NOVEL DICARBOXYLIC ACID DIESTERS

Abstract

The present invention relates to novel dicarboxylic diesters as well as the use thereof as for inhibiting the methane production in ruminants

Claims

1. Dicarboxylic acid diesters of formula (I) ##STR00011## wherein n is an integer selected in the range from 1 to 15, and X is a C.sub.1-24-alkylene group, a C.sub.2-24-alkenylene group or a C.sub.6-24-arylene group, with the proviso that when n is >3 the resulting hydrocarbon chain may be interrupted by —O— or —NH—.

2. The dicarboxylic diester according to claim 1, wherein n is an integer selected in the range from 1 to 15 and X is a C.sub.1-24-alkylene group, a C.sub.2-24-alkenylene group or a C.sub.6-24-arylene group.

3. The dicarboxylic diester according to claim 1, wherein n is selected in the range from 3 to 9, preferably in the range from 3 to 6, most preferably n is 3.

4. The dicarboxylic diester according to claim 1, wherein X is selected from the group of a C.sub.1-10-alkylene group, a C.sub.2-5-alkenylene group and a C.sub.6-10-arylene group.

5. The dicarboxylic diester according to claim 1, wherein X is selected from the group of an unsubstituted C.sub.1-5-alkylene group, an unsubstituted C.sub.2-4-alkenylene group, 1,2-phenylene, 1,3-phenylene and 1,4-phenylene, most preferably from the group of an unsubstituted linear C.sub.1-3-alkylene group and vinylene.

6. The dicarboxylic diester according to claim 1, which are bis(3-(nitrooxy)propyl) succinate, bis(3-(nitrooxy)propyl) malonate and bis(3-(nitrooxy)propyl) fumarate.

7. A process for the preparation of a dicarboxylic diester according to claim 1, said process comprising the step of reacting reacting a dicarboxylic acid of formula (II) respectively an acid chloride or a (cyclic) anhydride thereof with a nitrooxyalcohol of formula (III) ##STR00012##

8. A process for the preparation of a dicarboxylic diester according to claim 1 said process encompassing the step of reacting a fatty acid of formula (II) respectively an acid chloride or a (cyclic) anhydride thereof with an alcohol of formula (IV), followed by reacting the obtained fatty acid monoester (V) with nitrosulfuric acid ##STR00013##

9. Use of at least one dicarboxylic diester according to claim 1 as an active compound in animal feeding for reducing the formation of methane emanating from the digestive activities of ruminants and/or for improving ruminant performance.

10. Use according to claim 9, wherein the ruminant animal is selected from the group consisting of: cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

11. Use according to claim 9, wherein the methane production in ruminants calculated in liters per kilogram of dry matter intake is reduced by at least 10% when measured in metabolic chambers.

12. A feed composition or feed additive comprising at least one dicarboxylic diester of formula (I) according to claim 1.

13. The feed composition according to claim 12 which composition is a mineral premix, a vitamin premix, or a premix including vitamins and minerals or a bolus.

14. A method for reducing the production of methane emanating from the digestive activities of ruminants and/or for improving ruminant animal performance comprising the oral administration of a sufficient amount of at least one dicarboxylic diester of formula (I) according to claim 1 to the animal.

15. A method according to claim 14, wherein the amount of the at least one dicarboxylic diester of formula (I) administered to the ruminant animal is from 1 mg to 10 g per kg feed.

Description

EXAMPLE 1

Synthesis of 3-nitrooxy-propyl diesters

[0063] a.) Preparation of bis(3-(nitrooxy)propyl) malonate (I-a)

[0064] 12.7 g 3-nitrooxypropanol, 60 ml methylenchloride and 6.2 ml triethylamine are placed in a three-necked round bottom flask and the clear solution is cooled to 0° C. 7.3 g malonylchlorid is dissolved in 10 ml dichloromethane, transferred to the dropping funnel and added dropwise to the 3-nitrooxypropanol solution over 15 minutes, Gas formation is observed. The temperature rose to 28° C. After finishing the addition, the mixture is left stirring for 20 hours at room temperature. Then 5 g anhydrous potassium carbonate is added (002-formation) and the orange suspension is stirred for 30 minutes. The solids are filtered off and washed with 20 mL of dichloromethane. The clear, orange filtrate is concentrated under reduced pressure (10 mbar, 40° C. bath temperature). The product is obtained in 15.6 g as orange oil. The crude product was purified by dilution in 50 ml methyl tert-butylether (MTBE) and washed ten times with 50 ml of water, dried over sodium sulfate and concentrated under reduced pressure. The pure product was obtained in 14.2 g in (purity: 93%; vapor pressure: 0.00133mbar)

[0065] b.) Preparation of bis(3-(nitrooxy)propyl) fumarate (I-b)

[0066] 4.89 g 3-nitrooxypropanol, 24.3 ml methylenchlorid and 6.2 ml triethylamine are placed in a three-necked round bottom flask and the clear solution is cooled to 0° C. Fumarylchloride (2.28 ml) is dissolved in 10 ml dichloromethane, transferred to the dropping funnel and added dropwise to the 3-nitrooxypropanol solution over 15 minutes, keeping the temperature below 5° C. The color changes immediately to dark brown. In the end the mixture is allowed to warm to 24° C. and left stirring for four hours. Then the viscous reaction mixture is diluted with 200 ml ethyl acetate and extracted with 200 ml 2N hydrochloric acid. The aqueous phase is then re-extracted with 100 ml ethyl acetate and the combined organic phases washed three times with 200 mL water each (total 600 mL). The organic phase is dried over sodium sulfate and concentrated under reduced pressure (40° C./<0.1 mbar). The product is obtained in 5.2 g as dark-brown oil. The crude product was purified by column chromatography (120 g SiO2-column, eluent cyclohexane with 30% MTBE). The pure product was obtained in 2.32 g (purity: 96%; vapor pressure 0.00133 mbar)

[0067] c.) Preparation of bis(3-(nitrooxy)propyl) succinate (I-c)

[0068] 5.14 g of 3-nitrooxypropanol was dissolved in 20 ml dichloromethane and 2.319 ml succinyl dichloride was added. The mixture was stirred at 23° C. for 16 hours. Solid potassium carbonate was added until no further evolution of carbon dioxide was visible, then the mixture was filtered, the filter residue washed with acetone and the filtrate was concentrated under reduced pressure yielding 6.35 g of a nearly colorless liquid. The crude product was dissolved in 50 ml t-butylmethylether and washed 10 times with water (50 ml each). The organic phase was dried with magnesium sulfate and evaporated under reduced pressure yielding 5.7 g of bis(3-(nitrooxy)propyl) succinate (yield: 83%; purity:94.7%, vapor pressure: 0.00133 mbar).

EXAMPLE 2

In Vitro Test for Methane Production

[0069] A modified version of the “Hohenheim Forage value Test (HFT)” was used for testing the effect of specific compounds on the rumen functions mimicked by this in-vitro system.

[0070] Principle: Feed (i.e. a TMR) (300 mg) is given into a syringe with a composition of rumen liquor and an appropriate mixture of buffers (i.e. rumen-fluid buffer mix: 25 ml) and the substances to be tested in the concentrations as outlined in table 2 (the inhibitors to be tested are diluted in ethanol to reach the respective concentration of dry matter in 50 μl). The solution is incubated at 39° C. for 8 h. The quantity of produced gas is measured and put into a formula for conversion. After the incubation the composition of gas is measured with a GC.

Reagents:

[0071] Mass element solution: [0072] 6.2 g potassium dihydrogen phosphate (KH.sub.2PO.sub.4) [0073] 0.6 g magnesium sulfate heptahydrate (MgSO.sub.4*7H.sub.2O) [0074] 9 ml concentrated phosphoric acid (1 mol/l) [0075] dissolved in distilled water to 1 l (pH about 1.6)

[0076] Buffer solution: [0077] 35.0 g sodium hydrogen carbonate (NaHCO.sub.3) [0078] 4.0 g ammonium hydrogen carbonate ((NH.sub.4)HCO.sub.3) [0079] dissolved in distilled water to 1 l

[0080] Trace element solution: [0081] 13.2 g calcium chloride dihydrate (CaCl.sub.2*2H.sub.2O) [0082] 10.0 g manganese(II) chloride tetrahydrate (MnCl.sub.2*4H.sub.2O) [0083] 1.0 g cobalt(II) chloride hexahydrate (CoCl.sub.2*6H.sub.2O) [0084] 8.0 g iron(III) chloride (FeCl.sub.3*6H.sub.2O) [0085] dissolved in distilled water to 100 ml

[0086] Sodium salt solution: [0087] 100 mg sodium salt [0088] dissolved in distilled water to 100 ml

[0089] Reduction solution: [0090] first 3 ml sodium hydroxide (c=1 mol/l), then 427.5 mg sodium sulfide hydrate (Na.sub.2S*H.sub.2O) are added to 71.25 ml H.sub.2O [0091] solution must be prepared shortly before it is added to the medium solution

Procedure:

[0092] Sample weighing:

[0093] TMR (44% concentrate, 6% hay, 37% maize silage and 13% grass silage) is sieved to 1 mm and weighed exactly into the syringes. One run contains 4 repetitions, each with 16 syringes and comprises substrate controls, which display the gas production without the effect of the tested compounds, carrier controls, which display the gas production in the presence of the carrier (solvent) only (i.e. EtOH used to dissolve the test compounds), and test samples (in the carrier), which contain the test substances in varying concentrations as outlined in table 2.

[0094] Preparation of the rumen-fluid-buffer-mix (medium solution):

[0095] The components are mixed in a Woulff bottle in following order: [0096] 711 ml water [0097] 0.18 ml trace element solution [0098] 355.5 ml buffer solution [0099] 355.5 ml mass element solution

[0100] The completed solution is warmed up to 39° C. followed by the addition of 1.83 ml sodium salt solution and the addition of reduction solution at 36° C.

[0101] The rumen liquor (750 ml) is added, when the indicator turns colorless under continued agitation and 002-gassing.

[0102] Filling the syringes, incubation and determining gas volumes:

[0103] The rumen-fluid-buffer-mix is added to the glass syringe prepared as outlined above containing the TMR and the active to be tested. The syringes are then incubated for 8 hours at 39° C. under gentle agitation. After 8 the volume of gas produced is measured, and the percentage of methane in the gas phase is determined by gas chromatography.

Results

[0104] Table 2 outlines the methane inhibiting effect of various dicarboxylic acid diesters according to the present invention at different concentrations.

TABLE-US-00002 TABLE 2 Methane inhibition* # Structure 10 μmol/l 5 μmol/l 2.5 μmol/l (I-a) [00007]   bis(3-(nitrooxy)propyl) malonate −99 −97 −72 (I-b) [00008]   bis(3-(nitrooxy)propyl) fumarate −99% −96 n.a. (I-c) [00009]   bis(3-(nitrooxy)propyl) succinate −99 n.a. n.a. Ref [00010]   3-nitrooxypropanol −99% −86 −32 *reduction versus substrate control

[0105] As can be retrieved from table 2, the dicarboxylic diesters according to the present invention are highly efficient methanogenese inhibitors, while exhibiting very low vapor pressures making them particularly storage stable.