Pyrazole derivatives as dual function urease and nitrification inhibitor

Abstract

Disclosed is a dual functional urease and nitrification inhibitor composition and method for inhibiting the hydrolysis of urea and nitrification of ammonia in soil. Methods of making the dual functional urease and nitrification inhibitor and / or a composition containing the dual functional urease and nitrification inhibitor are also disclosed.

Description

PYRAZOLE DERIVATIVES AS DUAL FUNCTION UREASE AND NITRIFICATION INHIBITORCross-Reference to Related Applications

[0001] This application claims priority to India Provisional Patent Application of number 202341048459 filed on July 19, 2023 and to European Patent Application of number EP23196435 filed on September 11, 2023; the entire contents of which are incorporated herein by reference.I. Field of the Disclosure

[0002] This disclosure generally concerns compositions for maintaining or reducing the loss of nitrogen content in soils. In particular, the invention concerns dual functional urease and nitrification inhibitor molecules for fertilizer application.II. Background

[0003] Nitrogen is one of the most important nutrients for plant growth and productivity. Nitrogen-use efficiency (NUE) is defined as the ratio of a crop’s nitrogen uptake to the total input of nitrogen fertilizer. In agricultural crops, NUE is notoriously poor, with about 50% of nitrogenous fertilizer applied to crop systems lost to the environment as ammonia (NH3), nitrate (NO3 ), and nitrous oxide (N2O, a greenhouse gas). Low NUE in the agriculture industry contributes to high agricultural production costs, environmental pollution, and climate change. These losses are driven by urea hydrolysis, nitrification, and subsequent denitrification processes catalyzed by soil microorganisms.

[0004] The hydrolysis of urea is catalyzed by ureases, that are found in numerous bacteria, fungi, algae, plants, and as well as in soils, as a soil enzyme. These enzymes catalyze the hydrolysis of urea into carbon dioxide and ammonia. Nitrification is traditionally considered to be a two-step process where ammonia (NH3) is first oxidized to nitrite (NCU) by ammonia oxidizers, and subsequently to nitrate (NO3 ) by nitrite-oxidizing bacteria (NOB). Recent evidence indicates complete nitrification by Nitrospira bacteria that are present in natural soil environments.

[0005] Due to its rapid hydrolysis and nitrification in the soil, nitrogen from urea can be quickly lost. This not only reduces the efficiency of N use, but also can contribute to climate change and loss of biodiversity through emission of greenhouse gases in the production of excess urea fertilizers. Also, using urea in fertilizer blends that contain other soil nutrients is difficult, as urea can undesirably react with other components in the fertilizer, such as organic fertilizers. These reactions can produce water that liquefies solid granules or dry mixture products, cause clumping and loss of product, and increase the rate at which these undesirable reactions take place. See Biskupski etal. (EP 2,774,907); see also Achard etal. (US 5,409,516). Further, the production of water increases the amount of water that has to be removed during production of urea containing fertilizers, making these blended fertilizers difficult and more expensive to make. See Schwob (FR 2,684,372).

[0006] Various governments throughout the world have imposed regulations that obligate the agricultural industry to reduce their environmental impacts. One method for reducing agricultural environmental impact involves the use of urease inhibitors and nitrification inhibitors. Urease inhibitors are compounds that can prevent or retard the hydrolyzing urea and nitrification inhibitors are compounds that can prevent or retard the nitrification of ammonia. These inhibitors help to reduce losses of nitrogen in soil that would otherwise be used by plants.

[0007] However, biological and synthetic urease and nitrification inhibitors are associated with a variety of shortcomings, including instability at high temperatures, instability at high or low pH, variable effectiveness, difficulties in application, high cost, environmental pollution, and food safety risks.SUMMARY OF THE INVENTION

[0008] A solution to at least some of the problems discussed above is disclosed herein. The solution in some instances lies in duel urease and nitrification inhibitors disclosed herein and methods for use of the dual inhibitors. The duel inhibitors disclosed herein can include small molecules that can, in some instances, be synthesized.

[0009] The duel urease and nitrification inhibitors, in some instances, have a high melting point, which facilitates formulating the urease and nitrification inhibitors with melted urea at a temperature of about 132 to 135 °C or greater. The duel urease and nitrification inhibitors in some instances exhibit little to no toxicity and are equally or more effective than urease and nitrificationinhibitors currently on the market. The duel urease and nitrification inhibitors may be used in a variety of soil types, and over broad ranges of temperature and pH. Certain aspects of the disclosure are therefore directed to a composition or a compound that provides reduced hydrolysis of urea and reduced nitrification of ammonium nitrogen, the composition or compound comprising a duel urease and nitrification inhibitor having a structure represented by Formula I or FormulaVII:wherein:X is sulfur or oxygen; R is a moiety having a structure represented by Formula II or Formula III;Formula II;Formula III; andRi, R2, R3, R4, Rs, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstituted unsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen; and optionally one or more of: an additional inhibitor comprising an additional urease inhibitor and / or an additional nitrification inhibitor; a solid and / or liquid carrier; and / or at least one nitrogen (N), phosphorus (P), and / or potassium (K) fertilizer component.

[0010] In some aspects, the composition or compound of Formula V Formula VII may containR as a pyrazole or -N(CH3)2; Ri as H or methyl; R2 as methyl, n-propyl, or n-butyl; 3 as H; R4 as H; Rs as H; Re as H; R7 as H; Rs as H; R9 as methyl; and / or Rio as methyl.

[0011] In some aspects, the dual urease and nitrification inhibitor has a structure represented by Formula IV.Formula IV

[0012] In some aspects, the dual urease and nitrification inhibitor has a structure represented by Formula V.Formula V

[0013] In some aspects, the dual urease and nitrification inhibitor has a structure represented by Formula VI.Formula VI

[0014] In some aspects, the composition contains a combination of dual urease and nitrification inhibitors having structures represented by Formulas IV, V, and / or VI.

[0015] Some aspects of the disclosure are directed to a method for inhibiting both urease hydrolysis and nitrification of ammonia, the method including applying to a soil, a crop, and / or a fertilizer a composition or a compound comprising the dual urease and nitrification inhibitor disclosed herein. In some instances, the dual urease and nitrification inhibitor has a structure represented by Formula I or Formula VII.Formula I Formula VII wherein:X is sulfur or oxygen;R is a moiety having a structure represented by Formula VIII or Formula IX:Formula VIII;RioFormula IX; andRi, R2, RS, R4, RS, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstituted unsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen.

[0016] The dual urease and nitrification inhibitor may be applied to soil by application directly to the soil, by tilling into a soil, by application to water that contacts the soil, etc. In some instances, the dual urease and nitrification inhibitor is applied to the soil at the same time as, or at a different time than, a fertilizer, water, additional soil, a soil supplement, a seed, a plant, etc. The dual urease and nitrification inhibitor may be applied to a plant by application directly to the plant, by contact of the plant with a soil or fertilizer containing the dual urease and nitrification inhibitor, by application to water that contacts the plant, etc. In some instances, the dual urease and nitrification inhibitor is applied to the plant at the same time as, or at a different time than, a fertilizer, water, soil, a soil supplement, etc.

[0017] The composition, in some instances, can consist or consist essentially of the dual urease and nitrification inhibitor. In some aspects, the composition further comprises at least one nitrogenous fertilizer component. The at least one nitrogenous fertilizer component can be selected from the group consisting of urea, an ammonium salt, anhydrous ammonia, and combinations thereof. In specific aspects, the at least one nitrogenous fertilizer components is urea. In some embodiments, the urea is a solidified urea melt. In some aspects, the composition further comprises a phosphate based fertilizer. In some aspects, the dual urease and nitrification inhibitor is comprised within the solidified urea melt.

[0018] In some aspects, the composition further comprises at least one additional nitrification inhibitor. The at least one additional nitrification inhibitor can be nitrapyrin, dicyandiamide, ammonium thiosulfate, methyl-3-(4-hydroxyphenyl)propionate, 1,9-decanediol, potassium azide, 2-amino-4-chloro-6-methylpyrimidine, sulfathiazole, 4-amino-l,2,4-triazole, 2,4-diamino-6- trichloromethyl-s-triazine, potassium ethylxanthate, guanylthiourea, 4-nitrobenzotrichloride, 4- mesylbenzotrichloride, sodium thiocarbonate, phenylmercuric acetate, 3, 4-dimethylpyrazole phosphate, or sorgoleone, or combinations thereof. In some aspects, the composition further comprises at least one additional urease inhibitor. The at least one additional urease inhibitor may be at least one of N-(n-butyl) thiophosphoric triamide (NBTPT) and N-(n-propyl) thiophosphoric triamide. In some aspects, the composition further comprises at least one additional nitrification inhibitor and at least one additional urease inhibitor.

[0019] In some aspects, the dual urease and nitrification inhibitor is comprised within a carrier. In some aspects, the carrier comprises plaster of paris, flour, chalk powder, starch, gluten, kaolin, bentonite, colloidal silica, silica, dried distillers grains with solubles, lignin, a synthetic polymer, a wax, chitin, glycoaminoglycans, pectins, hyaluronic acid, chondroitin sulphate, dermatan sulphate, alginic acid, polymannuronic acid, polyguluronic acid, polyglucuronic acid, amylose, amylopectin, callose, chitosan, poly- galactomannan, dextran, xanthan, keratan sulphate, MgO, CaO, bone mill powder, rice husk, CaCCh, Na2COs, K2CO3, KH2PO4, NaHCCh, and / or MgCCh. In some embodiments, the synthetic polymer comprises poly butylene succinate adipate, poly lactic acid, poly butylene succinate, cellulose triacetate, cellulose diacetate, cellulose acetate, a starch acetate, poly(caprolactone), a poly(butylene terephthalate adipate), hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, methylcellulose, ethylcellulose, ethyl methyl cellulose, hydroxy ethyl cellulose, hydroxypropyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl cellulose, polyethylene glycol, cyclodextrin, an alcohol, water, a liquid buffer, a chelating agent, or a combination thereof.

[0020] In some aspects, the composition increases nitrogen use efficiency (NUE) of or for a crop. In some aspects, the method further comprises applying a nitrogenous fertilizer component to the soil, wherein a nitrogenous fertilizer component and the composition are comprised in two separate compositions. In certain aspects, the two separate compositions are applied to the soil within one hour of each other. In other aspects, the two separate compositions are applied to thesoil separately and not within one hour of each other. In other aspects, the nitrogenous fertilizer component and the dual urease and nitrification inhibitor are comprised in a single composition.

[0021] Also disclosed are the following aspects 1 to 15 of the present invention.

[0022] Aspect 1 is a method of inhibiting urease activity and nitrification of ammonia, the method comprising applying a dual urease and nitrification inhibitor and / or a composition comprising the dual urease and nitrification inhibitor to a soil, a crop, and / or fertilizer, wherein the dual urease and nitrification inhibitor has a structure represented by Formula I:Formula I wherein:X is sulfur or oxygen;R is a moiety having a structure represented by Formula II or Formula III:Formula II;RioFormula III; andRi, R2, RS, R4, RS, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstituted unsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen.

[0023] Aspect 2 is the method of aspect 1, wherein the composition further comprises at least one nitrogenous fertilizer component.

[0024] Aspect 3 is the method of aspect 2, wherein the at least one nitrogenous fertilizer component comprises urea, an ammonium salt, anhydrous ammonia, or any combination thereof .

[0025] Aspect 4 is the method of any of aspects 1 to 3, wherein the composition further comprises at least one additional nitrification inhibitor, wherein the at least one additional nitrification inhibitor preferably comprises nitrapyrin, dicyandiamide, ammonium thiosulfate, methyl-3-(4-hydroxyphenyl)propionate, 1,9-decanediol, potassium azide, 2-amino-4-chloro-6- m ethylpyrimidine, sulfathiazole, 4-amino-l,2,4-triazole, 2,4-diamino-6-trichloromethyl-s- triazine, potassium ethylxanthate, guanylthiourea, 4-nitrobenzotrichloride, 4- mesylbenzotrichloride, sodium thiocarbonate, phenylmercuric acetate, 3, 4-dimethylpyrazole phosphate, sorgoleone, or any combination thereof.

[0026] Aspect 5 is the method of any one of aspects 1 to 4, wherein the composition further comprises at least one additional urease inhibitor, wherein the additional urease inhibitor preferably comprises N-(n-butyl) thiophosphoric triamide, or N-(n-propyl) thiophosphoric triamide, or a combination thereof.

[0027] Aspect 6 is the method of any one of aspects 1 to 5, wherein the composition increases nitrogen use efficiency (NUE) of a crop.

[0028] Aspect 7 is the method of any one of aspects 1 to 6, wherein the dual urease and nitrification inhibitor is (i) distributed throughout a fertilizer, (2) coated on a fertilizer, and / or (iii) is encapsulated.

[0029] Aspect 8 is the method of any one aspects 1 to 7, wherein: R is a pyrazole or -N(CH3)2; Ri is H or methyl; R2 is methyl, / / -propyl, or / / -butyl; R3 is H; R4 is H; Rs is H; Re is H; R7 is H;Rs is H; R9 is methyl; and / or Rio is methyl.

[0030] Aspect 9 is the method of any one aspects 1 to 8, wherein the dual urease and nitrification inhibitor has a structure represented by Formula IV, Formula V, or Formula VI:Formula IV Formula V ; or Formula VI

[0031] Aspect 10 is a fertilizer composition comprising:A dual urease and nitrification inhibitor having a structure represented by Formula VII:Formula VII wherein:R is a moiety having a structure represented by Formula VIII or Formula IX;Formula IX; andRi, R2, R3, R4, Rs, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstituted unsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), amethoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen; and one or more of: an additional inhibitor comprising an additional urease inhibitor and / or an additional nitrification inhibitor; a solid and / or a liquid carrier; and / or at least one nitrogen (N), phosphorus (P), and / or potassium (K) fertilizer component.

[0032] Aspect 11 is the fertilizer composition of aspect 10, wherein: the additional nitrification inhibitor comprises nitrapyrin, dicyandiamide, ammonium thiosulfate, methyl-3-(4-hydroxyphenyl)propionate, 1,9-decanediol, potassium azide, 2-amino-4-chloro-6-methylpyrimidine, sulfathiazole, 4-amino- 1,2,4- triazole, 2,4-diamino-6-trichloromethyl-s-triazine, potassium ethylxanthate, guanylthiourea, 4-nitrobenzotrichloride, 4-mesylbenzotrichloride, sodium thiocarbonate, phenylmercuric acetate, 4-dimethylpyrazole phosphate, sorgoleone, or any combinations thereof; and / or the additional urease inhibitor comprises N-(n-butyl) thiophosphoric triamide, N-(n- propyl) thiophosphoric triamide, or a combination thereof;

[0033] Aspect 12 is the fertilizer composition of any one of aspects of 10 or 11, wherein the solid and / or liquid carrier comprises plaster of paris, flour, chalk powder, starch, gluten, kaolin, bentonite, colloidal silica, silica, dried distillers grains with solubles, lignin, a synthetic polymer, a wax, chitin, glycoaminoglycans, pectins, hyaluronic acid, chondroitin sulphate, dermatan sulphate, alginic acid, polymannuronic acid, polyguluronic acid, polyglucuronic acid, amylose, amylopectin, callose, chitosan, poly- galactomannan, dextran, xanthan, keratan sulphate, MgO, CaO, bone mill powder, rice husk, CaCCh, Na2COs, K2CO3, KH2PO4, NaHCCh, MgCCh, an alcohol, water, a liquid buffer, a chelating agent, or any combination thereof.

[0034] Aspect 13 is the fertilizer composition of any one of aspects 10 to 12, wherein the at least one nitrogen (N), phosphorus (P), and / or potassium (K) fertilizer component comprises at least one nitrogenous fertilizer component.

[0035] Aspect 14 is the fertilizer composition of any one of aspects 10 to 13, wherein: R is a pyrazole or -N(CH3)2; Ri is H or methyl; R2 is methyl, w-propyl, or / / -butyl; R3 is H; R4 is H; Rs is H; Re is H; R7 is H; Rs is H; R9 is methyl; and / or Rio is methyl.

[0036] Aspect 15 is The fertilizer composition of any one of aspects 10 to 14, wherein the dual urease and nitrification inhibitor has a structure represented by Formula IV, Formula V, or Formula VI:

[0037] The term “nitrification inhibitor” is a compound that inhibits bacteria in soil from converting nitrogen, such as the nitrogen provided in a fertilizer compound, into nitrate.

[0038] The term “urease inhibitor” is a compound that inhibits activity of the enzyme urease, an enzyme which converts urea into ammonia.

[0039] The term “fertilizer” is defined as a material applied to soils or to plant tissues to supply one or more plant nutrients essential or beneficial to the growth of plants and / or stimulants or enhancers to increase or enhance plant growth.

[0040] The term “granule” can include a solid material. A granule can have a variety of different shapes, non-limiting examples of which include a spherical, a puck, an oval, a rod, an oblong, or a random shape.

[0041] The term “particle” can include a solid material less than a millimeter in its largest dimension.

[0042] The terms “particulate” or “powder” can include a plurality of particles.

[0043] The terms “about” or “approximately” as used herein are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment, the terms aredefined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0044] The terms “wt. %,” “vol.%,” or “mol.%” refers to a weight, volume, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, that includes the component. In a non-limiting example, 10 grams of component in 100 grams of the material is 10 wt. % of component.

[0045] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0046] The phrase “and / or” means “and” or “or.” To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0047] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0048] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0049] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0050] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0052] FIG. 1 : FIG. 1A shows urease enzymatic steps of urea hydrolysis. FIG. IB is a chemical pathway of ammonia to nitrate and N2O.

[0053] FIG. 2 : is a representation of the active site of an ammonium oxidase enzyme. FIG. 2A is a schematic depicting a purported ammonia oxidase copper metalloenzyme active site with two copper atoms. FIG. 2B is a ball and stick model of ammonia oxidase active site.

[0054] FIG. 3 : is a representation of the active site of an urease enzyme. FIG. 3A is a schematic depicting a purported urease enzyme active site with two nickel atoms. FIG. 3B is a ball and stick model of a urease enzyme active site.DETAILED DESCRIPTION OF THE INVENTION

[0055] Human activity has had the single largest influence on the global nitrogen cycle by introducing unprecedented amounts of reactive nitrogen into ecosystems. The agricultural industry, in particular, is the single largest human activity that affects the global nitrogen-cycle. In agricultural systems, rapid and unchecked urea hydrolysis and nitrification results in inefficient nitrogen-use by crops, leading to nitrogen-leakage and environmental pollution.

[0056] Hydrolytic decomposition of urea is the process by when urea is applied to the soil, where urea rapidly hydrolyzes to ammonium carbonate. Ammonium carbonate is unstable and breaks down to NH3 and CO2. The NH3 is either absorbed by the soil or volatilizes. The hydrolysis reaction is facilitated by urease enzymes. Urease inhibitors block this enzyme to prevent the conversion of urea to NH3, in some instances for a period of 1 to 2 weeks, allowing time for the incorporation of urea into the soil by rainfall or other means.

[0057] Nitrification is the process by which ammonia is converted to nitrites (NO2 ), nitrates (NO3 ), and nitrous oxide (N2O) (FIG. 1). This process naturally occurs in the environment, where it is carried out by specialized bacteria. Most nitrification is performed by two groups of ammoniaoxidizing bacteria, Nitrosomonas sp. and Nitrobacter spp. These bacteria are generally ubiquitouscomponents of soil microbial populations. Bacterial enzymes that are key to the nitrification process include ammonia monooxygenase, hydroxylamine dehydrogenase, and nitrite oxidoreductase. Inhibition of the nitrification enzyme ammonia monooxygenase, the first enzyme in the nitrification process, retards the conversion of NH4+into NH2OH, and slows fertilizer loss due to nitrification.

[0058] Certain aspects of the present disclosure are directed to compositions that inhibit the hydrolysis of urea and the nitrification process, thereby increasing nitrogen longevity in soil. This is accomplished by applying one of more of the dual urease and nitrification inhibitor compounds disclosed herein to a plant, soil, crop, and / or fertilizer. The dual urease and nitrification inhibitors disclosed herein may be combined with an additional nitrification inhibitor and / or an additional urease inhibitor. The dual urease and nitrification inhibitors disclosed herein include small, low molecular weight organic compounds. These compounds are relatively inexpensive to synthesize and can be produced in large quantities at a fraction of the cost of other, more structurally-complex urease inhibitors, nitrification inhibitors, and dual inhibitors.I. Duel Urease and Nitrification Inhibitor

[0059] Certain embodiments herein concern a composition comprising a duel urease and nitrification inhibitor having a structure represented by Formula VII:wherein:R is a moiety having a structure represented by Formula VIII or Formula IX;Formula IX; andRi, R2, RS, R4, RS, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstituted unsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen.

[0060] The composition may further include at least one nitrogenous fertilizer component, at least one additional nitrification inhibitor, at least one urease inhibitor and / or at least one additional dual urease and nitrification inhibitor. The additional urease and nitrification inhibitors may be used to inhibit the hydrolysis of urea and / or nitrification of ammonia in soil by applying the composition to the soil.

[0061] In some aspects, a fertilizer nutrient can be included or excluded in the dual urease and nitrification inhibitor composition. If included, fertilizer nutrients can be chosen based on the particular needs of certain types of soil, climate, or other growing conditions to maximize the efficacy of the fertilizer in enhancing plant growth and crop yield. Additional additives may also be included or excluded in the dual urease and nitrification inhibitor composition. Non-limiting examples of additives that can be included or excluded from the dual urease and nitrificationinhibitor composition of the present invention include nitrogen nutrients, phosphorus nutrients, potassium nutrients, micronutrients, and / or secondary nutrients. The micronutrient can be copper, iron, chloride, manganese, molybdenum, or nickel, or any combinations thereof. The nitrogen nutrient can be urea, ammonium nitrate, ammonium sulfate, diammonium phosphate, monoammonium phosphate, urea-formaldehyde, ammonium chloride, and / or potassium nitrate. In some aspects, the secondary nutrients may include calcium, magnesium, and / or sulfur, such as lime and / or superphosphate.

[0062] In some instances, the dual urease and nitrification inhibitor composition can include at least one additional nitrification inhibitor and / or at least one additional urease inhibitor. Suitable nitrification inhibitors include, but are not limited to, 3,4-dimethylpyrazole phosphate (DMPP), dicyandiamide (DCD), thiourea (TU), 2-chloro-6-(trichloromethyl)-pyridine (Nitrapyrin), 5- ethoxy-3-trichloromethyl-l,2,4-thiadiazol, which is sold under the tradename Terrazole®, by OHP Inc., USA, 2-amino 4-chloro 6-methyl pyrimidine (AM), 2-mercaptobenzothiazole (MBT), or 2- sulfanilamidothiazole (ST), and any combination thereof. In one aspect, a nitrification inhibitor can comprise DMPP, DCD, TU, nitrapyrin, 5-ethoxy-3-trichloromethyl-l,2,4-thiadiazol, AM, MBT, or ST, or a combination thereof. In some embodiments, a fertilizer composition can comprise NBTPT, DMPP, TU, DCD, PPDA, nitrapyrin, 5-ethoxy-3-trichloromethyl-l,2,4- thiadiazol, AM, MBT, ST, or a combination thereof.

[0063] In some instances, a binder may be included in the dual urease and nitrification inhibitor composition. Exemplary binders include a phosphate, a polyphosphate, a biodegradable polymer, or a wax, or a combination thereof. Suitable waxes include, but are not limited to, vegetable waxes, high melt waxes, ethylene bis(stearamide) wax, paraffin waxes, polyethylene based waxes, and olefin waxes. Suitable phosphates include, but are not limited to, diammonium phosphate, and monoammonium phosphate. Suitable polyphosphates include, but are not limited to, ammonium polyphosphate. Suitable biodegradable polymers include, but are not limited to, polyacrylamide, polyacrylic acid, polyacrylonitrile, biodegradable polylactic acid and other biodegradable polymeric material such as polylactic acid, poly(3 -hydroxypropionic acid), polyvinyl alcohol, polyethylene glycol, poly e-caprolactone, poly L-lactide, poly butylene succinate, and biodegradable starch based polymers. The binder can include plaster of Paris, flour, starch, gluten, kaolin, bentonite, colloidal silica, a cyclodextrin, or combinations thereof. Suitable flours include,but are not limited to, rice flour, wheat flour, and bleached wheat flour. Suitable starches include, but are not limited to, dextrin modified starches.

[0064] In some instances, the dual urease and nitrification inhibitor composition can include a pH buffer. Exemplary pH buffers include MgO, KH2PO4, NaHCCh, chalk powder, aluminum, magnesium hydroxide, aluminum hydroxide / magnesium hydroxide co-precipitate, aluminum hydroxide / sodium bicarbonate co-precipitate, calcium acetate, calcium bicarbonate, calcium borate, calcium carbonate, calcium bicarbonate, calcium citrate, calcium gluconate, calcium hydroxide, dibasic sodium phosphate, dipotassium hydrogen phosphate, dipotassium phosphate, disodium hydrogen phosphate, magnesium acetate, magnesium borate, magnesium bicarbonate, magnesium carbonate, magnesium hydroxide, magnesium lactate, magnesium oxide, magnesium phosphate, magnesium silicate, magnesium succinate, magnesium tartrate, potassium acetate, potassium carbonate, potassium bicarbonate, potassium borate, potassium citrate, potassium metaphosphate, potassium phthalate, potassium phosphate, potassium polyphosphate, potassium pyrophosphate, potassium succinate, potassium tartrate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium gluconate, sodium hydrogen phosphate, sodium hydroxide, sodium lactate, sodium phthalate, sodium phosphate, sodium polyphosphate, sodium pyrophosphate, sodium tartrate, sodium tripolyphosphate, synthetic hydrotalcite, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, tripotassium phosphate, trisodium phosphate, and trometamol, and combinations thereof.

[0065] The dual urease and nitrification inhibitor composition described herein can be comprised in a composition useful for application to soil, a crop, and / or a fertilizer. In some aspects, in addition to the dual urease and nitrification inhibitor composition described herein, the composition may include fertilizer compounds, micronutrients, primary nutrients, urea, nitrogen nutrients, insecticides, herbicides, or fungicides, or combinations thereof. The dual urease and nitrification inhibitor composition described herein can also be included in a blended composition comprising fertilizers. The fertilizer can be urea, monoammonium phosphate (MAP), diammonium phosphate (DAP), muriate of potash (MOP), monopotassium phosphate (MKP), triple super phosphate (TSP), rock phosphate, single super phosphate (SSP), ammonium sulfate, and the like.

[0066] A dual urease and nitrification inhibitor composition formed into a granule can have desirable physical properties such as desired levels of abrasion resistance, granule strength,pelletizability, hygroscopicity, granule shape, and size distribution. In some aspects, the dual urease and nitrification inhibitor composition granule can have a crush strength above 1.5 kgf, such as above 1.8 kgf, such as 2 kgf to 6 kgf, or at least any one of, equal to any one of, or between any two of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 kgf. Bulk density of the fertilizer granules can be 1 g / cc to 1.2 g / cc, or at least any one of, at most any one of, equal to any one of, or between any two of 1, 1.02, 1.04, 1.06, 1.08, 1.1, 1.12, 1.14, 1.16, 1.18, and 1.2 g / cc. In some aspects, 10 mg or more, such as 10 mg to 40 mg, or at least any one of, at most any one of, equal to any one of, or between any two of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 mg of the fertilizer granule can dissolve in 1 ml of water at a pH 7, under stirring at a rate 90 to 110 rpm, and at an ambient temperature, within 5 minutes of adding 100 mg of the fertilizer granules to the water. In some aspects, the fertilizer granule is capable of losing less than 0.13 wt. %, such as 0.02 % to 0.12 wt. %, or at most any one of, equal to any one of, or between any two of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, and 0.12 wt. % in an attrition loss test. Attrition loss can be measured using the following attrition loss test.

[0067] Attrition loss test: A plurality of sieved dual urease and nitrification inhibitor granules with individual size 2 to 4 mm, and total volume 100 cm3is weighed (Wl) and is placed into the test drum along with the 50 stainless steel balls having a total weight of 100 gm. The drum is closed and rotated for 10 min at 30 rpm. Then, the steel balls are separated from the sample and the material is screened over 2 mm sieve using a sieve shaker. The total weight of the granules over 2 mm are then re-weighed (W2). Results are calculated in terms of % weight loss using the formula:. . . , . . , . .. weight of sample remained on 2 mm sieve ((W2y) . > _Weight loss due to attrition (wt. %) = — - — - - — — - — — — - — - X 100 intial wei ht of the sample (Wl)

[0068] In some instances, the dual urease and nitrification inhibitor composition can be extruded before drying. The extruder can extrude the dual urease and nitrification inhibitor composition at pressures at least, at most, equal to, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 bar. The extruder can extrude the dual urease and nitrification inhibitor composition at temperatures at least, at most, equal to, or between any two of -20, -15, -10, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 °C. The extrudate can be sliced or divided before drying, such as by a die.

[0069] In certain aspect, the desired size of a final dual urease and nitrification inhibitor product, such as a granule, can be 0.5 to 5 mm, or 1 to 4 mm, and having a size lower than 0.5 mm or 1 mm, and / or having a size bigger than 4 mm or 5 mm can be obtained using one or more size screens. In certain aspects, the dual urease and nitrification inhibitor composition can be rounded and / or spheronized in a spheronizer. In some instances, the dual urease and nitrification inhibitor composition is contacted with a spheronizer for at least any one of, at most any one of, equal to any one of, or between any two of 5, 10, 15, 20, 25, 30, 35, 40, 50, or 60 seconds. The spheronizer in some instances has a rotating or rotatable disk capable of rotating at least any one of, at most any one of, equal to any one of, or between any two of 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 rpm. In certain aspects, the dual urease and nitrification inhibitor composition can be dried in a dryer at a temperature 40 °C to 85 °C, or at least any one of, at most any one of, equal to any one of, or between any two of 40, 45, 50, 55, 60, 65, 70, 75, 80 and 85 °C. In some aspects, the dryer can be a fluid bed dryer, drum dryer, or flash dryer. In some aspects, the dual urease and nitrification inhibitor composition can be dried in the dryer with an hot air flow having a flow rate of at least any one of, at most any one of, equal to any one of, or between any two of 100, 150, 200, 250, 300, 350, 400, 450, and 500 m3 / hr and / or a rotation of at least any one of, at most any one of, equal to any one of, or between any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 rpm.II. Methods of Using the Duel Urease and Nitrification Inhibitor

[0070] The duel urease and nitrification inhibitor, including duel urease and nitrification inhibitor granules and compositions containing the duel urease and nitrification inhibitor, can be used in methods of inhibiting the hydrolysis of urea and nitrification of ammonia in soil. Such methods can include applying to the soil, a plant, and / or a fertilizer an effective amount of a composition and / or blend containing the duel urease and nitrification inhibitor of the present invention having a structure represented by Formula I.Formula I wherein:X is sulfur or oxygen;R is a moiety having a structure represented by Formula II or Formula III:Formula III; andRi, R2, R3, R4, Rs, Re, R7, Rs, R9, and Rio are each independently H, substituted or unsubstituted Cl to C9 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted saturated heterocyclic, substituted or unsubstitutedunsaturated heterocyclic, substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen.

[0071] The method may include increasing the growth and yield of crops, trees, ornamentals, etc., such as, for example, palm, coconut, rice, wheat, corn, barley, oats, and / or soybeans. The method can include applying the duel urease and nitrification inhibitor of the present invention to at least one of a soil, an organism, a liquid carrier, a liquid solvent, a solid carrier, a fertilizer, etc. The duel urease and nitrification inhibitor and / or composition(s) containing the duel urease and nitrification inhibitor can be applied to plants, soil, and / or fertilizer through any suitable method of application.

[0072] Non-limiting examples of plants that can benefit from the duel urease and nitrification inhibitor of the present invention include vines, trees, shrubs, stalked plants, ferns, etc. The plants may include orchard crops, vines, ornamental plants, food crops, timber, and harvested plants. The plants may include Gymnosperms, Angiosperms, and / or Pteridophytes.

[0073] The effectiveness of compositions comprising the duel urease and nitrification inhibitor of the present invention can be ascertained by analyzing nitrification activity as described below. It is understood that different soils have different characteristics, which can affect the effectiveness of the duel urease and nitrification inhibitor compositions. The effectiveness of a duel urease and nitrification inhibitor composition can also be directly compared to other urease inhibitors and nitrification inhibitor compositions by doing a side-by-side comparison in the same soil under the same conditions.III. Methods of Making the Duel Urease and Nitrification Inhibitor

[0074] The dual urease and nitrification inhibitor can be manufactured by means known in the field, such as by organic synthesis. In some instances, the manufacture can be performed at industrial scale at a manufacturing facility. In some instances, the dual urease and nitrification inhibitor is manufactured at a facility that manufactures the dual urease and nitrification inhibitor and other products.Examples

[0075] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit theinvention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.Example I Molecular Modeling Study

[0076] A molecular modeling approach was used to predict the relative binding affinity of Pyrazole derivatives of Formulas IV, V, and VI, as compared to benchmarking nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) in the active site of modeled ammonia monooxygenase enzyme, also known as nitrification enzyme (FIG. 2A). The crystal structure of ammonia monooxygenase is unknown, however, literature reveals information by homology modelling and other indirect approaches, which indicates that it is a copper metalloenzyme. Ammonia monooxygenase Cu site modelling shows two Cu(II) ions separated by 2.55 A. The atoms of the 12S2 rhombus deviate from the plane by 0.2 A, and the angle between the two 1S2 planes is 170.1°. Cui is bound to Met-A, His-A, and Cys-A from both chains in a distorted tetrahedral geometry, while Cu2 is bound to residues, Met-B, His-B, and Cys58-B from both chains, again in a distorted tetrahedral geometry (FIG. 2A). Both copper atoms depicted in FIG.2 A are in +1 -oxidation states. In light of available information of the active site, density functional theory (DFT) modelling was used to examine ammonia monooxygenase active site geometry. The active site geometry was minimized (FIG. 2B) using B3LYP functional as implemented in the Gaussian 09 package in conjunction with 6-3 lG(d,p) basis sets for the nonmetal elements such as C, H, N, and O, while for metal Cu an effective core pseudopotentials (SDD) and the associated basis sets with a polarization function was used. An analytical frequency calculation verified all the stationary points. The Gibbs energies were computed at 298.15° K and at atmospheric pressure (unless specified otherwise), assuming an ideal gas behavior using unsealed harmonic vibrational frequencies obtained within the rigid rotor approximation.

[0077] A similar molecular modeling approach was used to predict the relative binding affinity of the Pyrazole derivatives of Formulas IV, V, and VI, as compared to benchmarking urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT), in the active sites of Urease enzyme (FIG3 A). The crystal structure of urease enzyme is known and the urease enzyme structure 5OL4, was obtained from Protein Data Bank. The active site of SPU enzyme comprising aHis222, carboxylated aLys220, aHis249, aHis275, aHis323, aHisl37, aHisl39, aAsp363 and aala366, along with both nickel atoms and three oxygen atoms connected to nickel was extracted. Hydrogenatoms were added to satisfy valency of different atoms. An overall charge of +1.0 was set on the system to obtain closed shell configuration. The active site geometry was minimized (FIG. 3B).

[0078] Compounds of Formulas IV to VI and the commercial inhibitors DMPP and NBPT (for benchmarking purpose) were introduced individually and the ligand receptor complex geometry minimized using DFT methods. The binding energies (BE) of the inhibitors within the active site were estimated using Equation (1):Ebinding=Energy (Enzyme-Inhibitor complex)—[Ener y (Active site) + Energy (Inhibitor)]Equation (1)Table 1 - Calculated DFT Binding Energies

[0079] During competitive inhibition, an effective inhibitor molecule, with a higher binding energy within the enzyme active site, would have lower ICso values. Binding Energy was estimated for candidate Formulas IV to VI and benchmarks as reported in Table 1. The binding energies of Formulas IV to VI were within close range of the respective benchmarks for binding urease enzyme and nitrification enzyme, demonstrating that Formulas IV to VI have comparable affinity for ammonia monooxygenase as well as urease. The proposed Formulas IV to VI shows negative binding energies against both the active sites, hence these molecules are expected to function as dual function inhibitors.* * *

[0080] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMS1. A method of inhibiting urease activity and nitrification of ammonia, the method comprising applying a dual urease and nitrification inhibitor and / or a composition comprising the dual urease and nitrification inhibitor to a soil, a crop, and / or fertilizer, wherein the dual urease and nitrification inhibitor has a structure represented by Formula I:Formula I wherein:X is sulfur or oxygen;R is a moiety having a structure represented by Formula II or Formula III:Formula II; orRioFormula III; andRi, R2, R3, R4, Rs, Re, R7, Rs, R9, and Rio are each independently H, a substituted or unsubstituted Cl to C9 alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted saturated heterocyclic, a substituted or unsubstituted unsaturated heterocyclic, a substituted or unsubstituted aryl, a hydroxy (-OH), a methoxy (-OCH3), a ethoxy (OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen.

2. The method of claim 1, wherein the composition further comprises at least one nitrogenous fertilizer component.

3. The method of claim 2, wherein the at least one nitrogenous fertilizer component comprises urea, an ammonium salt, or anhydrous ammonia, or any combination thereof.

4. The method of any one of claims 1 to 3, wherein the composition further comprises at least one additional nitrification inhibitor, wherein the additional nitrification inhibitor preferably comprises nitrapyrin, dicyandiamide, ammonium thiosulfate, methyl-3-(4- hydroxyphenyl)propionate, 1,9-decanediol, potassium azide, 2-amino-4-chloro-6- methylpyrimidine, sulfathiazole, 4-amino-l,2,4-triazole, 2,4-diamino-6-trichloromethyl-s- triazine, potassium ethylxanthate, guanylthiourea, 4-nitrobenzotrichloride, 4- mesylbenzotri chloride, sodium thiocarbonate, phenylmercuric acetate, 3, 4- dimethylpyrazole phosphate, or sorgoleone, or any combination thereof.

5. The method of any one of claims 1 to 4, wherein the composition further comprises at least one additional urease inhibitor, wherein the additional urease inhibitor preferably comprises N-(n-butyl) thiophosphoric triamide, or N-(n-propyl) thiophosphoric triamide, or a combination thereof.

6. The method of any one of claims 1 to 5, wherein the method increases nitrogen use efficiency (NUE) of a crop.

7. The method of any one of claims 1 to 6, wherein the dual urease and nitrification inhibitor is (i) distributed throughout a fertilizer, (2) coated on a fertilizer, and / or (iii) is encapsulated.

8. The method of any one of claims 1 to 7, wherein: R is a pyrazole or -N(CH3)2; Ri is H or methyl; R2 is methyl, w-propyl, or w-butyl; R3 is H; R4 is H; Rs is H; Re is H; R7 is H; Rs is H; R9 is methyl; and Rio is methyl.

9. The method of claim 8, wherein the dual urease and nitrification inhibitor has a structure represented by Formula IV, Formula V, or Formula VI:Formula IVFormula VI10. A fertilizer composition comprising: a dual urease and nitrification inhibitor having a structure represented by Formula VII:Formula VII wherein:R is a moiety having a structure represented by Formula VIII or Formula IX;Formula IX; andRi, R2, R3, R4, Rs, Re, R7, Rs, R9, and Rio are each independently H, a substituted or unsubstituted Cl to C9 alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted saturated heterocyclic, a substituted or unsubstituted unsaturated heterocyclic, a substituted or unsubstituted aryl, a hydroxy (-OH), amethoxy (-OCH3), a ethoxy (-OC2H5), a nitro (-NO2), an amino (-NH2) or a halogen; and optionally one or more of: an additional inhibitor comprising an additional urease inhibitor and / or an additional nitrification inhibitor; a solid and / or a liquid carrier; and / or at least one nitrogen (N), phosphorus (P), and / or potassium (K) fertilizer component.

11. The fertilizer composition of claim 10, wherein: the additional nitrification inhibitor comprises nitrapyrin, dicyandiamide, ammonium thiosulfate, methyl-3-(4-hydroxyphenyl)propionate, 1,9-decanediol, potassium azide, 2-amino-4-chloro-6-methylpyrimidine, sulfathiazole, 4-amino- 1,2,4- triazole, 2,4-diamino-6-trichloromethyl-s-triazine, potassium ethylxanthate, guanylthiourea, 4-nitrobenzotrichloride, 4-mesylbenzotrichloride, sodium thiocarbonate, phenylmercuric acetate, 4-dimethylpyrazole phosphate, or sorgoleone, or any combination thereof; and / or the additional urease inhibitor comprises N-(n-butyl) thiophosphoric triamide, or N-(n- propyl) thiophosphoric triamide, or a combination thereof;12. The fertilizer composition of any one of claims 10 or 11, wherein the solid and / or liquid carrier comprises plaster of paris, flour, chalk powder, starch, gluten, kaolin, bentonite, colloidal silica, silica, dried distillers grains with solubles, lignin, a synthetic polymer, a wax, chitin, glycoaminoglycans, pectins, hyaluronic acid, chondroitin sulphate, dermatan sulphate, alginic acid, polymannuronic acid, polyguluronic acid, polyglucuronic acid, amylose, amylopectin, callose, chitosan, poly- galactomannan, dextran, xanthan, keratan sulphate, MgO, CaO, bone mill powder, rice husk, CaCCh, Na2COs, K2CO3, KH2PO4, NaHCCh, MgCCh, an alcohol, water, a liquid buffer, a chelating agent, or any combination thereof.- 33 -13. The fertilizer composition of any one of claims 10 to 12, wherein the at least one nitrogen (N), phosphorus (P), and / or potassium (K) fertilizer component comprises at least one nitrogenous fertilizer component.

14. The fertilizer composition of any one of claims 10 to 13, wherein: R is a pyrazole or -N(CH3)2; RI is H or methyl; R2 is methyl, w-propyl, or w-butyl; R3 is H; R4 is H; Rs is H;Re is H; R? is H; Rs is H; R9 is methyl; and / or Rio is methyl.

15. The fertilizer composition of any one of claims 10 to 14, wherein the dual urease and nitrification inhibitor has a structure represented by Formula IV, Formula V, or Formula VIFormula V ; orFormula VI

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