyrrolidone, and the copolymers thereof. Examples of thickeners (i.e., compounds which impart modified fluidity to formulations, i.e., high viscosity under static conditions and low viscosity under agitation) are polysaccharides and organic and inorganic clays such as xanthan gum.

A "pesticide" is anything that prevents, destroys or controls a harmful organism ("pest") or disease, or protects plants or plant products during production, storage and transportation.

The term includes, but is not limited to: herbicides, fungicides, insecticides, acaricides, nematicides, molluscicides, rodenticides, growth regulators, repellents, rodenticides and biocides, as well as plant protection products.

Plant protection products are "pesticides" that protect crops or desired or intended plants. They are mainly used in the agricultural sector, but also in forestry, horticulture, in recreational areas and in private gardens. They contain at least one active substance and have one of the following functions: - to protect plants or plant products against pests/diseases, before or after harvest; -affect the life processes of plants (such as substances that influence their growth, with the exception of nutrients), -preserve plant products;

25 BE2022/5162 - destroying or preventing the growth of unwanted plants or parts of plants.

They may also contain other ingredients, including safeners and synergists.

An active substance is a chemical, plant extract, pheromone or micro-organism (including viruses) that is active against "harmful organisms" or on plants, parts of plants or plant products.

The most common use of pesticides is in the form of plant protection products (GBPs).

The term "pesticide" is often used interchangeably with "plant protection product", but pesticide is a broader term that includes applications other than plants/crops, for example biocides.

Biocides, such as herbicides, bactericides, molluscicides, algicides, phytotoxic agents, fungicides, and mixtures thereof may be added.

Bactericides may be added to preserve and stabilize the formulation. Examples of suitable bactericides are those based on dichlorophene and benzyl alcohol hemi-formal (Proxel® from ICI or Acticide® RS from Thor Chemie and

Kathon® MK from Rohm & Haas) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® M BS from Thor Chemie). Examples of suitable antifreezes are ethylene glycol, propylene glycol, urea and glycerine. Examples of anti-foaming agents are silicone emulsions (such as, for example, Silicon® SRE,

Wacker, Germany or Rhodorsil®, Rhodia, France), long chain alcohols, fatty acids, salts of fatty acids, fluoroorganic compounds and agrochemical formulations containing the compounds of formula (I) thereof.

Suitable dyes are pigments of low water solubility and solvent soluble, e.g. water-soluble dyes.

Examples of adhesion promoters, such as tackifiers or binders, are polyvinylpyrrolidones, polyvinyl acetates, polyvinyl alcohols, and cellulose ethers (Tylose®,

Shin Etsu, Japan).

26 BE2022/5162

Granulates, for example coated granules, impregnated granules and homogeneous granules, can be prepared by binding the active substances to solid carriers. Examples of solid carriers are mineral earths such as silica gels, silicates, talc, kaolin, attaclay, limestone, chalk, chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium sulphate, magnesium sulphate, magnesium oxide, ground synthetic substances, fertilizers, such as, for example, ammonium sulphate , ammonium phosphate, ammonium nitrate, urea, and products of plant origin, such as cereal flour, bark flour, wood flour and nut shell flour, cellulose powders and other solid carriers.

Anti-caking agents such as oils and/or waxes can be added.

The agrochemical formulations generally contain between 0.01 and 95%, preferably between 0.1 and 90%, and most preferably between 0.5 and 90%, by weight of active substances. The compounds of the agrochemical formulations containing the compounds of formula (1) are used in a purity of from 90% to 100%, preferably from 95% to 100% (according to their NMR spectrum).

The compounds of the agrochemical formulations containing the compounds of formula (I) can be used as such or in the form of their agricultural compositions, e.g. in the form of directly sprayable solutions, powders, suspensions, dispersions, emulsions, oil dispersions, pastes, dustable products, scattering agents or granules, by means of atomising, spraying, spraying, spreading, brushing, dipping or pouring. The forms of application depend entirely on the intended purposes; in any case, the intention is to ensure the best possible distribution of the compounds present in the agrochemical formulations containing the compounds of formula (I).

Aqueous application forms can be prepared from emulsion concentrates, pastes or wettable powders (sprayable powders, oil dispersions) by adding water.

For the preparation of emulsions, pastes or oil dispersions, the substances, as such or dissolved in an oil or solvent, can be homogenized in water using a wetting agent, tackifier, dispersant or emulsifier. Alternatively, concentrates can be prepared consisting of an active substance, a wetting agent, a tackifier, a dispersant or an emulsifier and optionally a solvent or oil, and such concentrates are suitable for dilution with water.

27 BE2022/5162

The concentrations of the active substances in the ready-to-use preparations can be varied within relatively wide margins. In general, they are between 0.0001 and 10%, preferably between 0.001 and 1%, by weight of the compounds of the agrochemical formulations containing the compounds of formula (I).

The agrochemical formulations containing the compounds of formula (1) can also be successfully used in the ultra-low volume process (ULV), where it is possible to use compositions containing more than 95% by weight of active substance, or even to apply the active substance without additives.

Different types of oils, wetting agents, additives, herbicides, fungicides, other pesticides or bactericides can be added to the active compounds, possibly just before use (tank mix). These agents can be mixed with the compounds of the agrochemical formulations containing the compounds of formula (I) in a weight ratio of 1:100 to 100:1, preferably 1:10 to 10:1.

The compositions of this invention may also contain fertilizers (such as ammonium nitrate, urea, potassium and superphosphate), phytotoxic agents and plant growth regulators (plant growth amendments) and safeners. These can be used sequentially or in combination with the compositions described above, possibly also added just before use (tank mix). For example, the plant(s) may be sprayed with a composition of this invention either before or after treatment with the fertilizers.

In the agrochemical formulations containing the compounds of formula (I), the weight ratio of the compounds generally depends on the properties of the compounds of the agrochemical formulations containing the compounds of formula (|).

The agrochemical formulations containing the compounds of formula (I) can be used individually or already wholly or partially mixed together to prepare the composition according to the invention. They can also be packaged and further used as a combination composition such as a kit of parts.

The user usually applies the composition of the invention from a pre-doser, a backpack sprayer, a spray tank or a spray plane. The agrochemical composition is hereby prepared with water and/or buffer to the desired level

28 BE2022/5162 application concentration, to which other adjuvants may optionally be added, thus obtaining the ready-to-use spray liquid or the agrochemical composition according to the invention. Usually 50 to 500 liters of ready-to-use spray liquid are applied per hectare of useful agricultural area, preferably 50 to 400 litres.

In a particular embodiment, the absolute amount of the active substances, represented by formula (I), used is between 1 mg/litre and 100 mg/litre, in particular between 1 mg/litre and 20 mg/litre, in particular between 1 mg/litre and 25 mg/litre, especially between 2 mg/litre and 200 mg/litre especially between 2 mg/l and 100 mg/l, especially between 2 mg/l and 50 mg/l, especially between 2 mg/l and 25 mg/l, especially between 4 mg/l and 40 mg/l, especially between 4 mg/l and 20 mg/l, especially between 4 mg/l and 16 mg/l, and especially between 4 mg /l and 12 mg/l.

In one embodiment, the individual compounds of the agrochemical formulations containing the compounds of formula (I), formulated as a composition (or formulation) such as parts of a kit or parts of the inventive mixture, may be mixed by the user in a spray tank and further excipients can be added, if applicable (tank mix).

The term "agrochemical" means here: any active substance that can be used in the agrochemical industry (including applications in agriculture, horticulture, floriculture and for use in the home and garden, but also products intended for non- crop-related applications such as applications for the control of unwanted insects and rodents in the context of public health/pest control, household use, such as fungicides and insecticides for household use, and products for the protection of plants or parts of plants, crops, bulbs, tubers , fruits (e.g. against 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 parts of plants that are harvested (e.g. the fruits, flowers, seeds, etc.).

An "agrochemical composition" as used herein is a composition for agrochemical use, as defined herein, containing at least one active substance of a compound of formula (|), optionally with one or more additives that provide optimum dispersion, atomization, deposition , wetting of the leaves, distribution,

29 BE2022/5162 promote retention and/or uptake of agrochemicals. Such additives are, for example, diluents, solvents, auxiliaries, surfactants, wetting agents, spreading agents, oils, stickers, viscosity regulators (such as thickeners, penetrants), pH regulators (such as buffers, acidifiers), anti-zinc agents, anti-freezing agents, photoprotectants, anti-foaming agents, biocides and/or drift inhibitors.

A "carrier", as used herein, is a solid, semi-solid or liquid carrier in or onto which an active agent can be suitably incorporated, immobilized, immobilized, adsorbed, absorbed, bound, encapsulated, embedded, attached or compressed. Non-limiting examples of such carriers are nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, a gel, weak ionic resin particles, liposomes, cochleate delivery vehicles, small granules, granules, nanotubes, bucky balls, water droplets that are part of a water-in-oil emulsion, oil droplets that are part of an oil-in-water emulsion, organic materials such as cork, wood or other materials of vegetable origin (e.g. in the form of seed husks, wood 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 fractions or fragments of them.

The terms "effective amount", "effective dose" and "effective amount" as used herein mean the amount necessary to achieve the desired result or results. More exemplary information on amounts, modes of application and appropriate ratios to be used is given below. Those skilled in the art are well aware of the fact that such amount may vary in a wide range and depends on various factors such as the cultivated plant treated and the climatic and soil conditions.

The terms "determine", "measure", "assess", "monitor" and "analyze" as used herein are interchangeable and include both quantitative and qualitative determinations.

It is clear that the agrochemical composition is stable both during storage and use, which means that the integrity of the agrochemical composition is maintained under storage and/or use conditions of the agrochemical composition, which include elevated temperatures, freeze-thaw cycles, changes in pH or in

30 BE2022/5162 ionic strength, UV radiation, presence of harmful chemicals and the like. More preferably, the compounds of formula (I) as described herein remain stable in the agrochemical composition, meaning that the integrity and activity of the compounds are maintained under storage and/or use conditions of the agrochemical composition, which include elevated temperatures, freezing thaw cycles, changes in pH or in ionic strength, UV radiation, presence of harmful chemicals and the like. Preferably, the compounds of formula (1) remain stable in the agrochemical composition when the agrochemical composition is stored at ambient temperature for a period of two years or when the agrochemical composition is stored at 54°C for a period of two weeks. Preferably, the agrochemical composition of the present invention retains at least about 70% activity, preferably at least about 70% to 80% activity, preferably about 80% to 90% activity or more. Examples of suitable carriers include alginates, gums, starches, cyclodextrins, celluloses, polyurea, polyurethane, polyester or clay.

The agrochemical compound can appear in any formulation; preference is given to powders, 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 liquefied concentrates.

The agrochemical composition according to the invention can be applied once to a crop, but can also be applied two or more times in succession with an interval between each two applications. The agrochemical composition according to the invention can be applied to the crop alone or in combination with other materials, preferably other agrochemical compositions; alternatively, the agrochemical composition according to the invention can be applied separately to the crop with other materials, preferably other agrochemical compositions, which are applied to the same crop at different times.

In yet another embodiment, the invention provides a method for the manufacture of ("or the production of" which is equivalent to wording) a

31 BE2022/5162 agrochemical composition according to the invention, consisting of the formulation of a molecule of formula (1) as defined above, together with at least one conventional 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, encapsulation, emulsification, coating, encrusting, pilling, extrusion granulation, fluid bed granulation, coextrusion, spray drying, spray cooling, atomization, addition or condensation polymerization, interfacial polymerization, in situ polymerization, coacervation, spray encapsulation, molten dispersion cooling, solvent evaporation, phase separation, solvent extraction, sol-gel polymerization, fluid bed coating, pan coating, melting , passive or active absorption or adsorption.

The common agrochemical adjuvants are well known and preferably include, but are not limited to, aqueous and/or organic solvents, pH adjusters (such as buffers, acidity regulators), surfactants, wetting agents, spreading agents, adhesion promoters (such as tackifiers, stickers), carriers, fillers, viscosity regulators (such as thickeners), emulsifiers, dispersants, sequestering agents, anti-zinc agents, coalescing agents, rheology enhancers, anti-foaming agents, photoprotectors, anti-freezing agents, biostimulants (including bacterial and/or fungal inoculants or micro-organisms), biocides (preferably selected from herbicides, bactericides, phytotoxic substances, fungicides, pesticides and mixtures thereof), plant growth regulators, protective substances, penetrating substances, anti-caking agents, mineral and/or vegetable oils and/or waxes, dyes and drift-preventing substances or a suitable combination thereof.

The insecticide may be an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biobased insecticide, or a combination thereof. The herbicide used to remove unwanted plants may contain a chlorophenoxy compound, a nitrophenol compound, a nitrocresol compound, a dipyridyl compound, an acetamide, an aliphatic acide, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinon dioxide, a thiocarbamate, a carbamate, a carbanilate, a chloropyridilate or a combination thereof, carbanilate, chloropyridinyl, a

32 BE2022/5162 cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acide, isopropylamine, an isopropylamine derivative, oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endohall, sodium chlorate, or a combination thereof. The fungicide may be a substituted benzene, a thiocarbamate, an ethylene bis-dithiocarbamate, a thiophthalidamide, a copper compound, an organic mercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon , triforine, or a combination thereof.

The fungal inoculant may be any fungal inoculant of the family

Glomeraceae, a fungal inoculant of the family Claroidoglomeraceae, a fungal inoculant of the family Acaulosporaceae, a fungal inoculant of the family Sacculospraceae a fungal inoculant of the family Entrophosporaceae, a fungal inoculant of the family Pacidsproraceae, a fungal inoculant of the family Diversisporaceae, a fungal inoculant of the family Paraglomeraceae, a fungal inoculant of the family Archaeosporaceae a fungal inoculant of the family Geosiphonaceae, a fungal inoculant of the family Ambisporacea, a fungal inoculant of the family Scutellosproaceae, a fungal inoculant of the family Dentiscultataceae, a fungal inoculant of the family Racocetraceae a fungal inoculant of the phylum Basidiomycota, a fungal inoculant of the phylum

Ascomycota, a fungal inoculant of the phylum Zygomycota, a fungal inoculant of 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 bacterial inoculant of the genus Burkholderia , bacterial inoculant of the genus Sinorhizobium, bacterial inoculant of the genus Kluyvera, bacterial inoculant of the genus Azotobacter, bacterial inoculant of the genus

Pseudomonas bacterial inoculant of the genus Azosprillium, bacterial inoculant of the genus Bacillus, bacterial inoculant of the genus Streptomyces, bacterial inoculant of the genus Paenibacillus, bacterial inoculant of the genus Paracoccus, bacterial inoculant of the genus Enterobacter bacterial inoculant of the genus Alcaligenes, bacterial inoculant of the genus Mycobacterium, bacterial inoculant of the genus

33 BE2022/5162

Trichoderma, bacterial inoculant of the genus Gliocladium, bacterial inoculant of the genus Klebsiella, or a combination thereof.

The mixture may additionally contain at least one microorganism selected from the list 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 vinelandii and Clostridium pasteurianum,

Glomus species.

The pyrazolo[3,4-b]pyridines used in the present invention can be used in combination with these excipients. The excipients used depend on the specific application form and the active substance and preferably include solvents, solid carriers, dispersants or emulsifiers, such as solubilizers, protective colloids, surfactants and adhesives. Furthermore, organic and inorganic thickeners, bactericides, antifreezes, antifoams, optionally colorants and tackifiers or binders can be used in combination with the nitrification inhibitors and in the fertilizer mixture. Suitable excipients are discussed in WO 2013/121384 on pages 25 to 26.

Other possible preferred ingredients are oils, humectants, adjuvants, biostimulants, herbicides, bactericides, other fungicides and/or pesticides. These are discussed, for example, in WO 2013/121384 at p. 28/29.

The fertilizer mixtures are preferably in solid form, including powders, prills and granules. It is furthermore possible to administer the nitrification inhibitor in the form of a formulation, solution or dispersion separately or simultaneously with a fertilizer.

In addition, the nitrification inhibitor of the present invention can be used to reduce nitrogen losses in organic fertilizers and also on crop waste and pasture land or during liquid manure storage and monitoring of ammonium load in livestock houses.

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

The present invention also relates to a method of fertilizing agricultural or horticultural soils, wherein a fertilizer mixture containing compounds A and B

34 BE2022/5162

A. an inorganic and/or organic and/or organomineral fertilizer and

B. 10 to 10000 % by weight, calculated on the fertilizer, of a pyrazolo[3,4-b]pyridine as defined above, or the compounds A and B separately, but within a period of 0 to 5 hours, preferably 0 to 1 hour, and preferably at about the same time, is applied to the bottom.

Parallel to the improvement of the nitrogen utilization in the mineral, organic and organomineral fertilizers containing ammonium or urea, the use of the nitrification inhibitors, according to the present invention, and of compositions containing them, has the effect of increasing the yield and the production of crop biomass , in some cases significantly, increases.

Likewise, the nitrification inhibitor according to the invention can be added to organic fertilizers, such as liquid fertilizers, for example during the actual storage of such fertilizers, to prevent nitrogen nutrients from being lost through delayed conversion of the individual nitrogen forms into gaseous, i.e. volatile, nitrogen compounds, and at the same time contribute to a reduction of the ammonia load in animal stables. In addition, the nitrification inhibitor or compositions containing the nitrification inhibitor can be used on agricultural stems and pastures to reduce gaseous nitrogen loss and prevent nitrate leaching.

In general, the pyrazole[3,4-b]pyramine-4-carboxamide of the general formula (A) can be used for reducing the nitrogen or carbon losses in inorganic and/or organic and/or organineral fertilizers or nitrogen or carbonaceous compounds or materials and also on harvest waste and pasture or during the storage of liquid manure and for reducing the ammonia load in animal houses.

Nitrogen losses are often due to N2O and/or NO emissions or NO3 leaching.

Nitrogen losses can occur in nitrogen-containing compounds or materials, such as roots, plants, fertilizers, animals, etc. Nitrogen losses usually occur in nitrogen-containing mineral fertilizers or organic nitrogen-containing substances. The term "nitrogen losses" includes all forms of nitrogen or nitrogen compounds that are lost through emissions or leaching, as indicated above. An example are

35 BE2022/5162 greenhouse gas emissions that can be reduced by using the N-heterocyclic compound of the present invention.

The same applies to carbon losses that can occur in carbon-containing compounds or materials. Carbon-containing compounds or materials are, for example, carbonate mineral fertilizers and carbon-containing organic matter, such as roots, plants, animals, organic fertilizers, etc. Carbon losses often occur in the form of CO2 emissions, which are part of greenhouse gas emissions.

Therefore, the 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 often contain nitrogen and/or carbon in covalently or ionically bonded form, e.g. in the form of ammonium, protein, nitrate, carbonate, carbohydrates, cellulose, or other organic carbonaceous compounds. Thus, the terms "carbon" and "nitrogen" may refer to the elemental form or to compounds or materials containing carbon and/or nitrogen atoms.

Nitrogen and/or carbon-containing compounds or materials may be present in fertilizers, in the soil, or in the environment. The most striking effect of the

N-heterocyclic compounds of the present invention is the reduction of greenhouse gas emissions from soil containing nitrogenous compounds or materials.

The most efficient is the reduction of greenhouse gas emissions from fertilized soil. These greenhouse gas emissions are often caused by processes after nitrification.

The plants to be treated or to be rooted in the soil according to the invention are preferably selected from the group consisting of agricultural, forestry, ornamental and horticultural crops, each in their natural or genetically modified form. Preferably, non-transgenic agricultural plants are treated.

Preferred agricultural crops are field crops selected from the group consisting of potatoes, sugar beets, wheat, barley, rye, oats, sorghum, rice, corn, cotton, oilseed rape, rapeseed, canola, soybeans, peas, field beans, sunflowers, sugarcane cucumbers, tomatoes, onions, leeks, lettuce, pumpkins; even more

Preferably, the plant is selected from the group consisting of wheat, barley, oats, rye, soybeans, maize, rapeseed, cotton, sugar cane, rice and sorghum.

In a preferred embodiment of the invention, the crop to be treated is selected from the group consisting of tomato, potato, wheat, barley, oats, rye, soybean, corn, rapeseed, canola, sunflower, cotton, sugar cane, sugar beet, rice, sorghum, meadow grass and pastureland.

In another preferred embodiment of the invention, the crop to be treated is selected from the group consisting of tomato, potato, wheat, barley, oats, rye, soybean, corn, rapeseed, canola, sunflower, cotton, sugar cane, sugar beet, rice and sorghum.

In a particularly preferred case of the invention, the plants to be treated are selected from the group consisting of tomato, wheat, barley, oats, rye, maize, rapeseed, canola, sugar cane and rice.

In one embodiment, the plant to be treated according to the method of the invention is an agricultural plant. "Agricultural crops" are plants, part or all of which (e.g., seeds) are harvested or grown on a commercial scale or serve as a major source of animal feed, foodstuffs, fibers (e.g., cotton, linen), fuels (e.g., wood, bio -ethanol, biodiesel, biomass) or other chemical compounds. Preferred agricultural crops 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 plums, stone fruits, or soft fruits, such as apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries, or gooseberries; legumes, such as lentils, peas, alfalfa, or soybeans; oilseed crops, such as rapeseed, rapeseed, canola, linseed, mustard, olives, sunflowers, coconut, cocoa beans, castor oil, oil palms, peanuts or soybeans; cucurbits, such as pumpkins, cucumbers or melons; fiber crops, such as cotton, flax, hemp or jute; citrus fruits, such as oranges, lemons, grapefruits (grapefruit), or tangerines; vegetables, such as spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, cucumbers or peppers; plants containing laura, such as avocados, cinnamon or camphor; energy and resource plants, such as corn, soybean, rapeseed, canola, sugar cane or oil palm; tobacco; nuts; coffee; tea; bananas; vines (table grapes and grape juice grapes); hop; peat; natural rubber plants.

37 BE2022/5162

Meadow grass and grassland consist of grasses or grass mixtures consisting of, for example, blue grass (Poa spp.), bent grass (Agrostis spp.), rye grass (Lolium spp.), fescue (Festuca spp, hybrids and cultivars), Zoysia grass (Zoysia spp.), Bermuda grass (Cynodon spp.), St. Augustine Grass, Bahig Grass (Paspalum), Yarrow Grass (Eremachloa),

Carpet Grass (Axonopus), Buffalo Grass and Grama Grass. Meadows can also consist of mixtures of the above grasses, e.g.

Melilotus albus.

In one case, the plant to be treated according to the method of the invention is a horticultural plant. By "horticultural crops" is meant plants that are commonly used in horticulture - for example, the cultivation of ornamental plants, herbs, vegetables and/or fruit. Examples of ornamental crops are sod, geraniums, pelargonias, petunias, begonias and fuchsias. Examples for vegetables are potatoes, tomatoes, peppers, cucurbits, cucumbers, melons, watermelons, garlic, onions, carrots, cabbage, beans, peas and lettuce and more preferably tomatoes, onions, peas and lettuce. Examples for fruits are apples, pears, cherries, strawberries, citrus fruits, peaches, apricots and blueberries. In horticulture, a substrate often replaces (part of) the soil.

In one case, the plant to be treated according to the method of the invention is an ornamental plant. "Ornamental plants" are plants commonly used in the garden, e.g. in parks, gardens and on balconies. Examples are sod, geraniums, pelargonias, petunias, begonias and fuchsias.

In one case, the plant to be treated according to the method of the invention is a forestry crop. The term "forestry crop" refers to trees, more specifically trees used in reforestation or industrial plantations. Industrial plantations are generally used for the commercial production of forest products, such as wood, pulp, paper, rubber trees, Christmas trees, or young trees for gardening purposes. Examples of forestry crops are coniferous trees, such as pines, in particular Pinus spec, spruce and spruce, eucalyptus, tropical trees, such as teak, rubber tree, oil palm, willow (Salix), in particular Salix spec, poplar (cottonwood), in particular Populus spec, beech, especially Fagus spec, birch, oil palm, and oak.

38 BE2022/5162

The following definitions apply:

The term "plants" should be understood as plants of economic importance and/or plants cultivated by humans. They are preferably selected from agricultural, forestry, ornamental and horticultural crops, each in their natural or genetically modified form. The term "plant" as used herein includes all parts of a plant, such as germinating seeds, emerging seedlings, herbaceous vegetation, as well as established woody plants, including all parts below ground (such as roots) and above ground.

The term "soil" should be understood as a natural body consisting of living (e.g. microorganisms (such as bacteria and fungi), animals and plants) and nonliving matter (e.g. minerals and organic matter (e.g. organic compounds in different degrees of decomposition ), liquid and gases) that resides on the land surface and is characterized by soil horizons that are distinct from the original material as a result of various physical, chemical, biological and anthropogenic processes.

"Nitrification Inhibitor" means: a chemical that retards or slows down the nitrification process that normally takes place in (fertilized) soil. Nitrification inhibitors slow down the natural conversion of ammonium to nitrate and target microorganisms, preferably ammonia-oxidizing bacteria (AOB) and/or ammonia-oxidizing archaea (AOA), through the activity of bacteria, e.g. AOB and AOA, such as Nitrosomonas spp., Nitrosospira spp. and/or Archaea. The nitrification inhibitor is usually combined with a fertilizer, preferably a (ammonium) nitrogen-containing fertilizer, for example a solid or liquid inorganic, organic and/or organomineral fertilizer, or manure.

"Nitrification" should be understood as the biological oxidation of ammonia (NH3) or ammonium (NH4+) with oxygen to nitrite (NO2-), followed by the oxidation of these nitrites to nitrate (NO3-) by micro-organisms. In addition to nitrate (NO3-), nitrification also produces nitrous oxide. Nitrification is an important step in the nitrogen cycle in the soil.

The term "denitrification" should be understood as the microbiological conversion of nitrate (NO3-) and nitrite (NO2-) into gaseous forms of nitrogen, generally N2 or

N20. This respiration process reduces oxidized forms of nitrogen in response to the oxidation of an electron donor such as organic matter. The

39 BE2022/5162 preferred nitrogen electron acceptors, in order from most to least thermodynamically favourable, are: nitrate (NO3-), nitrite (NO2-), nitric oxide (NO) and nitrous oxide (N20). Within the general nitrogen cycle, denitrification completes the cycle by returning N2 to the atmosphere. This process is mainly carried out by heterotrophic bacteria (such as Paracoccus denitrificans and various pseudomonads), although autotrophic denitrifiers have also been identified (e.g.

Thiobacillus denitrificans). Denitrifiers are represented in all major phylogenetic groups. When deprived of oxygen, many bacterial species can switch from oxygen to nitrates to aid respiration in a process called denitrification, which converts the water-soluble nitrates to gaseous products, including nitrous oxide, that are emitted into the atmosphere.

Nitrous oxide", commonly referred to as "happy gas" or "laughing gas", is a chemical compound with the chemical formula N20. At room temperature, it is a colorless, non-flammable gas. Nitrous oxide is produced naturally in the soil by the microbial processes nitrification and denitrification These natural emissions of nitrous oxide can be increased through a variety of agricultural practices and activities, including, for example, a) the direct addition of nitrogen to the soil through the use of mineral and organic fertilizers, b) the cultivation of nitrogen-fixing crops, c ) the cultivation of soils with a high organic content.

The term "fertilizers" should be understood as (chemical) compounds that are applied to promote the growth of plants and fruits. Fertilizers are usually applied through the soil (for uptake by the roots of the plant) or via foliar feeding (for uptake through the leaves). The term "fertilizers" can be divided into two broad categories: a) organic fertilizers (composed of decaying plant/animal matter) and b) inorganic fertilizers (composed of chemicals and minerals). Organic fertilizers include slurry, worm casting, peat, seaweed, sewage and guano. Manufactured organic fertilizers include compost, blood meal, bone meal, and seaweed extracts. Other examples are enzymatically digested proteins, fish meal and feather meal. The decomposing crop residues of previous years and manure are another source of fertility.

In addition, minerals occurring in nature, such as phosphate from minestone, potassium sulphate and limestone, are also considered to be inorganic fertilizers. Inorganic fertilizers are usually manufactured through chemical processes (eg

40 BE2022/5162 the Haber-Bosch process), also using naturally occurring deposits while chemically modifying them (e.g. concentrated triple superphosphate). Naturally occurring inorganic fertilizers include

Chilean sodium nitrate, minestone phosphate and limestone. As a third category, organineral fertilizers can be mentioned, a combination of inorganic and organic fertilizers.

"Fertilizer containing urea" (urea fertilizer) means synthetic fertilizers containing urea, excluding naturally occurring fertilizers 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 produced in the form of granules or prills. Urea fertilizer can be produced by dropping the liquid urea from a prilling tower and drying the product. Urea can also be obtained in liquid form, which can be used for foliar fertilization, e.g. on potatoes, wheat, vegetables and soybeans, and for liquid field application. Usually it is mixed with ammonium nitrate to form UAN with 28% N.

The term "locus" (plant habitat) should be understood as any type of environment, soil, area or material where the plant grows or is intended to grow. According to the invention, particular preference is given to soil.

The invention is further illustrated by the following examples.

Examples 1. Pyrazolo[3,4-b]pyridines

Several pyrazolo[3,4-b]pyridines were obtained from Enamine (SIA Enamine,

Vestienas iela 2 B, LV-1035 Riga, Latvia - https://enamine.net/) or MolPort (SIA MolPort,

Lacplesa street 41, LV-1011 Riga, Latvia - https://www.molport.com/). The respective compounds are listed in Table 1 below.

41 BE2022/5162 2. Material and methods 2. 1Nitrite measurements

Nitrite (NO2-) was measured using the Griess reagent (Product number:

G4410, Griess reagent (modified), Sigma-Aldrich). Equal volumes of the sample or a diluted sample and the Griess reagent were mixed in a transparent flat bottom multi-well plate and incubated in the dark at room temperature for 10 minutes. Absorbance values at 540 nm were measured spectrophotometrically (EnVision, Perkin Elmer®) and used to calculate the [NO2-] using a standard curve. 2. 2Ammonium measurements

Ammonium (NH4+) was measured using a modified Berthelot's reagent protocol. 8 µl culture sample, 35 µl reagent A (0.5 g NaOH with 8 ml NaClIO (2.5%) in 92 ml MilliQ) and 33 µl reagent B (1 g salicylic acid, 0.5 g NaOH and 1.0237 qg sodium nitroprussidedinydrate in 100 ml MilliQ) were added sequentially to 160 µl MIlliQ in 96-well flat bottom plates (cat. no. 353072, Falcon® 96 Well

Clear Microplate, Corning). Absorbance values at 635 nm were measured spectrophotometrically (EnVision, Perkin Elmer®) after a 30 min incubation and used to calculate [NH4+] using a standard curve. 2. 3 Culture conservation 2.3.1 Nitrososphaera viennensis

A liquid culture sample of Nitrososphaera viennensis EN/6T was kindly provided by Prof. Dr. Christa Schleper (University of Vienna). The cells were cultured in fresh water medium (FWM) as previously described by Tourna et al. (2011). All necessary materials were rinsed with 1% HCl and washed at least 3 times with MilliQ water. FWM (pH 7.5 (KOH)) contained 17 mM NaCl, 3 mM MgCl2 x 2

H2 O, 680 HM CaCl2 x 2 H2 O, 1.47 MM KH2 PO4 and 6.71 mM KCl. After autoclaving, 1 mL non-enamelled trace element mixture (101 mM

HCl, 488 uM H3BO3, 508 uM MnCl2 x 4 H20O, 803 uM CoCl2 x 6 H20, 101 uM NiCl2 x 6 H20, 12 uM CuCl2 x 2 H20, 503 uM ZnSO4 x 7 H20 and 150 uM Na2MoO4 x 2 H20), 1 mLFe-NaEDTA (7.5 mM), 1 ML vitamin solution (20 mg/l biotin, 20 mg/l folic acid, 100 mg/l pyridoxine HCI, 50 mg/l thiamine HCl, 50 mg/l riboflavin, 50 mg/l nicotinic acid,

42 BE2022/5162 50 mg/L DL-pantothenic acid, 50 mg/L P-aminobenzoic acid, 2 g/L choline chloride and 10 mg/L vitamin B12), 2 mL NaHCO3 (1 M), 2 mL kanamycin (100 mg/mL ), 2 mL of carbenicillin (100 mg/ML), 3 mL of NH4Cl (1 M), 10 mL of HEPES buffer (1 M, pH 7.6) and 1 mL of Na-pyruvate (0.1 M) were added aseptically. All stock solutions were sterilized through a filter, except the autoclaved, non-enamelled trace element mixture. The cultures were incubated aerobically at 42°C without shaking in sterile 250 ml Erlenmeyer flasks or 30 ml culture tubes (product number: 216-1290, Quickstart universal containers STERILIN®, VWR) filled with 100 or 20 ml

FWM and sealed with tape (Micropore TM Surgical Tape 1530-1, 3MTM). Late-log phase cultures ([NO 2 -] 2 1 mM) were grown by dilution (1:400) in fresh FWM. 2.3.2ABIL

Ammonia Binding Liquid Inoculum (ABIL) is a nitrifying microbial enrichment containing both AOA and AOB and was obtained from Avecom nv (Belgium). The suspension was sieved through a 0.5 mm diameter iron mesh to remove large particles, supplemented with 2.5 mM (NH 4 ) 2 SO 4 and poured into a sterile Erlenmeyer flask.

The culture flasks were sealed with tape (Micropore™ Surgical Tape 1530-1, 3M™) and incubated in the dark at 30°C with shaking (+160 rpm). Growth was assessed by NH4+ measurements. 2.4Nitrification Inhibition Assays 2.4.1Nitrososphaera viennensis 7-day old 100 ml cultures, grown in 250 ml Erlenmeyer flasks with a [NO2-] 2 1 mM, were overconcentrated 5 times in fresh FWM by centrifugation (4000 rpm, 15 min, 5°C ) in 50 ml centrifuge tubes (Cat. No. 430829, CentriStar"" Conical Centrifuge Tubes,

Corning®). All cultures were pooled per batch in one sterile Schott flask.

Then 200 μl per well was dispensed at low pipetting speed into transparent 96-well flat-bottom plates (cat. no. 353072, Falcon® 96 Well Clear Microplate,

Corning®) using 150 µl wide-orifice tips (two steps of 100 µl) on a Tecan robot (Freedom EVO®, Tecan). 0.5 µl 99.99% DMSO (negative control - final concentration 0.25%) and 0.5 µl 0.04 M PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-0xyl 3-oxide , positive control - final concentration 100 µM) were manually added to the first and last column, respectively. Finally, 0.5 µl of candidate nitrification inhibitors (1 mM stock solutions in 99.99% DMSO -

43 BE2022/5162 final concentration 2.5 µM) was added to the central wells using pincers on a Tecan robot (Freedom EVO®, Tecan). Between additions, the pins were washed sequentially with 99.5% DMSO, MilliQ water and 100% ethanol and air dried. All plates were sealed with tape (Micropore TM

Surgical Tape 1530-1, 3M TM), stacked by 4 on top of a 96-well plate filled with 100 µl of MilliQ per well and covered with aluminum foil. The positional effects of the stacks in the incubator were taken into account. The plates were incubated at 42°C without shaking. 24 hours later, NO 2 was measured spectrophotometrically at 540 nm (EnVision, Perkin Elmer®). To this end, the samples were first diluted 25-fold by pipetting x µl samples into x µl fresh growth medium in intermediate plates (cat. no. 353077,

Falcon® 96-well Clear Round Bottom Microplate, Corning®) and then mix 15 µl of the diluted samples with 15 µl of Griess Reagent in the wells of a 384-well transparent flat bottom plate (Cat. No. X7001, Low Profile micro plate,

Molecular Devices), which was measured spectrophotometrically at 540 nm (EnVision, Perkin

Elmer®). 2.4.2ABIL 600 ml ABIL cultures grown in 1-L Erlenmeyer flasks that completely consumed 5 MM NH4+ (measured via the modified Berthelot reagent protocol) within 24 hours were used for a miniaturized, high-throughput nitrification inhibition assay. First, a standard cassette with enough wide tubes in the dispenser (Multidrop TM

Combi Reagent Dispenser, Thermo Scientific") to prevent clogging of the system during dispensing. The tubes were then primed with 100 ml of MilliQ and the ABIL culture (with sediment) was evenly distributed in transparent 96-well flat plates. bottom (cat. no. 353072, Falcon® 96 Well Clear

Microplate, Corning®). The culture was continuously stirred using a magnetic stirrer (speed 600/min) for homogeneous distribution. The stirrer and tube (continuously covered with broth) were placed on opposite sides of the flask to avoid obstruction. All wells in columns 2 through 12 were filled with 135 µl of ABIL.

The first column was used for an ammonium standard curve per plate: the wells

B1 through D1 and wells F1 through H1 each received 135 µl of MilliQ followed by 15 µl of either 5 mM, 12.5 MM or 25 mM (NH 4 ) 2 SO 4 . Wells A1 and E1 received 150 µl

44 BE2022/5162

MilliQ. The wells in column 12 received alternately 1.5 µl 10 mM PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl, positive control - final concentration 100 µM) or 1.5 µl 99 .99% DMSO (solvent of the compound and negative control - final concentration 1%). The different controls were added with a JANUS

Mini VariSpan 4-tip, Perkin Elmer®. After adding all controls, the MDT (Janus Mini MDT, Perkin Elmer ®) added 1.5 µl of compounds (final concentration 10 HM) from the library plates to all central wells. Finally, the dispenser (Multidrop TM

Combi Reagent Dispenser, Thermo Scientific") Add 15 µl of a 25 mM (NH4 )2 SO4 solution (N source - final concentration 5 mM) to columns 2 through 12.

The test plates were sealed with tape (Micropore™ Surgical Tape 1530-1, 3M™), stacked 4 and wrapped in aluminum foil before incubation in the dark at 28°C with vigorous shaking (+210 rpm). 24 hours later, an 8 µl culture sample was taken from each well of the test plates and diluted in 160 µl MilliQ water in readable transparent flat bottom 96-well plates (cat. no. 353072, Falcon® 96 Well Clear

Microplate, Corning). Then 35 µl of Reagent A and 33 µl of Reagent B were successively added to the wells. Finally, the read plates were incubated for 30 min at room temperature and measured spectrophotometrically at 635 nm (EnVision, Perkin Elmer®). 2.4.3 Quantification of nitrification inhibition

To assess the effectiveness of the compounds (in terms of nitrification inhibition) and to allow comparison between plates and batches of cultures, we calculated relative nitrification. In more detail, all nitrite results were normalized to both a negative control, which contained no compound, and a positive control, which contained 100 µM PTIO, using Equation 1. This normalization was performed per multi-well plate, and each plate contained 8 positive and 8 negative controls.

Relative ratio =D, (Vergalikino 1)

This means that compounds that allowed complete nitrification (no nitrification inhibition) show a relative nitrification of 1 (or 100%). A connection that

45 BE2022/5162 shows the same nitrification inhibition as the positive control shows a nitrification inhibition of 0.

For the nitrifying community ABIL, relative nitrification was calculated by comparing the ammonium consumed (equation 2), with 100 μM PTIO as a positive control.

Relative nitrification _ Ammonium(positive control) Ammonium(compoung) (Equation

Ammonium(positive control) -Ammonium(negative control) 2) 2.51Analysis performed in the soil

Soil samples were taken on different plots in Belgium or Germany. Vegetation was removed and samples were taken from several plots.

All soil samples were mixed and sieved (mesh size 2.8 mm) to filter large debris and homogenize the soil.

The moisture content of the soil was determined by drying the soil in an oven at 60°C for 248 hours and measuring the weight before and after drying. Based on soil water content, compound solutions were prepared such that addition of nitrification inhibitor and NH4Cl solution results in a final compound concentration of 50 µM (unless stated otherwise) and a final concentration of NH4+ of 10 mM. The soil was first treated with the respective nitrification inhibitor solution and then with the NH4Cl solution. Extracts with 1M KCl were used to measure NH4+ concentrations. The nitrification percentage was calculated as the ratio of the ammonium consumed in the treated soil to the ammonium consumed in the untreated soil; the nitrification inhibition percentage was calculated as "100% - the nitrification percentage". 3. Results

The results presented in Table 1 are based on the use of the nitrifying archaeon N. viennensis and the nitrifying community ABIL (ammonia-binding

46 BE2022/5162 liquid inoculum - Avecom nv, Belgium). Table 1 shows that all tested substances clearly inhibit nitrification in at least one of the tested systems.

Table 1: Examples of structures with a pyrazolo[3,4-b]pyridine backbone that inhibit nitrification in nitrifying archaea.

The nitrification is expressed relative to the control without inhibitors, where the nitrification is 100%.

Example | Structure ABIL - | N. viennensis relative - nitrification relative (%) nitrification (%) micromolar | 2.5 micromolar 1 A Z1601242688 33

N

N ç

NN N U

2 OR z90278803 15

Now SD 4

F F

3 ; F; z98638976 36 76

N° €

N NN 5

Da 0-4 4 Y Z1697078538 | 36

NT A

N N

47 BE2022/5162

L Z55426831 40 75

F T F

+Z56944137 45

Fa

Table 2 lists pyrazolo[3,4-b]pyridines that have been tested in several experiments for their nitrification inhibition in soil. Table 2 shows one example resulting in strong nitrification inhibition in 4 different soils. The level of nitrification inhibition 5 of example 7 is high, in one case even higher than that of the commonly used nitrification inhibitor DMP. More importantly, in all cases the combination of DMP with example 7 results in a stronger nitrification inhibition than separate application of the substances. This confirms that the new substance (Example 7) targets a group of microorganisms not targeted by DMP.

Table 2: Examples of structures with a pyrazolo[3,4-b]pyridine backbone that inhibit nitrification in soil.

Nitrification inhibition % 14 or 21 days after ammonium supplementation and treatment with the different examples pr ee 7] 14 d 14 d 21 d 21 d a Be #8] #| € 7 HC 63 51 34 53 \ N. CHs

NTA

AJ

© HN oO 7+C1 HC 79 88 81 63 \ N CH: / CS

IN N

HC

HN Oo + DMP

Claims (11)

Conclusions A pyrazolo[3,4-b]pyridine compound of formula (A) © ; # \ R5 La R1 R3 R2 (A) with the following definitions: R1 hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide, R2 hydrogen, methyl, carboxylic acid, carboxamide, methoxycarboxamide or methylcarboxamide, where at least R1 or R2 is hydrogen or methyl R3 is hydrogen or a C1-C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and is preferably hydrogen, methyl, ethyl or isopropyl, R4 is hydrogen or a C1- C4 hydrocarbon residue which may contain one or two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and which is preferably hydrogen, methyl, ethyl or isopropyl and R5 is hydrogen or a C1-C4 hydrocarbon residue which contains one or may contain two heteroatoms selected from the group consisting of fluorine, nitrogen, oxygen and sulfur and is preferably hydrogen, methyl, ethyl or iso-propyl. as a nitrification inhibitor, preferably acting on an inhibitory AOA and/or comammox. 49 BE2022/5162 The use of a pyrazolo[3,4-b]pyridine according to claim 1, wherein the compound of formula (A) is a compound selected from the group consisting of the compounds of formulas (I) (II), ( I), (IV), (V), (VI), (VID, (VID, (IX), (X), (XD), (KID, (KID, (XIV), (XV), (XVI) , (XVID, (XVII, (XVIV), and (XX): CH H,C 3 N HN / Ss 7 > N | N \ A0 N A0 / N CH, H,C (I) (Il) CH, H.C \ O NH N N CH, IT N S \ N N / N A CH, H,C N O OH H:C CH: CHa (IN) (IV) ea N CH: N 3 HC / A N \ | N 5 CH, NN i Q | u N N 3 HC NS Ne 7 O H.C 0 CH; (V) (VI) HO O H;C \ N CH, D a 7 \ A CH, N N H,C i CH; HNO CH3 (VII) (VIII)CH2 CH; ACH; Ken CH; N; N/C7| CH3/C7| CH N N NN Nd H 3 C D NH, D NH, (IX) (X) CH; A CH3 CH A CH3 N N / CE | CH J N N DA CH LE 3 N N N x > CH (XI) (XII) 51 BE2022/5162 HC X \ N CH , N CH N 3 N 3 PF LC N | à A \ > 9 NS 0 H,C H3C 3 HN NH, © SCH; (XIII) (XIV) CH, 64"—CH3 ; N CH N 3 LC N > N \ | Á 7 CH3 > D N du 0 H3C OR OH (XV) (XVI) CH, CH3 N N N N / pi CH, Ç / Ce | CH, N| A . À = 5 > G HN NH, 2 Nen, (XVII) (XVIII) 52 BE2022/5162 H,C CH HC CH, \, N \ N / (7 CH3 / A CH3 N | N AR NN H3C H3CO NH, © OH (XVIV) (XX) The use of any one of the preceding claims, wherein the pyrazolo[3,4-b]pyridine compound inhibits microorganisms selected from the group consisting of ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB), or combinations thereof. The use of any one of the preceding claims, wherein the pyrazolo[3,4-b]pyridine compound is a pyrazolo[3,4-b]pyridine compound of formula (|) and wherein the pyrazolo[3,4-b]pyridine -4-carboxamide of formula (I) inhibits ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB). The use of any one of the preceding claims, wherein the pyrazolo[3,4-b]pyridine compound of formula (Il) to (VIII) has an inhibitory effect on ammonia-oxidizing archaea (AOA). The use of a pyrazolo[3,4-b]pyridine compound according to any one of the preceding claims, wherein the pyrazolo[3,4-b]pyridine compound is administered in combination or in admixture with a compound selected from the group consisting of 2 -(3,4-dimethyl-pyrazol-1-yl)-succinic acid (DMPSA), 3,4-dimethylpyrazole (DMP), 3,4-dimethylpyrazole phosphate (DMPP). The use of a pyrazolo[3,4-b]pyridine compound according to any one of the preceding claims as an additive or coating material for inorganic and/or organic and/or organomineral fertilizers, preferably inorganic fertilizers, more preferably ammonium and/or urea-containing nitrogen fertilizers. The use as claimed in any one of the preceding claims, wherein the pyrazolo[3,4-b]pyridine-4 compound is administered in the form of a 53 BE2022/5162 formulation, solution or dispersion, alone or simultaneously with a fertilizer, or incorporated into the fertilizer or applied to the fertilizer. The use of a pyrazolo[3,4-b]pyridine compound according to any one of the preceding claims for reducing nitrogen or carbon losses in inorganic and/or organic and/or organineral fertilizers or nitrogen or carbon containing compounds or materials and also on harvest waste and pasture land or during the storage of liquid manure and for reducing the ammonia load in animal stables. 10. A fertilizer mixture, consisting of A. an inorganic and/or organic and/or organomineral fertilizer and B. 10 to 10000 weight proppm, based on the fertilizer, of a pyrazolo[3,4-b]pyridine according to any one of the preceding claims. 11. A method of fertilizing agricultural or horticultural soils, in which a fertilizer mixture containing compounds A and B A. an inorganic and/or organic and/or organomineral fertilizer and B. 10 to 10000 weight proppm, calculated on the inorganic fertilizer, of a pyrazolo[3,4-b]pyridine as defined in any one of claims 1 to 5, or compounds A and B separately, but within a period of 0 to the bottoms for up to 5 hours, preferably 0 to 1 hour, more preferably about the same time.