Method for producing α-hydroxy esters by esterification of α-hydroxy acids

Abstract

The present disclosure provides methods for producing alpha-hydroxy esters from the corresponding alpha-hydroxy acids by transesterification. Also provided are alpha-hydroxy esters produced according to the methods disclosed herein, and compositions containing the alpha-hydroxy esters.

Description

[Technical Field]

[0001] Related Applications This application claims priority to International Application No. PCT / CN2019 / 104692, filed September 6, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure provides methods for producing alpha-hydroxy esters from the corresponding alpha-hydroxy acids by esterification, such as transesterification. Also provided are alpha-hydroxy esters produced according to the methods disclosed herein, compositions comprising the alpha-hydroxy esters, and methods of using the compositions. [Background technology]

[0003] Alpha-hydroxyester analogs of natural amino acids are useful as dietary supplements and in the study of enzymatic processes and protein function. The synthesis of such esters typically involves acid-catalyzed Fischer esterification of the corresponding acid with an alcohol in the presence of a strong acid, such as H2SO4 or Amberlyst® cation exchange resin, acid-mediated hydrolysis of the corresponding nitrile in the presence of a strong acid, or enzyme-mediated methods. However, acid-catalyzed procedures result in decomposition of the starting materials and products, as well as contamination of the products with dimeric and oligomeric components. Such methods generally result in low yields, and complex purification techniques are required to isolate the target compound from polymeric by-products. Enzymatic methods require expensive and unstable reactants and special reaction conditions.

[0004] A particularly important α-hydroxy ester is isopropyl 2-hydroxy-4-(methylthio)butanoate (HMBi). HMBi is the isopropyl ester of 2-hydroxy-4-(methylthio)butanoic acid (HMBA), a hydroxy analog of methionine. HMBi is used to aid in methionine supplementation in ruminants, including cows. Ensuring sufficient levels of methionine in dairy cows helps maintain desirable levels of milk protein synthesis and, therefore, milk production. However, the methionine content in animal feeds is largely inadequate, making it a major limiting factor in dairy cow diets. HMBi is a chemical derivative of methionine that readily and rapidly diffuses through the rumen wall, avoiding degradation by ruminal microorganisms. Once HMBi penetrates the rumen wall, it is metabolized by the liver and becomes available for milk protein synthesis in dairy cows.

[0005] There is a need for additional methods for synthesizing α-hydroxy esters, such as HMBi, that use inexpensive reactants and mild reaction conditions and provide product esters in high yield and purity. Summary of the Invention

[0006] In one aspect, the present disclosure provides a compound of formula (I): [ka] (In the formula, R 1 is H; optionally -OH, -SH, -SC 1~4 C substituted with alkyl, -CONH2, or guanidino 1~4 Alkyl; optionally -OH or C 1~4 selected from alkyl-substituted phenyl; indolyl; and imidazolyl; However, R a and R b are each independently H or C 1~4 is alkyl, R 2 is C 1~8 Alkyl or C 4~7cycloalkyl) A method for producing a compound of the formula Formula (II): [ka] a compound of formula (A) or formula (B) or formula (C): [ka] (In the formula, R x is H, C 1~4 alkyl, and CH=CH-; R y is C 1~3 alkyl) The present invention is directed to the above method, which comprises reacting the compound of formula (I) with a reactant of formula (II).

[0007] In another aspect, the present disclosure provides a compound of formula (I): [ka] (In the formula, R 1 is H; optionally -OH, -SH, -SC 1~4 Alkyl, -CONH2, -NR a R b , or guanidino-substituted C 1~4 Alkyl; optionally -OH or C 1~4 selected from alkyl-substituted phenyl; indolyl; and imidazolyl; However, R a and R b are each independently H or C 1~4 is alkyl, R 2 is C 1~8 Alkyl or C 4~7 cycloalkyl) A method for producing a compound of the formula Formula (III): [ka] (In the formula, R3 is H or -C(O)R x and However, R x is C 1~4 alkyl) The compound R 2 -OH.

[0008] In another aspect, the present disclosure provides: Formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] with acetyl chloride to form a compound of formula (III-A): [ka] and (b) Reacting the compound of formula (III-A) with isopropanol to obtain a compound of formula (IV-A): [ka] and providing a compound of (c) treating the compound of formula (IV-A) with an aqueous base to give the compound of formula (IA); The present invention relates to the above method, which comprises:

[0009] In another aspect, the present disclosure provides: Formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] and n-heptane; (b) dehydrating the compound of formula (II-A) by azeotropic distillation; (c) adding n-heptane as a non-polar solvent to the dehydrated compound of formula (II-A); (d) Treating the above dehydrated compound of formula (II-A) in heptane with acetyl chloride to give compound of formula (III-A): [ka] and providing a compound of (e) reacting the compound of formula (III-A1) with isopropanol in a two-phase reaction mixture containing a hydrophilic phase and a hydrophobic phase to produce a compound of formula (IV-A): [ka] and providing a compound of (f) treating the compound of formula (IV-A) with an aqueous base to provide the compound of formula (IA) in the hydrophobic phase of the reaction mixture; (g) separating the hydrophobic phase from the hydrophilic phase base to provide the compound of formula (IA). The present invention relates to the above method, which comprises:

[0010] In another aspect, the present disclosure relates to a method for isolating the compound of formula (IA) (HMBi) by partitioning a mixture of HMBi and at least one impurity selected from HMBA, HMBA dimer, HMBA oligomer, HMBi dimer, and HMBi oligomer between an n-heptane phase and a basic aqueous phase. In some embodiments, the mixture comprises HMBi and HMBA.

[0011] In another aspect, the disclosure relates to a method for isolating HMBi by partitioning a mixture of HMBi and at least one impurity selected from HMBA, HMBA dimer, HMBA oligomer, HMBi dimer, and HMBi oligomer between a hydrophobic phase and a hydrophilic phase in a two-phase reaction.

[0012] In another aspect, the disclosure is directed to a compound of Formula (I) or Formula (IA) prepared according to any of the methods described herein.

[0013] In another aspect, the present disclosure is directed to a compound of Formula (I) or Formula (IA) having a purity by weight (and / or by GC or HPLC) of at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, which is a crude compound, unpurified, and / or purified only by fractional distillation.

[0014] In another aspect, the present disclosure is directed to a composition comprising at least 95% by weight and / or by GC or HPLC analysis of a compound of formula (IA) above, and about 1 to about 4999 ppm of n-heptane as an impurity.

[0015] In another aspect, the present disclosure is directed to an animal feed composition comprising a compound of formula (I) or (IA) described herein.

[0016] In another aspect, the present disclosure is directed to an animal feed composition comprising a compound of Formula (I) or Formula (IA) described herein. In some embodiments, the animal feed is a cattle feed, such as a dairy cattle feed.

[0017] In another aspect, the present disclosure is directed to a method of providing bioavailable methionine to a dairy cow, the method comprising administering to the cow a compound of Formula (I) or Formula (IA) or an animal feed composition described herein. In another aspect, the present disclosure is directed to a method of providing at least about 50% bioavailable methionine to a dairy cow, the method comprising administering to the cow a compound of Formula (I) or Formula (IA) or an animal feed composition described herein. In another aspect, the present disclosure is directed to a method of improving milk obtained from a dairy cow, the method comprising administering to the cow a compound of Formula (I) or Formula (IA) or an animal feed composition described herein. In another aspect, the present disclosure is directed to a method of improving the condition of a cow, the method comprising administering to the cow a compound of Formula (I) or Formula (IA) or an animal feed composition described herein. [Brief explanation of the drawings]

[0018] [Figure 1A] FIG. 1A is an HPLC chromatogram showing the results of the reaction of HMBA with isopropyl acetate in the presence of 5% aqueous HCl, as described in Example 3. [Figure 1B] FIG. 1B is a HPLC chromatogram showing the results of HPLC analysis of the reaction mixture during the reaction of HMBA with isopropyl acetate in the presence of 5% aqueous HCl, as described in Example 3. [Figure 2A] FIG. 2A is an HPLC chromatogram showing the results of the uncatalyzed transesterification reaction of 1.0 equivalent of HMBA (1001.5 g) with 2.54 equivalents of iPrOAc (2000 mL) at 90-95° C. for 18 hours without added catalyst, as described in Example 3. [Figure 2B] FIG. 2B is an HPLC chromatogram showing the results of HPLC analysis of the reaction mixture during the reaction of 1.0 equivalent of HMBA (1001.5 g) with 2.54 equivalents of iPrOAc (2000 mL) without added catalyst at 90-95° C. for 2-18 hours, as described in Example 3. [Figure 3A]FIG. 3A is an HPLC chromatogram showing the results of the reaction of 1.0 equivalent of HMBA (1010 g) with 2.54 equivalents of iPrOAc (2000 mL) in the presence of 5% aqueous HSO at 90-95 °C for 18 hours, as described in Example 3. [Figure 3B] FIG. 3B is an HPLC chromatogram showing the results of HPLC analysis of the reaction mixture during the reaction of 1.0 equivalent of HMBA (1010 g) with 2.54 equivalents of iPrOAc (2000 mL) in the presence of 5% aqueous HSO at 90-95°C for 18 hours, as described in Example 3. [Figure 4] FIG. 4 is an exemplary process flow chart for the synthesis of HMBi according to Example 4 disclosed herein. [Figure 5A] FIG. 5A is an HPLC chromatogram showing the results of HPLC analysis of the products of the HMBi synthesis disclosed in Example 13. [Figure 5B] FIG. 5B is an HPLC chromatogram showing the results of HPLC analysis of the products of HMBi synthesis using the two-phase reaction disclosed in Example 13. [Figure 6] FIG. 6 is an exemplary process flow chart for the synthesis of HMBi using the two-phase reaction disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise specified, terms in this disclosure have their plain and ordinary meanings as understood by those skilled in the art. The following terms used in the specification and claims are defined for purposes of this disclosure and have the following meanings:

[0020] As used herein, the terms "isopropyl 2-hydroxy-4-(methylthio)butanoate," "HMBi," and "isopropyl ester of 2-hydroxy-4-(methylthio)butanoic acid" refer to the compound having the following structure (Formula IA): [ka] This refers to a compound of the formula:

[0021] As used herein, the terms "2-hydroxy-4-(methylthio)butanoate," "2-hydroxy-4-(methylthio)butanoic acid," and "HMBA" refer to the compound having the following structure (Formula II-A): [ka] This refers to a compound of the formula:

[0022] The compounds described herein may exist in racemic form, as a single enantiomer, or as a mixture of enantiomers. Thus, for example, HMBi refers to racemic HMBi (i.e., "DL-HMBi"), or D-HMBi or L-HMBi, or mixtures thereof.

[0023] The compounds described herein may also exist in the form of salt.It should be understood that the chemical formulas shown herein include the structures shown as well as their salt forms.For example, if a compound contains carboxylic acid, the formula also includes the salt form (carboxylate) of its conjugate base, such as sodium salt, potassium salt, magnesium salt or calcium salt.If a compound contains indole group or imidazole group, the formula also includes the salt of its conjugate acid, for example, HCl salt.

[0024] "Alkyl" means a linear, saturated, monovalent hydrocarbon radical of 1 to 8 carbon atoms (e.g., 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms) or a branched, saturated, monovalent hydrocarbon radical of 3 to 8 carbon atoms (e.g., 3 to 6 carbon atoms, 3 to 4 carbon atoms, or 3 carbon atoms), such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl (including all isomeric forms), and the like.

[0025] "Cycloalkyl" means a cyclic, saturated, monovalent hydrocarbon radical of three to ten carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0026] "Optional" or "optionally" means that the subsequently described event or circumstance may occur, but need not necessarily occur, and the statement encompasses both cases where the event or circumstance occurs and cases where it does not occur. For example, an alkyl group "optionally substituted with -OH" means that -OH may be present, but need not necessarily be present, and the statement encompasses both cases where the alkyl group is substituted with an -OH group and cases where the alkyl group is not substituted with an -OH group.

[0027] The term "reaction solvent" refers to an organic liquid used to carry dissolved reactants. In some embodiments, one of the reactants in the reaction functions as both a reactant and a reaction solvent. In other embodiments, the reactants are diluted with a different reaction solvent.

[0028] The term "acid catalyst" refers to an acid that is added to a reaction in a substoichiometric amount and functions to catalyze the reaction. The acid catalyst may be a Bronsted acid (such as an acid with a pKa less than 7, e.g., HCl, H2SO4, KHSO4, acetic acid, etc.) or a Lewis acid (such as a boronic acid). In some embodiments, the acid is generated in situ, for example, by reaction of acetyl chloride or TMSCl with water or an alcohol.

[0029] The term "strong acid" refers to an acid that completely dissociates into its component ions. Strong acids include, but are not limited to, HCl, HNO, HSO, HBr, HI, HClO, and HClO.

[0030] The term "concentration" refers to the amount of solute in a solvent. As used herein, concentration may be expressed in terms of weight percent or in terms of molarity (M) or normality (N).

[0031] The term "reflux temperature" or "reflux" refers to the temperature at which the reaction solvent boils; a condenser is usually used to cool and condense the solvent vapors back into the reactor. The exact temperature at which a given solvent reaches reflux can vary depending on environmental factors.

[0032] The terms "heptane" or "n-heptane" refer to pure n-heptane or n-heptane in a mixture with other C7 isomers (e.g., at least 90% n-heptane and at least 95% total C7 isomers).

[0033] The term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values ​​(e.g., ±5-10% of the recited numerical value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having a similar function or result). When terms such as "at least" and "about" precede a recitation of numerical values ​​or ranges, the term modifies all values ​​or ranges set forth in the recitation. In some cases, the term "about" may encompass rounded numerical values.

[0034] The terms "extract," "extraction," or "extracting" refer to the process of partitioning a substance between an organic phase and an aqueous phase. In some embodiments, the extraction is performed on the reaction mixture or a concentrated residue of the reaction mixture. An "extract" is the organic phase once separated from the aqueous phase. As used herein, extraction techniques can be used to isolate the final product. As used herein, extracting does not include purification methods performed on the crude reaction product, such as simple distillation, vacuum distillation, azeotropic distillation, fractional distillation, sequential distillation, flash chromatography, HPLC, or recrystallization.

[0035] As used herein, "purification" or "purifying" refers to a method of isolating the product of a reaction after the reaction is complete. Purification methods include simple distillation, vacuum distillation, azeotropic distillation, fractional distillation, continuous distillation, flash chromatography, HPLC, or recrystallization.

[0036] The term "substantially", e.g., "substantially in monomeric form", refers to the purity of the compound of formula (I) or formula (IA), or the purity of HMBA, with respect to dimeric and / or oligomeric analogs.

[0037] As used herein, the term "dimer" or "dimeric compound" refers to a compound in which two molecules of a given monomer structure, or one molecule each of two different monomer structures, are condensed into a single molecule. In the case of HMBA, an HMBA dimer may, for example, be in the following form: [ka] In the case of HMBA and HMBi, HMBA / HMBi dimers (e.g., heterodimers) may exist, for example, in the following form: [ka] As used herein, the term "oligomer" or "oligomeric compound" refers to a compound in which three or more molecules of a given monomer structure, or three or more molecules of at least two different monomer structures, are condensed into a single polymeric structure. HMBA and HMBi may form homo-oligomers (e.g., HMBA trimers or tetramers) or hetero-HMBA / HMBi oligomers (containing at least one HMBA monomer unit and at least one HMBi monomer unit). In commercially available samples of "88% HMBA," the above-described "HMBA dimer" and "HMBA oligomer" structures are typically present along with water.

[0038] The term "purity" or percentage of a compound (e.g., x% HMBA) refers to the purity of a compound in a sample as measured by weight, by GC analysis, and / or by HPLC analysis. In some embodiments, the purity by weight is measured by GC or HPLC analysis with UV detection.

[0039] The term "purity by weight" refers to the purity of a compound in a sample with respect to other components in the sample, expressed as a ratio of the mass of the compound to the mass of the sample, expressed as a percentage.

[0040] The term "purity," with respect to gas chromatography (GC) purity or HPLC purity, refers to the calculated purity (expressed as a percentage) of the peak area of ​​the compound of interest relative to the sum of all peak areas in a chromatogram. In some embodiments, purity is measured by HPLC with UV detection.

[0041] In some embodiments, the purity is that required to comply with regulations for the sale of regulated products. For example, in the case of HMBi, the compound contains 0.5% or less water (e.g., as measured by Karl Fischer analysis). (See Commission Implementing Regulation (EU) No. 469 / 2013 of May 22, 2013.)

[0042] The terms "crude," "crude product," and "crude compound" refer to a sample of compound obtained from a reaction mixture after concentration of the reaction mixture and / or after extraction of the reaction mixture into an organic solvent and concentration of the organic extract.

[0043] The term "not produced during the reaction" refers to a reaction that does not produce a given compound as a product. In some embodiments, "not produced during the reaction" means that the substance is not produced in a stoichiometric, catalytic, or detectable amount. In some embodiments, "not produced during the reaction" means that the substance may be present at the beginning of the reaction (e.g., in a mixture with the starting materials), but the amount of the substance does not increase substantially during the reaction. In some embodiments, certain reactions described herein do not produce isopropyl alcohol and / or water. Detection of isopropyl alcohol can be performed by GC or other methods known in the art, and detection of water can be performed by Karl Fischer analysis or other methods known in the art.

[0044] As used herein, the term "reducing the amount of water" refers to partial or complete removal of water from the reaction mixture, where complete water removal indicates that no water is detectable using standard detection methods such as Karl Fischer analysis.

[0045] The term "animal feed composition" refers to a product suitable for use in animal nutrition. In some embodiments, the animal feed composition is an animal feed (e.g., food or drinking water containing a nutritional supplement), and in some embodiments, the animal feed composition is a feed additive. The feed additive is suitable for mixing with animal feed or drinking water.

[0046] The term "carrier" refers to a carrier suitable for animal feed additives. Suitable carriers include water (for liquid or solid feed additives) or silica (for solid feed additives). In some embodiments, the carrier is silica (silicon dioxide). In some embodiments, the feed additive comprises the compound and silica in a 3:2 ratio.

[0047] In some embodiments, the animal feed comprises a granulated, protein-rich feed (e.g., peanut-based, rapeseed meal-based, and / or soybean meal-based) supplemented with 2.5% or 1% by weight of HMBi. In some embodiments, the animal feed comprises about 45% and about 50% grain (corn, barley, wheat, and / or wheat by-products) supplemented with 0.5% or 3.0% by weight of HMBi. In some embodiments, the animal feed comprises a meal or granulated feed containing molasses, supplemented with 2.5% or 1% by weight of HMBi, respectively.

[0048] The term "administering" refers to providing a nutritional supplement to a subject animal. Administering may be performed orally, for example, through ingestion of food or drinking water containing the compound, or by injection or other modes of administration.

[0049] As used herein, "improving milk" refers to improving the quality and / or quantity of milk produced by a treated cow or group of treated cows compared to milk produced by untreated control animals. Milk improvements include, for example, increasing the protein content of the milk (e.g., increasing alpha, beta, and / or kappa protein), increasing the fat content of the milk, and / or increasing the volume of milk produced.

[0050] As used herein, "improving cattle condition" refers to an improvement in the health index of a treated cattle or a group of treated cattle compared to the health index of untreated control animals. Improvement in cattle condition may refer, for example, to an increase in some characteristic, such as weight, compared to untreated animals.

[0051] As used herein, improving fertility includes, for example, shortening the interval between birth and reproduction and / or increasing fertility during insemination.

[0052] As used herein, improved liver function includes, for example, a reduction in metabolic problems, an improvement in very low density lipoprotein levels, a reduction in hyperketonemia, and / or a reduction in the incidence of fatty liver.

[0053] As used herein, "increased energy" refers to, for example, stimulation of fermentation processes in the rumen, leading to an increase in digestible organic matter and thus more energy for the animal.

[0054] Transesterification In some embodiments, the present disclosure provides a compound of formula (I): [ka] (In the formula, R 1 is H; optionally -OH, -SH, -SC 1~4 C substituted with alkyl, -CONH2, or guanidino 1~4 Alkyl; optionally -OH or C 1~4selected from alkyl-substituted phenyl; indolyl; and imidazolyl; However, R a and R b are each independently H or C 1~4 is alkyl, R 2 is C 1~8 Alkyl or C 4~7 cycloalkyl) A method for producing a compound of the formula Formula (II): [ka] a compound of formula (A) or formula (B) or formula (C): [ka] (In the formula, R x is H, C 1~4 alkyl, and CH=CH-; R y is C 1~3 alkyl) with a reactant of formula (I).

[0055] In some embodiments, a reactant of Formula (A) (e.g., isopropyl acetate, isopropyl formate, isopropyl acrylate), or a reactant of Formula (B) (e.g., isopropyl methanesulfonate), or a reactant of Formula (C) (e.g., triisopropyl borate) functions as a reaction solvent. In some embodiments, a reactant of Formula (A), (B), or (C) is used in an amount of about 1 molar equivalent ("equivalent" or "eq." or "equiv.") to about 20 equivalents, or about 1 equivalent to about 10 equivalents, or about 1 equivalent to about 5 equivalents, or about 1 equivalent to about 3 equivalents relative to the HMBA starting material. In some embodiments, the reactant of Formula (A) is isopropyl acetate, and the isopropyl acetate functions as a reaction solvent (i.e., no solvent is added, neat isopropyl acetate). In some embodiments, the reactant of Formula (B) is isopropyl methanesulfonate, and the isopropyl methanesulfonate functions as a reaction solvent. In some embodiments, the reactant of formula (C) is triisopropyl borate, which functions as the reaction solvent. In some embodiments, the reaction is carried out in at least one separate organic solvent that is not a reactant of formula (A), (B), or (C). In some embodiments, the at least one separate organic solvent is R 2 -OH (e.g., methanol, ethanol, isopropyl alcohol, etc.), diisopropyl ether, THF, dichloromethane, methyl-THF, toluene, and dioxolane. In some embodiments, the reaction solvent is isopropyl alcohol. In some embodiments, the reaction solvent is isopropyl alcohol, the reactant is a reactant of formula (A) (e.g., isopropyl acetate), and about 1 equivalent to about 10 equivalents, or about 1 equivalent to about 5 equivalents, or about 1 equivalent to about 3 equivalents of the reactant relative to the HMBA starting material.

[0056] In some embodiments, the reactant is a reactant of formula (A), such as isopropyl acetate, and the reactant is a reactant of formula R, such as acetyl chloride. x C(O)Cl is added to a compound of formula R, such as isopropyl alcohol.2 The resulting mixture of reactants of formula (A), such as isopropyl acetate, is mixed with a compound of formula (II) or formula (II-A) and reacted.

[0057] In some embodiments, R 1 is H. In some embodiments, R 1 is optionally -OH, -SH, -SC 1~4 Alkyl, -CONH2, -NR a R b , or guanidino-substituted C 1~4 In some embodiments, R 1 is optionally -OH, -SH, or -SC 1~4 Alkyl-substituted C 1~4 In some embodiments, R 1 is methyl, ethyl, isopropyl, isobutyl, sec-butyl, -CH2-OH, and -CH2CH2-SC 1~4 In some embodiments, R 1 is -CH2CH2-S-CH3. In some embodiments, R 1 is optionally -OH or C 1~4 It is selected from alkyl-substituted phenyl; indolyl; and imidazolyl.

[0058] In some embodiments, R 2 is selected from methyl, ethyl, and isopropyl. 2 is isopropyl.

[0059] In some embodiments, the reactant is a reactant of formula (A). x is selected from H, methyl, and CH=CH-. In some embodiments, R x is methyl. In some embodiments, the reactant is of formula (B). In some embodiments, R yis methyl. In some embodiments, the reactant is a reactant of formula (C). In some embodiments, the reactant is a reactant of formula (C), and R 2 is isopropyl.

[0060] In some embodiments, R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl. In some embodiments, R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl, the reactant is a reactant of formula (A), R x is methyl. In some embodiments, R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl. In some embodiments, R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl, the reactant is a reactant of formula (B), R y is methyl.

[0061] In some embodiments, the present disclosure provides: Formula (IA): [ka] A method for producing a compound of the formula Formula (II-A): [ka] with isopropyl acetate or isopropyl methanesulfonate. In some embodiments, the reacting is with isopropyl acetate. In some embodiments, the reacting is with isopropyl acetate in isopropyl alcohol as a reaction solvent.

[0062] In some embodiments, the methods for preparing compounds of Formula (I) and (IA) disclosed herein further include adding at least one nonpolar solvent to a reaction mixture, such as the reaction between a compound of Formula (II) and a compound of Formula (A), (B), or (C); or the reaction between a compound of Formula (II-A) and isopropyl acetate or isopropyl methanesulfonate. In some embodiments, the reaction is a two-phase reaction comprising a hydrophobic phase and a hydrophilic phase of the reaction mixture. In some embodiments, the nonpolar solvent is selected from petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene. In some embodiments, the nonpolar solvent is selected from hexane, hexanes, n-heptane, octane, nonane, decane, benzene, toluene, and methyl tert-butyl ether. In some embodiments, the nonpolar solvent is n-heptane.

[0063] In some embodiments, the reacting is carried out in the absence of an acid catalyst. In some embodiments, the reacting is carried out in the presence of at least one acid catalyst. In some embodiments, the at least one acid catalyst is selected from H2SO4, HCl, and p-toluenesulfonic acid (p-TsOH). In some embodiments, the at least one acid catalyst is HCl. In some embodiments, the acid catalyst is HCl generated in situ by the reaction of acetyl chloride or TMSCl with water or an alcohol (e.g., Formula (II) or (II-A)). In some embodiments, the reacting is carried out at a pH of about 1 or greater. In some embodiments, the reacting is carried out at a pH of about 3 or greater.

[0064] In some embodiments, the compound of Formula (II) or Formula (II-A) (starting material) is present in a sample containing water prior to the reacting, and the method further comprises converting the sample to a compound of Formula (D): C 1~3 Alkyl-C(O)Cl (D) to form a catalyst mixture containing HCl, and mixing the reactants with the catalyst mixture. In some embodiments, at least a molar equivalent, or a molar excess, of the acid chloride of Formula (D) is used relative to the amount of water in the sample (e.g., as determined by Karl Fischer analysis). In some embodiments, the HCl is generated in situ by the above.

[0065] In some embodiments, the compound of Formula (II) or Formula (II-A) (the starting material) is present in a sample containing water prior to reacting, and the method includes treating the sample with at least one dehydrating agent prior to reacting. In some embodiments, the at least one dehydrating agent is selected from MgSO4, Na2SO4, PO5, diatomaceous earth, CaCl2, molecular sieves, or an azeotropic solvent (e.g., a solvent such as n-propanol or benzene). In some embodiments, the at least one dehydrating agent is selected from MgSO4 and Na2SO4. In some embodiments, the dehydrating agent is added neat to the sample. In other embodiments, the sample is diluted with a polar or non-polar solvent such as diethyl ether, ethyl acetate, or dichloromethane and dehydrated over the dehydrating agent. In some embodiments, the dehydrating agent is removed from the sample by filtration. In other embodiments, the dehydrating agent is an azeotropic solvent and is removed by distillation (e.g., azeotropic removal of water). In some embodiments, the azeotrope solvent is selected from hexane, n-heptane, n-propanol, isopropyl acetate, ethyl acetate, toluene, and benzene.

[0066] In some embodiments, the reacting provides a reaction mixture containing a compound of Formula (I) or Formula (IA), and the method further includes optionally reducing the reactant of Formula (A) (e.g., isopropyl acetate) or the reactant of Formula (B) (e.g., isopropyl methanesulfonate) or the reactant of Formula (C) (e.g., triisopropyl borate) by distillation to provide a crude residue. In some embodiments, the method further includes optionally adding a base to the reaction mixture or the crude residue, such as sodium acetate, aqueous NaOH, e.g., 0.1-10N aqueous NaOH, or 5N aqueous NaOH, aqueous NaHCO3, aqueous K2CO3, or aqueous Na3PO4, preferably 0.1-10N aqueous NaOH, or 5N aqueous NaOH, to raise the pH to about 5 to about 10, or about 5 to about 9, or about 5 to about 8, to provide a basic mixture; and extracting the compound of Formula (I) or Formula (IA) from the basic mixture into at least one nonpolar solvent to provide an extract. In some embodiments, the at least one nonpolar solvent is selected from hexane, hexanes, n-heptane, octane, nonane, and decane. In some embodiments, the at least one nonpolar solvent is n-heptane. In some embodiments, the volume of n-heptane used in the extraction is about 1-10 mL, or about 1-5 mL, or about 1-3 mL, or about 2 mL per kilogram of calculated HMBi yield or crude residue mass (e.g., the calculated or estimated amount of HMBi in the mixture). In some embodiments, the n-heptane used in the extraction is at a temperature of about 25°C to 50°C, or about 30°C to about 50°C, or about 30°C to about 40°C prior to the extraction. In some embodiments, the extract comprises a compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight. In some embodiments, the method includes removing the at least one non-polar solvent from the extract, optionally by distillation, to provide a compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight.

[0067] In some embodiments, the reacting provides a reaction mixture comprising a compound of Formula (I) or Formula (IA), and the method further includes adding a base, optionally solid sodium acetate, to the reaction mixture to raise the pH to a range of about 5 to about 10, or about 5 to about 9, or about 5 to about 8 to provide a basic mixture; dehydrating the basic mixture using a dehydrating agent to provide a dehydrated basic mixture; and purifying the compound of Formula (I) or Formula (IA) from the dehydrated basic mixture by distillation to provide the compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight.

[0068] In some embodiments, R 2 No -OH, e.g., isopropyl alcohol, is produced during the reacting. In some embodiments, no water is produced during the reacting.

[0069] In some embodiments, the reacting is carried out at a temperature of at least about 20°C, or at least about 30°C, or at least about 40°C, or at least about 50°C, or at least about 60°C, or at least about 70°C, or at least about 80°C, or at least about 90°C, or from about 20°C to about 150°C, or from about 20°C to about 100°C, or from about 20°C to about 90°C, or from about 60°C to about 150°C, or from about 60°C to about 100°C, or from about 60°C to about 95°C, or from about 75°C to about 90°C, or from about 80°C to about 150°C, or from about 80°C to about 100°C, or from about 80°C to about 90°C, or at a temperature of about 89°C, or the reflux temperature of the reactants of Formula (A) or Formula (B). In some embodiments, the reactant is of Formula (A) and the reacting is carried out at a temperature of at least about 60°C, or at least 75°C, or at least about 80°C, or at least about 90°C, or at least about 100°C, or at a temperature in the range of about 60°C to about 150°C, or about 60°C to about 100°C, or about 60°C to about 95°C, or about 75°C to about 90°C, or about 80°C to about 150°C, or about 80°C to about 100°C, or about 80°C to about 90°C, or the reflux temperature of the reactant of Formula (A); or, the reactant is of Formula (B) and the reacting is carried out at a temperature of about 20°C to about 90°C, or about 20°C to about 60°C, or about 20°C to about 30°C.

[0070] In some embodiments, the reacting is carried out for a time ranging from about 1 hour to about 24 hours, or from about 2 hours to about 15 hours, or from about 3 hours to about 14 hours, or from about 4 hours to about 12 hours, or from about 4 hours to about 10 hours, or from about 10 hours to about 20 hours, or from about 14 hours to about 16 hours.

[0071] In some embodiments, following the reacting, the compound of Formula (I) or Formula (IA) is extracted using at least one suitable solvent, such as petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene. In some embodiments, following the reacting, the compound of Formula (I) or Formula (IA) is extracted using at least one suitable solvent, such as hexane, hexanes, n-heptane, octane, nonane, decane, benzene, toluene, or methyl tert-butyl ether. In some embodiments, prior to reacting the compound of Formula (I) or Formula (IA), at least one suitable solvent, such as n-heptane or toluene, is added to the reaction mixture. In some embodiments, the solvent is a nonpolar solvent. In some embodiments, the solvent is a nonpolar solvent. In some embodiments, the solvent is hexane, hexanes, n-heptane, octane, nonane, or decane, or a mixture of isomers thereof. In some embodiments, the solvent is n-heptane. In some embodiments, following the reaction, the compound of Formula (I) or Formula (IA) is extracted into n-heptane. In some embodiments, the n-heptane extraction is performed immediately after the reaction in the reactor. In some embodiments, the reaction is a two-phase reaction, and the n-heptane extraction is performed after the compound of Formula (I) is formed during the reaction in the reactor. In some embodiments, the reaction mixture is adjusted to a pH of about 5 to about 10, or about 5 to about 9, or about 5 to about 8, and the resulting mixture is extracted with n-heptane. In some embodiments, the reaction is a two-phase reaction, and the reaction mixture is adjusted to a pH of about 5 to about 10, or about 5 to about 9, or about 5 to about 8, thereby increasing the amount of the compound of Formula (I) that partitions into n-heptane.In some embodiments, the volume of n-heptane is about 1-10 mL, or about 1-5 mL, or about 1-3 mL, or about 2 mL per kilogram of calculated HMBi yield or crude residue mass (e.g., the calculated or estimated amount of HMBi present in the mixture). In some embodiments, the extracting is carried out with n-heptane at a temperature of about 25°C to 50°C, or about 30°C to about 50°C, or about 30°C to about 40°C prior to the extracting. In some embodiments, n-heptane selectively extracts HMBi over HMBA or HMBA and / or HMBi dimer or oligomer species.

[0072] In some embodiments, the method further comprises combining isopropyl alcohol with acetyl chloride to form a solution of isopropyl acetate in isopropanol, and reacting the compound of Formula (II) or Formula (II-A) with the reactant of Formula (A) that is isopropyl acetate comprises adding the compound of Formula (II) or Formula (II-A) to the solution of isopropyl acetate in isopropanol.

[0073] Esterification by stoichiometric acetylation In one aspect, the present disclosure provides a compound of formula (I): [ka] (In the formula, R 1 is H; optionally -OH, -SH, -SC 1~4 Alkyl, -CONH2, -NR a R b , or guanidino-substituted C 1~4 Alkyl; optionally -OH or C 1~4 selected from alkyl-substituted phenyl; indolyl; and imidazolyl; However, R a and R b are each independently H or C 1~4 is alkyl, R 2 is C 1~8 Alkyl or C4~7 cycloalkyl) A method for producing a compound of the formula Formula (III-A) or Formula (III-B): [ka] (In the formula, R 3 HA-C(O)R x and However, R x is C 1~4 alkyl) The compound R 2 -OH.

[0074] In some embodiments, R 2 -OH is a solvent for reacting the compound of formula (III-A) or (III-B).

[0075] In some embodiments, R 1 is H. In some embodiments, R 1 is optionally -OH, -SH, -SC 1~4 Alkyl, -CONH2, -NR a R b , or guanidino-substituted C 1~4 In some embodiments, R 1 is optionally -OH, -SH, or -SC 1~4 Alkyl-substituted C 1~4 In some embodiments, R 1 is methyl, ethyl, isopropyl, isobutyl, sec-butyl, -CH2-OH, and -CH2CH2-SC 1~4 In some embodiments, R 1 is -CH2CH2-S-CH3. In some embodiments, R 1 is optionally -OH or C 1~4 It is selected from alkyl-substituted phenyl; indolyl; and imidazolyl.

[0076] In some embodiments, R 2 is methyl, ethyl, or isopropyl. 2 is isopropyl.

[0077] In some embodiments, the method comprises reacting a compound of formula (III-A) with R 2 In some embodiments, the method comprises reacting the compound of formula (III-B) with R 2 This involves reacting with -OH.

[0078] In some embodiments, the R 2 The reacting with -OH may be carried out at a temperature of at least about 20°C, or at least about 30°C, or at least about 40°C, or at least about 50°C, or at least about 60°C, or at least about 70°C, or at least about 80°C, or at a temperature in the range of about 20°C to about 90°C, or about 60°C to about 95°C, or about 75°C to about 90°C, about 82°C, or R 2 In some embodiments, the reacting is carried out for a time period ranging from about 1 hour to about 24 hours, or from about 2 hours to about 15 hours, or from about 3 hours to about 14 hours, or from about 4 hours to about 12 hours.

[0079] In some embodiments, the present disclosure provides a compound of Formula (III-A) or a compound of Formula (III-B) comprising R 2 -OH (where R 1 and R 2 is as defined herein), formula (IV): [ka] (In the formula, R 1 and R 2 In some embodiments, the present method for preparing a compound of Formula (I) relates to providing a compound of Formula (III-A) or Formula (III-B) by reacting the compound of Formula (III-A) with R 2-OH to provide a compound of formula (IV) in a reaction mixture of formula (IV), The reacting further includes treating the compound of formula (IV) with an aqueous base to provide the compound of formula (I).

[0080] In some embodiments, the method comprises: (a) concentrating the reaction mixture of formula (IV) to form a concentrated reaction mixture; or (b) extracting the compound of formula (IV) from the reaction mixture of formula (IV) into an organic solvent to form an extract, and concentrating the extract to form a concentrated extract. further comprising Treating the compound of formula (IV) in the concentrated reaction mixture or the concentrated extract with an aqueous base comprises treating a concentrate of formula (IV) with the aqueous base.

[0081] In some embodiments, the compound of Formula (III-A) or the compound of Formula (III-B) is R 2 The above process for reacting a compound of formula (IV) with a compound of formula (III-A) or (III-B) with R 2 -OH. In some embodiments, the method further comprises adding at least one non-polar solvent to the reaction between the -OH. In some embodiments, the method is a two-phase reaction comprising a hydrophobic phase and a hydrophilic phase of the reaction mixture. In some embodiments, the non-polar solvent is selected from petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene. In some embodiments, the non-polar solvent is selected from hexane, hexanes, n-heptane, octane, nonane, decane, benzene, toluene, and methyl tert-butyl ether. In some embodiments, the non-polar solvent is n-heptane.

[0082] In some embodiments, the compound of formula (I) partitions into the hydrophobic phase of the reaction mixture.

[0083] In some embodiments, the process for preparing a compound of Formula (I) further comprises treating a compound of Formula (IV) with an aqueous base to provide a compound of Formula (I) in the hydrophobic phase of the reaction mixture, followed by separating the hydrophobic and hydrophilic phases of the reaction mixture, and concentrating Formula (I) in the hydrophobic phase to provide a compound of Formula (I).

[0084] In some embodiments, the aqueous base solution is an aqueous NaOH solution, such as 0.1 N NaOH, an aqueous NaHCO solution, an aqueous KCO solution, or an aqueous NaPO solution. In some embodiments, the aqueous base solution is an aqueous NaOH solution, such as 0.1 N NaOH, or an aqueous NaHCO solution.

[0085] In some embodiments, the method further includes treating the compound of Formula (IV) with the aqueous base to provide the compound of Formula (I), followed by extracting the compound of Formula (I) into an organic solvent to form an extract of Formula (I), and concentrating the extract of Formula (I) to provide the compound of Formula (I). In some embodiments, the treating with the aqueous base is carried out at a temperature of at least about 0° C., or at least about 20° C., or at least about 25° C., or at least about 30° C., or at least about 40° C., or from about 0° C. to about 70° C., or from about 20° C. to about 50° C., or from about 20° C. to about 30° C. In some embodiments, the treating with the aqueous base is carried out for a time period ranging from about 1 hour to about 24 hours, or from about 1 hour to about 5 hours, or from about 1 hour to about 3 hours.

[0086] In some embodiments, the present method for preparing a compound of formula (I) comprises preparing a compound of formula (II): [ka] with an acylating agent to provide a compound of formula (III-A) or (III-B). In some embodiments, the acylating agent is acetyl chloride or acetic anhydride.

[0087] In some embodiments, the method further comprises first adding a dehydrating agent to the compound of Formula (II) and subsequently dehydrating the compound of Formula (II) prior to reacting the compound of Formula (II) with the acylating agent. In some embodiments, the at least one dehydrating agent is an azeotrope solvent. In some embodiments, the at least one dehydrating agent is n-heptane.

[0088] In some embodiments, the acylating agent is acetyl chloride, and optionally, acetyl chloride is the reaction solvent.

[0089] In some embodiments, the method comprises reacting a compound of formula (III-A) with R 2 -OH, wherein the acylating agent is present in an amount ranging from about 1.0 to about 1.5 molar equivalents, or about 1.0 to about 1.2 molar equivalents, or about 1.0, 1.05, 1.1, or 1.2 molar equivalents relative to the compound of Formula (II). In some embodiments, the method comprises reacting a compound of Formula (III-B) with R 2 -OH, wherein the acylating agent is present in an amount ranging from about 1.9 to about 2.5 molar equivalents, or from about 1.9 to about 2.1 molar equivalents, or in an amount of about 2.0 molar equivalents relative to the compound of formula (II).

[0090] In some embodiments, treating the compound of Formula (II) with the acylating agent comprises adding the acylating agent to the compound of Formula (II) at a temperature ranging from about 0° C. to about 20° C. to form an acylation mixture, and warming the acylation mixture to a temperature ranging from about 21° C. to about 80° C., or from about 40° C. to about 80° C., or to about 52° C., or to the reflux temperature of the acylating agent. In some embodiments, the acylating agent is acetyl chloride, and warming comprises warming the acylation mixture to the reflux temperature of acetyl chloride or to about 52° C.

[0091] In some embodiments, the method provides a crude compound of Formula (I), unpurified or purified only by fractional distillation, having a purity by weight (and / or by GC or HPLC) of at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%. In some embodiments, the method provides a crude compound of Formula (I), unpurified or purified only by fractional distillation, that is substantially in monomeric form or contains less than about 5% by weight, or less than about 3% by weight, of dimeric and / or oligomeric compounds.

[0092] In some embodiments, a compound of formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] with acetyl chloride to give a compound of formula (III-A): [ka] and providing a compound of (b) reacting a compound of formula (III-A1) with isopropanol to give a compound of formula (IV-A): [ka] and providing a compound of (c) treating the compound of formula (IV-A) with an aqueous base to give a compound of formula (IA); There is a method as described above, which includes:

[0093] In some embodiments, a compound of formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] and n-heptane; (b) dehydrating the compound of formula (II-A) by azeotropic distillation; (c) adding n-heptane as a non-polar solvent to the dehydrated compound of formula (II-A); (d) Treating the above dehydrated compound of formula (II-A) in heptane with acetyl chloride to give compound of formula (III-A): [ka] and providing a compound of (e) reacting a compound of formula (III-A1) with isopropanol in a two-phase reaction mixture containing a hydrophilic phase and a hydrophobic phase to produce a compound of formula (IV-A): [ka] and providing a compound of (f) treating the compound of formula (IV-A) with an aqueous base to provide a compound of formula (IA) in the hydrophobic phase of the reaction mixture; (g) separating the hydrophobic phase from the hydrophilic phase base to provide a compound of formula (IA). There is a method as described above, which includes:

[0094] In some embodiments, a compound of formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] with acetyl chloride to obtain a compound of formula (III-B1): [ka] and providing a compound of (b) reacting a compound of formula (III-B1) with isopropanol to give a compound of formula (IV-A): [ka] and providing a compound of (c) treating the compound of formula (IV-A) with an aqueous base to give a compound of formula (IA); There is a method as described above, which includes:

[0095] In some embodiments, a compound of formula (IA): [ka] A method for producing a compound of the formula (a) Formula (II-A): [ka] and n-heptane; (b) dehydrating the compound of formula (II-A) in n-heptane by azeotropic distillation; (c) adding n-heptane as a non-polar solvent to the dehydrated compound of formula (II-A); (d) Treating the above dehydrated compound of formula (II-A) in heptane with acetyl chloride to obtain a compound of formula (III-B1): [ka] and providing a compound of (e) reacting a compound of formula (III-B1) with isopropanol in a two-phase reaction mixture containing a hydrophilic phase and a hydrophobic phase to produce a compound of formula (IV-A): [ka] and providing a compound of (f) treating the compound of formula (IV-A) with an aqueous base to provide a compound of formula (IA) in the hydrophobic phase of the reaction mixture; (g) separating the hydrophobic phase from the hydrophilic phase base to provide a compound of formula (IA). There is a method as described above, which includes:

[0096] In some embodiments, the method provides a reaction mixture containing a compound of Formula (I) or Formula (IA), and the method further includes concentrating the reaction mixture to provide a crude residue. In some embodiments, the method further includes adding a base to the reaction mixture or crude residue, optionally including sodium acetate, aqueous NaOH, e.g., 0.1-10N aqueous NaOH, or 5N aqueous NaOH, aqueous NaHCO3, aqueous K2CO3, or aqueous Na3PO4, preferably 0.1-10N aqueous NaOH, or 5N aqueous NaOH, to raise the pH to about 5 to about 10, or about 5 to about 9, or about 5 to about 8, to provide a basic mixture; and extracting the compound of Formula (I) or Formula (IA) from the basic mixture into at least one nonpolar solvent to provide an extract. In some embodiments, the at least one nonpolar solvent is selected from petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene. In some embodiments, the at least one nonpolar solvent is selected from hexane, hexanes, n-heptane, octane, nonane, and decane. In some embodiments, the at least one nonpolar solvent is n-heptane. In some embodiments, the volume of n-heptane used in the extracting step is about 1-10 mL, or about 1-5 mL, or about 1-3 mL, or about 2 mL per kilogram of calculated HMBi yield or mass of crude residue (e.g., the calculated or estimated amount of HMBi in the mixture). In some embodiments, the extracting step is performed with n-heptane at a temperature of about 25°C to 50°C, or about 30°C to about 50°C, or about 30°C to about 40°C prior to the extracting step. In some embodiments, the extract comprises a compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight.In some embodiments, the method includes removing the at least one non-polar solvent from the extract, optionally by distillation, to provide a compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight.

[0097] In some embodiments, the reacting provides a reaction mixture comprising a compound of Formula (I) or Formula (IA), and the method further includes adding a base, optionally solid sodium acetate, to the reaction mixture to raise the pH to a range of about 5 to about 10, or about 5 to about 9, or about 5 to about 8 to provide a basic mixture; dehydrating the basic mixture using a dehydrating agent to provide a dehydrated basic mixture; and purifying the compound of Formula (I) or Formula (IA) from the dehydrated basic mixture by distillation to provide a compound of Formula (I) or Formula (IA) in at least about 95% purity by GC, by HPLC, or by weight.

[0098] compound products In some embodiments, the reacting provides a crude compound of Formula (I), unpurified or purified only by fractional distillation, that is at least about 80% pure by weight (and / or by GC or HPLC), or at least about 90% pure by weight, or at least about 95% pure by weight, or at least about 96% pure by weight, or at least about 97% pure by weight, or at least about 98% pure by weight. In some embodiments, the reacting provides a crude compound of Formula (I) or Formula (IA), unpurified or purified only by fractional distillation, that is substantially in monomeric form or contains less than about 5% by weight or less than about 3% by weight of dimeric and / or oligomeric compounds.

[0099] In some embodiments, the disclosure relates to compounds of Formula (I) or Formula (IA) prepared as described herein. In some embodiments, the disclosure relates to compounds of Formula (I) or Formula (IA) that are at least about 80%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98% pure by weight (and / or by GC or HPLC), and that are unpurified or purified only by fractional distillation. In some embodiments, the compounds are substantially in monomeric form or contain less than about 5% by weight, or less than about 3% by weight, of dimeric and / or oligomeric compounds.

[0100] In some embodiments, the HMBi (Formula (IA)) product has one or more of the following specifications: (a) an HMBi monomer content and chemical purity of at least about 95% by weight or by HPLC analysis; (b) a water content of less than about 0.5% by Karl Fischer analysis; (c) a pH of less than about 6.0 (measured at a 1% concentration in water); and (d) an n-heptane content by HPLC of less than about 5000 ppm, or in the range of about 1 to about 4999 ppm, or about 100 to about 4000 ppm, or about 250 to about 3000 ppm, or about 400 to about 2000 ppm, or about 500 to about 1900 ppm.

[0101] When a compound of Formula (II) or (II-A) is present in a starting material mixture containing HMBA dimer / oligomer material (e.g., commercially available 88% HMBA), the esterification reaction described herein can depolymerize the dimer / oligomer and convert the resulting monomer to HMBi. Thus, the reaction can achieve a yield of HMBi greater than 100% based on the amount of monomeric Formula (II) / (II-A) in the starting material. In some embodiments, HMBA is used as the starting material at a purity of about 95%. In some embodiments, the HMBA starting material is 95% pure and anhydrous. In some embodiments, the HMBA starting material is 88% pure and contains monomeric, dimeric, and oligomeric material, as well as water. In some embodiments, the HMBA starting material is not the direct product of the hydrolysis of 2-hydroxy-4-(methylthio)butanenitrile (HMBN). In some embodiments, the HMBA starting material is derived from α-hydroxy-γ-butyrolactone or 2-hydroxy-4-(methylthio)butanamide.

[0102] Also disclosed herein are compounds of Formula (I) or Formula (IA) prepared by any of the methods described herein. In some embodiments, compounds of Formula (I) or Formula (IA) are present that are at least about 95% pure by weight (and / or by GC or HPLC), or at least about 96%, or at least about 97%, or at least about 98% pure, crude, unpurified, and / or purified only by fractional distillation. In some embodiments, the compounds are present in a form comprising R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl, or the compound is a compound of formula (IA). In some embodiments, the compound is substantially in monomeric form or contains less than about 5% by weight, or less than about 3% by weight, of dimeric and / or oligomeric compounds.

[0103] In another aspect, the disclosure relates to a composition comprising at least 95% of a compound of Formula (IA) by weight or by HPLC analysis and about 1 to about 4999 ppm of n-heptane as an impurity. In some embodiments, the composition comprises about 1 to about 1000 ppm of n-heptane as an impurity.

[0104] Animal feed compositions and uses In some aspects, the present disclosure relates to an animal feed composition comprising a compound of Formula (I) or Formula (IA) described herein. In some embodiments, the animal feed composition is suitable for administration to ruminants such as cattle, cows, sheep, antelope, deer, giraffe, bovine animals (e.g., bison, buffalo, or yak), goats, and / or gazelles. In some embodiments, the animal feed composition is a cattle feed composition, such as a dairy cattle feed composition, or an additive for cattle feed, such as dairy cattle feed. In some embodiments, the animal feed composition is a dairy cattle feed composition.

[0105] In some embodiments, the animal feed composition is an animal feed or an animal feed additive. In some embodiments, the animal feed additive is in liquid or solid form, the liquid form comprising the compound and optionally a liquid carrier, and the solid form comprising the compound mixed with a solid carrier, optionally the solid carrier being silica (silicon dioxide), and optionally the ratio of the compound to the solid carrier is about 5:1 to about 1:5, or about 3:2. In some embodiments, the feed composition is a liquid feed additive or a solid feed additive. In some embodiments, the animal feed composition is a drinking water additive. In some embodiments, the pH of the liquid feed additive or drinking water additive is in the range of about 4.0 to about 7.5.

[0106] In some embodiments of the animal feed composition, R 1 is -CH2CH2-S-CH3, and R 2 is isopropyl. In some embodiments, the compound is of formula (IA).

[0107] In some embodiments, the disclosure relates to a method for providing bioavailable methionine to a dairy cow, the method comprising administering to the cow a compound or animal feed composition described herein. In some embodiments, administering comprises feeding the cow a feed composition comprising the compound. In some embodiments, the disclosure relates to a method for providing at least about 50% bioavailable methionine to a dairy cow, the method comprising administering to the cow a compound or animal feed composition described herein. In some embodiments, the disclosure relates to a method for improving milk obtained from a dairy cow, the method comprising feeding the cow a compound or animal feed composition described herein. In some embodiments, the improvement in the milk comprises increasing the protein content in the milk. In some embodiments, the improvement in the milk comprises increasing the fat content in the milk. In some embodiments, the disclosure relates to a method for improving the condition of a cow, the method comprising feeding the cow a compound or animal feed composition described herein. In some embodiments, the improvement in the condition of the cow comprises improving fertility. In some embodiments, the improvement in the condition of the cow comprises improving liver function. In some embodiments, the improvement in the condition of the cow comprises increasing energy.

[0108] HMBi isolation / extraction methods Also disclosed herein is a method for extracting or isolating HMBi by partitioning a mixture of HMBi and at least one impurity selected from HMBA, HMBA dimer, HMBA oligomer, HMBi dimer, and HMBi oligomer between an n-heptane phase and a basic aqueous phase. In some embodiments, the pH of the basic aqueous phase is about 5 to about 10, or about 5 to about 9, or about 5 to about 8.

[0109] In some embodiments, the partitioning of HMBi into the n-heptane phase occurs when n-heptane is added to the reaction mixture after the reaction is complete, nearly complete, or stopped, ie, after 12 to 24 hours, or within 16 to 24 hours, or within 12 to 16 hours, or within 14 to 18 hours.

[0110] Also disclosed herein are methods for extracting or isolating HMBi by partitioning a mixture of HMBi and at least one impurity selected from HMBA, HMBA dimer, HMBA oligomer, HMBi dimer, and HMBi oligomer between the hydrophobic and hydrophilic phases of a two-phase reaction. In some embodiments, the hydrophobic phase comprises n-heptane. In some embodiments, the hydrophilic phase comprises isopropanol. In some embodiments, during the reaction, HMBi partitions into the hydrophobic phase and the at least one impurity partitions into the hydrophilic phase.

[0111] In some embodiments, partitioning of HMBi into the n-heptane phase is achieved during the course of the reaction by adding n-heptane to the reaction mixture along with HMBA, acetyl chloride, isopropyl alcohol, and / or any other combination of reactants described herein. In some embodiments, partitioning of HMBi into the n-heptane phase is achieved when an aqueous base solution is added to the reaction mixture. In some embodiments, adding the aqueous base solution to the reaction mixture increases the amount of HMBi in the n-heptane phase.

[0112] Without wishing to be bound by any particular theory, it is believed that when n-heptane is added to the reaction mixture to produce a two-phase reaction mixture, the more hydrophobic product migrates (based on partition coefficients and / or distribution coefficients) from the hydrophilic phase of the reaction mixture across the hydrophobic / hydrophilic phase boundary into the hydrophobic phase of the two-phase reaction mixture. Based on Le Chatelier's principle, partitioning the product from the hydrophilic phase to the hydrophobic phase shifts the equilibrium of the reaction in favor of product formation while simultaneously protecting the product from undesirable side reactions in the hydrophilic phase.

[0113] In some embodiments, the non-polar solvent is selected from petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene. In some embodiments, other non-polar solvents, such as petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, octane, decane, and benzene, can be used as the hydrophobic phase.

[0114] In some embodiments, the volume of n-heptane is about 1-10 mL, or about 1-5 mL, or about 1-3 mL, or about 2 mL per kilogram of calculated HMBi yield or crude residue mass (e.g., the calculated or estimated amount of HMBi in the mixture). In some embodiments, the extracting is performed with n-heptane at a temperature of about 25°C to 50°C, or about 30°C to about 50°C, or about 30°C to about 40°C prior to the extracting.

[0115] In some embodiments, any of the reactions described herein may be carried out using a continuous flow apparatus.

[0116] Aspects of the present disclosure can be further understood in light of the following examples, which should not be construed as in any way limiting the scope of the disclosure.

[0117] Those skilled in the art will recognize that many modifications, alternatives, and equivalents are possible, and all such modifications, alternatives, and equivalents are intended to be encompassed herein. [Example]

[0118] Apparatus: All millimole-scale experiments were carried out using a 100 mL or 250 mL three-neck round-bottom flask equipped with a magnetic rotor, dropping funnel, and thermometer. The reaction flask was equipped with a condenser and a thermometer to monitor the reaction temperature. When the reaction was carried out at reflux, a silicone oil bath was used to heat the reaction mixture. For experiments at temperatures below room temperature, a salt / ice-cooled mixture bath was used. All kg-scale experiments were carried out using a 5 L glass-lined reactor or a 5 L jacketed three-neck vessel flask equipped with two condensers and an overhead stirrer. Concentration and / or purification of intermediates and crude products was carried out using a laboratory-scale vacuum distillation apparatus or column chromatography.

[0119] For Examples 1-12, as indicated, HMBA (minimum 95% purity) containing monomeric HMBA (possibly containing a mixture of dimers and / or oligomers); or HMBA (88% purity) containing a mixture of monomers, dimers, and / or oligomers and 12% water was used as the starting material.

[0120] General Scheme 1: Transesterification [ka]

[0121] Example 1: Screening-scale synthesis of HMBi by transesterification of HMBA with various isopropyl esters [ka]

[0122] To a 100 mL reaction vessel containing 10 equivalents of isopropyl formate (10 g) was added HMBA (95.6%; 1.70 g, 1.0 equivalent). No acid catalyst was used in this reaction. The reaction mixture was stirred vigorously and then heated to 100°C (or 150°C) for 7 hours. Reactions at high temperatures were carried out in a sealed tube. Samples of the reaction mixture were taken before and after the reaction and analyzed by HPLC. HMBi conversion was monitored by HPLC. Reactions using other isopropyl reagents were similarly carried out on a 1-2 g scale at the indicated temperatures for 7 hours. The reaction may be carried out at the reflux temperature of the isopropyl reagent.

[0123] Table 1 shows the results of a screening experiment in which six forms of isopropyl esters were reacted with HMBA under uncatalyzed transesterification. These results indicate that HMBi was produced using isopropyl formate, isopropyl acetate, isopropyl acrylate, and isopropyl methanesulfonate, but not diisopropyl carbonate or diisopropyl oxalate. For example, the reaction of HMBA with isopropyl methanesulfonate at 25°C for 12 hours produced HMBi (59% conversion, sequence number 15). Reactions of HMBA with other esters at elevated temperatures also produced HMBi (sequence numbers 2, 5, and 11). [Table 1]

[0124] Example 2. Transesterification of HMBA by catalytic transesterification with isopropyl acetate [ka]

[0125] HMBA (95%, 247 g) was diluted with dichloromethane, dried over MgSO4, filtered, and concentrated under reduced pressure to give dried HMBA, which was treated with isopropyl acetate (500 mL). The reaction mixture was heated to reflux (approximately 80°C) for 12 hours. This reaction was repeated N=3 (N=number of experiments). The conversion of the reaction was monitored by gas chromatography to observe the conversion to HMBi over the reaction period. After the reaction was complete, the reaction mixture was cooled to room temperature and partitioned between 250 mL of ethyl acetate and 250 mL of water (2x). The collected organic phase was further washed with 300 mL of saturated NaHCO3 (2x) and 300 mL of saturated NaCl solution. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give crude HMBi (210 g, 75%). The crude product was purified by distillation to determine the isolated yield. The purity of the product was confirmed by gas chromatography and HPLC. 1 1 H NMR was used to determine the

[0126] In some experiments, the HMBi product (50% yield) was isolated after column chromatography. In other experiments, the reaction was carried out for 4-12 hours.

[0127] Example 3 Transesterification of HMBA with Isopropyl Acetate with or without a catalyst on a kilogram scale [ka]

[0128] HMBA (88%, 1009.95 g) was diluted with dichloromethane (1.13 L), dried over MgSO4, filtered, and concentrated under reduced pressure to obtain dehydrated HMBA. This dehydrated HMBA was charged to a reaction vessel containing 2 L of isopropyl acetate (2.54 equiv.). For the uncatalyzed transesterification reaction, the reaction mixture was heated at reflux (approximately 90–95 °C) for 18 h. For the catalytic transesterification reaction, 5 wt. % (57.35 mL) of HCl (36%) or 5 wt. % H2SO4 (98%) was added, and the reaction mixture was then heated to reflux with stirring. The reaction conversion in each case was monitored by HPLC (offline measurement). No isopropyl alcohol formation was detected by HPLC analysis during the uncatalyzed reaction, the reaction with HCl, and the reaction with H2SO4 (see Figure 1A and IB). HPLC analysis was performed as follows: Eluent: 650 mL MilliQ water + 2 mL 85% phosphoric acid + 350 mL acetonitrile Column: OOG-4633-EO, Kinetex 5 μm EVO C18, 100 Å, LC column size: 250 × 4.6 mm Detector: Spectrosystem UV2000, 210 nm; Flow rate: 1 mL / min; Oven temperature: 30°C

[0129] Upon completion of the reaction, the reaction mixture was cooled to a temperature below 50°C, and isopropyl acetate (approximately 700 mL) was distilled off under reduced pressure. The residue was treated with 100 g of sodium acetate or saturated NaHCO3 solution (250 mL) to adjust the pH to approximately 8-10. The mixture was diluted with preheated n-heptane (1500 mL), and the resulting mixture was stirred for 60 minutes (to extract HMBi). The organic phase was separated, and the aqueous layer was extracted with another 500 mL of preheated n-heptane. The combined organic phases were washed with an additional 500 mL of water (3x) and 1000 mL of saturated NaCl solution. The organic layer was concentrated under reduced pressure to obtain crude HMBi. The crude material was then treated with 5% by weight (based on the mass of the crude material) of activated carbon at 50°C for 2 hours to obtain a decolorized crude material. The mixture was filtered, and the filtrate was distilled to remove n-heptane and then concentrated under reduced pressure. The yield and purity of the product was determined by HPLC and showed a crude purity of 78% before workup using n-heptane extraction.

[0130] The product is obtained as a pale yellow to light yellow oil with a product purity of ≥95% monomeric ester, which allows for the determination of the product structure. 1 This was confirmed by H-NMR. 1 H NMR (400 MHz, CDCl3) δ 5.08 (hept, J = 6.3 Hz, 1 H), 4.24 (dd, J = 7.9, 3.8 Hz, 1 H), 2.97 (br, 1 H), 2.67-2.55 (m, 2 H), 2.10-2.01 (m, 4 H), 1.93-1.84 (m, 1 H), 1.27 (d, J = 1.9 Hz, 3 H), 1.26(d, J = 2.2 Hz, 3 H).

[0131] Reactions using isopropyl acetate and an acid catalyst or using isopropyl methanesulfonate were carried out similarly to the above procedure, and the experimental results are shown in Table 2. [Table 2]

[0132] The HPLC results shown in Figures 1A, 1B, 2A, 2B, 3A, and 3B are summarized in the table below.

[0133] [Table 3]

[0134] [Table 4]

[0135] [Table 5]

[0136] [Table 6]

[0137] [Table 7]

[0138] [Table 8]

[0139] Example 4: Catalytic transesterification of HMBA with isopropyl acetate on a 500 kg scale [ka]

[0140] HMBA (88%, 500 kg) was charged into a 3000 L stainless steel reactor containing 1000 kg of isopropyl acetate. 14.5 kg of H2SO4 (98%) was added as a catalyst. The reaction mixture was then heated to 80-90 °C and refluxed for 6 h. This reaction was repeated N = 3 (N = number of experiments). Upon completion of the reaction, the reaction mixture was cooled to a temperature below 50 °C, and then NaOH solution (20 wt%, 560 kg) was added to adjust the pH to 6.5-7.5. After that, the organic and aqueous layers were clearly separated. The organic phase was then washed with a small amount of water (100 kg) to remove salts. The organic phase was then added with 40 kg of NaOH solution (20 wt%) to adjust the pH to 6.5-7.5, and then concentrated in the 3000 L stainless steel reactor to obtain the crude HMBi product. The yield and purity of the product were determined by HPLC (57.5% yield, 95% purity). Figure 4 shows an example of a process flow chart for the synthesis of HMBi.

[0141] Example 5 Synthesis of HMBi by reaction of HMBA with in situ generated isopropyl acetate [ka]

[0142] Procedure A: To a stirred sample of isopropyl alcohol (2000 g) was added acetyl chloride (110 mol % relative to HMBA), and the mixture was stirred at room temperature for 30 minutes. This mixture was treated with HMBA (1000 g), and the resulting mixture was heated at 80-90°C for 2 hours. The progress of the reaction was monitored by gas chromatography. Gas chromatography (GC) revealed that the reaction products were HMBA (0.4% residual) and HMBi (GC purity 93.0%).

[0143] Procedure B: Further reactions were carried out similarly at 80-90°C using 110 mol% acetyl chloride for 4 hours (0% HMBA remaining; 95.8% HMBi), 6 hours (0.5% HMBA remaining; 95.4% GC purity), 8 hours (0.3% HMBA remaining; 95.7% GC purity), 10 hours (0% HMBA remaining; 97.0% GC purity), 24 hours (0% HMBA remaining; 97.0% GC purity), or 48 hours (0% HMBA remaining; 96.4% GC purity); or at 150-160°C using 110 mol% acetyl chloride for 48 hours (73% GC purity); or at 50-60°C for 5 hours using 105 mol% acetyl chloride (91% GC purity) or 110 mol% acetyl chloride (95% GC purity). The reaction using 101 mol% acetyl chloride at 50-60°C proceeded more slowly.

[0144] Work-up procedure: The reaction was carried out using 105 mol% acetyl chloride at 50-60°C for 16 hours. (a) Dilution with ethyl acetate and washing with water until the organic phase had a neutral pH (GC purity of 81% of the reaction mixture); (b) Concentration under reduced pressure at 50–60°C for 2 h (81% HMBi in the reaction mixture before workup; 89% GC purity after workup); (c) Concentration under reduced pressure followed by stirring of the residue at 50-60°C for 8 hours (GC purity before workup: 89%; purity after workup: 86%); (d) Concentration under reduced pressure followed by keeping the residue at room temperature for 3 days (GC purity before workup 89%; purity after workup 83%); (e) Concentration under reduced pressure followed by stirring of the residue at 120-130°C for 6 hours (89% purity before workup; 69% purity after workup); (f) Neutralization with NaOH solution (to pH 10) and concentration under reduced pressure for 3 hours (GC purity of HMBi in the reaction mixture before workup 96%; purity after workup 79%) The mixture was then post-treated by

[0145] Procedure C: To isopropyl alcohol (160 kg), AcCl (5.0 kg) was added in 10 portions over 10 minutes (internal temperature below 40°C) with stirring, and stirring was continued at room temperature for 2 hours. Then, HMBA (100 kg) and isopropyl alcohol (40 kg) were added sequentially. The resulting reaction mixture was heated to 80-90°C over 1 hour and stirred at this temperature for 14-16 hours. The reaction mixture was monitored by GC. After cooling to 40-50°C, isopropyl alcohol (100 L) was recovered by vacuum distillation (-0.07 MPa) for 6-8 hours. The temperature was maintained at 50-65°C, and sodium acetate (6.0 kg) was added to adjust the pH to 5-6. Vacuum distillation was then continued until no more isopropyl alcohol was recovered. The resulting HMBi (approximately 130 kg) was cooled with recycled water and the pH was adjusted to 8-9 by adding 40 kg of 5N NaOH solution. The mixture was diluted with n-heptane (140 kg) and stirred for 15 minutes. The organic layer was separated, and the aqueous layer was extracted with another 70 kg of n-heptane. The n-heptane was removed under reduced pressure (60-70 °C, -0.07 MPa) to give crude HMBi as a pale yellow oil in 70-75% yield.

[0146] The resulting HMBi may be purified and decolorized by optional treatment with activated carbon, followed by distillation to obtain HMBi in at least 95% purity.

[0147] Example 6 Reaction of anhydrous HMBA with isopropyl acetate on a pilot scale [ka]

[0148] Neat HMBA (88%, 100 kg) was added with anhydrous Na2SO4 (10 kg) or MgSO4 (10 kg), and the resulting mixture was stirred for 2–3 hours to remove most of the water from the HMBA. This mixture was filtered and optionally washed with isopropyl alcohol or isopropyl acetate. Acetyl chloride (9.14 kg, 0.2 equiv.) was added to the filtrate in 10 portions over 10 minutes, maintaining the reaction mixture temperature below 40 °C to react with the remaining water and generate HCl in situ. The appropriate amount of acetyl chloride was determined by Karl Fischer analysis after the Na2SO4 dehydration step. The resulting mixture was stirred at room temperature for 2 hours. Karl Fischer analysis was used to confirm that no detectable water remained. (In laboratory scale, the dehydration process can be accomplished by diluting with dichloromethane, drying over MgSO4 or Na2SO4, filtering, and concentrating the residue with acetyl chloride to remove residual water, or by treating the filtrate with acetyl chloride to remove residual water and then concentrating under reduced pressure to remove dichloromethane.) Isopropyl acetate (200 kg) was then added to the reactor. The resulting reaction mixture was heated to 80-90°C over 1 hour and stirred at this temperature for 14-16 hours. Conversion to HMBi was monitored by GC or HPLC. The reaction mixture was cooled to approximately 50°C, and the isopropyl acetate was recovered by vacuum distillation (-0.07 MPa) for 6-10 hours (or until no isopropyl acetate remained). The resulting HMBi was then quenched with recycled water.

[0149] Workup 1: 5N NaOH was added to the cooled HMBi to adjust the pH to 8-9. n-Heptane (140 kg) was added to the mixture, and the mixture was vigorously stirred for 15-30 minutes. The organic phase was separated, and the aqueous layer was extracted with another 70 kg of n-heptane. n-Heptane was removed from the combined organic layers at 60-70°C and -0.07 MPa to yield crude HMBi with a purity of at least 95%. (If the material is brown in color, activated carbon (2-5 kg) can be added directly to the crude HMBi.) The mixture was stirred for 2-3 hours and filtered to yield a pale-colored product without rinsing.

[0150] Workup 2: To the cooled HMBi (from one or more batches), sodium acetate was added to adjust the pH to 5-7. The mixture was then dried over NaSO or MgSO and filtered (without rinsing) to remove most of the water. The resulting crude material was purified by fractional distillation to yield purified HMBi with a purity of at least 95%.

[0151] Example 7 Reaction of HMBA with in situ generated HCl and in situ generated isopropyl acetate on a pilot scale [ka]

[0152] Anhydrous MgSO4 (10 kg) was added to HMBA (88%, 100 kg), and the resulting mixture was stirred for 2–3 hours to remove most of the water from the HMBA. The mixture was filtered and rinsed with isopropyl alcohol. Acetyl chloride (2.6 kg, 0.05 equiv.) was added to the filtrate in five portions over 10 minutes, and the temperature of the reaction mixture was maintained below 40 °C to react with residual moisture and generate HCl in situ to form a dehydrated HMBA / HCl mixture. Karl Fischer analysis confirmed the absence of residual water.

[0153] Acetyl chloride (55 kg; 1.05 equivalents relative to HMBA) was added in 20 portions to stirred isopropyl alcohol (160 kg), maintaining the internal temperature below 40 °C. The reaction mixture was stirred at room temperature for 2 hours to produce isopropyl acetate. The dehydrated HMBA / HCl mixture (assumed to be 100 kg) was added to the mixture, followed by 30 kg of isopropyl alcohol. The resulting reaction mixture was heated to 80–90 °C over 1 hour and stirred at this temperature for 14–16 hours. Conversion to HMBi was monitored by GC or HPLC. The reaction mixture was cooled to approximately 50 °C, and the isopropyl alcohol was recovered by vacuum distillation (-0.07 MPa) for 6–10 hours (or until no isopropyl alcohol remained), followed by fractional distillation to separate the residual isopropyl alcohol and isopropyl acetate. The resulting HMBi was then quenched with recycled water. The cooled HMBi was subjected to workup 1 or 2 as in Example 6 to obtain HMBi with a purity of at least 95%.

[0154] Example 8 Pilot-scale synthesis of HMBi using in situ generated HCl [ka]

[0155] Anhydrous Na2SO4 (10 kg) was added to HMBA (88%, 100 kg), and the resulting mixture was stirred for 2–3 hours to remove most of the water from the HMBA. The mixture was filtered and optionally washed with isopropyl alcohol or isopropyl acetate. Acetyl chloride (4.57 kg, 0.1 equiv.) was added to the filtrate in 10 portions over 10 minutes to react with residual moisture and generate HCl in situ, while maintaining the reaction mixture temperature below 40°C. The resulting mixture was stirred at room temperature for 2 hours. Isopropyl alcohol (200 kg) was then added to the reactor. The resulting mixture was heated to 80–90°C over 1 hour and stirred at this temperature for 14–16 hours. Conversion to HMBi was monitored by GC or HPLC. The reaction mixture was cooled to approximately 50°C, and the isopropyl alcohol was recovered by vacuum distillation (-0.07 MPa) for 6–10 hours (or until no isopropyl alcohol remained). The resulting HMBi was then cooled with recycled water. The cooled HMBi was subjected to work-up 1 or work-up 2 as described in Example 6 to obtain HMBi with a purity of at least 95%.

[0156] Example 9 Transesterification of HMBA with triisopropyl borate [ka]

[0157] HMBA (88%) was dehydrated as described in the previous example. Dehydrated HMBA (250 g, 1 equivalent) was treated with triisopropyl borate (626 g, 2 equivalents) at 95°C for 10 hours, resulting in 77% conversion to HMBi. The product was extracted into n-heptane and washed as above to give HMBi in 72% yield (229.1 g) and 98% purity by HPLC analysis.

[0158] General Scheme 2: Esterification by Stoichiometric Acetylation [ka]

[0159] Example 10 Synthesis of HMBi using stoichiometric acetyl chloride and isopropanol [ka]

[0160] Acetyl chloride (10 g, 1.1 equiv.) was added dropwise to HMBA (5 g) while maintaining the temperature below room temperature. The reaction mixture was stirred at room temperature for 3 hours until 2-acetoxy-4-(methylthio)butanoic acid (Ac-HMBA) was formed. Isopropyl alcohol (10 g) was added to the reaction mixture, and the reaction solution was heated at 55°C for 4-12 hours or at 80°C / reflux for 12 hours. The mixture was cooled to room temperature and concentrated. The residue was treated with aqueous base (0.1 N NaOH), extracted with ethyl acetate (50 mL), washed with water (30 mL), saturated NaHCO3 solution, and saturated NaCl solution, filtered, and concentrated to give crude HMBi. The crude product was further purified by distillation and analyzed by gas chromatography and HPLC using 4-iodoanisole as an internal standard. 1 Analyzed by H NMR (95.2% GC purity; 88.1% yield).

[0161] Further experiments were carried out using procedures similar to those described above, using 1.0, 1.05, 1.2, and 2.0 equivalents of acetyl chloride at reaction temperatures of 25, 60, 90, and 120° C. Under all of these conditions, HMBi was obtained in purity of at least 94% and crude yields of at least 75%. [Table 9]

[0162] Example 11 Synthesis of HMBi using stoichiometric acetyl chloride and isopropanol on a kilogram scale [ka]

[0163] HMBA (95%, 1000 g) was reacted with 1.1 equivalents of acetyl chloride under reflux with stirring for 3 hours. Isopropyl alcohol (2000 mL) was slowly added, and the reaction mixture was heated to reflux for 12 hours. The mixture was cooled and concentrated. The residue was treated with 0.1 N NaOH (200 mL), and the product was extracted with ethyl acetate (2 × 400 mL), washed with water (2 × 1000 mL) and saturated NaCl solution (1000 mL), filtered, and concentrated under reduced pressure. The crude product was purified by fractional distillation under reduced pressure to give HMBi in at least 75% yield.

[0164] Example 12A Synthesis of HMBi using 2 equivalents of acetyl chloride and isopropanol [ka]

[0165] Step 1, Option A: Acetyl chloride (2 equiv.) was added dropwise to HMBA (1 equiv.) either neat or in the presence of an organic solvent with stirring. The reaction temperature was maintained at 10-15°C and then slowly increased to room temperature or until 2-acetoxy-4-(methylthio)butanoic anhydride was formed (as indicated by GC). This reaction was also carried out using 2.2 equiv. of acetyl chloride at 55°C for 3 hours, resulting in crude yields of at least 93% Ac-HMBA.

[0166] Step 1, Option B: Continuous Flow Acylation. The reaction was carried out in a flow chemistry environment (a coiled PFA R&D-scale reactor). A solution of the acylation reagent, 2 equivalents of AcCl in THF, was pumped by a syringe pump into the inlet of a T / Y mixer (PTFE) and combined with 1 equivalent of HMBA flow solution (neat or organic solvent). The reagents were circulated at controlled flow rates by a dosing pump. The reaction temperature was maintained at room temperature, or the mixture was heated until 2-acetoxy-4-(methylthio)butanoic anhydride was formed (as indicated by GC analysis).

[0167] Step 2: The intermediate from Step 1 was reacted with 1 equivalent of isopropyl alcohol solution by stirring the reaction mixture at room temperature overnight. After the addition of the alcohol (in this case, isopropyl alcohol), the reaction temperature was raised to approximately 40°C (or higher) and maintained at this condition until Ac-HMBi was formed (as indicated by GC analysis). In this case, Steps 1 and 2 were carried out independently. Optionally, the above two steps can be carried out in one pot without isolating the product of Step 1. The intermediate hydroxyacylating anhydride (2-acetoxy-4-(methylthio)butanoic anhydride) is the direct product from Step 1 and is used in Step 2 in the same reaction vessel without isolation.

[0168] Step 3 After the reaction in Step 2 was complete, the reaction mixture was cooled to room temperature and concentrated. The crude residue was treated with a deacylating agent (aqueous base).

[0169] Workup and isolation of HMBi product The mixture from step 3 was then diluted with ethyl acetate and washed with 30 mL of water. The collected organic phase was further processed by washing twice with saturated NaHCO3 and saturated brine solutions. The organic fraction was then filtered and concentrated under reduced pressure to give crude HMBi. This crude product was optionally purified by distillation. The purity of the crude product was confirmed by GC and 1 1 H NMR was used to determine the

[0170] Step 2 was also carried out using 2 equivalents of isopropyl alcohol, giving a 94% conversion to HMBi and a crude yield of at least 87%.

[0171] Example 12B Synthesis of HMBi using 2 equivalents of acetyl chloride and isopropanol [ka]

[0172] Step 1: Acetyl chloride (2 equiv.) was added dropwise to HMBA (1 equiv.) either neat or in the presence of an organic solvent with stirring. The reaction temperature was maintained between 10 and 15°C, slowly increased to room temperature over time, and then heated at 55°C for 3 hours to obtain a crude yield of at least 93% Ac-HMBA.

[0173] Step 2: The intermediate from Step 1 was reacted with one equivalent of isopropyl alcohol solution. After the addition of the alcohol (in this case, isopropyl alcohol), the reaction temperature was raised to approximately 40 °C (or higher under reflux) and maintained at this temperature for 12–15 h until Ac-HMBi was formed (as indicated by GC analysis). In this case, Steps 1 and 2 are carried out independently. Optionally, the two steps above can be carried out in one pot without isolating the product from Step 1. The intermediate, 2-acetoxy-4-(methylthio)butanoic acid (Ac-HMBA), is the direct product from Step 1 and is used in Step 2 in the same reaction vessel without isolation.

[0174] Step 3 After the reaction in Step 2 was complete, the reaction mixture was cooled to room temperature and concentrated. The crude residue was treated with a deacylating agent (aqueous base).

[0175] Workup and isolation of HMBi product The mixture from step 3 was then diluted with ethyl acetate and washed with 30 mL of water. The collected organic phase was further processed by washing twice with saturated NaHCO3 and saturated brine solutions. The organic fraction was then filtered and concentrated under reduced pressure to give crude HMBi. This crude product was optionally purified by distillation. The purity of the crude product was confirmed by GC and 1 1 H NMR was used to determine the

[0176] Step 2 was also carried out using 2 equivalents of isopropyl alcohol, yielding a reaction mixture with a GC purity of 80% HMBi after the reaction (12-15 hours) and 94% HMBi after workup.

[0177] Materials for Examples 13 and 14: 88% HMBA (monomer, dimer, and oligomer) and 12% water were pre-dehydrated to a minimum water content. Isopropyl alcohol (IPA) (99.8%), n-heptane (AR), AcCl (99%), NaOH (99%), concentrated HCl (37%), sulfuric acid (98%), IPA (99.9%), n-heptane (99.9%), AcCl (99%), NaOH (99%), and sulfuric acid (98%) were used. Lab-scale two-phase reactions were performed on 50 g or 100 g scales.

[0178] Examples 13-14: One-pot synthesis and extraction of HMBi with acetyl chloride (AcCl) and isopropanol using a two-phase reaction [ka]

[0179] Example 13 One-pot synthesis and extraction of HMBi with acetyl chloride (AcCl) and isopropanol using a two-phase reaction

[0180] Dehydration step: Before starting the reaction, a dehydration step was performed using azeotropic distillation. HMBA (100 g) was mixed with n-heptane (200 mL) in a 500 mL flask. This was stirred and distilled at 100-110 °C until all the n-heptane was distilled into the collection vessel. This distillation was carried out for more than 3 hours. Approximately 10-11 mL of water was visible and settled at the bottom of the collection vessel.

[0181] Esterification: Next, a specific number of equivalents (eq) of IPA, a specific amount of n-heptane, and 0.10 equivalents of AcCl were added to HMBA or dehydrated HMBA in a flask, as shown in Table 4. The mixture separated into two phases. The mixture was then heated to 80–90°C and refluxed for 8–20 hours for esterification. After refluxing, the mixture was cooled to 40–50°C. Sodium hydroxide solution (20% wt / wt) was added dropwise to adjust the pH to 8–9, allowing for clear separation of the organic and aqueous layers. 0.5 mL of each sample was analyzed for HMBA and HMBi content. The conversion rate was calculated by dividing the sum of the HMBi contents in the organic and aqueous layers by the theoretical HMBi production. Some reaction mixtures were further processed to calculate the isolated yield. Briefly, the aqueous layer was back-extracted twice with n-heptane (0.5 volumes each). All organic phases were combined, washed with a small amount of water (10 mL), and then concentrated on a rotary evaporator (Heidolph Hei-VAP Precision ML / G3) to give the crude HMBi product.

[0182] Table 4 shows the results for each of the biphasic reaction conditions tested, with reflux at 80–90 °C and 0.1 equivalents of AcCl fixed for all reactions. The IPA equivalents, n-heptane volume, and reaction time were screened variables. To recycle unreacted HMBA, the aqueous phases from reactions 2b, 4a, 4b, 4c, and 4d were combined and acidified with concentrated hydrochloric acid to a pH of 2.0–2.5. After pH adjustment, two phases were formed. The upper organic layer, containing unreacted HMBA and a small amount of HMBi, was separated and directly applied to the HMBi synthesis process as described above, except without the dehydration step (referred to as reaction 5a in Table 4). All organic phases from reactions 2b, 4a–4d, and 5a were combined and evaporated to obtain the crude product.

[0183] [Table 10]

[0184] Results: All conversions of HMBi from laboratory-scale reactions are listed in Table 5. Conversions were found to increase with the number of equivalents of IPA and the volume of n-heptane. When two volumes of n-heptane were used as solvent (Table 5, columns 2a–2d), the conversions (71.9%, 76.4%, 78.3%, and 80.2%) increased with the number of equivalents of IPA (1.5, 2.0, 2.5, and 3.0 equivalents). When 3.0 equivalents of IPA were used (Table 5, columns 1c, 2d, 3b, and 3c), the conversions of HMBi (56.4%, 80.2%, 81.9%, and 83.7%) increased with the volume of n-heptane (0, 2, 3, and 4 volumes). A similar trend was observed when 2.0 equivalents of IPA was used in the reaction (Table 5, series numbers 2b and 3a). The reaction time did not significantly affect the conversion rate as long as it was 8 hours or longer (Table 5, series numbers 4a to 4d).

[0185] [Table 11]

[0186] Compared to the single-phase process, the biphasic reaction improved the conversion of HMBi. The conversion in the single-phase process was 76.2% (Table 5, sequence 1a), which decreased without pre-dehydration of HMBA and the use of n-heptane (Table 5, sequence 1b, 73.7%; sequence 1c, 56.4%). The use of ≥ 2.0 equivalents of IPA and ≥ 2 volumes of n-heptane improved this to 76.4-86.8%.

[0187] HPLC Analysis of Final Products. The HPLC method used for the quantification of HMBA and HMBi was based on the EURL Evaluation Report on the Analytical Methods (JRC.D.5 / FSQ / CvH / SB / ag / Ares(2012)240861) presented in connection with the Application for Authorization of a Feed Additive in accordance with Regulation (EC) No. 1831 / 2003 and adapted to our laboratory. The mobile phase contained 0.2% aqueous phosphoric acid (85%) (v / v, channel A) and acetonitrile (channel B). After washing and separation, 100 μL of the organic and aqueous layers were dissolved in 10 mL of acetonitrile, respectively. A 10 μL sample was injected per assay. The optimal detection wavelength (210 nm) was selected for simultaneous quantification of these two molecules. The percentages of the components were calculated by integrating the peak areas.

[0188] Analysis of dimeric and oligomeric impurities: As shown in Figure 5A (Table 5A) and 5B (Table 5B), the dimeric and oligomeric impurities in the reaction mixture were reduced or eliminated by the two-phase reaction. The HPLC purity of the reaction mixture improved from 92.8% to 97.9%. [Table 12] [Table 13]

[0189] Example 14 Pilot-scale reaction of HMBi with acetyl chloride (AcCl) and isopropanol using a biphasic reaction

[0190] Two 1500 L glass-lined reactors, one 3000 L stainless steel reactor, and one 3000 L stainless steel storage tank were used. Briefly, 600 kg of HMBA (88% HMBA and 12% water) and 1200 L (820.8 kg) of n-heptane were charged into the 3000 L reactor and heated to 90 °C to distill off the n-heptane. The distilled n-heptane was placed in another 3000 L storage tank and allowed to stand for 3 hours. A water layer of 49–55 kg of water formed at the bottom. The remaining HMBA was then divided into two equal parts, and each of the two HMBA volumes was transferred to one 1500 L glass-lined reactor. To each 1500 L reactor, HMBA (300 kg), IPA (2.5 equiv / 264 kg for the first batch and 1.5 equiv / 158.4 kg for the second batch using recycled solvent), n-heptane (2 volumes, 600 L, 410.4 kg, fresh for the first batch and recycled for the second batch), and AcCl (0.1 equiv, 15.0 kg) were added. The mixture was heated to 80-90 °C and refluxed for 10-12 h. It was then cooled to 40-50 °C, and the pH was adjusted to 8-9 by adding NaOH solution (20% wt / wt). After this, the organic and aqueous layers became clear. Both layers were sampled and analyzed for HMBA and HMBi content. The conversion was calculated by dividing the total HMBi content of the organic and aqueous layers by the theoretical HMBi production. The aqueous layer was back-extracted with two portions of n-heptane (0.5 volumes each, 102.6 kg). All organic phases were combined and washed with a small amount of water (60 L), which was then concentrated in a 3000 L stainless steel reactor to give the crude HMBi product.

[0191] A total of four batches were prepared, including one recycle batch. For the recycle batch, the aqueous phases from the three previous batches were combined and acidified with concentrated sulfuric acid to lower the pH to 2.0-2.5. Phase separation was observed. The upper organic layer, containing approximately 265 kg of HMBA and a small amount of HMBi, was separated. This organic layer was then directly applied to the HMBi synthesis process as described above, but without the dehydration step. The reaction conditions for all three batches and one recycle batch are listed in Table 6.

[0192] [Table 14]

[0193] Results: The conversions and corrected yields of HMBi from the pilot-scale synthesis are shown in Table 7. Using the same reaction conditions, the conversions were very close to those from the laboratory-scale synthesis, indicating that this process is scalable. A total of 1823.5 kg of crude HMBi product was obtained from 1800 kg of HMBA, for an overall corrected yield of 81.5%. Assays of the crude products were all above 90%. [Table 15]

[0194] FIG. 6 shows an example process flow chart for the synthesis of HMBi using a two-phase reaction.

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, R １ is -S-C １～４ Alkyl-substituted C １～４ is alkyl, R ２ is C １～８ Alkyl or C ４～７ cycloalkyl) A method for producing a compound of the formula Formula (II): 【Chemistry 2】 with a compound of formula (A) or formula (B): 【Transformation 3】 (In the formula, R ｘ is H, C １～４ Alkyl, and CH ２ ═CH—; R ｙ is C １～３ alkyl) The method comprising reacting the compound of formula (I) with a reactant of formula (II).

2. 10. The method of claim 1, wherein the reactant of formula (A) or formula (B) functions as a reaction solvent.

3. R １ But -CH ２ CH ２ -S-C １～４ 3. The method of claim 1 or claim 2, wherein the alkyl is alkyl.

4. R １ Ga-CH ２ CH ２ -S-CH ３ The method of claim 3, wherein

5. R ２ 5. The method of claim 1, wherein is selected from methyl, ethyl, and isopropyl.

6. R ２ The method of claim 5 , wherein is isopropyl.

7. R １ Ga-CH ２ CH ２ -S-CH ３ and R ２ 3. The method of claim 1 or claim 2, wherein is isopropyl.

8. Formula (IA): 【Chemistry 4】 A method for producing a compound of the formula Formula (II-A): 【Transformation 5】 with a reactant which is isopropyl acetate or isopropyl methanesulfonate.

9. 9. The method of any one of claims 1 to 8, wherein the reacting is carried out in the absence of an acid catalyst.

10. 9. The method of any one of claims 1 to 8, wherein the reacting is carried out in the presence of at least one acid catalyst.

11. The at least one acid catalyst is H ２ SO ４ HCl, and p-TsOH.

12. Prior to the reaction, the compound of formula (II) or formula (II-A) is present in a sample containing water; The sample was treated with the formula (D): C １～３ Alkyl-C(O)Cl (D) reducing the amount of water in the sample by contacting the sample with an acid chloride of mixing said reactants with said catalyst mixture; The method of claim 11 further comprising:

13. Prior to the reaction, the compound of formula (II) or formula (II-A) is present in a sample containing water; 12. The method of claim 1, further comprising treating the sample with a dehydrating agent prior to the reacting.

14. 14. The method of claim 13, wherein the at least one dehydrating agent is an azeotrope solvent selected from hexane, n-heptane, n-propanol, isopropyl acetate, ethyl acetate, toluene, and benzene.

15. said reacting providing a reaction mixture comprising said compound of formula (I) or formula (IA); removing the isopropyl acetate or the reactant of formula (A), or the reactant of formula (B) or isopropyl methanesulfonate to provide a crude residue; The crude residue was treated with sodium acetate, aqueous NaOH, 0.1 to 10N aqueous NaOH, 5N aqueous NaOH, NaHCO ３ aqueous solution, K ２ CO ３ Aqueous solution, or Na ３ P.O. ４ adding an aqueous base to raise the pH to a range of 5 to 10 to provide a basic mixture; extracting the compound of formula (I) or formula (IA) from the basic mixture into at least one non-polar solvent to obtain an extract; 15. The method of claim 1, further comprising:

16. 16. The method of claim 15, wherein the at least one non-polar solvent is selected from petroleum ether, toluene, methyl tert-butyl ether, hexane, cyclohexane, hexanes, n-heptane, octane, nonane, decane, and benzene.

17. 17. The method of claim 16, wherein the at least one non-polar solvent is n-heptane.

18. 18. The method of claim 17, wherein the extract comprises the compound of formula (I) or formula (IA) in a purity of at least 95% by GC, HPLC, or weight.

19. 19. The method of any one of claims 16 to 18, comprising removing the at least one non-polar solvent from the extract to provide the compound of Formula (I) or Formula (IA) in a purity of at least 95% by GC, HPLC, or weight.

20. 20. A process according to any one of claims 1 to 19, wherein the reacting is carried out at a temperature of at least 20°C, or at least 30°C, or at least 40°C, or at least 50°C, or at least 60°C, or at least 70°C, or at least 80°C, or at least 90°C, or a temperature in the range of from 20°C to 150°C, or from 20°C to 100°C, or from 20°C to 90°C, or from 60°C to 150°C, or from 60°C to 100°C, or from 60°C to 95°C, or from 75°C to 90°C, or from 80°C to 150°C, or from 80°C to 100°C, or from 80°C to 90°C, or at a temperature of 89°C, or the reflux temperature of the reactants of Formula (A) or Formula (B).

21. 21. The method of any one of claims 1 to 20, wherein the reacting is carried out for a time period ranging from 1 hour to 24 hours, or from 2 hours to 15 hours, or from 3 hours to 14 hours, or from 4 hours to 12 hours, or from 4 hours to 10 hours, or from 10 hours to 20 hours, or from 14 hours to 16 hours.

22. further comprising combining isopropyl alcohol with acetyl chloride to form a solution of isopropyl acetate in isopropanol; 22. The method of any one of claims 1 to 21, wherein reacting the compound of Formula (II) or Formula (II-A) with the reactant of Formula (A) that is isopropyl acetate comprises adding the compound of Formula (II) or Formula (II-A) to a solution of the isopropyl acetate in isopropanol.

23. 23. The method of any one of claims 1 to 22, wherein the reacting provides a crude compound of Formula (I) or Formula (IA) that is at least 95%, or at least 96%, or at least 97%, or at least 98% pure by weight, GC, and / or HPLC, wherein the crude compound is unpurified or purified only by fractional distillation.

24. 24. The method of any one of claims 1 to 23, wherein the reacting provides a crude compound of formula (I) or formula (IA) that is substantially in monomeric form or contains less than 5% by weight, or less than 3% by weight, of dimeric and / or oligomeric compounds, and which is unpurified or purified only by fractional distillation.

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