Methods and compositions for reducing gas emission and / or methane emission and / or improving feed utilisation of ruminants

Abstract

The present invention relates to a method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I) or a salt thereof: The present invention also relates to a feed additive comprising the one or more compounds as defined in the present invention and at least one selected from the group consisting of probiotica, bacteria, enzymes (lactate), fatty acids, herbs, spices or essential oils, vitamins, minerals, trace elements, flavour enhancers and preservatives.

Description

[0001] New PCT-Patent Application

[0002] Ludwig-Maximilians-Universitat Munchen, in Vertretung des Freistaates Bayern

[0003] Vossius Ref.: AJ2811 PCT

[0004] Methods and compositions for reducing gas emission and / or methane emission and / or improving feed utilisation of ruminants

[0005] The present invention relates to a method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I) as defined below or a salt thereof. The present invention also relates to a feed additive comprising the one or more compounds as defined in the present invention and at least one selected from the group consisting of probiotica, bacteria, enzymes, fatty acids, herbs, spices or essential oils, vitamins, minerals, trace elements, flavour enhancers and preservatives.

[0006] Background of the invention

[0007] Arguably the biggest man-made challenge of the century is to halt the progression of the climate change. Methane (CPU) is a potent greenhouse gas that accounts for a third of net global warming since the industrial revolution. A rapid reduction of methane emissions is regarded as the single most effective strategy to keep the goal of limiting global warming to 1.5 °C within reach.

[0008] While methane has a considerably higher short-term global warming potential than carbon dioxide (CO2), its atmospheric half-life of 9.1 ± 0.9 years is relatively brief. Nevertheless, one ton of methane is equivalent to 25 tons of CO2 in terms of its long-term climate impact over 100 years and the concentration of methane in the atmosphere has more than doubled since the beginning of the 19thcentury. One of the primary sources of anthropogenic methane is agriculture, particularly ruminant farming. The enteric fermentation process in cattle results in 2.8 Gt CO2e (CO2 equivalent), representing 5.63% of total greenhouse gas emissions in 2019. While the reduction of methanogenesis in ruminants through feed additives has been demonstrated, highly potent, affordable, and widely available compounds for inhibition are not yet established. Methanogenesis during enteric fermentation in cattle

[0009] Methanogenic archaea are ubiquitous in nature and play an important role in the global carbon cycle. The following three pathways for methanogenesis are known.

[0010] 1. The hydrogenotrophic pathway, in which H2 reduces CO2 to CH4.

[0011] 2. the methylotrophic pathway, in which the substrates methanol, methylamines or methylated sulfides act as methyl group donors for Coenzyme-M formation.

[0012] 3. The acetoclastic pathway, in which acetate is used to form acetyl-CoA, which then methylates tetrahydromethanopterin and is then incorporated into the metabolic pathway.

[0013] The hydrogenotrophic pathway is the predominant pathway for methanogenesis in ruminants. In the bovine digestive system resides a complex microbiome consisting of anaerobic bacteria, archaea and fungi, which enables its host to draw nutrients from otherwise mostly indigestible parts of plants. In this symbiosis, ruminal microbes break down substrates that are high in polymerized structural carbohydrates of otherwise low nutritional value. The efficiency of digestion and fermentation for different feeds is a genetically determined trait but depends largely on the quality and type of the feed. The main fermentation end products are short-chain fatty acids, including propionate, butyrate, acetate, ammonia, microbial protein and the gases CO2 and hydrogen (H2).

[0014] The methanogenesis pathway in archaea, such as Methanobrevibacter ruminantium, is called the Wolfe cycle. The process is based on H2 and CO2, and, to a lesser extent formate, as substrates. In the first step, CO2 is bound to methanofuran. It is then transferred and enzymatically processed with the help of reduced cofactor F420 to form tetrahydromethanopterin. In the next step, the methyl group is transferred to coenzyme-M. Methyl-coenzyme-M takes part in an enzymatic reaction unique to archaea. The protein that catalyzes this reaction is the methyl-coenzyme-M reductase (MCR). With the help of cofactor F430, methane is produced and the two substrates, methyl-coenzyme-M, and coenzyme-B, form a dimer. Regeneration of the coenzymes by ferrodoxin reduction completes the cycle. The inhibitor classes of halogenated compounds can target the F420 and F430 cofactors by reducing their active metal centers. The well-studied inhibitors 3-nitrooxypropanol (3-NOP) and coenzyme M analogs can also specifically bind to the MCR and oxidize the active center.

[0015] Through close ecto- and endosymbiontic association with protozoa, methanogens, such as M. ruminantium, act as the main H2 sink in the cow rumen. The H2 concentration in the rumen is largely under kinetic control, with a feedback loop regulating its accumulation. Reductive acetogenesis is one of the alternative H2 sinks, but usually strongly suppressed by the competing activity of methanogens. Elevated H2 concentrations result in a shift from acetate production towards propionate, butyrate and reductive acetogenesis, which are volatile fatty acids with high energy content. It is estimated that cows lose between 2% and 12% of their energy intake through the eructation of methane.

[0016] These facts suggest that methods for a reduction of enteric methane production can also enhance feed utilisation, improve diet digestibility, and ultimately increase livestock productivity. However, research has shown that an inhibition of methanogenesis by at least 50% is necessary for the hydrogen to be redirected to other metabolic pathways.

[0017] State of the art regarding feed additives for the reduction of ruminal methanogenesis

[0018] Different types of feed additives can be used to manipulate the ruminal composition and affect methane production, including oils, algae (Machado et al., 2014), haloalkanes, nitrates, minerals, ionophores, protozoa control, bacteriocins, phytochemicals (Soliva et al., 2011), 3-nitrooxypropanol (Hristov et al., 2015), acetogens, organic acids and other approaches. A combination of agents that target different parts or aspects of the methanogenesis pathway is likely the most potent solution with the least side effects.

[0019] For 3-nitrooxypropanol a long-term reduction of methane production of up to 27% with an average dose of 1,44 g per day / cow has been reported (Hristov et al., 2015). Asparagopsis taxiformis supplementation over a long period can result in reduction of methane emissions of up to 80%, which was achieved with an average daily dose of 48,5 g per day / cow (Roque et al., 2021). However, the active agents in A. taxiformis are cause for concern, as well as the limited availability of this biological food additive. Object of the invention

[0020] An object of the present invention is to provide a method of reducing methane production in animals of the order artiodactyla and methods for enhancing feed utilization, improving diet digestibility, or improving livestock productivity in animals of the order artiodactyla.

[0021] Summary of the invention

[0022] The present inventors could establish small-molecule compounds which act as inhibitors of methane production in cattle. The rate of methane production is preferably inhibited by at least 15 %, more preferably at least 20 %, even more preferably at least 25 %, further more preferably at least 30 %, most preferably at least 50% at a concentration of less than 10 grams per day / animal, preferably less than 5 grams per day / animal, most preferably less than 1 gram per day / animal. Due to their low complexity, it is possible to produce the molecular compounds on an industrial scale. The active ingredients are also considered to be well tolerated, as they target an enzymatic pathway that is unique to archaea and therefore have only minor off-target effects.

[0023] Specifically, the present invention relates to the following aspects:

[0024] In a first aspect, the present invention provides a method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I) or a salt thereof: n is an integer from 0 to 2; m is an integer from 0 to 2; each of X1, X2and X3is individually selected from the group consisting of a hydrogen atom, a halogen atom (-F, -Cl; -Br, -I), a hydroxy group (-OH), a thiol group (-SH), an azido group (-N3), a mesylate group (-O-SO2-CH3), a cyano group (-CN), wherein at least one of X1, X2and X3is not hydrogen, and not more than one of X1, X2and X3can be a hydroxy group (-OH) or a thiol group (-SH);

[0025] V is selected from the group consisting of a hydrogen atom (-H), a hydroxy group (-OH), an alkoxy group, and an amine group (-NH2);

[0026] Z is selected from the group consisting of a carbonyl group (-C(=O)-R1), a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), a sulfinamide group (-NH-S(=O)-R8), a phenyl group, and a tetrazol-5-yl group; wherein

[0027] R1is selected from the group consisting of -OCH3, -NH2, -NH-C1-C3 alkyl, -N(CI-C3 alkyl , -NH-CH2-C(Hal)3, and -NH-CH(OH)-C(Hal)3;

[0028] R2is selected from the group consisting of -C1-C3 alkyl, -C0-C2 alkyl-C(Hal)3, -F, -NH2, -NH-(CH(OH)-C(Hal)3, -NH-CH2-C(Hal)3;

[0029] R3is selected from the group consisting of -C1-C3 alkyl, -NH2 and -NH-CH(OH)-C(Hal)3;

[0030] R4is selected from the group consisting of -C1-C3 alkyl, -NH2 and -(4-methylphenyl);

[0031] R5is selected from the group consisting of -C(Hal)3, -CH(Hal)2, -O-CH2-C(Hal)3, -CO-NH-CH(OH)-C(Hal)3 and -NH-RX, wherein Rxis selected from the group consisting of -H, -C1-C3 alkyl, -CH(OH)-C(Hal)3;

[0032] R6is selected from the group consisting of -C1-C3 alkyl, -NH2, -F; and

[0033] R7is selected from the group consisting of -NH2, -NH-CH2-C(Hal)3;

[0034] R8is -C1-C4 alkyl.

[0035] For all embodiments of the present invention, it is to be understood that compounds of all formulas can be in any stereoisomeric form such as enantiomers and diastereomers, and racemates thereof. The racemates may contain the respective enantiomers in any ratio, but preferably in a 50:50 molar ratio. The compounds are preferably present as pure enantiomers, i.e., with an enantiomeric excess (ee) of at least 70 %, more preferably 80 %, even more preferably 90 %, most preferably 95 %.

[0036] In a second aspect, the present invention provides a method of enhancing feed utilisation in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof.

[0037] In a third aspect, the present invention provides a method of improving diet digestibility in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof.

[0038] In a fourth aspect, the present invention provides a method of improving livestock productivity in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof.

[0039] In a fifth aspect, the present invention provides the use of one or more compounds for reducing methane production, enhancing feed utilization, improving diet digestibility, or improving livestock productivity in animals of the order artiodactyla, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof.

[0040] In a sixth aspect, the present invention provides a method of reducing methane production in a bioreactor, the method comprising introducing an additive into the reactor, wherein the additive comprises one or more compounds selected from the above general formula (I) or a salt thereof.

[0041] In a seventh aspect, the present invention provides the use of one or more compounds for reducing methane production in a bioreactor, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. In an eighth aspect, the present invention provides the use of one or more compounds for reducing methane production in a process for the clarification and / or degradation of polyhalogenated effluents, and other processes that rely on the availability or production of hydrogen or volatile fatty acids; or in processes of composting, landfilling and manure spreading, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof.

[0042] In a ninth aspect, the present invention provides a composition comprising a nutritious feed and one or more compounds selected from the above general formula (I) or a salt thereof.

[0043] In a tenth aspect, the present invention provides a feed additive comprising one or more compounds selected from the above general formula (I) or a salt thereof and at least one selected from the group consisting of probiotica, bacteria, enzymes (such as lactase), fatty acids, herbs, spices or essential oils, vitamins, minerals, trace elements, flavour enhancers and preservatives.

[0044] In an eleventh aspect, the present invention provides a compound according to the general formula (III): wherein

[0045] W is selected from -SO2, -C(=O)- or -C(=O)-O-;

[0046] V is selected from -H or -OH; each E is individually selected from H, Cl or F, wherein at least two E's are a halogen atom selected from Cl and F, and k is an integer from O to 2. Brief description of the Figures

[0047] Figure 1 shows the inhibitory activity of 2-Bromoethyl mesylate.

[0048] Figure 2 shows the inhibitory activity of l-lodo-3-(methylsulfonyl)propane.

[0049] Figure 3 shows the inhibitory activity of 2-lodoethanesulfonamid, l-lodo-2-(methylsulfonyl) ethane and 3-lodopropanesulfonamide.

[0050] Figure 4 shows the inhibitory activity of / V-(2-lodoethyl)methanesulfonamide.

[0051] Figure s shows the inhibitory activity of 2-Bromoethanesulfonamide and l-Chloro-2- (methylsulfonyl)ethane.

[0052] Figure 6 shows the inhibitory activity of 2-Cyanoethanesulfonamide and 3-(Methylsulfonyl) propanenitrile.

[0053] Figure 7 shows the inhibitory activity of l,l,l-Trichloro-3-(methylsulfonyl)propane.

[0054] Figure 8 shows the inhibitory activity of 2-lodoethylsulfonyl fluoride.

[0055] Figure 9 shows the inhibitory activity of (2-Bromo-2,2-difluoroethyl)mesylate and 2,2,2-Trichloroethyl urea.

[0056] Figure 10 shows the inhibitory activity of 5-(2-lodoethyl)-lH-l,2,3,4-tetrazole, 2,2,2-Trichloro- N-(2,2,2-trichloro-l-hydroxyethyl)acetamide and 2,2,2-Trifluoro-N-(2,2,2-trichloro- l-hydroxyethyl)acetamide.

[0057] Figure 11 shows the inhibitory activity of (2,2,2-Tribromoethyl)sulfamate, (2,2,2-Trichloroethyl)sulfamate and (2,2,2-Tribromoethyl)mesylate.

[0058] Figure 12 shows the inhibitory activity of 2,2-Dichloro-N-(2,2,2-trichloro-l-hydroxyethyl) acetamide.

[0059] Figure 13 shows the inhibitory activity of 2,2,2-Trichloro-N-(2,2,2-trichloroethyl)acetamide.

[0060] Figure 14 shows the inhibitory activity of N-(2,2,2-trichloroethyl)sulfamide, 1,1,1-Trichloro- 3-methylsulfonylpropan-2-ol and N,N-bis(2,2,2-trichloroethoxy)urea.

[0061] Figure 15 shows the inhibitory activity of l-Azido-2-(methylsulfonyl)ethane, 2-Chloro- / V-(2,2,2-trichloroethyl) acetamide and 4-Methyl-N-(2,2,2-trichloroethyl)benzene- sulfonamide.

[0062] Figure 16 shows the inhibitory activity of 3,3,3-Trichloropropanesulfonamide, N-(2,2,2-Trichloroethyl)methanesulfonamide and (3,3,3-Trichloropropyl)mesylate.

[0063] Figure 17 shows the inhibitory activity of (3,3,3-Trichloropropyl)sulfamate and N-(3,3,3-Trichloropropyl)methanesulfonamide. Figure 18 shows the inhibitory activity of 4-Methyl-N-(2,2,2-trichloro-l-hydroxyethyl) benzenesulfonamide and N-(2-Bromo-2,2-difluoroethyl)sulfamide.

[0064] Figure 19 presents the ruminal methane concentration in Kathe following the administration of a high dose (3 g per day) of 3-iodopropanesulfonamide as an inhibitor.

[0065] Figure 20 presents the ruminal methane concentration in Hanni following the administration of a high dose (3 g per day) of 3-iodopropanesulfonamide as an inhibitor.

[0066] Figure 21 presents the ruminal methane concentration in Hanni following the administration of a placebo.

[0067] Figure 22 presents the ruminal methane concentration in Kathe following the administration of a placebo.

[0068] Figure 23 presents the ruminal methane concentration in Kathe following the administration of a low dose (1 g per day) of l-lodo-2-(methylsulfonyl)ethane as an inhibitor.

[0069] Figure 24 presents the ruminal methane concentration in Hanni following the administration of a low dose (1 g per day) of l-lodo-2-(methylsulfonyl)ethane as an inhibitor.

[0070] Figure 25 presents the ruminal methane concentration in Hanni following the administration of a placebo.

[0071] Figure 26 presents the ruminal methane concentration in Kathe following the administration of a placebo.

[0072] Figure 27 illustrates the changes in body weight for Hanni and Kathe over time, together with a corresponding linear regression.

[0073] Detailed description of the invention

[0074] The compounds selected from the above general formula (I) or a salt thereof for inhibition of methanogenesis were inspired by the chemical structures of two known inhibitors that are closely related to coenzyme M (Goenrich et al., 2004). Both of these inhibitors,

[0075] 2-bromoethansulfonate (Balch and Wolfe, 1979) and the strongest inhibitor found to date,

[0076] 3-bromopropanosulfonate (Ellermann et al., 1989), have the disadvantage that they lack the ability to passively penetrate the cell wall. Administration of these compounds leads to rapid development of resistance in animals and in in vitro culture systems. Therefore, they cannot be used as feed additives to reliably suppress methanogenesis. We suggest that a neutralisation of the negative charge on the sulfonic acid leads to improved cell permeability and therefore passive cell penetration. The compounds differ substantially from state of the art inhibitors since the sulfonate group is replaced by a methylsulfonyl, methylsulfonate, methylsulfinyl, sulfonamide, sulfamate or sulfinamide group. Additionally, different functional groups were added. For the functional groups, a selection was made from several useful groups, including imidazole as a nickel chelator.

[0077] Disclosed herein is a method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I*) or a salt thereof: wherein each of X1, X2and X3is individually selected from the group consisting of a hydrogen atom, a halogen atom (-F, -Cl; -Br, -I), a hydroxy group (-OH), a thiol group (-SH), an azido group (-N3), a mesylate group (-O-SO2-CH3), and a cyano group (-CN); preferably, at least one of X1, X2and X3is not hydrogen, and not more than one of X1, X2and X3can be a hydroxy group (-OH) or a thiol group (-SH); p is 0, 1, 2 or 3; q is 1, 2 or 3; a is 0 or 1; each V is individually selected from the group consisting of a hydrogen atom (-H), a hydroxy group (-OH), an oxo group (=0), an alkoxy group, and an amine group (-NH2); when V is an oxo group, then a is 0; when V is a hydrogen atom (-H), a hydroxy group (-OH), an alkoxy group, or an amine group (-NH2), then a is 1;

[0078] A is selected from the group consisting of -CH2-, -CH(OH)-, -C(=O)NH-, -C(=NH)NH-, -C(NH2)=N-, -C(=NH)-, -NH-, -O- or -N(CI-C3alkyl)- (preferably -N(CH3-), and -S(=O)O-; W is selected from the group consisting of a single bond, -C(=O)-, -S(=O)2-, -S(=O)-, -C(=S)-, -CH(NH2)-, -C(=NH)-, and -C(=O)-C(=O)-; b is 0 or 1; when B is selected from the group consisting of H, F, -CX3, -NH2, -NH-C1-C3 alkyl (preferably -NH-CH3), -N(CI-C3 alkyl)2(preferably -N(CH3)2, 4-methylphenyl, tetrazol-5-yl, and -C1-C4 alkyl (preferably ethyl, propyl or tert-butyl), then b is 0; when B is selected from the group consisting of -CH2-, -CH(OH)-, -C(=O)NH-, -C(=NH)NH-, -C(=NH)-, -NH-, -O-, and -N(Ci-Cs alkyl)- (preferably -N(CH3-), then b is 1.

[0079] The compounds of general formula (I) below are a preferred embodiment of the compounds of general formula (I*). Preferred embodiments of X1, X2, X3and V described below with respect to general formula (I) are also preferred embodiments of general formula (I*). The second to tenth aspects of the present invention may also use the compounds of general formula (I*) instead of the compounds of general formula (I).

[0080] In the first aspect, the present invention relates to a method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I) or a salt thereof: n is an integer from 0 to 2; m is an integer from 0 to 2; each of X1, X2and X3is individually selected from the group consisting of a hydrogen atom, a halogen atom (-F, -Cl; -Br, -I), a hydroxy group (-OH), a thiol group (-SH), an azido group (-N3), a mesylate group (-O-SO2-CH3), a cyano group (-CN), wherein at least one of X1, X2and X3is not hydrogen, and not more than one of X1, X2and X3can be a hydroxy group (-OH) or a thiol group (-SH); V is selected from the group consisting of a hydrogen atom (-H), a hydroxy group (-OH), an alkoxy group, and an amine group (-NH2);

[0081] Z is selected from the group consisting of a carbonyl group (-C(=O)-R1), a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), a sulfinamide group (-NH-S(=O)-R8), a phenyl group, and a tetrazol-5-yl group; wherein

[0082] R1is selected from the group consisting of -OCH3, -NH2, -NH-C1-C3 alkyl (preferably -NH-CH3), -N(CI-C3 alkyl (preferably -NfCHsh), -NH-CH2-C(Hal)3 (preferably -NH-CH2-CCI3), and -NH-CH(OH)-C(Hal)3(preferably -NH-CH(OH)-CCI3);

[0083] R2is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -C0-C2 alkyl-C(Hal)3 (preferably -CCI3), -F, -NH2, -NH-(CH(OH)-C(Hal)3 (preferably -NH-(CH(OH)-CCI3), -NH-CH2-C(Hal)3(preferably -NH-CH2);

[0084] R3is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2 and -NH-CH(OH)-C(Hal)3(preferably -NH-CH(OH)-CCI3);

[0085] R4is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2 and -(4-methylphenyl);

[0086] R5is selected from the group consisting of -C(Ha 1)3 (preferably -CCI3 or -CF3), -CH(Hal)2 (preferably -CHCI2), -O-CH2-C(Hal)3 (preferably -O-CH2-CCI3), -CO-NH-CH(OH)-C(Hal)3(preferably -CO-NH-CH(OH)-CCI3) and -NH-RX, wherein Rxis selected from the group consisting of -H, -C1-C3 alkyl (preferably -CH3 or -C2H5), -CH(OH)-C(Hal)3(preferably -CH(OH)-CC );

[0087] R6is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2, -F; and

[0088] R7is selected from the group consisting of -NH2, -NH-CH2-C(Hal)3 (preferably -NH-CH2-CCI3);

[0089] R8is -C1-C4 alkyl (preferably tert-butyl).

[0090] Reducing methane production in animals preferably refers to a reduction of at least 15 %, more preferably at least 20 %, even more preferably at least 25 %, further more preferably at least 30 %, most preferably at least 50%. Methane reductions as referred to herein can be measured in individual animals by laser methane detectors by methods known in the art (Chagunda et al., 2009). Alternatively, methane produced by a ruminant can also be assessed at barn level, e.g. by making use of commercially available solutions with the GreenFeed system (GreenFeed system C-Lock, Rapid City, SD USA etc.).

[0091] According to the present invention, animals of the order artiodactyla are preferably animals of the suborder ruminantia or are animals of the family Camelidae (such as dromedary, Bactrian camel, llama, alpaca, guanaco, and vicuna), more preferably animals of the family Cervidea (such as muntjac, elk, wapiti, red deer, fallow deer, reindeer, caribou, white-tailed deer, roe deer, and moose) or Bovidae, even more preferably of the tribus caprini (such as sheep and goat) or bovini (such as cow), most preferably of the genus bos (cows, Cattle, bos taurus). The animals are preferably lifestock, i.e., domesticated animals raised in an agricultural setting in order to provide labour and / or produce products for consumption such as meat, eggs, milk, fur, leather, and wool. Preferably, the lifestock is selected from cows, sheeps, goats, alpacas, llamas, and camels, most preferably cows.

[0092] The compounds can be administered individually or in combination with each other. The compound(s) can be administered in the form of an emulsion or a solution. The compounds can be administered bound to a matrix, as a polymer consisting of one or more different compounds, or conjugated to other polymers. The feed additive can be administered as a solid, compressed as pellets with feed and feed additives. The feed additive can be administered in a liquid solvent, in capsules or as a gel. The feed additive can be administered via a bolus over a long period of time in one administration, with the bolus matrix comprising minerals, vitamins, trace elements and additives that preserve the active compounds. The feed additive can be administered as a "salt lick" or "lick mix" in combination with sodium chloride and / or other salts, and optionally vitamins, minerals, trace elements and / or additives that preserve the active compound. The feed additive can be administered with desired amounts of fatty acids or other substances that improve absorption, effect, and shelf life. The administration can be performed with substances that improve the feeding behaviour of the animals, in particular to achieve the intake of the desired quantities of the active substance ("flavour enhancer"). For example, by a combination with herbs, spices, or essential oils. The administration can be performed with other supplements including vitamins, minerals, trace elements, flavour enhancers and preservatives. The at least one compound of general formula (I), or a salt thereof can be administered in combination with at least one additional active substance which shows similar effects with regard to methane formation in the rumen and which is selected from the group consisting of diallyl disulfide, garlic oil, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid and derivatives thereof. Further components may be given together with the compound according to the present invention include, for example, plants, plant parts, or extracts thereof, including but not limited to herbs, leaves, and grasses, as well as algae, yeasts, fungi, essential oils, and ionophores such as monensin or rumensin.

[0093] The amount of the at least one active compound as defined in formula (I) administered to the animals of the order artiodactyla is from 1 mg to 10 g per kg of feed, preferably from 10 mg to 1 g per kg of feed, more preferably, from 50 mg to 500 mg per kg of feed.

[0094] The salts of the one or more compounds selected from the general formula (I) may be any type of salts. If intended for animals, the salts are preferably veterinarily acceptable, i.e., harmless for ingestion. Examples of the salts include inorganic acid salts, such as a hydrochloride salt, a sulfate salt, a nitrate salt, and the like; organic acid salts, such as an acetate salt, a methanesulfonic acid salt, and the like; alkali metal salts, such as a sodium salt, a potassium salt, and the like; alkaline earth metal salts, such as a magnesium salt, a calcium salt, and the like; quaternary ammonium salts, such as dimethylammonium, triethylammonium, and the like; and the like.

[0095] Preferred embodiments of the one or more compounds selected from general formula (I) of the present invention or a salt thereof will be described below: n is an integer from 0 to 2, preferably 0 or 1. m is an integer from 0 to 2. Preferably, at least on of m and n is 0, and preferably m and n are 0. Preferably, m or n is 1 and the other of m and n is 0.

[0096] Each of X1, X2and X3is individually selected from the group consisting of a hydrogen atom, a halogen atom (-F, -Cl; -Br, -I), a hydroxy group (-OH), a thiol group (-SH), an azido group (-N3), a mesylate group (-O-SO2-CH3), a cyano group (-CN), wherein at least one of X1, X2and X3is not hydrogen, and not more than one of X1, X2and X3can be a hydroxy group (-OH) or a thiol group (-SH). Preferably, each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), an azido group (-N3), or a cyano group (-CN). More preferably each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I).

[0097] V is selected from the group consisting of a hydrogen atom (-H), a hydroxy group (-OH), an alkoxy group, and an amine group (-NH2). Preferably, V is selected from the group consisting of a hydrogen atom (-H) and a hydroxy group (-OH).

[0098] Z is selected from the group consisting of a carbonyl group (-C(=O)-R1), a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), a sulfinamide group (-NH-S(=O)-R8), a phenyl group, and a tetrazol-5-yl group; wherein

[0099] R1is selected from the group consisting of -OCH3, -NH2, -NH-C1-C3 alkyl (preferably -NH-CH3), -N(Ci-Cs alkyl)2 (preferably -NfCHsh), -NH-CH2-C(Hal)3 (preferably -NH-CH2-CCI3), and -NH-CH(OH)-C(Hal)3(preferably -NH-CH(OH)-CCI3);

[0100] R2is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -C0-C2 alkyl-C(Hal)3, (preferably -CCI3), -F, -NH2, -NH-(CH(OH)-C(Hal)3(preferably -NH-(CH(OH)-CCI3), -NH-CH2-C(Hal)3(preferably -NH-CH2-CCI3);

[0101] R3is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2 and -NH-CH(OH)-C(Hal)3(preferably -NH-CH(OH)-CCI3);

[0102] R4is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2 a- d -(4-methylphenyl); R5is selected from the group consisting of -C(Hal)3 (preferably -CCI3 or -CF3), -CH(Hal)2 (preferably -CHCI2), -O-CH2-C(Hal)3(preferably -O-CH2-CCI3), -CO-NH-CH(OH)-C(Hal)3(preferably -CO-NH-CH(OH)-CCl3) and -NH-RX, wherein Rxis selected from the group consisting of -H, -C1-C3 alkyl (preferably -CH3 or -C2H5), -CH(OH)-C(Hal)3 (preferably -CH(OH)-CCl3);

[0103] R6is selected from the group consisting of -C1-C3 alkyl (preferably -CH3), -NH2, -F; and

[0104] R7is selected from the group consisting of -NH2, -NH-CH2-C(Hal)3 (preferably -NH-CH2-CCI3);

[0105] R8is -C1-C4 alkyl (preferably tert-butyl).

[0106] Preferably, Z is selected from the group consisting of a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), and a tetrazol-5-yl group; wherein

[0107] R2is selected from the group consisting of -CH3, -CCI3, -NH2, -NH-CH2-CCI3;

[0108] R3is selected from the group consisting of -CH3, -NH2;

[0109] R4is selected from the group consisting of -CH3, -NH2;

[0110] R5is selected from the group consisting of -CCI3, -CF3, -CHCb, -O-CH2-CCI3, -CO-NH-CH(OH)-CCl3 and -NH-RXwherein Rxis selected from the group consisting of -H, -CH3, -C2H5, -CH(OH)-CCI3;

[0111] R6is selected from the group consisting of -CHs, -NH2, -F; and

[0112] R7is selected from the group consisting of -NH2, -NH-CH2-CCI3.

[0113] More preferably, Z is selected from the group consisting of a sulfone group (-SO2R2), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), and a tetrazol-5-yl group; wherein

[0114] R2is selected from the group consisting of -CH3, -NH2, -NH-CH2-CCI3;

[0115] R4is selected from the group consisting of -CH3, -NH2;

[0116] R5is selected from the group consisting of -CCI3, -CF3, -CHCb, -O-CH2-CCI3, -CO-NH-CH(OH)-CCl3 and -NH-RXwherein Rxis selected from the group consisting of -H, -CH3, -C2H5, -CH(OH)-CCI3; and

[0117] R6is selected from the group consisting of -CHs, -NH2, -F. Even more preferably, Z is selected from the group consisting of a sulfone group (-SO2R2), an N-sulfonamide group (-NH-SO2-R4), a sulfonate group (-O-SO2-R6);

[0118] R2is -CH3or -NH2;

[0119] R4is -CH3 or -NH2; and

[0120] R6is -CH3or -NH2.

[0121] In a preferred embodiment of the first aspect of the present invention, the one or more compounds selected from general formula (I) or a salt thereof are one or more compounds selected from the following general formula (II) or a salt thereof:

[0122] X-(CHr)s-Y (II) wherein X is a heteroatom-containing group, Y is a non-ionic functional group containing an S atom, r is each independently an integer from 0 to 2 as valency permits; and s is an integer from 1 to 4.

[0123] Preferably, in the one or more compounds selected from general formula (II) or a salt thereof, X is selected from the group consisting of a halogen atom, an amino group (-NR21R22), an azido group (-N3), a cyano group (-CN), a formamide group (-CO-NH2), an N-formamide group (-NH-CHO), a hydroxy group (-OH), an alkoxy group (-OR24), an aldehyde group (-CHO), an O-hydroxylamine group (-O-NH2), a hydroxylamine group (-NH-OH),an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 18 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S; and

[0124] Y is selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfinamide group (-SO-NR32R33), a sulfoxide group (-S(=O)-R28)), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37). wherein n and m are as defined in claim 1; R21and R22are each independently selected from the group consisting of H, -OH and -C1-C3 alkyl;

[0125] R24is selected from the group consisting of -C1-C3 alkyl;

[0126] R28is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;

[0127] R29and R30are each independently selected from the group consisting of H and -C1-C3 alkyl;

[0128] R31is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;

[0129] R32and R33are each independently selected from the group consisting of H and -C1-C3 alkyl;

[0130] R34is selected from the group consisting of H, -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;

[0131] R35and R36are each independently selected from the group consisting of H and -C1-C3 alkyl; and

[0132] R37is selected from the group consisting of H and -C1-C3 alkyl.

[0133] The heteroatom-containing group X can be selected from the group consisting of a halogen atom, an amino group (-NR21R22), an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), an alkoxy group (-OR24), an O-hydroxylamine group (-O-NH2), an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 10 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S. More preferably, X is selected from the group consisting of a halogen atom, an amino group (-NR21R22), an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), an alkoxy group (-OR24), an O-hydroxylamine group (-O-NH2), an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 10 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S. Even more preferably, X is selected from the group consisting of a chlorine atom, a bromine atom, an iodine atom, an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), a methoxy group (-O-CH3), an O-hydroxylamine group (-O-NH2), an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 10 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S. Most preferably, X is selected from the group consisting of a chlorine atom, a bromine atom, an iodine atom, an azido group (-N3), a cyano group (-CN).

[0134] The halogen atom (abbreviated as "Hal") can preferably be selected from the group consisting of a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), and an iodine atom (I). More preferably the halogen atom is selected from the group consisting of a chlorine atom, a bromine atom, and an iodine atom, most preferably, the halogen atom is a bromine atom or an iodine atom. In the group "C(Hal)3" in the definitions of R1, R2, R3, R4, R5, R6, and R7, each Hal is individually selected from the group consisting of F, Cl, Br and I. Preferably, each Hal is the same selected from the group consisting of F, Cl, Br and I. More preferably, "C(Hal)3" is CF3 of CCI3, most preferably CCI3.

[0135] In the amino group (-NR21R22), R21and R22are each independently selected from the group consisting of H, -OH and -C1-C3 alkyl. Preferably, R21and R22are each independently selected from the group consisting of H, -OH and a methyl group, most preferably, each of R21and R22is H.

[0136] In the alkoxy group (-OR24), R24is selected from the group consisting of -Ci-Cs alkyl. Preferably, R24is a methyl group.

[0137] The heterocyclic ring system having 5 to 10 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N (nitrogen), O (oxygen) and S (sulfur) includes aromatic and non-aromatic groups. Preferably, the heterocyclic ring system has 5 or 6 ring atom members and comprises 1 to 4 heteroatoms selected from the group consisting of N, O and S, more preferably the heterocyclic ring system has 5 or 6 ring atom members and comprises 1 to 4 N-atoms. Most preferably, the heterocyclic ring system has 5 ring atom members and comprises 2 to 4 N-atoms.

[0138] Exemplary heterocyclic ring systems include pyrrolyl, furyl, thienyl, diazolyl (imidazolyl, pyrazol), triazolyl (1,2,3-triazole or 1,2,4-triazole), tetrazolyl, thiazolyl, oxazolyl, pyrazolyl, isoxazolyl, pyrrolidinyl, tetra hydrofuryl, tetrahydrothienyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, pyrazolidinyl, isoxazolidinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,3,5- triazinyl, morpholinyl, piperazinyl, thiopyranyl, piperidinyl, dioxanyl, tetrahydropyranyl, dithianyl, azepinyl, oxepinyl, thiepinyl, diazepinyl, azocinyl, oxocinyl, thiocinyl, dioxocinyl, azoninyl, oxoninyl, thioninyl, diazoninyl, azecinyl, oxecinyl, thiecynyl, and dioxecinyl. More preferably, the heterocyclic ring system is a diazolyl, triazolyl, or tetrazolyl group, most preferably, an lH-imidazol-l-yl group or a tetrazol-5-yl group.

[0139] The non-ionic functional group Y is a group that does not carry a net electrical charge under physiological conditions, i.e., at neutral pH. The non-ionic functional group Y contains an S atom. Y is preferably selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfinamide group (-SO-NR32R33), a sulfoxide group (-S(=O)-R28)), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37). More preferably, Y is selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37). Even more preferably, Y is selected from the group consisting of a sulfonamide group (-SO2-NH2), a methylsulfone group (-SO2-CH3), a methylsulfonate group (-OSO2-CH3), a sulfamate group (-OSO2-NH2), a methanesulfonamide-N-yl group (-NH-SO2-CH3). Most preferably, Y is selected from the group consisting of a sulfonamide group (-SO2-NH2), a methylsulfone group (-SO2-CH3), a methylsulfonate group (-OSO2-CH3), a sulfamate group (-OSO2-NH2), and an N-methanesulfonamide group (-NH-SO2-CH3).

[0140] In the sulfoxide group (-S(=O)-R28), R28is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom. Preferably R28is selected from the group consisting of methyl, trifluoromethyl and a fluorine atom.

[0141] In the sulfonamide group (-SO2-NR29R30), R29and R30are each independently selected from the group consisting of H and -C1-C3 alkyl. Preferably, R29and R30are each independently selected from H and methyl, more preferably, each of R29and R30is H. In the sulfone group (-SO2R31), R31is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom. Preferably, R31is selected from the group consisting of methyl, trifluoromethyl and a fluorine atom.

[0142] In the sulfinamide group (-SO-NR32R33), R32and R33are each independently selected from the group consisting of H and -C1-C3 alkyl. Preferably, R32and R33are each independently selected from H and methyl, more preferably, each of R32and R33is H.

[0143] In the sulfonate group (-OSO2R34), R34is selected from the group consisting of H, -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom. Preferably, R34is selected from the group consisting of H, methyl, trifluoromethyl and a fluorine atom, more preferably, R34is selected from the group consisting of methyl, trifluoromethyl and a fluorine atom, most preferably, R34is methyl.

[0144] In the sulfamate group (-OSC>2-NR35R36), R35and R36are each independently selected from the group consisting of H and -C1-C3 alkyl. Preferably, R35and R36are each independently selected from the group consisting of H and methyl. Most preferably, each of R35and R36is H.

[0145] In the N-sulfonamide group (-NH-SO2-R37), R37is selected from the group consisting of H and -C1-C3 alkyl. Preferably, R37is selected from the group consisting of H and methyl, most preferably R37is methyl.

[0146] The -C1-C3 alkyl group in the above definitions of R1, R2, R3, R4, R5, R6, R7, R21, R22, R24, R28, R29, R30, R31, R32, R33, R34, R35, R36, and R37can be selected from a methyl group (-CH3), an ethyl group (-CH2-CH3), an n-propyl group (-CH2-CH2-CH3), an isopropyl group (-CHfCHsh). Preferably, the -C1-C3 alkyl group is a methyl group or an ethyl group, most preferably a methyl group. Furthermore, it is preferable that the -C1-C3 alkyl group in each of the above definitions of R1, R2, R3, R4, R5, R6, R7, R21, R22, R24, R28, R29, R30, R31, R32, R33, R34, R35, R36, and R37is a methyl group.

[0147] The -C1-C3 haloalkyl group in the above definitions of R28, R31, R34, and R37is a -C1-C3 alkyl group as defined above, wherein 1 or more hydrogen atoms, preferably all of the hydrogen atoms are replaced by halogen atoms. The halogen atoms are each the same or different selected from the group consisting of a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), and an iodine atom (I). More preferably the halogen atoms are each the same or different selected from the group consisting of a fluorine atom, a chlorine atom, and a bromine atom, even more preferably, the halogen atoms are each the same or different selected from a fluorine atom and a chlorine atom, most preferably, each of the halogen atoms is a fluorine atom. The -C1-C3 haloalkyl group is preferably a perhaloalkyl group, i.e., each of the hydrogen atoms of the corresponding -C1-C3 alkyl group is replaced by a halogen atom. Most preferably, the -C1-C3 haloalkyl group is a trifluoromethyl group.

[0148] In the second aspect, the present invention relates to a method of enhancing feed utilisation in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect.

[0149] Enhancing feed utilization encompasses, without being limited thereto, increasing the average daily weight gain, carcass yield, and / or milk yield, and / or energy corrected milk yield. In the alternative, enhancing feed utilization encompasses decreasing the average daily feed uptake while maintaining positive, animal specific average daily weight gain, and / or milk yield, and / or energy corrected milk yield. Enhancing feed utilization can be evaluated by combining direct measurements of animal performance with indicators of nutrient use efficiency. Daily feed intake is recorded to calculate dry matter intake, which is then related to milk yield or body weight gain as measures of productive efficiency (National Academies of Sciences, 2021). To account for metabolic processes, milk can be standardized for protein and fat content and further categorized according to the composition of milk fats, yielding the energy-corrected milk (ECM) value (Van Soest, 1994). Feed conversion efficiency, expressed as ECM per kilogram of dry matter intake, provides an integrated measure of utilization under controlled feeding conditions (Hall, 2023). In the third aspect, the present invention relates to a method of improving diet digestibility in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect.

[0150] Improving diet digestibility means in accordance with the present invention an enhancement in the conversion of feed into energy. Diet digestibility can be assessed by the use of established in vitro and in situ approaches. The Hohenheimer Futterwerttest represents a standardized gas production technique that allows estimation of organic matter digestibility of feed substrates under controlled laboratory conditions (Menke and Steingass, 1988). Complementary to this, the in situ nylon bag method can be applied, where weighed feed samples are inserted into the rumen of fistulated cows and retrieved after 48 h of incubation. The recovered bags are then washed, dried weighed and analyzed to determine the proportion of degraded material (0rskov and McDonald, 1979). The results from these assays provide an estimate of the potential nutrient availability of feedstuffs and indicate how effectively dietary components can be utilized by the rumen microbiota. Higher values reflect improved digestibility, which can translate into enhanced feed efficiency and animal performance under practical feeding conditions.

[0151] In the fourth aspect, the present invention relates to a method of improving livestock productivity in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect.

[0152] Improving livestock productivity encompasses, without being limited thereto, increasing the average daily weight gain, and / or decreasing the average daily feed uptake while maintaining a positive, animal specific average daily weight gain. Improved livestock productivity can result either from greater feed conversion efficiency, where more energy-corrected milk or body weight gain is obtained per unit of dry matter intake, or from higher overall ECM yield or weight gain at unchanged efficiency, where animals achieve greater output without altering conversion rates (Seiko, 2025).

[0153] In the fifth aspect, the present invention relates to the use of one or more compounds for reducing methane production, enhancing feed utilization, improving diet digestibility, or improving livestock productivity in animals of the order artiodactyla, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect. The expressions "reducing methane production", "enhancing feed utilization", "improving diet digestibility", and "improving livestock productivity" and "animals of the order artiodactyla" have the same meaning as in the first to fourth aspects described above.

[0154] The methods and uses of the present invention, in which one or more compounds or a feed additive comprising the one or more compounds are administered to animals are non-therapeutic methods. Specifically, it is clear that "methane production" is not an illness of the animals. Therefore, "reduction of methane production" does not relate to the treatment of a medical condition of the animals. Furthermore, "reduction of methane production" can lead to an enhanced feed utilization, an improved diet digestibility, and / or an improved livestock productivity. Therefore, the methods and uses of the first to fifth aspect of the present invention do not relate to methods for treatment of the animal body by therapy.

[0155] Anaerobic fermentation in bioreactors and environment

[0156] Methane is the predominant gaseous product of the complete anaerobic degradation of organic matter. It is a potent greenhouse gas, the concentration of which in the atmosphere is on the increase. Hydrogen, a fermentation product of numerous facultative and strictly anaerobic bacteria, as well as an intermediate in methane production, is also a combustible gas that could be recovered and utilized as a fuel. Hydrogen represents an attractive energy source with the potential to replace conventional fossil fuels from both an economic and an environmental standpoint. The advantages of hydrogen are manifold. In addition to a wider range of industrial applications than methane, it can be used in the synthesis of ammonia, alcohols and aldehydes, as well as for hydrogenation of edible oil, petroleum, coal and shale oil. Furthermore, hydrogen is an ideal fuel, producing only water as the sole product of combustion. It can be used directly in internal combustion engines or to generate electricity through fuel cells.

[0157] Bioreactors or fermenters can be found in a variety of sizes and forms. They encompass a range of versions, from laboratory-sized glassware to steel tanks used in breweries and sewage treatment facilities. There is also a vast variety in applications. In some applications a certain substrate, environmental condition or microbial community is needed, to enhance the efficiency of the output of the desired products. In the context of waste and wastewater treatment, the utilisation of established microbial communities is preferable to that of pure cultures of hydrogen-producing bacteria. However, in a mixed culture system operating under anoxic conditions, the hydrogen produced by bacteria is subject to rapid consumption by other hydrogen-consuming bacteria. Consequently, to effectively harness hydrogen from a mixed culture system, a pretreatment process is required to suppress the majority of hydrogen-consuming bacterial activity while maintaining the viability of the hydrogenproducing bacteria. Other examples of bioreactors or anaerobic fermentation systems where the presence of hydrogen-consuming bacteria or methanogens is depreciated are: acetate reduction for biofermentation of ethanol; production of volatile fatty acids (Steinbusch et al., 2009); production of polyhydroxyalkanoates (Costa et al., 2023); clarification and degradation of polyhalogenated effluents (Lin et al., 2021) and other processes that rely on the availability of hydrogen or volatile fatty acids. Composting, landfilling and manure spreading are other examples where the inhibition of methanogenesis is environmentally beneficial.

[0158] Effective pretreatment processes include heating, acidic or basic treatment, aeration, chemicals, electric current, and more (Guo et al., 2010). The chemicals used for the selective cultivation of hydrogen-producing bacteria include 2-bromoethanosulfonate, acetylene and chloroform (Sparling et al., 1997).

[0159] By inhibiting methane production, the efficiency of hydrogen production and the availability of volatile fatty acids can be improved (see Guo et al., 2010 for an overview of this technology). In the sixth aspect, the present invention relates to a method of reducing methane production in a bioreactor, the method comprising introducing an additive into the reactor, wherein the additive comprises one or more compounds selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect. The bioreactor is preferably a hydrogen production bioreactor, an ethanol production bioreactor, or a volatile fatty acid production bioreactor.

[0160] Preferably, the method of the sixth aspect comprises introducing the additive for the pretreatment of the inoculum, effluents sludge or wastes.

[0161] In the seventh aspect, the present invention relates to the use of one or more compounds for reducing methane production in a bioreactor, wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect. The bioreactor is the same as in the above sixth aspect.

[0162] In the eighth aspect, the present invention relates to the use of one or more compounds for reducing methane production in a process for the clarification and / or degradation of polyhalogenated effluents, and other processes that rely on the availability of hydrogen or volatile fatty acids; or in processes of composting, landfilling and manure spreading (including feeding the compounds to animals of the order artiodactyla that produces the manure, which in turn produces less methane), wherein the one or more compounds are selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect.

[0163] In the ninth aspect, the present invention relates to a composition comprising a nutritious feed and one or more compounds selected from the above general formula (I) or a salt thereof. The compounds selected from the above general formula (I) or a salt thereof are the same as defined above with respect to the first aspect. A nutritious feed is a feed which is a source of at least one of carbohydrates, proteins or fat for the animals. Examples of the nutritious feed include forage such as grasses, legumes, and silage, including alfalfa, clover, and hay; grains such as corn, barley, oats, and wheat; protein supplements such as soybean meal, canola meal, and cottonseed meal; by-products such as beet pulp, citrus pulp, and brewers' grains; and concentrates, which are high-energy feeds that include grains and / or protein supplements. A nutritious feed is usually a feed that provides essential nutrients required for growth, health, and overall well-being. It typically contains a balanced mix of carbohydrates, proteins, fats, vitamins, minerals, and water, tailored to meet the specific dietary needs of the species being fed. Nutritious feed promotes proper development, supports reproductive health, enhances immune function, and ensures optimal production, such as milk, eggs, or meat, in livestock. The composition and quality of nutritious feed can vary depending on the animal's age, activity level, and purpose (e.g., for maintenance, growth, or production). Additionally, the nutritious feed may comprise the feed additive mentioned in the eleventh aspect below. As is known by those skilled in the art, the term „nutritious feed" in this context refers to products used in animal nutrition for purposes of improving the quality of feed and the quality of food from animal origin, or to improve the animals' performance, e.g. providing enhanced digestibility of the feed materials. Nonlimiting examples include technological additives such as preservatives, antioxidants, emulsifiers, stabilising agents, acidity regulators and silage additives; sensory additives, especially flavours and colorants; (further) nutritional additives, such as vitamins, amino acids and trace elements; and (further) zootechnical additives, such as digestibility enhancers and gut flora stabilizers.

[0164] For feed compositions for ruminants such as cows, the ruminant diet is usually composed of an easily degradable fraction (concentrate) and a fiber-rich less readily degradable fraction (hay, forage, or roughage).

[0165] Hay is made of dried grass, legume, or whole cereals (grasses: timothy, ryegrasses, fescues; legumes: clover, lucerne / alfalfa, peas, beans, vetches; whole cereals: barley, maize, oat, sorghum). Other forage crops include sugarcane, kale, rape, and cabbage. Root crops such as turnips, swedes, mangles, fodder beet, and sugar beet (including sugar beet pulp and molasses) are also used. Tubers such as potatoes, cassava, and sweet potato are further feed sources. Silage is an ensiled version of the fiber-rich fraction prepared by controlled anaerobic fermentation.

[0166] The concentrate fraction is largely cereals (barley including brewers' and distillers' grains, maize, wheat, sorghum) but also often contains protein-rich feed ingredients such as soybean, rapeseed, palm kernel, cotton seed, and sunflower.

[0167] Cows may also be fed total mixed rations (TMR), in which forage, silage, and concentrate are mixed before serving. Premixes are an example of feed additives that may comprise the active compounds according to the invention. The compounds can also be included in a bolus placed in the rumen, releasing defined dosages continuously over a specific period.

[0168] In the tenth aspect, the present invention relates to a feed additive comprising one or more compounds selected from the above general formula (I) or a salt thereof and at least one selected from the group consisting of probiotica, bacteria, enzymes (such as lactase), fatty acids, herbs, spices or essential oils, vitamins, amino acids, minerals, trace elements, sensory additives, especially flavour enhancers and colorants, antioxidants, (further) zootechnical additives, such as digestibility enhancers and gut flora stabilizers, emulsifiers, stabilising agents, acidity regulators and silage additives, and preservatives.

[0169] Preferably, the animal feed additive comprises (a) at least one compound of formula (I) and (b) at least one fat-soluble vitamin, (c) at least one water-soluble vitamin, (d) at least one trace mineral, and / or (e) at least one macro mineral.

[0170] So-called premixes are examples of animal feed additives of the invention. A premix designates a preferably uniform mixture of one or more micro-ingredients with diluents and / or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.

[0171] Apart from the active ingredients of the invention (i.e., the compounds of formula (I)), the premix of the invention contains at least one fat-soluble vitamin, and / or at least one water- soluble vitamin, and / or at least one trace mineral, and / or at least one macro mineral. In other words, the premix of the invention comprises the at least one compound according to the invention together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

[0172] Macro minerals may be separately added to the feed. Therefore, in a particular embodiment, the premix comprises the active ingredients of the invention together with at least one additional component selected from the group consisting of fat-soluble vitamins, water- soluble vitamins, and trace-minerals.

[0173] Examples of Additional Components of the feed additive:

[0174] • Fat-soluble vitamins: vitamin A, vitamin D3, vitamin E, and vitamin K (e.g. vitamin K3).

[0175] • Water-soluble vitamins: vitamin BI2, biotin, choline, vitamin Bi, vitamin B2, vitamin B6, niacin, folic acid, and Ca-D-panthothenate.

[0176] • Trace minerals: manganese, zinc, iron, copper, iodine, selenium, and cobalt.

[0177] • Macro minerals: calcium, phosphorus, and sodium.

[0178] In an eleventh aspect, the present invention relates to a compound according to the general formula (III): wherein

[0179] W is selected from -SO2, -C(=O)- or -C(=O)-O-;

[0180] V is selected from -H or -OH; each E is individually selected from H, Cl or F, wherein at least two E's are a halogen atom selected from Cl and F, and k is an integer from O to 2.

[0181] In a preferred embodiment of the eleventh aspect, the present invention relates to a compound selected from the group consisting of l-chloro-2-(methylsulfonyl)ethane, 1-iodo- 2-(methylsulfonyl)ethane, 2-bromoethanesulfonamide, 2-iodoethanesulfonamide, 1-iodo- 3-(methylsulfonyl)propane, 3-iodopropanesulfonamide, 2-bromoethyl methanesulfonate, / V-(2-iodoethyl)methanesulfonamide, l-azido-2-(methylsulfonyl)ethane, 2,2-dichloro- N-(2,2,2-trichloro-l-hydroxyethyl)acetamide, N-(2,2,2-trichloroethyl)-3,3,3-trifluoro- propanesulfonamide, N-(2,2,2-trichlorethyl)-(2,2,2-trichlorethyl)carbamate and N-(2,2,2-trichloro-l-hydroxyethyl)-(2,2,2-trichloroethyl)carbamate.

[0182] Preferred compounds of general formula (I) used in the present invention are listed below:

[0183] Table 1

[0184]

[0185]

[0186] Table 2

[0187]

[0188] Test for anti-methanogenic efficacy

[0189] The compounds were initially tested in an in vitro rumen assay. This assay setup was specifically designed for easy reproducibility. The primary reaction vessel was a 500 mL bottle (DURAN®pressure plus, Schott AG, Mainz) with a bromobutyl stopper and a corresponding screw cap. Two holes were drilled into the stoppers, and 5 mL serological pipettes were cut to length so that one pipette extended 15 cm from the outside of the stopper into the vessel, while the other just penetrated the stopper. The external ends were attached to 5 cm long silicone tubes with an internal diameter of 6 mm. The shorter tube was attached via luer-lock connectors (neoLab, Heidelberg, Germany) and a luer-lock valve (B. Braun, Melsungen, Germany) to a fitted 500 mL gas bag (Daklapack, Lelystad, Netherlands) and the long tube was sealed with a 5 mm rubber core (Carl Roth, Karlsruhe, Germany). The setup was reinforced and sealed with epoxy resin (DIPON, Dortmund, Germany). Subsequently, 10 porose ceramic cylinders, ca. 1 cm in diameter and length, were placed in each bottle to serve as attachment surface for the microbes as well as l g of dried, ground silage (high fibre silage, Stroh, Hamburg, Germany).

[0190] Rumen fluid was obtained from the local slaughterhouse, from randomly selected cattle that had been slaughtered immediately before. Each experiment consisted of a batch of 16 bottles, comprising test cultures and negative control cultures which originated from the same inoculum. For preparation of one batch, samples from the solid contents and fluid from the rumen of four individual animals were pooled, blended, strained, and mixed 1 / 4 with rumen medium that was prepared according to established protocols (Czerkawski and Beckenridge, 1977). Each bottle was filled with 250 mL of the inoculum. Subsequently, anaerobic conditions were established by flushing the bottle with nitrogen (Nitrogen 5.0, Linde, Dublin, Ireland). With this setup, rumen fluid was cultivated in a closed container, at a constant temperature of 39 °C (Incubation hood M, Neolab, Heidelberg, Germany), and with regular shaking in an orbital shaker (WS-1500, Wiggens, Straubenhardt, Germany) for 5 minutes at 100 rpm with pause intervals of 5 minutes. Rumen cultures are kept alive for 11 days by a daily addition of nutrients in the form of 2 g of dried, ground silage homogenized in 50 mL of saliva buffer. Each bottle produces ca. 300 mL gas per day, which is collected in the attached gas bag. Gas bags are exchanged daily, and their molecular composition is analysed using FT-IR spectroscopy (SpectrumTwo, Perkin Elmer, Massachusetts, USA) with a mounted gas cell (Storm 10 cm Pyrex, Specac Ltd., Orpington, UK) with calcium fluoride windows (CaF2 Window-Pair, Specac Ltd., Orpington, UK). To evaluate the quantities of methane and CO2 produced, a calibration curve was generated by fitting volumetrically diluted methane concentrations ranging from 0 to 25% and CO2 concentrations ranging from 20 to 80% into a standard curve. Subsequently, the spectra of the individual gas bag contents were fitted and evaluated. Daily measurements of gas volume and composition were conducted starting at day one of the experiment.

[0191] The chemical compounds used as test substances were purchased either as available product or synthesized on demand by Enamine (Enamine Ltd., Kyiv, Ukraine), BioSynth (Biosynth, Nobelova, Slowakia), and Ambinter (Greenpharma, Orleans, France). Pure test substances were shipped as liquids, gel-like substances or powders and were stored according to the manufacturer's advice either at room temperature, at 4 °C or at -20 °C. Immediately before use, test substances were aliquoted and dissolved in ethanol, DMSO or water to maximize solubility.

[0192] Test substances dissolved in 1 mL of solvent were added once on day four of the experiment to result in defined concentrations in the range of (5 pM to 3 mM) in the overall rumen culture volume. The substances' efficacy of methanogenesis inhibition is quantified by comparison with a negative control stimulated by 1 mL of solvent without test substance. A separate control base line is calculated for every used solvent in the current batch. The volume of methane, CO2 and residual gasses produced daily before and after addition of the test substances were recorded for 11 days. This data was normalized to the gas volumes produced by the negative control samples in the same batch. Maximum inhibition of methanogenesis typically occurred a few days after stimulation with test substances. The efficiency of methanogenesis inhibition is quantified at the point of minimal methane production as percentage of the methane production in the negative control. Viability of the bacterial cultures is ensured by measurement of overall gas volume production, visual inspection, and via a highly characteristic olfactory signature. Extensive microbiome characterizations by gene sequencing were done previously during establishment of the setup.

[0193] Examples

[0194] The following examples illustrate the present invention but should not be construed as limiting the scope of the invention.

[0195] Production Examples

[0196] The following chemicals listed in Table 3 are commercially available:

[0197] Table 3 The following compounds listed in Table 4 were synthesized according to literature procedures:

[0198] Table 4 Synthetic procedures

[0199] Further substances were synthesized through new synthetic routes. The following Table 5 summarizes chemicals, which were used for the synthesis and are commercially available.

[0200] Table 5

[0201]

[0202] In the following the synthesis of the further substances is described.

[0203] 2.2-Dichloro-N-(2,2,2-trichloroethyl)acetamide

[0204] Published without synthesis route (Brovarets et al., 1994)

[0205] New synthesis route

[0206] A solution of 37.9 mg (0.257 mmol) dichloroacetyl chloride in 1.5 mL DCM was treated with 26.3 mg (0.257 mmol) triethylamine and stirred for 15 min. Then 2,2,2-trichloroethylamine (50 mg, 0.26 mmol) was added dropwise and the mixture was stirred at room temperature for 20 h. The mixture was treated with sodium carbonate solution and extracted with DCM. The organic phase was dried and evaporated to give 10 mg (15%) of the final product 2,2-dichloro- N-(2,2,2-trichloroethyl) acetamide as a colourless solid.1H-NMR (DMSO-De): 4.35 (d, CH2), 6.65 (s, CH), 9.50 (br. S, NH).

[0207] 2.2-Dichloro-N-(2,2,2-trichloro-l-hydroxyethyl)acetamide

[0208] A mixture of 50 mg (0.371 mmol) dichloroacetamide and 624 mg (4.23 mmol) chloral was treated with a catalytic amount of cone. Hydrochloric acid and stirred at room temperature for 18 h. Excess chloral was removed in vacuo, the residue disperged in water and kept in a refrigerator for 12 h. The precipitate was collected by filtration, washed with cold water and cold toluene and dried to give 63 mg (62%) of the final product 2,2-dichloro-N-(2,2,2-trichloro- 1-hydroxyethyl) acetamide as a pale yellow solid.TH-NMR (DMSO-De): 5.70 (s, CH), 6.65 (s, CH), 8.15 (br. S, NH), 9.45 (br. S, OH). N,N'-Bis(2,2,2-trichloro-l-hydroxyethyl)oxamide

[0209] Published in (Chattaway and James, 1934)

[0210] New synthesis route

[0211] 90.8 mg (1.00 mmol) oxamide was dissolved in a small amount of DMF and 442 mg (3.00 mmol) chloral was added. The mixture was heated in a closed vial under microwave irradiation at 100 °C for 8 h. After cooling, the precipitate was collected by filtration and washed with water to give the final product N,N'-bis(2,2,2-trichloro-l-hydroxyethyl) oxamide as a white solid (175 mg, 46%).XH-NMR (DMSO-D6): 5.72 (dd, 2 CH), 8.01 (d, 2 NH), 8.92 (d, 2 OH).

[0212] N-(2,2,2-Trichlorethyl)-3,3,3-trifluoropropanesulfonamide

[0213] A suspension of 110 mg (0.565 mmol) 2,2,2-trichlorethylamine hydrochloride in 1.2 mL DCM was treated with diisopropylamine (114 mg, 1.13 mmol) and cooled to 0 °C. Then 3,3,3-trifluoropropane-l-sulfonyl chloride (106 mg, 0.514 mmol) was added dropwise and the mixture was stirred for 14 h. The mixture was washed with 1 M hydrochloric acid, sodium bicarbonate solution, water, and brine, then dried and evaporated to give the product as an off-white solid (32 mg, 20%).XH-NMR (DMSO-D6): 2.74 (m, CH2), 3.44 (m, CH2), 4.09 (s, CH2), 8.58 (br. S, NH).

[0214] N-(2,2,2-Trichloro-l-hydroxyethyl)thiourea

[0215] Substance mentioned in (Goetzschel et al., 1988)

[0216] New synthesis route

[0217] A solution of 761 mg (10.0 mmol) thiourea and 1650 mg (10.0 mmol) chloral hydrate in 5 mL water was stirred for 2 days at room temperature. The precipitate was collected by filtration, washed with water and dried under vacuum to give the product as a colourless solid (102 mg, 4.6%).XH-NMR (DMSO-D6): 6.31 (m, CH), 7.29 (br. S, NH), 7.70 (d, OH), 7.93 (br. S, NH), 8.14 (br. D, NH). N-(2,2,2-Trichloroethyl)-(2,2,2-trichloroethyl)carbamate

[0218] A suspension of 100 mg (0.514 mmol) 2,2,2-trichloroethyl-l-amine hydrochloride in 0.6 mL dioxane was treated with 0,65 mi l M sodium hydroxide solution, then 114 mg (0.54 mmol) 2,2,2-trichloroethyl chloroformate was added slowly was added under ice-cooling. The mixture was stirred for 12 h, then treated with water and extracted with diethyl ether. The organic layers were washed with water and brine, then dried and evaporated to dryness. The residue was crystallized from ethyl acetate / isohexane to give the product as colourless crystals (55 mg, 33%).XH-NMR (DMSO-D6): 4.14 (d, CH2), 4.90 (s, CH2), 8.87 (s, NH).

[0219] N-(2,2,2-Trichloro-l-hydroxyethyl)-(2,2,2-trichloroethyl)carbamate

[0220] A mixture of 109 mg (0.565 mmol) 2,2,2-trichloroethyl carbamate and 150 mg (1.02 mmol) chloral was heated at 80 °C under nitrogen atmosphere until a clear melt formed. After 2 more h at 80 °C the mixture was allowed to cool down to room temperature. The formed precipitate was collected by filtration and crystallized from ethyl acetate / isohexane to give the product as a colourless solid (86 mg, 45%).XH-NMR (DMSO-D6): 4.90 (d, CH2), 5.52 (dd, CH), 7.84 and 8.90 (2d, OH and NH).

[0221] General synthetic routes, based on exemplary protocols shown below, are applicable to the preparation of the following groups of compounds. l,l,l-Trichloro-2-(methylsulfonyl)ethane (Sirrenberg, 1972; Lu et al., 2009)

[0222] To a 100 mL three-neck flask equipped with mechanical stirring and a 60 °C oil bath was charged 1,1,1,2-tetrachloroethane (4.20 g, 25.0 mmol). A solution of sodium methanethiolate (20 wt% in water) (17.5 g of solution, contains 3.50 g NaSMe, 50.0 mmol) and tetrabutylammonium bromide (97 mg, 0.30 mmol) was added, and the biphasic mixture was stirred at 60 °C for 12 h under N2. The mixture was cooled to rt, the layers were separated, and the organic phase was washed with H2O (2 x 20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo to provide 2,2,2-trichloroethyl methyl sulfide as a crude oil. To 30% H2O2(8.50 g, =7.7 mL, 75.0 mmol) in a 100 mLflask was added Na2WO4-2H2O (165 mg, 0.50 mmol, 2.0 mol%) with stirring at 10 °C (ice bath). A solution of 2,2,2-trichloroethyl methyl sulfide (4.49 g, 25.0 mmol) in CH2CI2(10.0 mL) was added dropwise while maintaining < 15 °C. The mixture was then warmed to 50 °C and stirred for 12 h. After cooling to rt, residual peroxide was quenched with NaHSO3. The phases were separated; the aqueous layer was extracted with CH2CI2(2 x 20 mL). The combined organic layers were washed with brine (2 x 20 mL), dried over Na2SO4, filtered, and concentrated to give crude 1,1,1-trichloro- 2-(methylsulfonyl)ethane. The crude product was refined by recrystallisation.

[0223] General procedure for l,l,l-Trihalo-2-(methylsulfonyl)ethanes

[0224] The described procedure is generally applicable to the preparation of other 1,1,1-trihalo- 2-(methylsulfonyl)ethanes by replacing 1,1,1,2-tetrachloroethane with alternative

[0225] 1.1.1-trihalo-2-chloroethane derivatives.

[0226] 2.2.2-Trichloroethanesulfonamide (Lu et al., 2009)

[0227] To a solution of 2,2,2-trichloroethane-l-sulfonyl chloride (100 mg, 0.431 mmol) in 3 mL THF was added 1.8 mL aqueous ammonia (25%) and the mixture was stirred for 12 h. The mixture was evaporated and dried under high vacuum, the residue extracted with ethyl acetate. The ethyl acetate layer was washed with water and brine, dried and evaporated to give the product.

[0228] General procedure for 2,2,2-Trihaloethanesulfonamides

[0229] The described procedure is generally applicable to the preparation of other 2,2,2-trihaloethylsulfonamides by replacing 2,2,2-trichloroethane-l-sulfonyl chloride with alternative 2,2,2-trihaloethane-l-sulfonyl chlorides. (2,2,2-Trichloroethyl)mesylate (Ahmed et al., 2002)

[0230] To a stirred solution of 2,2,2-trichloroethanol (0.50 g, 3.00 mmol, l.OO equiv) and triethylamine (0.50 mL, 0.363 g, 3.59 mmol, 1.20 equiv) in anhydrous CH2CI2(40 mL) at room temperature was added methylsulfonyl chloride (0.23 mL, 0.344 g, 3.00 mmol, l.OO equiv). The mixture was warmed to reflux and stirred for 2 h. The reaction mixture was poured onto ice-water and the organic phase was separated, then washed with H2O (3 x 20 mL), saturated NaHCO3(3 x 20 mL), and H2O (3 x 20 mL), dried over MgS04, filtered, and concentrated in vacuo to give (2,2,2-trichloroethyl)mesylate.

[0231] General procedure for (2,2,2-Trihaloethyl)mesylates

[0232] The described procedure is generally applicable to the preparation of other (2,2,2-trihaloethyl) mesylates by replacing 2,2,2-trichloroethanol with alternative

[0233] 2.2.2-trihaloethanol derivatives.

[0234] (2,2,2-Trichloroethyl)sulfamate (Yamamoto et al., 2011)

[0235] Chlorosulfonyl isocyanate (5.0 mL, 57.0 mmol, 2.20 equiv) was dissolved in CH3CN (8.0 mL) under N2and cooled to 0 °C. Formic acid (2.2 mL, 58.0 mmol, 2.24 equiv) was added dropwise via syringe, causing immediate gas evolution. The mixture was stirred for 30 min at 0 °C, then diluted with CH3CN (16 mL) and allowed to warm to rt, stirring for 2 h. After re-cooling to 0 °C, a solution of 2,2,2-trichloroethanol (3.87 g, 25.9 mmol, 1.0 equiv) in DMA (12.0 mL) was added dropwise via cannula, followed by DMA (9.0 mL) to complete the transfer. The reaction mixture was warmed to room temperature and stirred for 16 h. The mixture was quenched with H2O (20 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic phases were washed with H2O (3 x 20 mL) and brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography on silica gel gave

[0236] 2.2.2-trichloroethyl sulfamate. General procedure for (2,2,2-Trihaloethyl)sulfamates

[0237] The described procedure is generally applicable to the preparation of other trihaloethyl sulfamates by replacing 2,2,2-trichloroethanol with alternative 2,2,2-trihaloethanol derivatives.

[0238] N-(2,2,2-Trichloroethyl)methanesulfonamide

[0239] A stirred solution of 2,2,2-trichloroethyl-l-amine hydrochloride (20.0 g, 108.2 mmol) and triethylamine (27.63 g, 270.4 mmol) in DCM (100 mL) was cooled to -10 °C and methanesulfonyl chloride (24.8 g, 216.4 mmol) was added dropwise at the temperature not exceeding 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for another 2 h, then washed with water (3 x 100 mL), dried over Na2SO4 and concentrated in vacuo to obtain N-(2,2,2-trichloroethyl)methanesulfonamide (11.35 g, 46.3%).

[0240] General procedure for N-(2,2,2-Trihaloethyl)methanesulfonamides

[0241] The above-described procedure for the preparation of N-(2,2,2-trichloroethyl) methanesulfonamide is generally applicable to the preparation of other N-trihaloethyl methanesulfonamides by replacing 2,2,2-trichloroethyl-l-amine hydrochloride with alternative 2,2,2-trihaloethyl-l-amine hydrochlorides.

[0242] N-(2,2,2-Trichloroethyl)sulfamide

[0243] 97.3 mg (0.50 mmol) 2,2,2-trichloroethyl-l-amine hydrochloride was dissolved in DMF-chloroform (5 mL) and 1580 mg (20 mmol) pyridine. Then 124 mg (0.50 mmol) 1,1,1,3,3,3-hexyfluoroisopropyl sulfamate (HEIPS) was added, and the mixture stirred for 4.5 h at 50 °C. Toluene (10 mL) was added and the mixture was evaporated, and the residue was freeze-dried. The residue was taken up in water and extracted with ethyl acetate. Evaporation of the organic layer gave the product N-(2,2,2-trichloroethyl) sulfamide. General procedure for N-(2,2,2-Trihaloethyl)sulfamides

[0244] The above-described procedure for the preparation of N-(2,2,2-trichloroethyl)sulfamide is generally applicable to the preparation of other N-trihaloethyl sulfamides by replacing 2,2,2-trichloroethyl-l-amine hydrochloride with alternative 2,2,2-trihaloethylamine hydrochlorides. l,l,l-Trichloro-3-(methylsulfonyl)propane

[0245] 1,1,1,3-Tetrachloropropane (4.00 g, 22.0 mmol) was added to a 21% w / w aqueous solution of sodium methanethiolate (33.0 g, 99.0 mmol) and 18-crown-6 ether (0.29 g, 1.1 mmol) in H2O:THF (30 mL:3 mL). The reaction mixture was warmed to 70 °C and stirred for 16 h. After completion, the mixture was poured into water and extracted with CH2CI2(3 x 40 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford crude l,l,l-trichloro-3-(methylthio)propane (1.25 g, 29%, 51% purity).

[0246] To a stirred solution of l,l,l-trichloro-3-(methylthio)propane (1.25 g, 6.46 mmol, 51% purity) in MeCN (5 mL), EtOAc (10 mL), and H2O (20 mL) was added RuCI3-H2O (2.5 mg, 0.011 mmol), followed by Nal04(3.36 g, 15.5 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting solids were filtered off, and the filtrate was diluted with H2O (50 mL), then extracted with EtOAc (150 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (CH2CI2:MTBE) to afford l,l,l-trichloro-3-(methylsulfonyl)propane (0.36 g, 48%).

[0247] General procedure for l,l,l-trihalo-3-(methylsulfonyl)propanes

[0248] The above-described procedure for the preparation of l,l,l-trichloro-3-(methylsulfonyl) propane is generally applicable to the preparation of other trihalogenated (methylsulfonyl) propanes by replacing 1,1,1,3-tetrachloropropane with alternative 1,1,1-trihalo- 3-chloropropane derivatives. 3,3,3-Trichloropropanesulfonamide

[0249] To a stirred solution of (3,3,3-trichloropropyl) mesylate (1.0 g, 4.14 mmol) in 8 mL of DMF was added pyrimidine-2-thiol (0.456 g, 4.55 mmol), potassium carbonate (562 mg, 4.55 mmol), and the mixture was stirred at 60 °C overnight. After that, the reaction mixture was poured into water (15 mL), extracted with EtOAc (2 x 15 mL), the combined organic layers washed with brine 4 x 50 mL, dried and evaporated to afford 2-[(3,3,3-trichloropropyl)thio]pyrimidine (0.92 g, 86%).

[0250] To a stirred solution of 2-[(3,3,3-trichloropropyl)thio]pyrimidine (920 mg, 3.57 mmol) in 10 ml of DCM at 10 °C was added m-CPBA (1.8 g, 7.85 mmol, 75% purity) and then stirred at room temperature overnight. The reaction mixture was quenched with NaHCCh sat. solution (15 mL), stirred for 20 min, then extracted with DCM (1 x 10 mL). The organic layer was washed with water (10 mL), dried and evaporated to afford 2-[(3,3,3-trichloropropyl) sulfonyl]pyrimidine (0.79 g, 76%).

[0251] To a stirred 10 mL of MeOH was added metallic sodium (69 mg, 2.87 mmol) and stirred until all of the metal was dissolved. Then, 2-[(3,3,3-trichloropropyl)sulfonyl]pyrimidine (793 mg, 2.74 mmol) was added to the prepared solution and it was stirred overnight at room temperature. After that, the reaction mixture was evaporated, MTBE (8 mL) was added, prepared mixture was stirred for 5 min and filtered to afford (3,3,3-trichloropropyl)sulfinate sodium salt (0.53 g, 83%).

[0252] To a stirred solution of (3,3,3-trichloropropyl)sulfinate sodium salt (530 mg, 2.27 mmol) in 10 ml of water at room temperature was added sodium acetate x 3 H2O (617 mg, 4.54 mmol) and hydroxylamine sulfonic acid (256 mg, 2.27 mmol) and stirred overnight at room temperature. After that, the reaction mixture was acidified to pH ~3 with cone. HCI, extracted with EtOAc (2 x 10 mL), the combined organic layers dried and evaporated to afford the crude product. It was purified by HPLC to give pure 3,3,3-trichloropropanesulfonamide (0.05 g, 9.7%). General procedure for 3,3,3-trihalopropanesulfonamides

[0253] The above-described procedure for the preparation of 3,3,3-trichloropropanesulfonamide is generally applicable to the preparation of other trihalogenated propanesulfonamides by replacing (3,3,3-trichloropropyl) mesylate with alternative (3,3,3-triha lopropyl) mesylates.

[0254] (3,3,3-Trichloropropyl) mesylate

[0255] To a stirred solution of 3,3,3-trichloropropanoic acid (2.60 g, 14.6 mmol) in THF (25 mL) at room temperature was added BH3-DMS complex (2.75 mL, 29.2 mmol), then stirred at reflux overnight. The reaction mixture was quenched with MeOH (50 mL) dropwise and evaporated, then diluted with potassium carbonate solution (2N, 30 mL), and extracted with MTBE (2 x 40 mL). The combined organic layers were dried and evaporated to afford

[0256] 3.3.3-trichloropropan-l-ol (2 g, 83%).

[0257] To a stirred solution of 3,3,3-trichloropropan-l-ol (2.0 g, 12.2 mmol) and triethylamine (2.47 g, 24.4 mmol) in 20 mL of DCM at 0 °C was dropwise added methanesulfonyl chloride (2.1 g, 18.3 mmol). After stirring for 30 minutes at room temperature, the reaction mixture was poured into water (30 mL), and extracted with MTBE. The organic extract was washed with water one time, dried and evaporated to afford the crude product, which was purified by flash chromatography (hexane-MTBE) to afford (3,3,3-trichloropropyl) mesylate (2.6 g, 84%).

[0258] General procedure for (3,3,3-trihalopropyl) mesylates

[0259] The above-described procedure for the preparation of (3,3,3-trichloropropyl) mesylate is generally applicable to the preparation of other 3,3,3-trihalopropyl mesylates by replacing

[0260] 3.3.3-trichloropropanoic acid or 3,3,3-trichloropropan-l-ol with alternative

[0261] 3.3.3-trihalopropanoic acid or 3,3,3-trihalopropan-l-ol derivatives.

[0262] (3,3,3-Trichloropropyl) sulfamate

[0263] To a stirred solution of 3,3,3-trichloropropan-l-ol (500 mg, 3.06 mmol) in dimethylacetamide (5 mL) at 0 °C was added sulfamoyl chloride (565 mg, 6.12 mmol) and stirred overnight at room temperature. The reaction mixture was poured into water (10 mL), extracted with EtOAc (2 x 10 mL). The combined organic layers were washed with brine 4 times, dried and evaporated to afford the crude product, that was purified by flash chromatography (hexane- MTBE) to give pure (3,3,3-trichloropropyl) sulfamate (0.05 g, 6.7%).

[0264] General procedure for (3,3,3-trihalopropyl) sulfamates

[0265] The above-described procedure for the preparation of (3,3,3-trichloropropyl) sulfamate is generally applicable to the preparation of other 3,3,3-trihalopropyl sulfamates by replacing

[0266] 3.3.3-trichloropropan-l-ol with alternative 3,3,3-trihalopropan-l-ol derivatives.

[0267] N-(3,3,3-Trichloropropyl)methanesulfonamide

[0268] To a stirred solution of (3,3,3-trichloropropyl) mesylate (2.6 g, 10.8 mmol) in DMF (15 mL) was added sodium azide (0.91 g, 14 mmol), then stirred at 90 °C until full completion (NMR control). The reaction mixture was poured into water (30 mL), extracted with EtOAc (2 x 20 mL), washed with brine 4 times, dried and evaporated to afford 3,3,3-trichloropropane- 1-azide (1.17 g, 57%).

[0269] To a stirred solution of 3,3,3-trichloropropane-l-azide (1.17 g, 6.2 mmol) in dioxane (15 mL) was added 2M HCI (9 mL) followed by triphenylphosphine (2.11 g, 8.06 mmol) and stirred at room temperature overnight. After that, the reaction mixture was poured into water (15 mL) and extracted with MTBE (2 x 10 mL). The water layer was evaporated, and isopropyl alcohol (8 mL) was added, the prepared mixture was stirred for 5 min and filtered to afford

[0270] 3.3.3-trichloropropane-l-amine as HCI salt (0.15 g, 12%).

[0271] To 3,3,3-trichloropropane-l-amine hydrochloride (150 mg, 0.754 mmol), suspended in DCM (2 mL) was added triethylamine (190 mg, 1.89 mmol). At 0 °C was dropwise added methanesulfonyl chloride (172 mg, 1.5 mmol). After stirring for 30 minutes at room temperature, the reaction mixture was poured into water (3 mL), and extracted with MTBE. The organic layer was dried and evaporated to afford the crude product that was purified by HPLC to give N-(3,3,3-trichloropropyl)methanesulfonamide (0.048 g, 21.6%). General procedure for N-(3,3,3-trihalopropropyl)methanesulfonamides

[0272] The above-described procedure for the preparation of N-(3,3,3-trichloropropyl) methanesulfonamide is generally applicable to the preparation of other trihalogenated N-(3,3,3-trihalopropyl)methanesulfonamides by replacing (3,3,3-trichloropropyl) mesylate with alternative (3,3,3-trihalopropyl) mesylates.

[0273] N-(3,3,3-Trichloropropyl) sulfamide (Bolli et al. 2012)

[0274] A solution of 3,3,3-trichloropropane-l-amine (1.62 g, 10.0 mmol) and triethylamine (1.53 mL, 1.11 g, 11.0 mmol) in anhydrous CH2CI2(50 mL) was cooled to 0 °C under N2. A solution of N-chlorosulfonyl(tert-butyl) carbamate (2.16 g, 10.0 mmol) in CH2CI2(50 mL) was added slowly at 0 °C. The mixture was stirred for 45-60 min at 0 °C and then for 2-3 h at rt. The mixture was diluted with CH2CI2(50 mL), washed with 0.1 N HCI (2 x 50 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give N'-Boc-N-(3,3,3-trichloropropyl)sulfamide, which was purified by flash chromatography and recrystallization to provide the N'-Boc sulfamide as a solid.

[0275] To a stirred solution of N'-Boc-N-(3,3,3-trichloropropyl)sulfamide (3.42 g, 10.0 mmol) in CH2CI2(50 mL) at 0 °C was added trifluoroacetic acid (3.80 mL, 50.0 mmol) as a 50% v / v solution in CH2CI2(total added 7.60 mL) dropwise at 0 °C. The mixture was stirred for 2 h at 0 °C and then 12 h at rt. The solvents were removed under reduced pressure, and the residue was purified by column chromatography to afford (3,3,3-trichloropropyl)sulfamide.

[0276] General procedure for N-(3,3,3-Trichloropropyl) sulfamides

[0277] The above-described procedure for the preparation of N-(3,3,3-trichloropropyl) sulfamide is generally applicable to the preparation of other N-(3,3,3-halopropropyl) sulfamides by replacing 3,3,3-trichloropropane-l-amine with alternative 3,3,3-trihalopropane-l-amines. In vitro experiments

[0278] The following compounds, which are representative compounds from each chemical class in general formula (I), were tested for their efficacy in reducing enteric gas production using the above-described in vitro rumen assay: l-Chloro-2-(methylsulfonyl)ethane,

[0279] 1-lodo-2-(methylsulfonyl)ethane,

[0280] 2-Bromoethanesulfonamide,

[0281] 2-lodoethanesulfonamide,

[0282] 1-lodo-3-(Methylsulfonyl)propane,

[0283] 3-lodopropanesulfonamide,

[0284] 2-Bromoethyl methanesulfonate,

[0285] / V-(2-lodoethyl)methanesulfonamide,

[0286] 1-Azido-2-(methylsulfonyl)ethane,

[0287] 2-Bromoethyl mesylate,

[0288] 3-lodoethylsulfonyl fluoride,

[0289] 3-(Methylsulfonyl)propanenitrile,

[0290] 2-Cyanoethanesulfonamide,

[0291] 5-(2-lodoethyl)-lH-l,2,3,4-tetrazole,

[0292] 2.2.2-Trichloro-N-(2,2,2-trichloroethyl)acetamide,

[0293] 2-Chloro- / V-(2,2,2-trichloroethyl)acetamide,

[0294] 4-Methyl-N-(2,2,2-trichloroethyl)benzenesulfonamide,

[0295] 2.2.2-Trichloro-N-(2,2,2-trichloro-l-hydroxy-ethyl)acetamide,

[0296] 2.2.2-Trifluoro-N-(2,2,2-trichloro-l-hydroxy-ethyl)acetamide,

[0297] 2.2- Dichloro-N-(2,2,2-trichloro-l-hydroxy-ethyl)acetamide,

[0298] 4-Methyl-N-(2,2,2-trichloro-l-hydroxyethyl)benzenesulfonamide,

[0299] 2.2.2-Trichloroethyl urea,

[0300] N,N-bis(2,2,2-trichloroethoxy)urea, l,l,l-Trichloro-3-(methylsulfonyl)propan-2-ol,

[0301] (2,2,2-Trichloroethyl) mesylate,

[0302] (2-Bromo-2,2-difluoroethyl) mesylate, (2,2,2-Trichlorethyl) sulfamate,

[0303] (2,2,2-Tribromoethyl) sulfamate, l,l,l-Trichloro-3-(methylsulfonyl)propane, N-(2,2,2-Trichloroethyl)methanesulfonamide, N-(2-Bromo-2,2-difluoroethyl)sulfamide, 3,3,3-Trichloropropanesulfonamide, (3,3,3-Trichloropropyl)mesylate, (3,3,3-Trichloropropyl)sulfamate, N-(3,3,3-Trichloropropyl)methanesulfonamide.

[0304] Figures 1 to 18 demonstrate the inhibitory activity of a selection of the tested compounds, among which 10 substances having a peak reduction of methane of more than 80% at a concentration of 10 pM are seen to be highly effective (marked in bold in Table 6 below). Substances are tested in the provided concentrations, respectively, with rumen cultures in 250 mL bottles. Deviating from the previously used protocol for in vitro experiments, the daily nutrients added to each culture flask consisting of 2 g of dried, ground silage homogenized in 50 mL of saliva buffer were supplemented with 0.081 g tryptone feed and 20 mg Milkivit (minerals). A single dose of each test substance is added at day 4. The produced methane volume per day is normalized against a negative control.

[0305] The maximum percentual reductions of methane production relative to the control are as follows, where (d7) indicates that data was collected until day 7 of the experiment:

[0306] Table 6

[0307] In vivo experiment

[0308] Selected substances that showed anti-methanogenic activity in vitro rumen assay were further tested in in vivo trials. The study followed a 2x2 Latin square design using two rumen cannulated lactating dairy cows (named Hanni and Kathe) maintained under controlled feeding conditions. Each animal was allocated to either a treatment or control group (placebo) and housed in individual pens to allow accurate recording of feed intake and collection of fermentation gases. After a day of baseline recording, the substances were administered for 6 days, followed by 2 weeks of washout. Basal diets consisted of a total mixed ration formulated according to NRC recommendations, with hay provided ad libitum. Supplementation with additional concentrate was adjusted according to the individual cow's milk yield. During the experiment a fixed amount of concentrate was supplemented for each individual cow. In parallel with feed administration, test substances dissolved in 500 mL of water were administered twice daily directly into the rumen via a valve system fitted to the fistula, ensuring complete uptake. In accordance with the crossover design, one cow received the test substance, while the other (placebo) received 500 mL of water as a placebo.

[0309] Dosage levels were calculated from the in vitro data, assuming an estimated rumen volume of 100 Liters. Guided by the screening results, the test substances were evaluated in vivo at three concentration levels: low, medium, and high. The low in vivo dose per administration was set to match the in vitro concentration that yielded nearly complete inhibition, reflecting reports that inhibitors generally exhibit greater efficacy under in vitro conditions. For practicality, the nearest whole numbers were chosen, resulting in daily doses of 1 (low), 2 (medium), and 3 (high) grams for the inhibitors l-lodo-2-(methylsulfonyl)ethane and 3-lodopropanesulfonamide. Each daily dose was divided into two equal portions, administered in the morning and at noon. As a positive control, 3-NOP (3-nitrooxypropanol) was administered at 1.5 g / day, corresponding to a ruminal concentration of 60 pM per half-day dose (750 mg), which was representative of the medium dose. Administration of the positive control 3-NOP thus corresponded to the feeding concentrations proposed in the literature (Hristov et al., 2015).

[0310] Methane emissions from the cow rumen were measured at 10 fixed time points between 6:30 a.m. and 5:00 p.m. to monitor diurnal methane production. Measurements were conducted with two independent methods, namely by using a handheld laser methane detector (Non-Dispersive Infrared, NDIR-Iaser) and by simultaneously collecting digestive gases directly through the fistula valve. Each handheld measurement lasted three minutes per cow and was performed consecutively for both animals. For gas collection, a 500 mL gas bag was attached to the fistula valve via a pneumatic quick-lock system connected to a water separator and a check valve. Priorto each measurement, the separator and valve were flushed with ruminal gas. During the measurement period, the bag inflated due to excess pressure and rumen contractions and was removed immediately afterward. The water separator prevented rumen fluid from entering the collection bags, whereas the check valve ensured consistent filling by eliminating in- and deflation artifacts resembling breathing. The collected digestive gases from the bag attached to the cannula valve were analysed for their molecular composition using FT-IR spectroscopy in the same manner as the in vitro gas bags as described in (Laric et al., 2025).

[0311] A baseline day (day 1) preceded each experiment, during which methane was measured at the same time points and samples were collected in the same manner as during the treatment phase. Following the baseline, test substances were administered on six consecutive days (days 2-7). No further substance was administered on day 8, but the measurement protocol was continued to capture the gradual decline of treatment effects. At the end of day 8, a transfaunation was performed by transferring 1 L of fresh rumen fluid including solids from the placebo cow to the treatment cow via the fistula, in order to minimize carryover effects. A washout period from day 9 to 21 allowed full recovery of the rumen microbiome before the next experiment. In addition to ruminal gas production, feed intake, milk yield, and general animal health parameters were monitored daily. Samples were taken on days 1, 3, 5, 8, and 11 of the trial to observe the progression of parameters and changes. Sampling included blood collection from the tail vein and examination of clinical chemical and hematological parameters, including metabolic parameters and inflammation markers. Rumen fluid samples were collected via the fistula to assess microbial viability and fatty acid profiles. Milk, feces, and urine were also sampled and immediately frozen for subsequent residue analysis. For the assessment of feed utilization and livestock productivity the feed intake and milk yield were compared.

[0312] The appended figures 19 to 27 demonstrate that the tested compounds exhibited in vivo dosedependent anti-methanogenic activity consistent with the predictions from the in vitro assays. Measurements of methane emissions showed that administration of 1 g of 1-lodo- 2-(methylsulfonyl)ethane reduced methane production by up to 38.76% compared to the placebo treatment. At higher levels of methane reduction, feed intake decreased by up to 4% (SE = 3.40%), while milk yield decreased by 3.71% (SE = 2.31%), without accounting for the gradual decline in yield expected during the lactation period.

[0313] Administration of 3-lodopropanesulfonamide reduced methane emissions in a clear dosedependent manner, with reductions of up to 98.49% relative to the placebo. At the highest level of methane reduction, feed intake declined by up to 30.16% (SE = 4.38%), while milk yield decreased by 4.73% (SE = 1.92%), again not accounting for the natural lactation-associated decline. Despite the reduction in feed intake, cows showed no clinical signs of catabolic metabolism. The average weight gain between the stimulations (3 weeks) was 6.51 (R2=0.97) kg for Hanni and 7.45 kg (R2=0.81) for Kathe.

[0314] At a dose of 3 g / day of 3-lodopropanesulfonamide, milk yield relative to feed intake increased by 36.42% (from 0.54 L / kg feed under placebo to 0.74 L / kg feed under treatment), which indicates improved productivity and feed utilization. Clinical examinations revealed no clear abnormalities other than reduced feed intake, and both milk composition and blood parameters remained within normal ranges. Literature

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Claims

New PCT-Patent ApplicationLudwig-Maximilians-Universitat Munchen, in Vertretung des Freistaates BayernVossius Ref.: AJ2811 PCTCLAIMS1. A method of reducing methane production in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are selected from the following general formula (I) or a salt thereof:wherein n is an integer from 0 to 2; m is an integer from 0 to 2; each of X1, X2and X3is individually selected from the group consisting of a hydrogen atom, a halogen atom (-F, -Cl; -Br, -I), a hydroxy group (-OH), a thiol group (-SH), an azido group (-N3), a mesylate group (-O-SO2-CH3), a cyano group (-CN), wherein at least one of X1, X2and X3is not hydrogen, and not more than one of X1, X2and X3can be a hydroxy group (-OH) or a thiol group (-SH);V is selected from the group consisting of a hydrogen atom (-H), a hydroxy group (-OH), an alkoxy group, and an amine group (-NH2);Z is selected from the group consisting of a carbonyl group (-C(=O)-R1), a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), a sulfinamide group (-NH-S(=O)-R8), a phenyl group, and a tetrazol-5-yl group; whereinR1is selected from the group consisting of -O-C1-C3 alkyl, -NH2, -NH-C1-C3 alkyl, -N(CI-C3alkyl)2, -NH-CH2-C(Hal)3, and -NH-CH(OH)-C(Hal)3;R2is selected from the group consisting of -C1-C3 alkyl, -C0-C2 alkyl-C(Hal)3, -F, -NH2, -NH-(CH(OH)-C(Hal)3, -NH-CH2-C(Hal)3;R3is selected from the group consisting of -C1-C3 alkyl, -NH2and -NH-CH(OH)-C(Hal)3;R4is selected from the group consisting of -C1-C3 alkyl, -NH2and -(4-methylphenyl);R5is selected from the group consisting of -C(Ha l)3, -CHCI2, -O-CH2-C(Hal)3, -CO-NH-CH(OH)-C(Hal)3and -NH-RX, wherein Rxis selected from the group consisting of -H, -C1-C3 alkyl, -CH(OH)-C(Hal)3;R6is selected from the group consisting of -C1-C3 alkyl, -NH2, -F; andR7is selected from the group consisting of -NH2, -NH-CH2-C(Hal)3;R8is -C1-C4 alkyl.

2. The method according to claim 1, wherein n is an integer from 0 to 1; m is an integer from 0 tol; each of X1, X2and X3is individually selected from the group consisting of a halogen atom (-F, -Cl; -Br, -I), an azido group (-N3), a cyano group ( -CN);V is selected from either a hydrogen atom (-H) or a hydroxy group (-OH);Z is selected from the group consisting of a carboxy group (-O-C(=O)-R7), a sulfone group (-SO2R2), a sulfoxide group (-S(=O)-R3), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), and a tetrazol-5-yl group; whereinR2is selected from the group consisting of -CH3, -CCI3, -NH2, -NH-CH2-CCl3;R3is selected from the group consisting of -CH3, -NH2;R4is selected from the group consisting of -CH3, -NH2;R5is selected from the group consisting of -CCI3, -CF3, -CHCI2, -O-CH2-CCl3, -CO-NH-CH(OH)-CCl3 and -NH-RXwherein Rxis selected from the group consisting of -H, -CH3, -C2H5, -CH(OH)-CCI3;R6is selected from the group consisting of -CHs, -NH2, -F; and R7is selected from the group consisting of -NH2, -NH-CH2-CCl3.

3. The method according to claim 1, wherein n is an integer from 0 to 1; m is an integer from 0 tol; each of X1, X2and X3is individually selected from the group consisting of a halogen atom (-F, -Cl; -Br, -I), an azido group (-N3), a cyano group ( -CN);V is selected from either a hydrogen atom (-H) or a hydroxy group (-OH);Z is selected from the group consisting of a sulfone group (-SO2R2), an N-sulfonamide group (-NH-SO2-R4), an amide group (-NH-C(=O)-R5), a thiourea group (-NH-C(=S)-NH2), a sulfonate group (-O-SO2-R6), and a tetrazol-5-yl group; whereinR2is selected from the group consisting of -CH3, -NH2, -NH-CH2-CCI3;R4is selected from the group consisting of -CH3, -NH2;R5is selected from the group consisting of -CCI3, -CF3, -CHCI2, -O-CH2-CCI3, -CO-NH-CH(OH)-CCl3 and -NH-RXwherein Rxis selected from the group consisting of -H, -CH3, -C2H5, -CH(OH)-CCI3; andR6is selected from the group consisting of -CHs, -NH2, -F.

4. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), at least one of m and n is 0,Z is selected from the group consisting of a sulfone group (-SO2R2), an N-sulfonamide group (-NH-SO2-R4), a sulfonate group (-O-SO2-R6);R2is -CH3 or -NH2;R4is -CH3 or -NH2; andR6is -CH3 or -NH2.

5. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is a sulfone group (-SO2R2), wherein R2is -CH3.

6. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m or n is 1 and the other of m and n is 0, andZ is a sulfone group (-SO2R2), wherein R2is -CH3.

7. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is a sulfone group (-SO2R2), wherein R2is -NH2.

8. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m or n is 1 and the other of m and n is 0, andZ is a sulfone group (-SO2R2), wherein R2is -NH2.

9. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is a sulfonate group (-O-SO2-R6), wherein R6is -CH3.

10. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m or n is 1 and the other of m and n is 0, andZ is a sulfonate group (-O-SO2-R6), wherein R6is -CH3.

11. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is a sulfonate group (-O-SO2-R6), wherein R6is -NH2.

12. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I),m or n is 1 and the other of m and n is 0, andZ is a sulfonate group (-O-SO2-R6), wherein R6is -NH2.

13. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is an N-sulfonamide group (-NH-SO2-R4), wherein R4is -CH3.

14. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m or n is 1 and the other of m and n is 0, andZ is an N-sulfonamide group (-NH-SO2-R4), wherein R4is -CH3.

15. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m and n are 0, andZ is an N-sulfonamide group (-NH-SO2-R4), wherein R4is -NH2.

16. The method of claim 3, wherein each of X1, X2and X3is individually selected from a halogen atom (-F, -Cl, -Br, -I), m or n is 1 and the other of m and n is 0, andZ is an N-sulfonamide group (-NH-SO2-R4), wherein R4is -NH2.

17. The method of claim 1, wherein one or more compounds are selected from the following general formula (II) or a salt thereof:X-(CHr)s-Y (II) whereinX is selected from the group consisting of a halogen atom, an amino group (-NR21R22), an azido group (-N3), a cyano group (-CN), a formamide group (-CO-NH2), an N-formamide group (-NH-CHO), a hydroxy group (-OH), an alkoxy group (-OR24),an aldehyde group (-CHO), an O-hydroxylamine group (-O-NH2), a hydroxylamine group (-NH-OH),an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 18 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S; andY is selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfinamide group (-SO-NR32R33), a sulfoxide group (-S(=O)-R28)), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37). wherein r is each independently an integer from 0 to 2 as valency permits; and s is an integer from 1 to 4.R21and R22are each independently selected from the group consisting of H, -OH and -C1-C3 alkyl;R24is selected from the group consisting of -C1-C3 alkyl;R28is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;R29and R30are each independently selected from the group consisting of H and -C1-C3 alkyl;R31is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;R32and R33are each independently selected from the group consisting of H and -C1-C3 alkyl;R34is selected from the group consisting of H, -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;R35and R36are each independently selected from the group consisting of H and -C1-C3 alkyl; andR37is selected from the group consisting of H and -C1-C3 alkyl.

18. The method of claim 17, whereinX is selected from the group consisting of a halogen atom, an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), a thiol (-SH), andY is selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfinamide group (-SO-NR32R33), a sulfoxide group (-S(=O)-R28)), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37), wherein n and m are as defined in claim 1;R28is selected from the group consisting of -C1-C3 alkyl, -C1-C3 haloalkyl and a fluorine atom;R29and R30are each H;R31is selected from the group consisting of methyl and a fluorine atom;R32and R33are each H;R34is selected from the group consisting of methyl, and a fluorine atom;R35and R36are each H; and R37is H.

19. The method of claim 17, whereinX is selected from the group consisting of a halogen atom, an amino group (-NR21R22), an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), an alkoxy group (-OR24), an O-hydroxylamine group (-O-NH2), an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or a heterocyclic ring system having 5 to 10 ring atom members and comprising 1 to 4 heteroatoms selected from the group consisting of N, O and S,Y is selected from the group consisting of a sulfonamide group (-SO2-NR29R30), a sulfone group (-SO2R31), a sulfonate group (-OSO2R34), a sulfamate group (-OSO2-NR35R36), and an N-sulfonamide group (-NH-SO2-R37), wherein n, m, R21, R22, R24, R25, R29, R30, R31, R34, R35, R36, and R37are as defined in claim 2;wherein preferablyX is selected from the group consisting of a chlorine atom, a bromine atom, an iodine atom, an azido group (-N3), a cyano group (-CN), a hydroxy group (-OH), a methoxy group (-O-CH3), an O-hydroxylamine group (-O-NH2), an isocyanato group (-NCO), a thiol (-SH), a thiocyanato group (-SCN), an isothiocyanato group (-NCS), or an lH-imidazol-l-yl group, and a tetrazol-5-yl group,Y is selected from the group consisting of a sulfonamide group (-SO2-NH2), a methylsulfone group (-SO2-CH3), a methylsulfonate group (-OSO2-CH3), a sulfamate group (-OSO2-NH2), a methanesulfonamide-N-yl group (-NH-SO2-CH3), wherein r is each independently an integer from 1 or 2 as valency permits; s is an integer from 1 to 3; wherein more preferablyX is selected from the group consisting of a chlorine atom, a bromine atom, an iodine atom, an azido group (-N3), a cyano group (-CN),Y is selected from the group consisting of a sulfonamide group (-SO2-NH2), a methylsulfone group (-SO2-CH3), a methylsulfonate group (-OSO2-CH3), a sulfamate group (-OSO2-NH2), and an N-methanesulfonamide group (-NH-SO2-CH3), wherein r is 2; and s is an integer from 1 to 3.

20. The method of claim 1, wherein the one or more compounds comprise a compound selected from the group consisting of l-chloro-2-(methylsulfonyl)ethane, 1-iodo- 2-(methylsulfonyl)ethane, 2-bromoethanesulfonamide, 2-iodoethanesulfonamide, l-iodo-3-(methylsulfonyl)propane, 3-iodopropanesulfonamide, 2-bromoethyl methanesulfonate, / V-(2-iodoethyl)methanesulfonamide, l-azido-2-(methylsulfonyl) ethane, 2-bromoethyl mesylate, 3-iodoethylsulfonyl fluoride, 3-(methylsulfonyl) propanenitrile, 2-cyanoethanesulfonamide, 5-(2-iodoethyl)-lH-l,2,3,4-tetrazole, 2,2,2-trichloro-N-(2,2,2-trichloroethyl)acetamide, 2-chloro- / V-(2,2,2-trichloroethyl) acetamide, 4-methyl-N-(2,2,2-trichloroethyl)benzenesulfonamide, 2,2,2-trichloro-N-(2,2,2-trichloro-l-hydroxy-ethyl)acetamide, 2,2,2-trifluoro-N-(2,2,2-trichloro- l-hydroxy-ethyl)acetamide, 2,2-dichloro-N-(2,2,2-trichloro-l-hydroxy-ethyl)acetamide, 2,2,2-trichloroethyl urea, N,N-bis(2,2,2-trichloroethoxy)urea, trichloro- 3-methylsulfonylpropan-2-ol, 2,2,2-trichloroethyl mesylate, 2,2,2-trichlorethyl sulfamate, 2,2,2-tribromoethyl sulfamate, trichloro-3-methylsulfonylpropane.

21. A method of enhancing feed utilisation in animals of the order artiodactyla, or a method of improving diet digestibility in animals of the order artiodactyla, or a method of improving livestock productivity in animals of the order artiodactyla, the method comprising administering to the animals one or more compounds or a feed additive comprising the one or more compounds, wherein the one or more compounds are as defined in anyone of claims 1 to 20.

22. Use of the one or more compounds as defined in anyone of claims 1 to 20 for reducing methane production, enhancing feed utilization, improving diet digestibility, or improving livestock productivity in animals of the order artiodactyla.

23. The method of anyone of claims 1 to 5 or the use of claim 22, wherein the animals of the order artiodactyla are animals of the suborder ruminantia, preferably are animals of the family camelidae, more preferably animals of the family Bovidae, even more preferably of the tribus caprini or bovini, most preferably of the genus bos.

24. A method of reducing methane production in a bioreactor, the method comprising introducing an additive into the reactor, wherein the additive comprises one or more compounds as defined in anyone of claims 1 to 20.

25. The method of claim 24, wherein the method comprises introducing the additive for the pre-treatment of the inoculum, effluents sludge or wastes.

26. Use of the one or more compounds as defined in anyone of claims 1 to 20 for reducing methane production in a bioreactor.

27. The method of claim 24 or 25 or the use of claim 26, wherein the bioreactor is a hydrogen production bioreactor, an ethanol production bioreactor, or a volatile fatty acid production bioreactor.

28. Use of the one or more compounds as defined in anyone of claims 1 to 20 for reducing methane production in a process for the clarification and / or degradation of polyhalogenated effluents, and other processes that rely on the availability of hydrogen or volatile fatty acids; or in processes of composting, landfilling and manure spreading.

29. A composition comprising a nutritious feed and the one or more compounds as defined in anyone of claims 1 to 20.

30. A feed additive comprising the one or more compounds as defined in anyone of claims 1 to 20 and at least one selected from the group consisting of probiotica, bacteria, enzymes, fatty acids, herbs, spices or essential oils, vitamins, minerals, trace elements, flavour enhancers and preservatives.

31. A compound according to the general formula (III):whereinW is selected from -SO2, -C(=O)- or -C(=O)-O-;V is selected from -H or -OH; each E is individually selected from H, Cl or F, wherein at least two E's are a halogen atom selected from Cl and F, and k is an integer from 0 to 2.

32. The compound according to claim 31, wherein one of the following three conditions applies:(i) k is 0, and the three E's are one H and two Cl,(ii) k is 1, and all three E's are Cl, and(ii) k is 2 and all three E's are F;W is as defined in claim X.

33. A compound selected from the group consisting of l-iodo-2-(methylsulfonyl)ethane, 2-bromo--ethanesulfonamide, 2-iodoethanesulfonamide, l-iodo-3-(methylsulfonyl) propane, 3-iodo--propane--sulfonamide, 2-bromoethyl methanesulfonate, / V-(2-iodoethyl)methanesulfonamide, l-azido-2-(methylsulfonyl)ethane, 2,2-dichloro- N-(2,2,2-trichloro-l-hydroxyethyl)acetamide, N-(2,2,2-trichloroethyl)-3,3,3-trifluoropropane-sulfonamide, N-(2,2,2-trichlorethyl)-(2,2,2-trichlorethyl) carbamate and N-(2,2,2-trichloro-l-hydroxyethyl)-(2,2,2-trichloroethyl carbamate.

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