AU2018383221A1

AU2018383221A1 - The use of acetylene derivatives in ruminants

Info

Publication number: AU2018383221A1
Application number: AU2018383221A
Authority: AU
Inventors: Ian Kenneth Boddy; Vincenzo Carbone; Robert Starr Ronimus; Michael Tavendale
Original assignee: Pastoral Greenhouse Research Ltd
Current assignee: Pastoral Greenhouse Research Ltd
Priority date: 2017-12-14
Filing date: 2018-12-14
Publication date: 2020-07-09
Also published as: WO2019117733A1

Abstract

The present invention provides for the use of a compound of Formula I or Formula II or a salt thereof for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.

Description

THE USE OF ACETYLENE DERIVATIVES IN RUMINANTS

FIELD OF INVENTION

The present invention relates to the use of acetylene derivatives in ruminants, in particular the use of phenyl acetylene derivatives in ruminants. The use of such derivatives in ruminants is to inhibit methanogens to either reduce methane production in the rumen and/or to enhance productivity in the ruminant.

BACKGROUND OF INVENTION

Methane is produced as a natural consequence of digestion of feed by bacteria, fungi and protozoa in ruminant animals. This fermentation leads to the production of volatile fatty acids and peptides for the host. Fermentation also produces copious quantities of CO2 and H2 which are utilised by methanogenic archaea within the rumen to produce methane, which is ultimately released from the rumen, mostly through eructation.

The methane forming methanogens are members of the Archaea, and are quite different in a number of features compared to bacteria, fungi and protozoa. Methanogens have a number of unusual and archaeal-specific features including cell wall structures, lipids, cofactors, and amino acid synthesis pathways as well as their signature energy metabolism that is linked to methane production. They represent only about 1-4% of the rumen microbial community. It is known that due to the unique metabolic pathways of methanogens and their low numbers that methanogen-specific inhibitors can be developed that do not adversely affect the fermentation of feed.

It has been recognised that the release of such methane is deleterious for two reasons. One is that methane is a greenhouse gas and the other is that the methane loss represents a loss of an energy source for the ruminant. It has been previously recognised that if one could inhibit or reduce the release of methane from ruminants that the impact of methane on the environment and atmosphere would be reduced and productivity gains might be achieved in the ruminants.

The use of acetylene (C2H4) for the reduction or inhibition of methanogens is described by Sprott et al. in Acetylene as an Inhibitor of Methanogenic Bacteria, J. of Gen. Microbiology (1982), 128, 2453- 2462. Six pure cultures of methanogens were inhibited by low concentrations of dissolved acetylene, whilst other archaea and bacteria were not similarly inhibited or affected. Similarly, it was reported by Elleway et al. in Archivfur Mikrobiologie (1971), 76, Issue 4, 277-291 that methane production by filtrates of rumen contents was found to be inhibited by acetylene. Similarly, an in vivo study was conducted in New Zealand using acetylene to inhibit methanogenesis in sheep whilst studying the effects on ruminal homoacetogenic activity. It was found that ruminal homoacetogenic activity increased when methane formation was inhibited with acetylene. The results of this work were published in a poster presented at the 2015 Congress on Gastrointestinal Function in Chicago April 13-15, 2015 by Preeti Raju et al and Preeti Raju (2016) Homoacetogenesis as an alternative hydrogen sink in the rumen. PhD thesis, Massey University, Palmerston North, New Zealand.

One of the difficulties with the use of acetylene, however, is that because acetylene is a gas, it is difficult to administer to the rumen of a ruminant. Furthermore, its effect is short lived and is therefore not suitable as a methane reducing agent.

It has also been reported by Ungerfeld et al. in the Journal of Applied Microbiology 2004, 97, 520- 526 that 2 mmol I ¹ propynoic acid (a simple straight chain liquid acetylene compound) was strongly inhibitory towards (i) a pure culture of Methanobrevibacter ruminantium and (ii) a pure culture of Methanomicrobium mobile and somewhat less inhibitory towards (ii) a pure culture of

Methanosarcina mazei. At 4 mmol 1 ¹ propynoic acid caused further inhibition of (i) a pure culture of Methanobrevibacter ruminantium but had minimal further effects on (ii) a pure culture of

Methanosarcina mazei and (iii) a pure culture of Methanomicrobium mobile. The inhibition of methane production by using propynoic acid at 2 and 4 mmol I ¹ on pure cultures of

Methanobrevibacter ruminantium and Methanomicrobium mobile was greater than the inhibition previously reported by Ungerfeld et al. 2003 (Reproduction Nutrition Development 43, 189-202) in mixed ruminal cultures at 6 mmol I ¹.

Ungerfeld et al. has also reported in Letters in Applied Microbiology 42 (2006) 567-572 of the use of propynoic acid at 4 mmol I ¹ decreasing methane formation by more than two thirds in withdrawn///! vitro ruminal fluid fermentations taken from non-lactating Holstein cows fed on lucerne hay.

Propynoic acid is a liquid, which is unstable to sunlight and has an odour similar to acetic acid or vinegar, which makes propynoic acid unsuitable for adding to feed or water supplies of ruminants.

It is therefore an object of the present invention to overcome the above mentioned difficulties or to at least provide the public with a useful alternative.

SUMMARY OF INVENTION

In one aspect the present invention provides the use of a compound of Formula I, Formula I wherein the - line optionally represents a double bond; and wherein each X independently represents -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NFI2, -NFI(C_I-C₆ lower alkyl), -N(C_I-C₆ lower alkyl)2, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; an -8-12 membered bicyclic; -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0FI; -C(0)-Fl, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, -C(halo)3, -SFI, -C1-C6 lower alkyl, - (C1-C6 lower alkyl)-OFH, -NFI2, -NFI(C_I-C₆ lower alkyl), -N(C_I-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Cr C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0FI; - C(0)-Fl, or -C(0)-(Ci-C₆ lower alkyl); n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; or a fused 8-12 membered bicyclic; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, - C(halo)3, -SFI, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OFH, -NFI2, -NFI(C_I-C₆ lower alkyl), -N(C_I-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), - C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(C C₆ lower alkyl); or a salt thereof as defined by formula (I) for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.

In one embodiment the invention provides the use of a compound of Formula II,

Formula wherein each X independently represents -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; an -8-12 membered bicyclic; -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, -C(halo)3, -SH, -C1-C6 lower alkyl, - (C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Cr C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; - C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; or a fused 8-12 membered bicyclic; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), - C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(C C₆ lower alkyl); or a salt thereof as defined by formula (II) for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.

In one embodiment of a compound of Formula I or of Formula II each X independently represents - halo, -OH, -CN, -N0₂, -CECH, -SH, -Ci-C₆ lower alkyl, -(C C₆ lower alkyl)-OH, -NH₂, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; -O (C1-C6 lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)OH; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -C(0)-0(- C1-C6 lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OH, -CN, -N0₂, -CECH, -SH, -C C₆ lower alkyl, -(C C₆ lower alkyl)-OH, -NH₂, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(O)-

H, or -C(0)-(Ci-C₆ lower alkyl).

In one embodiment, n is selected from 0, 1, 2 or 3.

In one embodiment, n is selected from 0, 1 or 2.

In one embodiment, n is selected from 1 or 2.

In one embodiment the X substituents are located in the ortho and/or para positions.

In another embodiment the X substituent is located in the para position.

In another embodiment the X substituents are located in the meta positions.

In another embodiment the X substituents are located in the ortho positions.

In one embodiment of the compound of Formula I or II, X is -OCH3 and n is 1.

In one embodiment of the compound of Formula I or II, X is— CH3 and n is 1.

In one embodiment of the compound of Formula I or II, X is -CN and n is 1.

In one embodiment of the compound of Formula I or II, X is— NH2 and n is 1.

In one embodiment of the compound of Formula I or II, X is -halo and n is 1. In one embodiment the

-halo is -F or -Br.

In one embodiment of the compound of Formula I or II, X is -NO2 and n is 1.

In one embodiment of the compound of Formula I or II, X is a fused phenyl ring and n is 1. In another embodiment of the compound of Formula II X is a phenyl ring fused at the ortho and meta positions.

In one embodiment of the compound of Formula II is l-ethynyl-4-nitrobenzene.

In one embodiment the compound of Formula II is 2-methoxyphenyl acetylene.

In one embodiment the compound of Formula II is 1-ethynyl-naphthylene. In one embodiment the compound of Formula I or II is selected from one or more of the following:

In one embodiment the use of a compound of Formula I or Formula II as defined above is for reducing the formation of methane from the digestive actions of ruminants.

In one embodiment the use of a compound of Formula I or Formula II as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 10%.

In another embodiment the use of a compound of Formula I or Formula II as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 15%.

In yet another embodiment the use of a compound of Formula I or Formula II as defined above is for reducing the formation of methane from the digestive actions of ruminants by at least 20%. In one aspect the present invention further provides a method for reducing the production of methane emanating from a ruminant and/or for improving ruminant animal performance, comprising administering orally to the ruminant an effective amount of at least one compound of Formula I or Formula II or a salt thereof to the ruminant. It is to be understood by oral

administration, a route involving drenching, addition to feed, water source or pasture or manual administration of a bolus or a capsule.

In one embodiment the effective amount of at least one compound of Formula I or Formula II or a salt thereof is administered at least once-daily to the ruminant.

In one embodiment the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 10% per day.

In one embodiment the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 15% per day. In one embodiment the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 20% per day.

In a further aspect of the present invention there is provided a composition comprising at least one compound of Formula I or Formula II or a salt thereof for reducing the production of methane emanating from a ruminant, and the composition further including at least one agriculturally and an orally acceptable excipient .

In one embodiment the composition is adapted for use as a feed additive.

In another embodiment the composition is adapted for use as a ruminant lick.

In one embodiment the composition is adapted for use as an oral drench. In another embodiment the composition is adapted for use as a rumen capsule or bolus.

In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 10% per day.

In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 15% per day. In one embodiment the composition is adapted to reduce the production of methane emanating from the ruminant by at least 20% per day.

In one embodiment the excipient may include one or more minerals and/or one or more vitamins.

In one embodiment the excipient may include one or more vitamins selected from vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3, vitamin B12, biotin and choline, vitamin Bl, vitamin B2, vitamin B6, niacin, folic acid or the like.

In one embodiment the excipient may include one or more minerals selected from calcium, phosphorus, sodium, manganese, zinc, iron, copper, chlorine, sulfur, magnesium, iodine, selenium, and cobalt or the like.

In one embodiment the composition may further include sunflower oil, electrolytes such as ammonium chloride, calcium carbonates, starch, proteins or the like.

In one embodiment the composition may further include one or more anthelmintic.

The compounds of the present invention have potential for use in a ruminant to reduce the formation of methane without affecting microbial fermentation in a way that would be detrimental to the ruminant.

The invention is now described, by way of a non-limiting example, with reference to the

accompanying drawings, in which:—

FIG. 1 shows a plot of inhibition of methane production by ENB using a 60 ml rumen in vitro assay system.

FIG. 2 shows a plot of the gas profile for three sheep (one control animal, two treatment animals) dosed twice-daily with feed with 1- ethynyl-4-nitrobenzene (ENB).

FIG. 3 shows a plot of the gas profile for one of the five sheep dosed once-daily with 4-ethynyl-anisole. FIG. 4 shows a plot of the gas profile for one of the five sheep dosed once-daily with 4-ethynyl aniline.

FIG 5 shows a plot of the gas profile for the two cattle dosed with 1- ethynyl-4-nitrobenzene (ENB) provided in feed (Y-axis is g CFU and X-axis is the date).

FIG 6 shows a plot of the gas profile for a control cow with no inhibitor in the l- ethynyl-4-nitrobenzene (ENB) cattle trial. DETAILED DESCRI PTION OF INVENTION

DEFINTIONS

The term "ruminant" as used herein is a mammal that is able to acquire nutrients from plant-based food by fermenting it in a specialized foregut (the rumen) prior to digestion, principally through microbial activity. Representative examples of ruminants and other foregut fermenters include cattle, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, and antelope.

The term "effective amount" as used herein refers to an amount of at least one compound of Formula I or Formula I I or a salt thereof that either reduces the production of methane emanating from the ruminant or improves ruminant performance.

The term "ruminant performance" as used herein refers to improving the productivity of the ruminant, such as increased muscle weight gain, milk yield or quality, wool growth or quality, surviving offspring per parturition, or the like.

The term "halo" as used herein refers to a halogen atom selected from Cl, Br, I, or F.

The term "Ci-Cg lower alkyl" as used herein refers to a straight or branched chain, saturated hydrocarbon having from to 6 carbon atoms. Representative Ci-Cg lower alkyl groups include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert butyl, pentyl, isopentyl, neopentyi, hexyl, isohexyl, neohexyl and the like. The CrCg lower alkyl group may be further substituted, for example, by -halo, -OFI, -CN, -NO2, -CECFI, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)- OFI, -NH2, -N FI(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), - SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(O)- (C1-C6 lower alkyl).

The term "C3-C7 monocyclic cydoalkyi" as used herein is a 3-, 4-, 5-, 6-, or 7-membered saturated non-aromatic monocyclic cydoalkyi ring. Representative C3-C7 monocyclic cydoalkyi groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cydoheptyl. In one embodiment, the C3-C7 monocyclic cydoalkyi group may be substituted with one or more of the following groups: -halo, -OFI, -CN, -NO2, -CECFI, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -N FI2, -NFI(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0FI; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl). The term "3- to 6-membered monocyclic heterocycle" refers to: (i) a 3- or 4-membered non aromatic monocyclic cycloalkyl in which 1 of the ring carbon atoms has been replaced with an N, O or 5 atom; or (ii) a 5-, or 6-membered aromatic or non-aromatic monocyclic cycloalkyl in which 1-4 of the ring carbon atoms have been independently replaced with a N, O or S atom. The non-aromatic 3- to 6-membered monocyclic heterocycles can be attached via one or more ring nitrogen, sulfur, or carbon atoms. The aromatic 3- to 6-membered monocyclic heterocycles may be attached via one or more ring carbon atoms. Representative examples of a 3- to 6-membered monocyclic heterocycie group include, but are not limited to furanyl, furazanyl, imieazo!ieinyl, imieazoiinyl, imieazolyl, isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolieinyl, oxazolyl, oxazolieinyi, pyrimieinyl, phenanthrieinyl, phenanthrolinyl, piperazinyi, piperieinyl, pyranyl, pyrazinyl, pyrazo!ieinyl, pyrazolinyl, pyrazo!yl, pyrieazinyi, pyrieooxazole, ipyrieoimieazole, pyrieothiazole, pyrieinyi, pyrimieinyl, pyrrolieinyl, pyrrolinyl, quinudieinyl, tetrahydrofuranyl, thiadiaziny!, tbiadiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimieazoiyl, thiomorpholinyl, thiopbenyl, triazinyl, triazolyl. in one embodiment, the 3- to 6-membered monocyclic heterocycie group may be substituted with one or more of the following groups-halo, -OH, -CN, -NO2, -CECH, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -IMH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl).

The term a "a -8-12 membered bicyclic group" as used herein refers to a 8 -12 membered aromatic or non-aromatic bicyclic in which one or both of the rings of the bicyclic ring system optionally have 1 to 3 of the ring carbon atoms has been optionally replaced with a N, O or S atom or a carbonyl - C(O) group. Included in this class are 3-6 membered monocyclic heterocydes, as defined above, that are fused to a benzene ring. Representative examples of 8- to 12-membered bicyclic heterocydes include napbtha!eny!, benzimieazolyl, benzofurany!, henzothiofurany!, benzothiopheny!, benzoxazolyl, benzthiazo!yl, benztriazolyl, benztetrzolyl, benzisoxazolyl, benzisothiazolyl, benzimieazo!iny!, cinno!iny!, decahydroquino!iny!, IH-indazoly!, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofurany!, isoindazo!y!, isoindo!yl, isoindo!iny!, isoquino!inyl, naphthyr!einyl, octahydroisoquino!inyl, pbtha!azinyl, pteridinyl, purinyl, quinoxa!iny!, tetrahydroisoquinolinyl, and tetrahydroquinolinyl. In one embodiment, each ring of a the -8- to 12-membered bicyclic heterocycie group may be substituted with one or more of the following groups: -halo, -OH, -CN, - N0₂, -CECH, -SH, -Ci-C₆ lower alkyl, -(Ci-C₆ lower alkyl)-OH, -NH₂, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl). The term "or a salt thereof," as used herein, is a salt of an acid or a basic nitrogen atom. Illustrative salts include, but are not limited, to sodium salt, potassium salt, lithium salt, calcium salt, ammonium salt, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, and the like.

The term anthelmintic means a compound that expels parasitic worms (helminths) and other internal parasites from the body of the ruminant by either stunning or killing them and without causing significant damage to the host. The one or more anthelmintic may be selected from benzimidazoles, including albendazole, mebendazole, thiabendazole, fenbendazole, triclabendazole, and flubendazole; abamectin, diethylcarbamazine, ivermectin, suramin, pyrantel pamoate, levamisole, salicylanilides (eg niclosamide), nitrazoxanide, praziquantel, octadepsipeptides (eg emodepside), aminoacetonitrile derivatives (eg monepantel), spiroindoles (eg derquantel), pelletierine sulphate and artemisinin.

In vitro Studies

The compounds of the present invention were screened for initial in vitro activity using a fully automated incubation system for the measurement of total gas production and gas composition (methane and hydrogen). The system used is described in detail in the paper by Muetzel et al.

Animal Feed Science and Technology 196, (2014) 1-11. The system in this specification is referred to as the rumen in vitro system or RIV. The RIV system provides a relatively simple tool for determining the rate and production of methane and also whether the rate and production of methane is inhibited or reduced by a particular compound or feed.

Rumen in vitro assay method.

Rumen in vitro assays utilise rumen fluid from donor animals (typically either sheep or cattle) which is typically combined with a buffer and then incubated in sealed fermentation vessels at 39 °C for either 24 or 48 h. These assays systems have been well-characterised and used by the scientific community over the last two decades (Rymer et al. 2005). The system that has been used here to characterise the effects of inhibitors on methane production and gas production, and the formation of H2, was described by Muetzel et al. 2014. Rumen in vitro assay systems reflect what can occur in the rumen in vivo, but only short-term, due to their short incubation times (typically not exceeding 48 h) which is limited by its buffering capacity and the fact that it is a closed system.

Stock solutions of inhibitors were re-suspended in dimethylformamide (DMF) at concentrations 1000- fold higher than the highest used for the assays. Any further dilutions were also prepared using DMF. The total amount of inhibitor solution added to the rumen fluid-buffer mixture (60 mL) was 60 mI. Incubations used 60 ml of medium containing 12 ml of filtered rumen fluid and 48 ml of buffer in serum bottles for 24-48 h, essentially as described by Muetzel et al. 2014. Two fistulated cattle were used as donor animals for rumen fluid and treatments were incubated in duplicate bottles. Sets of duplicate incubation vials that contained rye grass (with rumen fluid-buffer mixture), and rye grass with 30 mM bromoethanesulfonate (BES) were also incubated as negative and positive controls, respectively.

ENB, for example, was tested using the 60 mL rumen in vitro assay system at 100 mM, 70 mM and 30 mM final concentrations and compared to a positive control with 30 mM BES (see Figure 1 for the effect on methane production). A negative control, with no added inhibitor (but with DMF) was also included. ENB produced BES-like effects with approximately 25% inhibition at 30 mM at 24 h. At 70 mM and 100 mM, inhibition levels were approximately 49% and 68% at 24 h, respectively. Other compounds of Formula I and Formula II were tested in the RIV using the same method described above for ENB. All the compounds were either sourced from commercially available stockists or synthesised by chemical synthesis companies. It can be seen from the results of the RIV experimentation tabulated below and the subsequent results of the in vivo testing that is described below that the RIV results for the compounds of Formula I and II are somewhat predictive of in vivo activity.

-

In vivo studies

The animal experiments described in this specification were all reviewed and approved by the Grasslands Animal Ethics Committee (Palmerston North, NZ) and all animals were cared for according the Code of Ethical Conduct (Animal Welfare Act, 1999) and its amendments.

Example 1

Use of l-ethynyl-4-nitrobenzene (ENB) in a 16 day sheep trial

ENB (l-ethynyl-4-nitrobenzene) was tested for its effectiveness in mitigating methane emissions in sheep over a 16 day treatment period. ENB was examined as a potential inhibitor because ENB was active in rumen in vitro assays at low concentrations (30 mM and 70 pM). ENB (250 g) was sourced from the company SYNthesis, Shanghai, China). NMR confirmed the synthesis; HPLC showed a single peak and the correct molecular mass was determined from mass spectrometry. l-ethynyl-4-nitrobenzene (ENB) To gain animal ethics approval, mice toxicity tests were required for ENB according to the standardised toxicity testing method OECD 423 (detailed below). The initial target concentration for mice testing was 300 mg/kg. ENB was not expected to be toxic based on its chemistry. In the case of ENB, the expectation of low toxicity was based on the evidence that it had been selected from a commercial chemical compound library (Chembridge, USA) that filters compounds for known reactive and toxic functional groups. ENB is considered to be non-toxic according to Regulation (EC) No. 1272/2008. In addition, ENB is not classified as dangerous according to Directive 67/548/EEC. Despite these indications of the compound being nontoxic, the toxicity was tested in mice acute oral toxicity tests performed according to OECD 423 prior to conducting this work to satisfy Animal Ethics requirements.

The toxicity of ENB was tested in mice under a contract research arrangement and no adverse effects were observed. It should be noted that the 300 mg/kg concentration used for the toxicity testing in mice for ENB was significantly higher (circa 19-fold) than that used for the sheep trials.

Mice testing

Mice testing used two groups with three mice (Female Swiss mice, non-pregnant) for each compound, all at approximately 8-9 weeks of age. On the day of testing, food was withdrawn 1-2 hours prior to dosing. Following fasting, the animals were weighed and the test substances administered. Access to food was resumed 1-2 hours after administration of ENB. ENB was administered in a single dose by oral gavage in corn oil (maximum of 0.3 ml per dose) at the selected dose and the time recorded. The mice were observed individually after dosing at least once during the first 30 minutes, periodically over the first 24 hours, with special attention given to the first 4 hours, and then daily thereafter for a total of 14 days. The mice were weighed every seven days (0, 7 and 14 days). All observations were systematically recorded for each individual animal. These observations included (if present): tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma, and changes in fur, skin, eyes, mucus membranes, respiration, motor activity and behaviour.

Trial Method

The trial was approved by the Grasslands Animal Ethics Committee and the use of ENB in animals was approved by the New Zealand Food Safety Authority, as required under ACVM legislation. Five sheep were used for the ENB trial treatment group and a control group with five untreated animals. Three further animals were dosed in the trial to determine if there were any adverse health effects caused by dosing with ENB (one animal was dosed first for two days and observed, then two new animals were dosed for two days and observed). This pre-testing was a condition of the AEC approvals (in addition to the mice toxicity testing).

The animals received a general purpose diet (GP) at 1.5 x maintenance energy requirements throughout the trials consisting of 500 g hay, 100 g soy bean meal, 290 g barley, 100 g molasses and 10 g mineral mix. During the time in the respiration chambers the animals had free access to water, with the feed offered twice a day at approximately 9:00 and 16:00 h in equal amounts. Feed refusals were weighed.

After the acclimatisation period, methane emissions were measured in open circuit respiration chambers as described in Chapter 1 of the technical manual on respiration chamber design (Pinares- Patifio CS, Hunt C, Martin R, West J, Lovejoy P, Waghorn GC. (2012) Chapter 1: New Zealand Ruminant Methane Measurment Centre, AgResearch, Palmerston North. In Technical Manual on Respiration Chamber Design'. Vol. 2013. (Eds. CS Pinares-Patifio and GC Waghorn). (Ministry of Agriculture and Forestry, Wellington, New Zealand).

Dry matter intake (DMI, kg/d) was recorded during methane measurements from the weight difference of feed offered and refused.

Rumen samples from stomach tubing were immediately subsampled for short chain fatty acid (SCFA) analysis (1.8 ml). The samples were centrifuged (20,000 g, 10 min, 4 °C). An aliquot of 0.9 ml of the supernatant was collected into 0.1 ml of internal standard (19.8 mM ethylbutyrate in 20% v/v phosphoric acid) for SCFA analysis and stored at -20 °C. In general, rumen contents were sampled by stomach tubing before methane emissions were measured in the respiration chambers and the day after being released from the chambers to examine any effects on rumen microbial populations. Volatile fatty acids (VFAs) and ammonia analyses were performed and are reported below.

SCFA samples were thawed and centrifuged (20,000 g, 10 min, 4 °C) and 800 pi of the supernatant was collected into a crimp cap glass vial. SCFA were analysed using a HP 6890 gas chromatograph (Attwood et al., 1998). Ammonia was analysed from the SCFA sample using a downscaled nitroprusside method (Weatherburn 1967).

Three sheep were used to test the potential toxicology of ENB, as described above, and no ill-effects were observed. A dose rate of 0.74 g/day/animal was used in the experiment (and assumed a 50 kg average animal weight) was based on the finding that 0.1 mM of the compound was inhibitory in rumen fluid-based assays, and that there is an approximate 10-fold dilution of effect between rumen in vitro assays and in vivo, and assumed a 5 litre rumen volume in sheep. For the continuation of the experiment, 10 animals were placed in crates for three days to adapt them to going into the chambers for the first measurements. On the last day in the crates the 10 animals were sampled for rumen fluid, blood and urine. The 10 animals entered the methane respiratory chambers for four days. On the start of the third day in the chambers, five animals were dosed with ENB in sunflower oil and five control animals were given only oil. The four days in chambers enable background methane levels to be assessed in all animals (control and treatment) and the initial knockdown in methane in the treatment animals. In addition, a treatment-control group comparison was possible, with n=5 for each group.

The five ENB-treated animals continued to be administered ENB orally in feed twice daily through to the end of the experiment (total of 16 days dosing). After 10 days in pens, all animals were re introduced into crates again for two days and then placed in the chambers for two days.

Results for ENB trial

ENB, with twice daily oral dosing with feed, produced a substantial decrease in methane emissions (~25% [g/d], P=0.010; 26% [g/kg DM I, P=0.008) during the first two days of the treatment (Table 1) the decrease in methane emissions led to a decrease in the proportion of acetate and an increase in butyrate reflecting a decrease in H production. These differences in the metabolic profiles were not observed after 16 d of dosing but the methane yield was still 16.6% lower in the treated animals compared to the control values from the same measurement period and the H production from these animals was increased. Daily profiles of methane and H are shown in Figure 3 for one control and two treated sheep. Dry matter intake was similar in the treated and the control sheep over the whole experimental period. No differences in any of the parameters measured were detected during the control period when no inhibitors were applied (Table 2).

Table 1. Differences between the treatment groups at the start of the treatment (Days 1 and 2) and at the end of the adaptation period (Day 13).

Days 1

Period and 2 Day 13

Treatment Control Treatment sed P value Control Treatment sed P-value

SCFA [mM] 85.1 82.2 7.79 0.721 85.1 81.8 10.31 0.757

Acetate

[%] 57.5 49.6 11.71 0.002 61.2 60.1 1.37 0.429

Propionate

[%] 25.0 27.0 2.48 0.450 21.5 21.7 2.45 0.929 Butyrate

[%] 13.3 19.0 1.65 0.008 12.8 13.8 1.78 0.592

Caproate

[%] 0.21 0.18 0.120 0.752 0.46 0.38 0.144 0.598

Isobutyrate

[%] 0.73 0.68 0.130 0.718 0.75 0.76 0.116 0.898

Isovalerate

[%] 0.81 0.76 0.181 0.784 0.85 0.74 0.151 0.514

Valerate

[%] 1.2 1.4 0.12 0.078 1.2 1.3 0.10 0.491

Minor [%] 3.0 3.0 0.26 0.726 3.3 3.2 0.41 0.828

DM I [kg/d] 0.9 1.0 0.08 0.754 0.9 1.1 0.14 0.292

CH₄ [g/d] 19.5 14.6 1.44 0.010 19.0 19.2 2.59 0.932

CH₄ [g/kg] 21.2 15.6 1.61 0.008 21.8 18.2 1.47 0.041

H₂ [g/d] 0.00 0.63 0.138 0.001 0.00 0.12 0.050 0.008

NH₄ [mM] 12.3 18.0 5.06 0.291 7.7 6.8 3.39 0.800

Statistics performed using GenStat using General Analysis of Variance.

Table 2. Differences of the groups before treatment started.

Period Control

Treatment Control Treatment sed P-value

SC FA [mM] 80.8 89.3 7.64 0.300

Acetate [%] 58.5 58.1 2.29 0.869

Propionate [%] 22.3 23.8 2.65 0.589

Butyrate [%] 14.7 13.8 1.07 0.439

Caproate [%] 0.19 0.19 0.062 0.938

Isobutyrate [%] 0.84 0.77 0.119 0.579

Isovalerate [%] 0.99 0.92 0.219 0.736

Valerate [%] 1.2 1.3 0.07 0.450

Minor [%] 3.2 3.1 0.34 0.814

DM I [kg/d] 1.0 1.0 0.05 0.476

CH₄ [g/d] 21.4 21.4 1.21 0.996

CH₄ [g/kg] 21.6 20.8 1.07 0.508

H₂ [g/d] -0.03 -0.04 0.023 0.769 NH₄ [mM] 15.0 17.6 4.77 0.599

As can be seen from the results, the level of methane inhibition in the first two days of dosing with ENB was circa 26.5% and after a further 14 days the inhibition of methane yield was still 16.6%. Dry matter intake was not affected by the treatment which would indicate that appetite was not affected. There were no observable toxic effects after 16 days of treatment. Finally, the applicant has shown that daily oral administration of ENB is a convenient and a safe method for dosing sheep.

Example 2 - Use of 4-ethynyl-anisole (4 ENA) and 4-ethynyl-aniline (4 EA) in 2 day sheep trial

The ethynyl derivatives 4-ethynyl-anisole (4 ENA) and 4-ethynyl-aniline (4 EA) are readily available from chemical supply companies, including Sigma-Aldrich. The liquid and solid acetylene derivatives 4-ethynyl-anisole and 4-ethynyl-aniline (respectively) have shown strong inhibition in rumen in vitro assays (at a concentration of ~100 mM).

To gain AEC approval, mice toxicity tests were required for all compounds according to the standardised toxicity testing method OECD 423. The initial target concentration for the two acetylene containing compounds for mice testing was 300 mg/kg of live weight. The compounds were not expected to be toxic based on their chemistries. A liquid acetylene derivative, 4-ethynyl-anisole is considered to be non-toxic according to Regulation (EC) No. 1272/2008 and is not classified as dangerous according to Directive 67/548/EEC. According to Regulation (EC) No 1272/2008 [EU- GHS/CLP] The solid acetylene derivative, 4-ethynyl-aniline, , exhibits toxicity: skin irritation (Category 2); Eye irritation (Category 2); specific target organ toxicity - single exposure (Category 3); Classification according to EU Directives 67/548/EEC or 1999/45/EC; and can be irritating to eyes, respiratory system and skin.

The toxicity of the two compounds were tested in mice at a Crown Research Institute in New Zealand. No adverse effects were noted at 300 mg/kg for 4-ethynyl-anisole. However, after dosing with 4- ethnyl-aniline the mice were observed to be lethargic and panted for 1-2 hours. No further effects were noted. It should be noted that the 300 mg/kg concentration used for mice testing for 4-ethynyl- aniline was significantly higher (25.6-fold) than that used for the sheep trial. The 300 mg/kg mice dosing for 4-ethynyl-anisole was also higher than that used for the sheep trials (22.7-fold).

The two acetylene derivative compounds were each tested at a rumen target concentration 10-fold higher levels in sheep (i.e. target rumen concentration of 1.0 mM for the acetylene derivatives). Materials and Methods

The trials were approved by the Grasslands Animal Ethics Committee, and the use of the compounds in animals by the New Zealand Food Safety Authority, as required under ACVM legislation. Eight sheep were used for each of the three trials. Each sheep was used as its own control (ie the first day in chambers without any added inhibitor was the control day). Three animals being dosed while in pens to determine if there were any adverse health effects due to dosing with the compounds (one animal was dosed first and observed, then two animals dosed and observed). This pre-testing was a condition of the AEC approval (as well as the mice toxicity testing). Five animals were then used for the treatment period with three days in chambers (Day 1, control day with no dosing; Day 2 and Day 3, with once-daily dosing).

The animals received a general purpose diet (GP) at 1.5 x maintenance energy requirements throughout the trial consisting of 500 g hay, 100 g soy bean meal, 250 g barley, 100 g molasses and 50 g mineral mix. The feed was analysed and contained 82% dry matter. The other components were (g per 100 g dry matter): crude protein (16.9); lipid (<1); ash (3.0); acid detergent fibre (23.1); neutral detergent fibre (50.5); soluble sugars and starch (24.9); and metabolisable energy (10.1 MJ/kg dry matter).

The trials were conducted as follows. After an adaptation period (minimum of 7 days of group feeding in pens) one animal was weighed and given a single dose of each compound (each mixed in sunflower oil) by drenching just prior to feeding. The dose rates were approximately 0.0132 g/kg and 0.0117 g/kg live weight (4-ethynyl-anisole, 4-ethynyl-aniline). These animals were monitored in pens for any overt adverse effects (overt health, behavioural and any effects on feeding). No effects were observed after two days, after which an additional two animals were similarly dosed and monitored in pens for two days.

For the remainder of the trial, five animals for each compound were adapted to feeding in crates for three days and then placed in the methane respiratory chambers for three days. For the first day in the chambers (Day 1, control with no dosing) the animals received no treatment and were simply adapted to the chambers. For the next two days (Days 2 and 3), the compounds were administered in sunflower oil by drenching as a single dose just before feeding. During the time in the respiration chambers the animals had free access to water, with the feed offered twice a day at approximately 9:00 and 16:00 h in equal amounts. Feed refusals were weighed. Results

The primary results regarding impact on CH4 and H2 levels are shown below in Table 3 for Day 1 (control) and Day 3 (second day of treatment) in the chambers.

Table 3 Summary of CH4 and H2 chamber data for Day 1 (first day in chamber, no dosing) and Day 3 (second day of dosing).

*[g/kg DMI]; Statistics performed using GenStat using General Analysis of Variance.

4-ethynyl-anisole

A typical gas profile from the treatment period (Day 3) in the chambers for one of the five sheep dosed once-daily with 4-ethynyl-anisole is shown in Fig. 2 below. Dosing with 4-ethynyl-anisole produced a rapid and near-total reduction in methane emissions, however, the effect was observed only for a limited number of hours post feeding (approximately six hours; up to approximately nine hours if the secondary peak is included) possibly due to washout or degradation of the product. Methane emissions did not recover to those observed before dosing (after feeding) in the respective animal controls. Overall reductions for Day 2 and 3 in the chambers were on average 29% and 28%, respectively (based on n=5 with methane emissions being compared to emissions from the same animal on the first non-treatment day in the chamber). The dosing almost entirely negated the typical large methane emissions that are usually present in the morning with feeding (which started 15-30 minutes after dosing) and was associated with a corresponding large spike in H2. In some cases the secondary peaks in H2 were observed after the dosing which may possibly be associated with rumination.

4-ethynyl-aniline

Similar to 4-ethynyl-anisole, 4-ethynyl-aniline was dosed as a suspension in sunflower oil and produced a rapid and near-total reduction in methane emissions. Methane emissions recovered over the next 24 hours after dosing, but not to pre-dosing levels. Overall reductions for Day 2 and 3 were on average 38% and 27%, respectively. Again, similar to 4-ethynyl-anisole, the dosing with 4-ethynyl- aniline almost entirely negated the typical large methane emissions that occur after feeding and it was associated with a large spike in H2.

Rumen parameters and volatile fatty acid profiles, ammonia metabolism, DMI, fermentation parameters (total short-chain fatty acids (SCFA), acetate, butyrate, propionate, minor VFAs) and gas profiles (CH4 and H2) were monitored during the trials (Table 4). The differences between the respective control periods and treatment periods (Day 3 only) for CH4 and H2 were all statistically significant. In addition, the ammonia was significantly increased with 4-ethynyl-aniline dosing.

Table 4 Rumen function parameters and VFAs before and after treatment with 4-ethynyl-anisole and 4-ethynyl-aniline. Values that were found to be statistically significant (e.g. at least <0.05) are in bold. The CH₄ and H₂ data are highlighted in grey.

* the control rumen samples were taken before treatment and prior to the morning feeding on Day 1

# the Day 3 rumen samples taken 24 hours after the last dosing and just prior to the morning feed.

Note: only Day 3 data shown for treatment group (i.e., last day in chamber, second day of dosing) sed, standard error of difference.

With dosing of 4-ethynyl-anisole and 4-ethynyl-aniline, large reductions in methane emissions were observed. Despite the effects being somewhat transient for 4-ethynyl-anisole and 4-ethynyl-aniline, the total inhibition for each treatment day was circa 30% for both compounds but this could reflect the fact that the animals were dosed only once-daily. The trials were run for two days to show that the compounds can reduce methane emissions in vivo. The compounds produced no noticeable toxic adverse effects on the animals during the two day exposure.

2-Ethynlphenyl)methylsulfane and 5-ethynylquinoline

A further 16 day sheep trial was conducted with two further compounds (2- ethynlphenyl)methylsulfane and 5-ethynylquinoline. Large reductions in methane emissions of more than 50% were seen in the first two days in chambers with increased hydrogen levels resulting. These reductions were not fully sustained in the subsequent two chamber measurements on dosing days 8 -9 and days 15-16 with a fall off in methane reduction in the later stages.

As described above, five acetylene derivative compounds have been tested in vivo and shown large reductions in methane emissions at least in the initial days of the study.

In vivo Example 3 - ENB cattle trial

This trial was approved by the Grasslands Animal Ethics Committee and the use of the ENB compounds in animals by the New Zealand Food Safety Authority, as required under ACVM legislation.

All animals received a general purpose diet (GP) at 1.5 x maintenance energy requirements throughout the trials consisting of (per kg feed) 500 g hay, 100 g soy bean meal, 290 g barley, 100 g molasses and 10 g mineral mix. During the time in the respiration chambers the animals had free access to water, with the feed offered twice a day at approximately 9:00 and 16:00 h in equal amounts. Feed refusals were weighed.

Rumen contents were sampled by stomach tubing before methane emissions were measured in the respiration chambers and the day after being released from the chambers to examine any effects on rumen microbial populations. Volatile fatty acids (VFAs) and ammonia analyses were performed and are reported below.

After the feed acclimatisation period, methane emissions were measured in open circuit respiration chambers as described in Chapter 1 of the technical manual on respiration chamber design (Pinares- Patifio et al., 2012). During methane measurements dry matter intake (DMI, kg/d) was recorded, from the weight difference of feed offered and refused.

Rumen samples from stomach tubing were immediately subsampled for short chain fatty acid (SCFA) analysis (1.8 ml). The samples were centrifuged (20,000 g, 10 min, 4 °C). An aliquot of 0.9 ml of the supernatant was collected into 0.1 ml of internal standard (19.8 mM ethylbutyrate in 20% v/v phosphoric acid) for SCFA analysis and stored at -20 °C.

SCFA samples were thawed and centrifuged (20,000 g, 10 min, 4 °C) and 800 pi of the supernatant was collected into a crimp cap glass vial. SCFA were analysed using a HP 6890 gas chromatograph (Attwood et al., 1998). Ammonia was analysed from the SCFA sample using a downscaled nitroprusside method (Weatherburn, 1967). The two-day cattle trial used the cattle chambers at the New Zealand Ruminant Methane Measurement Centre facility, AgResearch, Palmerston North according to standard procedures (Pinares-Patino et al., 2012). Three animals were used for the cattle trial with ENB. The animals were pre-adapted to feed (GP diet) for at least 10 days. Three cattle (one control, two treatment) were checked for their background methane emissions in chambers for one day, followed by dosing for two days in the treated animals (ENB). Their rumens were sampled prior to entering the chambers and after they were released. ENB was added to feed just prior to the experiment starting by first adding to soy meal and then to the bulk GP diet. The animals were in chambers for a total of three days.

Results for ENB cattle trial The gas profiles from the treatment period in the chambers for both sheep dosed with ENB in their feed are shown below in Fig. 4 (typical control group profile shown in Fig. 5), and are summarised for all animals in Table 5. ENB decreased methane yield (g CFU / kg DMI) by 19%. Along with a decreased methane yield for ENB an increase in hydrogen production was observed. No refusals were left by any cows during the experiment. Table 6 summarises the short chain fatty acid production and composition of cattle fed ENB compared to the control group.

Table 5. Dry matter intake and gas emissions from cattle fed ENB compared to the control group.

DMI CH₄ CH₄ H₂

Treatment [kg/d] [g/d] [g/kg] [g/d]

Control 5.4 148.1 27.6 0.2

ENB 4.1 91.7 22.3 4.8

SED NA 10.22 2.37 0.75

P value NA 0.027 0.089 0.015

Significance of differences tested using GenStat using General Analysis of Variance.

NA, not applicable

Table 6. Total short chain fatty acid production and composition of cattle fed ENB compared to a control group.

SCFA Acetic Propionic Butyric Valeric Caproic

Treatment [mM]

Control 43.9 67.3 13.3 14.7 0.9 0.1

ENB 59.0 69.0 13.1 13.4 0.9 0.4

SED 9.91 2.56 1.86 1.95 0.117 0.046 P value 0.209 0.526 0.512 0.823 0.466 0.011

ENB, when tested in cattle over two days, produced reductions in methane yield of circa 20%. These reductions were similar to those observed in the previous 16-day sheep trial for ENB. ENB dosing also caused an obvious increase in H2 emissions (a positive indicator of successful inhibition of methanogens). There were no apparent toxicity effects and no feed refusals.

The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to the particular embodiments of the invention described in the specification. Various modifications, substitutions, and variations can be made to the disclosed material without departing from the spirit and/or essential characteristics of the present invention. Accordingly, one of ordinary skill in the art will readily appreciate from the disclosure that later modifications, substitutions, and/or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilized according to such related embodiments of the present invention. Thus, the following claims are intended to encompass within their scope modifications, substitutions, and variations to the embodiments of the invention disclosed herein.

References

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Claims

1. The use of a compound of Formula I

Formula I wherein the - line optionally represents a double bond; and wherein each X independently represents -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NFI2, -NFI(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; an -8-12 membered bicyclic; -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0FI; -C(0)-Fl, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, -C(halo)3, -SFI, -C1-C6 lower alkyl, - (C1-C6 lower alkyl)-OFH, -NFI2, -NFI(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Cr C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0FI; - C(0)-Fl, or -C(0)-(Ci-C₆ lower alkyl); n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; or a fused 8-12 membered bicyclic; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OFI, -CN, -NO2, -CECFI, -CFhihalo), -CFI(halo)2, - C(halo)3, -SFI, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OFH, -NFI2, -NFI(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), - C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(C C₆ lower alkyl); or a salt thereof as defined by formula (I) for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.

2. The use of a compound of Formula II Formula wherein each X independently represents -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; an -8-12 membered bicyclic; -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, -C(halo)3, -SH, -C1-C6 lower alkyl, - (C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -S(Cr C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; - C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; or a fused 8-12 membered bicyclic; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -CH2(halo), -CH(halo)2, - C(halo)3, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)-OH, -NH2, -NH(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, -O (C1-C6 lower alkyl), -S(Ci-C₆ lower alkyl), -SO-(Ci-C₆ lower alkyl), -S0₂-(Ci-C₆ lower alkyl), - C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(C C₆ lower alkyl); or a salt thereof as defined by formula (II) for reducing the formation of methane from the digestive actions of ruminants and/or for improving ruminant performance.

3. The use as claimed in claim 1 or claim 2 wherein each X independently represents -halo, -OH, -CN, -NO2, -CECH, -SH, -Ci-C₆ lower alkyl, -(Ci-C₆ lower alkyl)-OH, -NH₂, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)2, a 3-7 membered monocyclic cycloalkyl, a 3-6 membered monocyclic heterocycle; -O (C1-C6 lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and wherein each -C1-C6 lower alkyl of X defined above may be further substituted by one or more substituents selected from -halo, -OH, -CN, -NO2, -CECH, -SH, -C1-C6 lower alkyl, -(C1-C6 lower alkyl)- OH, -NH2, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl^, -O (C1-C6 lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(0)-H, or -C(0)-(Ci-C₆ lower alkyl); and n represents 0, 1, 2, 3 or 4 and where n is 1, 2, 3 or 4, each X may further independently represent a fused 3-7 membered monocyclic cycloalkyl, a fused 3-6 membered monocyclic heterocycle; the one or more fused cyclic systems may be further substituted with one or more substituents selected from -halo, -OH, -CN, -N0₂, -CECH, -SH, -Ci-C₆ lower alkyl, -(Ci-C₆ lower alkyl)-OH, -NH₂, -N H(CI-C₆ lower alkyl), -N(CI-C₆ lower alkyl)₂, -O (C1-C6 lower alkyl), -C(0)-0(-Ci-C₆ lower alkyl), -C(0)0H; -C(O)- H, or -C(0)-(Ci-C₆ lower alkyl).

4. The use as claimed in any one of claims 1 to 3 wherein n is selected from 0, 1, 2 or 3.

5. The use as claimed in any one of claims 1 to 4 wherein n is selected from 0, 1 or 2.

6. The use as claimed in any one of claims 1 to 5 wherein n is selected from 1 or 2.

7. The use as claimed in any one of claims 1 to 6 wherein the one or more X substituent(s) are located in the ortho and/or para position(s).

8. The use as claimed in any one of claims 1 to 7 wherein one X substituent is located in the para position.

9. The use as claimed in any one of claims 1 to 6 wherein the one or more X substituent(s) are located in the meta position(s).

10. The use as claimed in any one of claims 1 to 7 wherein the one or more X substituent(s) are located in the ortho position(s).

11. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -OCH3 and n is 1.

12. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -CH3 and n is 1.

13. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -CN and n is 1.

14. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -NH₂ and n is 1.

15. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -halo and n is 1.

16. The use as claimed in claim 15 wherein the -halo is -F or -Br.

17. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is -NO2 and n is 1.

18. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula I or II, X is a fused phenyl ring and n is 1.

19. The use as claimed in any one of claims 1 to 10 wherein in the compound of Formula II X is a phenyl ring fused at adjacent ortho and meta positions and n is 1.

20. The use as claimed in any one of claims 2 to 4 wherein the compound is l-ethynyl-4- nitrobenzene.

21. The use as claimed in any one of claims 2 to 4 wherein the compound is 2-methoxyphenyl acetylene.

22. The use as claimed in any one of claims 2 to 4 wherein the compound is 1-ethynyl- naphthylene.

23. The use of a compound of Formula I or II as defined in claim 1 or claim 2, the compound being selected from one or more of the following:

24. The use of a compound of Formula I or Formula II as claimed in any one of claims 1 to 23 for reducing the formation of methane from the digestive actions of ruminants.

25. The use of a compound of Formula I or Formula II as claimed in any one of claims 1 to 23 for reducing the formation of methane from the digestive actions of ruminants by at least 10%.

26. The use of a compound of Formula I or Formula II as claimed in any one of claims 1 to 23 for reducing the formation of methane from the digestive actions of ruminants by at least 15%.

27. The use of a compound of Formula I or Formula II as claimed in any one of claims 1 to 23 for reducing the formation of methane from the digestive actions of ruminants by at least 20%.

28. A method for reducing the production of methane emanating from a ruminant and/or for improving ruminant animal performance, comprising the step of administering orally to the ruminant an effective amount of at least one compound of Formula I or Formula II or a salt thereof, as defined in any one of claims 1 to 23, to the ruminant.

29. The method as claimed in claim 28 wherein the oral administration step is achieved by one or more of the following:

- drenching the ruminant,

- adding to the ruminant feed,

- adding to the ruminant;

- adding to the ruminant water source

- adding to the ruminant pasture; and/or - manually administering a capsule or bolus to the ruminant.

30. The method as claimed in claim 28 or claim 29, wherein the effective amount of at least one compound of Formula I or Formula II or a salt thereof is administered at least once-daily to the ruminant.

31. The method as claimed in any one of claims 28 to 30 wherein the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 10% per day.

32. The method as claimed in any one of claims 28 to 30 wherein the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 15% per day.

33. The method as claimed in any one of claims 28 to 30 wherein the effective amount of at least one compound of Formula I or Formula II or a salt thereof reduces the production of methane emanating from the ruminant by at least 20% per day.

34. A composition comprising at least one compound of Formula I or Formula II or a salt thereof as claimed in any one of claims 1 to 23 for reducing the production of methane emanating from a ruminant, and the composition further including at least one agriculturally and an orally acceptable excipient.

35. A composition as claimed in claim 34 wherein the composition is adapted for use as a feed additive.

36. The composition as claimed in claim 34 wherein the composition is adapted for use as a ruminant lick.

37. The composition as claimed in claim 34 wherein the composition is adapted for use as an oral drench.

38. The composition as claimed in claim 34 wherein the composition is adapted for use as a rumen capsule or bolus.

39. The composition as claimed in any one of claims 34-38 wherein the composition is adapted to reduce the production of methane emanating from the ruminant by at least 10% per day.

40. The composition as claimed in any one of claims 34-38 wherein the composition is adapted to reduce the production of methane emanating from the ruminant by at least 15% per day.

41. The composition as claimed in any one of claims 34-38 wherein the composition is adapted to reduce the production of methane emanating from the ruminant by at least 20% per day.

42. The composition as claimed in any one of claims 34-41 wherein the excipient may include one or more minerals, and/or one or more vitamins.

43. The composition as claimed in claim 42 wherein the one or more vitamins are selected from vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3, vitamin B12, biotin and choline, vitamin Bl, vitamin B2, vitamin B6, niacin folic acid or the like.

44. The composition as claimed in claim 43 wherein the excipient may include one or more minerals selected from calcium, phosphorus, sodium, manganese, zinc, iron, copper, chlorine, sulfur, magnesium, iodine, selenium, and cobalt or the like.

45. The composition as claimed in any one of claims 34-44 wherein the composition may further include sunflower oil, electrolytes such as ammonium chloride, calcium carbonates, starch, proteins or the like.

46. The composition as claimed in any one of claims 34-45 wherein the composition further includes one or more anthelmintics.