WO2008154743A1

WO2008154743A1 - Methods for the synthesis of 4-hydroxyisoleucine, stereoisomers and analogs thereof

Info

Publication number: WO2008154743A1
Application number: PCT/CA2008/001169
Authority: WO
Inventors: Trevor Laird; Anh Thu Pham; Felix Spindler; Andreas Schumacher; Richard A. Sachleben
Original assignee: Innodia Inc.
Priority date: 2007-06-19
Filing date: 2008-06-19
Publication date: 2008-12-24

Abstract

A method for synthesizing 4-hydroxyisoleucine, stereoisomers and analogs thereof having a general formula I is disclosed herein. The method comprises reacting an alkyl isocyanoacetate of formula III with an acetoin or an acetoin analog of formula II to give an unsaturated N-formyl lactone of formula IV, hydrogenating the unsaturated N-formyl lactone to give N-formyl lactone of formula V and hydrolysing said lactone to yield the desired product of formula I.

Description

TITLE OF THE INVENTION

METHODS FOR THE SYNTHESIS OF 4- HYDROXYISOLEUCINE, STEREOISOMERS AND ANALOGS THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of US Provisional

Application No. 60/944,953 filed June 19, 2007, the entire contents of which are incorporated by reference.

FIELD OF THE INVENTION

(0002] The present disclosure relates to methods for the synthesis of 4- hydroxyisoleucine (4-HIL), stereoisomers and analogs thereof. More specifically, but not exclusively, the present disclosure relates to methods for the synthesis of (25',3Λ,45)-4-hydroxyisoleucine and analogs thereof.

BACKGROUND OF THE INVENTION

]0003] In 1973, Fowden et al., in Phytochemistry 12: 1707-171 1, 1973, reported the presence of the chiral amino acid (2S,3Λ,4Λ)-4-hydroxy-3- methylpentanoic acid (4-hydroxyisoleucine) in the seeds of fenugreek (Trigonella foenumgraecum), an annual herbaceous plant that is widespread in regions of Asia, Africa, and Europe. Its absolute configuration was subsequently restudied and corrected as being (2S,3RAS) by Alcock et al. in Phytochemistry 28:1835-1841 , 1989. It has been demonstrated that (25¹ ₎3/?,4,5)-4-hydroxyisoleucine possesses insulinotropic and insulin sensitizing activities (Broca et al., Am. J, Physiol. 277:E617-E623, 1999; Broca et al., Eur. J. Pharmacol. 390:339-345, 2000). (25,3/?,45)-4-Hydroxyisoleucine has since been developed for the treatment of diabetes (U.S. Patent No. 5,470,879; PCT publication No's WO 97/32577, WO 01/15689, and WO 2005/039626). [0004] The main setback with (25,3/?,45)-4-hydroxyisoleucine, is that it is difficult to obtain in large quantities. Due to its low content in fenugreek seeds (-1%) it can only be isolated in low yields. The presence of three adjacent chiral centers (2S,3R,4S configuration) makes it a structurally complex molecule posing a great challenge to synthetic chemists. The reported chemical syntheses of (25,3Λ,45)-4-hydroxyisoleucine and its isomers (e.g. Inghardt et al, Tetrahedron, 47, No. 32, 6469-6482, 1991 ; Dong et al, J. Org. Chem. 64, 2657-2666, 1999; Kassem et al, Tetrahedron: Asymmetry, 12, 2657-2661, 2001 ; and Wang et al, Euro. J. Org. Chem. 834-839, 2002) are either long (10-12 steps) or involve specialized, dangerous and/or non-scaleable chemistry.

[0005] Additional methods for synthesizing 4-hydroxyisoleucine have been reported in U.S. Patent Application Publication No. US 2003/0219880; and by Rolland-Fulcrand et al, Eur. J. Org. Chem. 873-877, 2004. More recent reports by Mioskowski et al, (WO 2004/052836 and WO 2004/099120) appear promising, but have not been tested from a scale-up perspective.

[0006] The present disclosure refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0007] The present disclosure relates to methods for the synthesis of 4- hydroxyisoleucine (4-HIL), stereoisomers, derivatives and analogs thereof. In an embodiment, the present disclosure relates to a method for the synthesis of (25^r,3Λ,4.S)-4-hydroxyisoleucine, as well as stereoisomers, derivatives and analogs thereof, comprising readily available starting materials and commonly used chemical modifications and reactions. In a further embodiment, the present disclosure relates to a scalable method for the synthesis of (2S,3/?,4S)-4- hydroxyisoleucine, as well as stereoisomers, derivatives and analogs thereof. In yet a further embodiment, the present disclosure relates to a method for the synthesis of (25',3Λ,45)-4-hydroxyisoleucine.

[0008] In an embodiment, the present disclosure relates to a method for synthesizing compounds of Formula I, including stereoisomers and analogs thereof:

Formula I

[0009] wherein X is selected from the group consisting of hydroxy, amino and thiol; the method comprising reacting a compound of Formula II:

Formula II

[0010] wherein X is selected from the group consisting of hydroxy, amino and thiol; with an alkyl isocyanoacetate of Formula III:

O

Il ⁺Φ

O ^^ Formula HI

[0011] wherein R is an alkyl group; providing a compound of Formula

IV:

Formula IV

|0012] wherein Z is selected from the group consisting of O, NH or S.

[0013] In an embodiment of the present disclosure, the method further comprises subjecting the compound of Formula IV to a hydrogenation step, providing a compound of Formula V:

Formula V

[0014] wherein Z is selected from the group consisting of O, NH or S.

[0015] In yet a further embodiment of the present disclosure, the method further comprises hydrolyzing the compound of Formula V, providing a compound of Formula I.

[0016] In an embodiment, the present disclosure relates to a method for synthesizing compounds of Formula I', including stereoisomers and analogs thereof:

I' [0017] the method comprising reacting a compound of Formula II':

OH Formula I!'

[0018] with an alkyl isocyanoacetate of Formula III:

O

Il ⁺<£^c

O ^^ Formula HI

[0019] wherein R is an alkyl group; providing a compound of Formula

IV:

Formula IV

[0020] In an embodiment of the present disclosure, the method further comprises subjecting the compound of Formula IV to a hydrogenation step, providing a compound of Formula V:

Formula V [0021] In yet a further embodiment of the present disclosure, the method further comprises hydrolyzing the compound of Formula V, providing a compound of Formula I'.

[0022] In an embodiment, the present disclosure relates to a method for synthesizing compounds of Formula I, including stereoisomers and analogs thereof:

Formula I

[0023] wherein X is selected from the group consisting of hydroxy, amino and thiol; the method comprising hydrolyzing a compound of Formula V:

Formula V

[0024] wherein Z is selected from the group consisting of O, NH or S.

]0025] In an embodiment, the present disclosure relates to a method for synthesizing compounds of Formula I', including stereoisomers and analogs thereof:

[0026] the method comprising hydrolyzing a compound of Formula V:

Formula V.

[0027] In an embodiment, the present disclosure relates to a method for the synthesis of the eight (8) possible stereoisomers of 4-hydroxyisoleucine starting from 3-hydroxy-2-butanone (i.e. acetoin) and alkyl isocyanoacetate. In yet a further embodiment, the present disclosure relates to a method for the synthesis of (25^r,3Λ,45)-4-hydroxyisoleucine starting from 3-hydroxy-2-butanone (i.e. acetoin) and alkyl isocyanoacetate. In yet a further embodiment, the present disclosure relates to a method for the diastereoselective synthesis of (2S,3R,4S)-4- hydroxyisoleucine starting from (5)-acetoin and alkyl isocyanoacetate.

[0028] In an embodiment, the present disclosure relates to a method for the synthesis of (25,3/?,45)-4-hydroxyisoleucine, its stereoisomers, derivatives and analogs thereof, from a condensation reaction comprising racemic unprotected acetoin and alkyl isocyanoacetate. In yet a further embodiment, the present disclosure relates to a method for the synthesis of (25,37?,45)-4-hydroxyisoleucine from a condensation reaction comprising racemic unprotected acetoin and alkyl isocyanoacetate.

[0029] In an embodiment, the present disclosure relates to a method comprising: (i) producing an unsaturated N-formyl lactone starting from racemic unprotected acetoin and alkyl isocyanoacetate; (ii) hydrogenating the unsaturated N- formyl lactone yielding saturated N-formyl lactone; and (iii) hydrolyzing the saturated N-formyl lactone producing (25,3Λ,45)-4-hydroxyisoleucine and (27?,3_lS',45)-4-hydroxyisoleucine as well as (27?,35',4/?)-4-hydroxyisoleucine and (25,3/?,4Λ)-4-hydroxyisoleucine. [0030] In an embodiment, the present disclosure relates to a method comprising: (i) producing an unsaturated (5)-N-formyl lactone starting from (S)- acetoin and alkyl isocyanoacetate; (ii) performing homogeneous hydrogenation on the unsaturated (5)-N-formyl lactone yielding saturated OS)-N -form yl lactone; and (iii) hydrolyzing the saturated (5)-N-formyl lactone producing (2S,3R,4S)-A- hydroxyisoleucine.

[0031) In an embodiment, the present disclosure relates to the use of acetoin and alkyl isocyanoacetate to prepare the various stereoisomers of A- hydroxyisoleucine. In an embodiment, the stereoisomer produced is (2S,3R,4S)-4- hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (25,3Λ,4/?)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (25,35, 45)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (25,35,4i?)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (2Λ,35,45)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (2Λ,35,4i?)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (2Λ,3Λ,45)-4-hydroxyisoleucine. In a further embodiment, the stereoisomer produced is (2R,3R,4R)-4- hydroxyisoleucine.

[0032] In an embodiment, the present disclosure relates to the use of

(S)-acetoin and alkyl isocyanoacetate to prepare (25,3i?,45)-4-hydroxyisoleucine. In a further embodiment, the present disclosure relates to the synthesis of {2S,3R,4S)-4- hydroxyisoleucine via an isolable saturated (5)-N-formyl lactone intermediate.

[0033] In an embodiment, the present disclosure relates to a method for the synthesis of analogs of 4-hydroxyisoleucine and/or for the synthesis of derivatives thereof. [0034] In an embodiment, the present disclosure relates to a method for the synthesis of analogs of (2<S^I,3Λ,45')-4-hydroxyisoleucine and/or for the synthesis of derivatives thereof.

[0035] In an embodiment, the present disclosure relates to pharmaceutical compositions comprising 4-hydroxyisoleucine and/or stereoisomers and/or analogs and/or derivatives thereof, prepared according to the methods described herein, and to the use of any of these compounds for the prevention and/or treatment of various diseases and conditions including but not limited to obesity and disorders of carbohydrate metabolism such as diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes and Metabolic Syndrome X.

[0036] In an embodiment, the present disclosure relates to pharmaceutical compositions comprising (25,3/?,45)-4-hydroxyisoleucine and/or analogs and/or derivatives thereof, prepared according to the methods described herein, and to the use of any of these compounds for the prevention and/or treatment of various diseases and conditions including but not limited to obesity and disorders of carbohydrate metabolism such as diabetes mellitus (type 1 and type 2 diabetes), pre-diabetes and Metabolic Syndrome X.

[0037] In an embodiment, the present disclosure relates to pharmaceutical compositions comprising neutraceutical properties; the compositions comprising a neutraceutically effective amount of 4-hydroxyisoleucine and/or stereoisomers and/or analogs and/or derivatives thereof, prepared according to the methods described herein.

[0038] In an embodiment, the present disclosure relates to prophylactic compositions comprising neutraceutical properties; the compositions comprising a neutraceutically effective amount of 4-hydroxyisoleucine and/or stereoisomers and/or analogs and/or derivatives thereof, prepared according to the methods described herein.

[0039] In an embodiment, the present disclosure relates to pharmaceutical compositions comprising neutraceutical properties; the compositions comprising a neutraceutically effective amount of (25,3/?,45)-4-hydroxyisoleucine and/or analogs and/or derivatives thereof, prepared according to the methods described herein.

[0040] In an embodiment, the present disclosure relates to prophylactic compositions comprising neutraceutical properties; the compositions comprising a neutraceutically effective amount of (25,3Λ,45)-4-hydroxyisoleucine, and/or analogs and/or derivatives thereof, prepared according to the methods described herein.

[0041] In an embodiment, the present disclosure relates to methods for the stereoselective synthesis of the stereoisomers of 4-hydroxyisoleucine. The synthetic processes for the enantio- and diastereoselective synthesis of the various stereoisomers comprise readily available starting materials and commonly used chemical modifications and reactions. The synthetic processes of the present disclosure are adaptable to the synthesis of analogs and/or derivatives thereof.

[0042] In an embodiment, the present disclosure relates to a method for the stereoselective synthesis of (2»S,3/?,4S)-4-hydroxyisoleucine. In a further embodiment, the present disclosure relates to a method for the enantioselective synthesis of (25^r,3i?,45)-4-hydroxyisoleucine. In a further embodiment, the present disclosure relates to a method for the diastereoselective synthesis of (2S,3R,4S)-4- hydroxyisoleucine. The synthetic processes for the enantio- and diastereoselective synthesis of (25,3Λ,45)-4-hydroxyisoleucine comprise readily available starting materials and commonly used chemical modifications and reactions. The synthetic processes of the present disclosure are adaptable to the synthesis of analogs and/or derivatives thereof.

[0043] In an embodiment, the present disclosure relates to a method for the synthesis of salts, solvates, crystal forms, active metabolites and/or prodrugs of the stereoisomers of 4-hydroxyisoleucine. Non-limiting examples of prodrugs of 4- hydroxyisoleucine comprise compounds wherein a suitable functionality such as, but not limited to, a hydroxy, an amino or a carboxyl group is derivatized with a biologically or chemically labile moiety that may be cleaved in vivo, to regenerate 4- hydroxyisoleucine or a pharmaceutically active analog of 4-hydroxyisoleucine.

[0044] In an embodiment, the present disclosure relates to a method for the synthesis of salts, solvates, crystal forms, active metabolites and/or prodrugs of (25,3Λ,45')-4-hydroxyisoleucine. Non-limiting examples of prodrugs of (2S,3R,4S)- 4-hydroxyisoleucine comprise compounds wherein a suitable functionality such as, but not limited to, a hydroxy, an amino or a carboxyl group is derivatized with a biologically or chemically labile moiety that may be cleaved in vivo, to regenerate (25,3Λ,45)-4-hydroxyisoleucine or a pharmaceutically active analog thereof.

[0045] Finally, the present disclosure relates to the use of any one or more of the stereoisomers of 4-hydroxyisoleucine and/or analogs and/or derivatives, thereof as active ingredients in pharmaceutical compositions for treatment and/or prophylactic purposes. In an embodiment, the present disclosure relates to the use of (25',3/?,45)-4-hydroxyisoleucine and/or analogs and/or derivatives thereof as active ingredients in pharmaceutical compositions for treatment and/or prophylactic purposes.

[0046] The foregoing and other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non- restrictive description of illustrative embodiments thereof, given by way of example only and which should not be interpreted as limiting the scope of the present disclosure.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0047] The present disclosure relates to methods for the synthesis of 4- hydroxyisoleucine, stereoisomers, derivatives and analogs thereof. In an embodiment, the present disclosure relates to a method for the synthesis of (25,3_JR,45)-4-hydroxyisoleucine, diastereomers, derivatives and analogs thereof. The methods provides for the preparation of large quantities of the stereoisomers, in optically pure quality and in an economically advantageous and practical manner. In an embodiment, the present disclosure relates to a method for the synthesis of the eight (8) possible stereoisomers of 4-hydroxyisoleucine starting from 3-hydroxy-2- butanone (i.e. acetoin) and alkyl isocyanoacetate. In an embodiment, the present specification relates to methods for the synthesis of derivatives and/or analogs of (25^r,3^,45)-4-hydroxyisoleucine, (2S,3#,4fl)-4-hydroxyisoleucine, (25,35,45)-4- hydroxyisoleucine, (2£3S,4Λ)-4-hydroxyisoleucine, (2R,3S,4S)-4- hydroxyisoleucine, (2/?,3S,4.K)-4-hydroxyisoleucine, (2R,3R,4S)-4- hydroxyisoleucine and (2/?,3Λ,47?)-4-hydroxyisoleucine.

[0048] In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this specification pertains.

[0049] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Similarly, the word "another" may mean at least a second or more. [0050] As used in this specification and claim(s), the words

"comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0051] The term "about" is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value.

[0052] As used in this specification, the term "alkyl" or "alk" refers to a monovalent group derived from a straight or branched chain saturated hydrocarbon comprising, unless otherwise specified, from 1 to 6 carbon atoms and is exemplified by methyl, ethyl, n- and /so-propyl, n-, sec-, iso- and

neopentyl and the like and may be optionally substituted with one, or more substituents.

[0053] As used in this specification, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers in which the connectivity of the atoms is the same but which differ in the arrangement of the atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers". Stereoisomers that are non-superimposable mirror images of each other are termed "enantiomers". When a compound comprises an asymmetric center (i.e. chiral center), for example, a carbon atom bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center, which is designated by the Cahn-Ingold-Prelog R- and 5-sequence rules. Alternatively, an enantiomer can be characterized by the manner in which it rotates the plane of polarized light, designated as dextrorotatory [(+)-enantiomer] or levorotatory [(-)-enantiomer]. A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0054] As used in this specification, the term "4-hydroxyisoleucine" or

"4-OH" refers to the compound 4-hydroxy-2-amino-3-methylpentanoic acid and to stereoisomers thereof. Typically, it refers to the (25,3Λ,45)-4-hydroxyisoleucine isomer.

[0055] As used in this specification, the term "analog(s) of 4- hydroxyisoleucine" or "analog(s) of 4-OH", refers to chemical analogs and derivatives of 4-hydroxyisoleucine. Non-limiting examples of such analogs and derivatives include lactones, salts, metabolites, solvates and prodrugs.

[0056] As used in this specification, the term "stereoisomer of 4- hydroxyisoleucine" refers to one of the following compounds: (2S,3RAS)-, (2S.3RAR)-, (2S,3SAR)-, (2S.3SAS)-, (2R.3SAS)-, (2R.3RAS)-, (2R.3SAR)-, or (2R, 3i?,4i?)-4-hydroxyisoleucine.

[0057] As used interchangeably in this specification, the term "carboxy" or "carboxyl" refers to a CO₂H group.

[0058] As used interchangeably in this specification, the term "carboxy protecting group" or "carboxyl protecting group" refers to those groups intended to protect a CO₂H group against undesirable reactions during synthetic procedures. Commonly used carboxyl -protecting groups are disclosed in Greene, "Protective Groups In Organic Synthesis, 4^th Edition" (Chapter 5) (John Wiley & Sons, New Jersey, 2007), which is incorporated herein by reference. [0059] As used in this specification, the term "amino" refers to an -NH₂ group.

|0060] As used in this specification, the term "N-protecting group" or

"nitrogen protecting group" refers to those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups In Organic Synthesis, 4^th Edition" (Chapter 7) (John Wiley & Sons, New Jersey, 2007), which is incorporated herein by reference. Non-limiting examples of N-protecting groups comprise acyl, aroyl, and carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, f-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, ø-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4- chlorobenzoyl, 4-bromobenzoyl and 4-nitrobenzoyl. Additional non-limiting examples of N-protecting groups comprise chiral auxiliaries such as protected or unprotected D or L-amino acids such as alanine, leucine, phenylalanine and the like; sulfonyl groups such as benzenesulfonyl, />-toluenesulfonyl and the like; carbamate forming groups such as benzyloxycarbonyl, /?-chlorobenzyloxycarbonyl, /^-methoxybenzyloxycarbonyl^-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, _jD-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5- dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,

3,4,5-trimethoxybenzyloxycarbonyl, l -(p-biphenylyl)-l-methylethoxycarbonyl, α,α- dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t- butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2, -trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Typical N-protecting groups comprise formyl, acetyl, benzoyl, pivaloyl, t- butylacetyl, alanyl, phenyl sulfonyl, benzyl, ?-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

[0061] As used in this specification, the term "N-protected amino" refers to an amino group protected by an N-protecting or nitrogen-protecting group.

[0062] As used in this specification, the term "carbonyl" refers to a

C(O) group. In an embodiment of the present specification, the C(O) group may be represented as C=O.

[0063] As used in this specification, the term "thiocarbonyl" refers to a

C(S) group. In an embodiment of the present specification, the C(S) group may be represented as C=S.

[0064] As used interchangeably in this specification, the term "halogen" or "halo" refers to F, Cl, Br or I.

[0065] As used interchangeably in this specification, the term

"hydroxy" or "hydroxyl" refers to an -OH group.

[0066] As used in this specification, the term "nitro" refers to an -NO₂ group.

[0067] As used in this specification, the term "thiol" refers to an SH group.

[0068] As used in this specification, the term "oxo" refers to =0.

[0069] As generally understood by those of skill in the art, an optically pure compound is one that is enantiomerically pure. As used in this specification, the term "optically pure" refers to a compound comprising a substantial excess of a single enantiomer. Typically, "optically pure" refers to a compound comprising at least 90% of a single isomer (80% enantiomeric excess (e.e.)). In an embodiment, "optically pure" refers to a compound comprising at least 95% of a single isomer (90% e.e.). In an embodiment, "optically pure" refers to a compound comprising at least 97.5% of a single isomer (95% e.e.). In an embodiment, "optically pure" refers to a compound comprising at least 99% of a single isomer (98% e.e.).

[0070] The compounds of the present disclosure comprise asymmetric

(i.e. chiral) centers. Unless indicated otherwise, the description or naming of a particular compound in the present disclosure and claims is intended to include all individual isomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well- known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 6th edition J. March, John Wiley and Sons, New Jersey, 2007). Individual stereoisomers of the compounds of the present disclosure can be prepared synthetically from commercially available starting materials comprising asymmetric (i.e. chiral) centers, or by the preparation of enantiomeric mixtures followed by resolution using methods well-known to those of ordinary skill in the art. Methods of resolution may be exemplified by: (a) attachment of the racemic mixture of enantiomers, designated (+/-), to a chiral auxiliary; (b) separation of the resulting diastereomers by recrystallization or chromatography; and (c) liberation of the optically pure product from the auxiliary. Alternatively, the mixture of enantiomers may be directly separated by means of chiral chromatographic columns. Chiral centers are designated herein by the symbols "/?" or "5," depending on the configuration of substituents around the chiral carbon atom, or are drawn by conventional means with a bolded line defining a substituent above the plane of the page in three-dimensional space and a hashed or dashed line defining a substituent beneath the plane of the page in three-dimensional space. [0071] It should be noted that throughout the present disclosure, when an atom is shown without hydrogen(s), but hydrogens are required or chemically necessary to form stable compounds, such hydrogens should be inferred as being part of the compound.

[0072] The present specification refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0073] Abbreviations: NMR: Nuclear Magnetic Resonance; MS: Mass

Spectrometry; m.p.: melting point; HRMS: High Resolution Mass Spectrometry; EtOAc: Ethyl acetate; CH₂Cl₂: Dichloromethane; CDCl₃: Chloroform-d; DBU: l ,8-diazabicyclo[5.4.0]undec-7-ene; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; TBDPS: f-Butyldiphenylsilyl; AcOH: Acetic acid; TLC: Thin Layer Chromatography; FAB: Fast Atom Bombardment; rt: room temperature; cat: catalyst; nbd: norbornadiene.

[0074] In accordance with an embodiment of the present disclosure, a general method for the synthesis of the various stereoisomers of 4- hydroxyisoleucine starting from acetoin and alkyl isocyanoacetate is illustrated hereinbelow in Scheme 1. Starting with either chiral acetoin, racemic acetoin or enantiomerically enriched acetoin, a portion of the desired (2S,3/?,4iS)-N-formyl lactone product can be found in the reaction mixture. Hydrolysis of the saturated lactone is typically achieved by first refluxing in the presence of a strong acid, drying the product, followed by treatment with a strong base. The condensation reaction is typically achieved under alkaline reaction conditions. Non-limiting examples of alkaline conditions comprise the use of bases such as KOz¹Bu, NaH, n- BuLi and amine bases such as DBU. Other bases are known in the art and are within the capacity of a skilled technician.

Scheme 1

[0075] In accordance with an embodiment of the present disclosure, a general method for the synthesis of (25,3/?,46)-4-hydroxyisoleucine starting from acetom and alkyl isocyanoacetate is illustrated hereinbelow in Scheme 2.

(2R,3S,4S) (2S,3R,4R)

Scheme 2

[0076] The condensation reaction between alkyl isocyanoacetate and acetoin provides access to an N-formyl lactone intermediate, which plays an important role in the synthesis of the vaπous stereoisomers of 4-hydroxyisoleucine The condensation reaction between alkyl isocyanoacetate 4 and unprotected racemic acetoin 7 was investigated and was shown to provide racemic unsaturated N-formyl lactone 8 in yields ranging from about 15 to about 40% following work-up and purification by flash chromatography on silica gel (Scheme 3) The presence of side product having the (Z)-geometry could not be discerned by either LC-MS or ¹H NMR analysis.

Scheme 3

[0077] Hydrogenation of racemic unsaturated N-formyl lactone 8 under homogeneous conditions, using an achiral catalyst, provided the desired saturated N- formyl lactone 9 as part of a mixture mainly comprising three other stereoisomers (Scheme 4). Trace amounts of the other possible stereoisomers were also observed following chiral GC analysis of the crude reaction mixture. In an embodiment of the present disclosure, the hydrogenation of racemic unsaturated N-formyl lactone 8 was performed by means of an achiral rhodium catalyst. In yet a further embodiment of the present disclosure, the hydrogenation of racemic unsaturated N-formyl lactone 8 was performed using the achiral rhodium catalyst [Rh(COD)(diPFc)]BF₄. The hydrogenation reaction proceeds with good stereoselectivity for stereoisomers 9 and 10. Other achiral catalysts are known in the art and are within the capacity of a skilled technician.

9; 2S,3R,4S 10; 2R,3S,4R

11 ; 2R,3S,4S 12; 2S,3R,4R

Scheme 4

[0078] Asymmetric hydrogenation of racemic unsaturated N-formyl lactone 8, under conditions of dynamic kinetic resolution (DKR) using a chiral catalyst, to prepare saturated N-formyl lactone 9 was also performed. In an embodiment of the present disclosure, the chiral catalyst was Rh/SL-F356-1 (SL- F356-1 is l-Dicylohexylphosphino-l'-[{(2"-N,N-dimethylaminoeth-l- yl)ferrocenyl}(phenyl)phosphino]ferrocene available from Solvias AG. Rh/SL- F356-1 is prepared by dissolving 11 mg (0.0148 mmol) of ligand SL-F356-1 and 5.4 mg (0.0144 mmol) of [Rh(nbd)₂]BF₄ in 0.8 ml of CD₃OD followed by stirring for 10 minutes (WO 2007/1 16081 ; see also WO 2007/051576 for a discussion on homogeneous catalysis). In yet a further embodiment of the present disclosure, the asymmetric hydrogenation was performed on an enantiomerically pure form of N- formyl lactone 8.

[0079] In accordance with an embodiment of the present disclosure, the condensation reaction can be performed using alkyl isocyanoacetate 4 and an enantiomer of acetoin 7 to provide a stereoisomer of N-formyl lactone 8. Thus the condensation reaction between (S)-acetoin 13 and alkyl isocyanoacetate 4 was investigated and provided (S)-lactone 16 in modest yields (-43%) (Scheme 5).

Scheme 5

[0080] (iS)-Acetoin 13 can be obtained, for example, by means of benzyl-protected (5)-ethyl lactate 14, prepared from commercially available (S)- ethyl lactate 1, followed by low temperature reaction with MeLi and deprotective hydrogenation (Scheme 6).

Scheme 6

[0081] Hydrogenation of (S)-lactone 16 under homogeneous conditions, using the achiral rhodium catalyst [Rh(COD)(diPFc)]BF₄, provided the desired lactone 9 as part of a reaction mixture comprising a product distribution substantially identical to that previously obtained for racemic N-formyl lactone 8, as confirmed by chiral GC analysis. While not wishing to be bound by any theory, this result would suggest that epimerization of 13 or 16 could have taken place and that N-formyl lactone 16 was in fact a racemic mixture. [0082] In accordance with an embodiment of the present disclosure, and as illustrated hereinbelow in Scheme 7, the condensation reaction between TBDPS- protected (S)-acetoin 3 and alkyl isocyanoacetate 4 was investigated.

5 (E)-lsomer 6 (Z)-lsomer

Scheme 7

[0083] Hydrogenation of the alkene moiety of 5 provides for the installment of the stereogenic centers comprising the 25" and 3R configuration respectively. An analogous hydrogenation reaction on the alkene moiety of substrate 6 provides for the installment of stereogenic centers having the 25 and 35 configuration respectively. As illustrated herein below in Table 1, and in accordance with the literature [Schδllkopf et al. Liebigs Ann. Chem. (1972), 766, 1 16-129; Chem. Ber. (1976), 109, 3964-5], the (Z)-isomer is predominantly obtained.

[0084] Table 1: Condensation reaction between TBDMS-protected (S)- acetoin (3) and ethyl isocyanoacetate (4; R = ethyl).

*For entries 4-7 only traces of product could be detected by LC-MS analysis, The Z/E ratio was determined by ¹H NMR analysis following aqueous work-up of the crude reaction mixture.

[0085] TBDPS-protected (S)-acetoin 3 can be readily obtained starting from commercially available (S)-ethyl lactate 1 (Scheme 8). In an embodiment of the present disclosure, as illustrated in Schemes 6 and 8, the acyl substitution is performed by means of an alkyl lithium (e.g. methyl lithium) reagent. Other organometallic reagents are known in the art (e.g. Grignard reagents), and are within the capacity of a skilled technician.

MeLi

DPS - 1 1 O ⁰C OTBDPS

Scheme 8

[0086] The hydrolysis of N-formyl lactone 9 to (25,3Λ,45)-4- hydroxyisoleucine was investigated. Treatment of (2.S,3/?,45)-4-hydroxyisoleucine with a stoichiometric mixture comprising formic acid and acetic anhydride afforded N-formyl lactone 9 as a crystalline solid comprising about 10% of the (2S,3R,4R)- stereoisomer (Scheme 9). Recrystallization afforded the desired N-formyl lactone 9 in a diastereomeric ratio > 98:2 as confirmed by GC analysis. Formic Acid

Acetic anhydride

Scheme 9

[0087] Refluxmg N-formyl lactone 9 with a mixture compπsing HCl

(2N) and dioxane afforded the free amino product ¹H NMR analysis of the crude reaction mixture confirmed the presence of large amounts of the free amino product Evaporating the reaction mixture to dryness, followed by treatment of the residue with an ethanolic solution compπsing aqueous NaOH (2 eq ) and subsequent neutralization using HCl (IN), afforded the desired (25,3i?,45)-4-hydroxyisoleucine having an optical puπty exceeding 99%

[0088] Treatment of N-formyl lactone 9 with an ethanolic solution compπsing aqueous NaOH (2 eq ), followed by neutralization with HCl (IN), afforded the desired (25',3i?,45)-4-hydroxyisoleucine in an optical puπty of about 73% It appears important that the formyl group be removed pπor to πng opening as the formylated product is prone to epimeπzation under basic conditions ¹H NMR analysis of the crude reaction mixture confirmed the presence of formyl-substituted ring opened product

[0089] Hydrogenation of N-formyl lactone 9 (H₂-Pd/C) in a mixture compπsing HCl (4N) and dioxane afforded the free amino product following filtration and evaporation of the crude reaction mixture Treatment of the residue with an ethanolic solution compπsmg aqueous NaOH (2 eq ) and subsequent neutralization using HCl (IN), afforded the desired (25,3Λ,45)-4-hydroxyisoleucine in an optical punty of about 79% [0090] As will be described in greater detail in the Experimental section hereinafter, the synthesis of (25,3/?,4>S)-4-hydroxyisoleucine, as well as stereoisomers, derivatives and analogs thereof was accomplished starting from a condensation reaction comprising either (S)-acetoin or racemic unprotected acetoin and alkyl isocyanoacetate. Moreover, as will be described in greater detail in the Experimental section hereinafter, a diastereoselective synthesis of (2S,3R,4S)-4- hydroxyisoleucine from a condensation reaction comprising (S)-acetoin and ethyl isocyanoacetate was accomplished.

|0091] Experimental

[0092] Condensation reaction between ethyl isocvanoaeetate (4) and

TBDPS-protected (S)-acetoin (3).

[0093] TBDPS-protected (S)-acetoin 3 was obtained from commercially available (5)-ethyl lactate 1. A solution of ethyl isocyanoacetate 4 (566 mg, 5 mmol) in THF (5ml) was slowly added under an argon atmosphere to a solution of KOtBu (5 ml of a IM solution in THF, 5 mmol) and additional THF (15 ml) at 0⁰C. The brown suspension was stirred for 5 minutes followed by the addition of a solution of 3 (1.63 g, 5 mmol) in THF (10 ml). The reaction mixture was stirred for 10 minutes at O⁰C followed by warming to room temperature. The reaction mixture was evaporated and the residue combined with ethyl acetate and water. Following neutralization with hydrochloric acid (IN), the layers were separated. The aqueous layer was subsequently extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na₂SO₄ and evaporated. The crude product (2.2 g as a yellow oil) was analyzed by ¹H NMR showing a Z/E ratio in excess of 4:1. The products were not separated by LC-MS or HPLC. [0094] Condensation reaction between ethyl isocvanoacetate (4) and raeemie unprotected acetoin (7).

[0095] Ethyl isocyanoacetate 4 (2.26 g, 20 mmol) was added under an argon atmosphere to a solution of KOtBu (20 ml of a 1 M solution in THF, 20 mmol) and additional THF (50 ml) at room temperature. The brown suspension was stirred for 5 minutes followed by the rapid addition of 7 (1.80 g, 20 mmol). The addition caused the solution to warm to 45°C. The reaction mixture was stirred for 15 minutes and was subsequently quenched by the addition of acetic acid (20 mmol). Ethyl acetate (100 ml) was subsequently added. The crude solution was washed with water (2 x 50 ml) and brine (50 ml). The organic layer was separated, dried over Na₂SO₄ and evaporated. The crude product was purified by flash chromatography on silica gel (EtOAc eluant) to yield the product as a yellow oil (450 mg, 15%).

[0096] Preparation of (SVacetoin (13).

[0097] The benzyl-protected (S)-acetoin 15 was prepared following the procedure reported by Denmark et al. (J. Am. Chem. Soc. 2000, 122(37), 8837- 8847). Compound 15 (6.6 g, 37 mmol) was dissolved in ethyl acetate (90 ml) and hydrogenated (H₂ pressure of 1 bar) over a period of 40 hours using palladium on charcoal as the catalyst (10% Pd/C, 3.35 g). The catalyst was removed by filtration and was thoroughly washed with ethyl acetate. The combined filtrates were carefully evaporated under reduced pressure (-150 mbar; water bath temperature <45°C). The crude product (3.26 g as a colorless oil), which still contained some toluene and ethyl acetate, was used without further purification. A sample of the so- obtained crude product was found to be chemically stable in CDCl₃ over a period of 10 days and exhibited the following specific optical rotation: [α]o²⁰⁼ + 68° [c=0.1 , CDCl₃]; Literature: [α]_D ²⁵= + 96° [c=5, CDCl₃] (Bel-Rhlid, Rachid; Renard, Michel F.; Veschambre, Henri; Bull. Soc. Chim. Fr. 1996, 133(10), 101 1-1022). [0098] Condensation reaction between ethyl isocyanoacetate (4) and

(S)-acetoin (13).

[0099] Ethyl isocyanoacetate 4 (13.57 g, 120 mmol) was added under an argon atmosphere to a solution of KOtBu (120 ml of IM solution in THF, 120 mmol) and additional THF (280 ml) at room temperature. The brown suspension was stirred for 5 minutes followed by the rapid addition of 13 (10.6 g, 120 mmol). The addition caused the mixture to warm to 43⁰C. The reaction mixture was stirred for 15 minutes and was subsequently quenched by the addition of acetic acid (120 mmol). Ethyl acetate (400 ml) and hydrochloric acid (0.5 M; 80 ml) were added and the mixture was stirred for an additional 45 minutes. The layers were then separated. The organic layer was washed with water (2 x 100 ml) and brine (100 ml), then dried over Na₂SO₄ and evaporated. The crude product was purified by flash chromatography on silica gel (EtOAc eluant) to yield the product as a yellow oil (8.1 g, 43 %).

[OOIOOJ Hvdrogenation of lactone 8.

[00101 J Racemic unsaturated N-formyl lactone 8 (155 mg, 1 mmol) was dissolved in MeOH (8 ml) and hydrogenated (H₂; pressure of 50 bar) over a period of 19 hours using Rh(COD)(diPFc)BF₄ (28.5 mg, 0.04 mmol) as the catalyst (diPFc being the achiral ligand). The reaction mixture was diluted using EtOAc and filtered using a pad of celite, followed by further filtration using a pad of silica gel. The filtrate was evaporated to dryness to yield the product as a yellow oil (150 mg, 96%). As observed by chiral GC analysis, the product was in fact a mixture of stereoisomers comprising 9 (2S,3R,4S), 10 (2R,3S,4R), H (2Λ,3S,45), and 12 (2S,3R,4R) in a 10:10:1 : 1 ratio. An essentially identical product distribution was observed when the hydrogenation was carried out using (S)-lactone 16. [00102] Preparation of qS^RΛS^-iV-formvI-SΛ-dimethvi- butanolide 9.

|00103] Acetic anhydride (9.4 ml, 100 mmol) was cooled to O⁰C under an argon atmosphere followed by the slow addition of formic acid (3.8 ml, 100 mmol) while stirring. The mixture was subsequently heated to 55°C over a period of 2 hours and then cooled to room temperature. The mixture (4.6 ml) was added to (25,3/?,45)-4-hydroxyisoleucine (670 mg, 4.6 mmol) followed by stirring at room temperature over a period of 20 hours. The reaction mixture was evaporated to dryness, followed by co-evaporation with toluene, EtOAc and CH₂Cl₂ (twice with each) to remove any residual acetic acid. The product was obtained as a colorless crystalline solid having a diastereomeric ratio > 91 :9. Recrystallization from CH₂Cl₂ / pentane afforded pure 9 (diastereomeric ratio > 98:2).

[00104] Hydrolysis of (2S,3R,4S)-2-N-formvI-3.4-dimethyl- butanolide 9.

[00105] Compound 9 (60 mg, 0.38 mmol, diastereomeric ratio > 98:2) was dissolved in 1,4-dioxane (5 ml). Following the addition of HCl (2N, 5 ml, 10 mmol), the reaction mixture was refluxed for 1 hour. The reaction mixture was cooled to room temperature and evaporated to dryness. The crude product was further dried under high vacuum over a period of 2 hours. The dried product was then dissolved in ethanol (1.5 ml) and treated with an aqueous NaOH solution (IN, 1.5 ml, 1.5 mmol) over a period of 20 hours. The reaction mixture was then carefully neutralized by the dropwise addition of HCl (IN) and evaporated to dryness. As observed by chiral HPLC analysis, (25',3/?,45)-4-hydroxyisoleucine was obtained having an optical purity exceeding 99%.

[00106] It is to be understood that the disclosure is not limited in its application to the details of construction and parts as described hereinabove. The disclosure is capable of other embodiments and of being practiced in various ways. It is also understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present disclosure has been described hereinabove by way of illustrative embodiments thereof, it can be modified, without departing from the spirit, scope and nature of the subject disclosure as defined in the appended claims.

REFERENCES

1. Fowden et al. Phytochemistry 12:1707-171 1 , 1973.

2. Pάco<± et al. Phytochemistry 28:1835-1841, 1989.

3. Broca et al. Am. J. Physiol. 277:E617-E623, 1999.

4. Broca et al. Eur. J. Pharmacol. 390:339-345, 2000.

5. U.S. Patent 5,470,879.

6. PCT Publications WO 97/32577, WO 01/15689, and WO 2005/039626.

7. Inghardt et al. Tetrahedron, 47, No. 32, 6469-6482, 1991.

8. Dong et al. J. Org. Chem. 64, 2657-2666, 1999.

9. Kassem et al. Tetrahedron: Asymmetry, 12, 2657-2661, 2001.

10. Wang et al, Euro. J. Org. Chem. 834-839, 2002.

11. U.S. Patent Application Publication 2003/0219880.

12. Rolland-Fulcrand et al. Eur. J. Org. Chem. 873-877, 2004.

13. PCT Publications WO 2004/052836 and WO 2004/099120.

14. Greene, T. W. and Wuts, P. G. M. Protective Groups In Organic Synthesis, 4^th Edition John Wiley & Sons, New Jersey, 2007 (Chapter 5).

15. Greene, T.W. and Wuts, P. G. M. Protective Groups In Organic Synthesis, 4^l Edition John Wiley & Sons, New Jersey, 2007 (Chapter 7).

16. March, J. Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, New Jersey, 2007 (Chapter 4).

17. PCT Publications WO 2007/116081 and WO2007/051576.

18. Schollkopf et al. Liebigs Ann. Chem. 766, 1 16-129, 1972; Chem. Ber. 109, 3964- 5, 1976.

19. Denmark et al. J. Am. Chem. Soc, 122(37), 8837-8847, 2000.

20. Bel-Rhlid, R.; Renard, Michel F.; Veschambre, H. Bull. Soc. Chim. Fr. 133(10), 1011-1022, 1996.

Claims

WHAT IS CLAIMED IS:

1. A method for synthesizing compounds of Formula I, including stereoisomers and analogs thereof:

Formula I

wherein X is selected from the group consisting of hydroxy, amino and thiol; said method comprising: a) reacting a compound of Formula II:

Formula II

wherein X is selected from the group consisting of hydroxy, amino and thiol; with an alkyl isocyanoacetate of Formula III:

Formula III

wherein R is an alkyl group; providing a compound of Formula IV:

wherein Z is selected from the group consisting of O, NH or S.

2. The method of claim 1, further comprising subjecting the compound of Formula IV to a hydrogenation step, providing a compound of Formula V:

Formula V

wherein Z is selected from the group consisting of O, NH or S.

3. The method of claim 2, further comprising hydrolyzing the compound of Formula V, providing a compound of Formula I.

4. The method of claim 1, wherein the compound of Formula II is selected from the group consisting of a racemic mixture, a non-racemic mixture, (S)-enantiomers and (R)-enantiomers.

5. The method of claim 4, wherein step a) is carried out under alkaline conditions.

6. The method of claim 5, wherein the alkaline conditions comprise using a base selected from the group consisting of K0?Bu, NaH, «-BuLi and amine bases such as DBU.

7. The method of claim 2, wherein said hydrogenation comprises using an achiral catalyst.

8. The method of claim 7, wherein the achiral catalyst comprises an achiral rhodium catalyst.

9. The method of claim 8, wherein the achiral rhodium catalyst comprises Rh(COD)(diPFc)BF₄.

10. The method of claim 2, wherein said hydrogenation comprises using a chiral catalyst.

11. The method of claim 10, wherein the chiral catalyst is selected from the group consisting of Rh, Ru and Ir-based catalysts comprising chiral di- phosphine or chiral phosphine-oxazoline ligands.

12. The method of claim 3, wherein said hydrolysis comprises treating the compound of Formula V with an acid providing a deformylated free amino product, followed by treatment of the free amino product with an alkaline solution.

13. The method of claim 1, wherein the compound of Formula I is selected from the group consisting of (2S,3R, 45)-, (25, 3R, 4R)-, (25,35,4#)-, (25,35,45)-, (2/?, 35, 45)-, (2R, 3R, 45)-, (2R,3S,4R)-, and (2R,3R,4R)-4- hydroxyisoleucine.

14. The method of claim 1, wherein the compound of Formula I is (25, 3 R, 45)-4-hydroxyisoleucine.

15. A method for synthesizing compounds of Formula I', including stereoisomers and analogs thereof:

said method comprising: a) reacting a compound of Formula II' :

OH Formula IF

with an alkyl isocyanoacetate of Formula III:

O

Il +^C

O ^^ Formula III

wherein R is an alkyl group; providing a compound of Formula IV:

16. The method of claim 15, further comprising subjecting the compound of Formula IV to a hydrogenation step, providing a compound of Formula V:

17. The method of claim 16, further comprising hydrolyzing the compound of Formula V, providing a compound of Formula I'.

18. The method of claim 15, wherein the compound of Formula IF IS selected from the group consisting of racemic acetoin, non-racemic acetoin, (S)-, and (R)-acetom

19. The method of claim 15, wherein step a) is earned out under alkaline conditions

20. The method of claim 19, wherein the alkaline conditions comprise using a base selected from the group consisting of KO?Bu, NaH, «-BuLi and amine bases such as DBU

21. The method of claim 16, wherein said hydrogenation compπses using an achiral catalyst

22. The method of claim 21, wherein the achiral catalyst compπses an achiral rhodium catalyst

23. The method of claim 22, wherein the achiral rhodium catalyst compπses Rh(COD)(diPFc)BF₄

24. The method of claim 16, wherein said hydrogenation compπses using a chiral catalyst

25. The method of claim 24, wherein the chiral catalyst is selected from the group consisting of Rh, Ru and Ir-based catalysts compπsing chiral di- phosphine or chiral phosphine-oxazohne hgands

26. The method of claim 17, wherein said hydrolysis compπses treating the compound of Formula V with an acid providing a deformylated free amino product, followed by treatment of the free amino product with an alkaline solution.

27. The method of claim 15, wherein the compound of Formula I is selected from the group consisting of (25, 3/?, 4S)-, (2S.3RAR)-, (2S.3SAR)-, (25,35,4S)-, (2R.3SAS)-, (2R.3RAS)-, (2R.3SAR)-, and (2/?,3/?,4/.)-4- hydroxyisoleucine.

28. The method of claim 15, wherein the compound of Formula I is (25, 3Λ,45)-4-hydroxyisoleucine.

29. A method for synthesizing compounds of Formula I, including stereoisomers and analogs thereof:

Formula I

wherein X is selected from the group consisting of hydroxy, amino and thiol; said method comprising hydrolyzing a compound of Formula V:

Formula V

wherein Z is selected from the group consisting of O, NH or S.

30. The method of claim 29, wherein said hydrolysis comprises treating the compound of Formula V with an acid providing a deformylated free amino product, followed by treatment of the free amino product with an alkaline solution.

31. The method of claim 29, wherein the compound of Formula I is selected from the group consisting of (25,3/?, 45)-, (2S.3RAR)-, (25,35,47?)-, (25,35,45)-, (2R, 35, 45)-, (2R,3R,4S)-, (2R.3SAR)-, and (2R.3 RAR)A- hydroxyisoleucine.

32. The method of claim 30, wherein the compound of Formula I is (25,3/?,45)-4-hydroxyisoleucine.

33. A method for synthesizing compounds of Formula I', including stereoisomers and analogs thereof:

said method comprising hydrolyzing a compound of Formula V:

Formula V

34. The method of claim 33, wherein said hydrolysis comprises treating the compound of Formula V with an acid providing a deformylated free amino product, followed by treatment of the free amino product with an alkaline solution.

35. The method of claim 34, wherein the compound of Formula I' is selected from the group consisting of (25, 3 R, 4S)-, (2S.3RAR)-, (25,35,4/?)-, (25,35,45)-, (2R.3SAS)-, (2R.3RAS)-, (2R.3SAR)-, and (2Λ,3Λ,4Λ)-4- hydroxyisoleucine.

36. The method of claim 35, wherein the compound of Formula I' is (25,3/?,4iS)-4-hydroxyisoleucine.