CZ20003986A3 - Antipicornaviral compounds and compositions, their pharmaceutical use and materials for their synthesis - Google Patents

Abstract

Peptide and peptidomimetic compounds of general formula (I), in which the individual symbols in the general formula have a specific meaning and which preferably inhibit or block the biological activity of picomaviral 3C protease. These compounds, as well as pharmaceutical compositions containing these compounds, are useful for the treatment of patients or hosts infected with one or more piconavirus species, such as RVP. Intermediates and methods for the preparation of such compounds.

Description

Technical area

The present invention relates to peptide-like and peptidomimetic compounds that advantageously inhibit the enzymatic activity of picornaviral 3C proteases, particularly rhinovirus 3C proteases (RVP), thereby slowing the growth of the virus in cell culture. The present invention also relates to the use of such compounds in pharmaceutical compositions and the therapeutic treatment of rhinoviral infections. The present invention further relates to methods for the synthesis of such compounds and compounds useful in such syntheses.

State of the art

Picornaviruses are a group of small, non-enveloped, positive-stranded RNA viruses that infect humans and other animals. These viruses include human rhinoviruses, human polioviruses, human coxsackieviruses, human echoviruses, human and bovine enteroviruses, encephalomyocarditis viruses, meningitis viruses, foot-and-mouth virus, hepatitis virus, and others. Human rhinoviruses are the main cause of the common cold. There is currently no effective therapy available for the treatment of the common cold, only symptomatic treatment.

Picornaviral infections can be treated by inhibiting proteolytic 3C enzymes. These enzymes are required for the natural maturation of picornaviruses. They are responsible for the autocatalytic cleavage of large genomic polyproteins into essential viral proteins. Members of the 3C protease family are cysteine proteases, where the sulfhydryl group most commonly cleaves the glutamine-glycine amide bond. It is believed that inhibition of 3C proteases blocks proteolytic cleavage of polyproteins, which may further delay viral maturation and replication by interfering with the formation of viral particles. Therefore, inhibition of the processing of these cysteine proteases by selective small molecules that are specifically recognized should represent an important and useful approach to the treatment of viral infections of this type, and especially the common cold.

Recently, several small molecule inhibitors of picornaviral 3C protease enzymatic activity (or antipicornaviral compounds) have been discovered. See, for example, U.S. Patent Application Serial No. 08/850,398, filed May 2, 1997, to Webber et al.; U.S. Patent Application Serial No. 08/991,282, filed December 16, 1997, to Dragovich et al.; and U.S. Patent Application Serial No. 08/991,739, filed December 16, 1997, to Webber et al. These U.S. patent applications, which are incorporated herein by reference, describe certain antipicornaviral compounds. However, there is still a need to find other small molecule compounds that are particularly potent antipicornaviral agents.

The essence of the invention

The present invention relates to small molecule compounds that are particularly potent antipicornaviral agents. The present invention further relates to the preparation of intermediates useful for the synthesis of said protease inhibitor compounds and synthetic methods useful for such syntheses. The present invention further relates to pharmaceutical compositions that are highly effective for the treatment of diseases mediated by the inhibition of picornaviral 3C proteases, such as the common cold.

activity. It was a peptidomimetic heterocyclic

The objects of the invention have been achieved by the discovery of compounds according to the present invention which are picornaviral 3C protease inhibitors and exhibit particularly potent antiviral activity. It has surprisingly been found that peptide and five-membered group-containing compounds have high rhinoviral protease inhibitory activity. It has further surprisingly been found that the rhinoviral protease inhibitory activity of peptide and peptidomimetic compounds can be significantly increased by replacing the glutamine-type group found in some rhinoviral protease inhibitory compounds with a side chain comprising a gamma or delta lactam.

The inhibitors according to the present invention are substances of the following general formula (I):

in which

Y represents -N(R _y )-C(R _y ) (R _y )- or -O-, wherein each R _y independently represents -H or lower alkyl;

R 1 represents -H, -F, -alkyl, -OH, -SH or an O-alkyl group;

R ₂ and R ₃ independently represent H;

O.

and ₄ \

(A ₃ )n /

and ₂ < (R41XR4· or ^r (R41XR41) , where n is an integer ranging from 0 to 5, Aχ represents CH or N, A ₂ and each A ₃ is independently selected from the group consisting of C(R ₄ i)(R ₄ x), N(R ₄₁ ), S, S (O) , S(0) ₂ and 0 and A ₄ represents NH or NR ₄₁ , where each R ₄₁ independently represents H or lower alkyl, provided that no more than two heteroatoms occur consecutively in the above ring formed by Aχ, A ₂ , (A ₃ ) _n , A ₄ and C=0 and at least one of R ₂ and R ₃ represents o.

NH, or

A4 \

(A ₃ )n (Ø41XR41)

R ₅ and R 8 independently represent H, F, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group;

R7 and Rg independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, _—OR17 , —SRx7, _—NRx7Rx8 , _{—NRi9NRi7Rx8} or —NRX7ORX8, where

R17, _R18 and Rxg independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group or an acyl group, provided that at least one of _R7 and _R8 represents an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group,

heteroaryl group, -0Ri ₇ , -SRx7, -NRx ₇ Rx8, -NR19NR17R18 or ^— NR17OR18; • R ₉ represents a suitable organic group; and • Z and Ζχ represent independently wearing H, F, alkyl group, cycloalkyl group,

heterocycloalkyl group, aryl group, heteroaryl group, -C(O)R ₂₁ , ^- CO ₂ R ₂₁ , ^- CN,

C(O)NR ₂₁ R ₂₂ , -C(O)NR ₂ iOR ₂₂ , -C(S)R ₂₁ , -C(S)NR ₂ iR ₂₂ , -no ₂ ,

-SOR ₂ i, “SO ₂ R ₂ i, -SO ₂ NR ₂ iR ₂₂ , -SO(NR ₂ i) (OR ₂₂ ) , -SONR21, SO ₃ R ₂ 1, -PO(OR ₂₁ ) ₂ , -PO(R ₂₁ ) (R ₂₂ ) , -PO(NR ₂ iR ₂₂ ) (OR ₂₃ ) , PO(NR ₂ iR ₂₂ ) (NR23R24) , “C (0) NR ₂ iNR ₂₂ R ₂₃ or C (S) NR _2:l NR ₂₂ R ₂₃ , where R ₂ i, R ₂₂ , R ₂₃ and R ₂₄ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or where any two of the substituents R ₂₁ , R ₂₂ , R 23 and R ₂₄ , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Z 1 do not both represent H;

or Z1 and Rx, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group, wherein Zχ and Rx have the meanings given above except for groups that cannot form a cycloalkyl or heterocycloalkyl group;

• or Z and Z1, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group, where Z and Z1 have the above meaning with the exception of groups that cannot form a cycloalkyl or heterocycloalkyl group.

The present invention also relates to prodrugs, pharmaceutically acceptable salts, pharmaceutically active metabolites and pharmaceutically acceptable solvates of compounds of the general formula

AND.

In preferred embodiments of the compounds of general formula I

O R ₂ and R ₃ independently represent H;

NH, or

where n is an integer ranging from 0 to 5, each R ₄ i independently represents H or lower alkyl, and the stereochemistry at the carbon atom marked with an asterisk may be R or S; provided that at least one of R ₂ and R ₃

(C(R ₄₁ )(R ₄₁ )) _n represents ^nh 2 or (Ø41XR41) ^(R41)(R ^

Preferably, R _g • 0 represents a five-membered heterocycle having one to three heteroatoms selected from the group consisting of O, N and S.

In other preferred embodiments, the variables of formula I have the following meanings: Z and Z _x are independently selected from the group consisting of H, F, lower alkyl, -CO2R21 and -C(0) _NR2iR22z , provided that Z and Zi do not both represent H, where _R2i and _R22 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group, or _R2i and _R22 , together with the atom or atoms to which they are attached, form a heterocycloalkyl group.

H,N,

At least one of R ₂ or R ₃ represents

O 'NH or ² and the other is H. R ₅ and R ₆ are independently selected from the group consisting of H, F, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, more preferably one of R ₅ and R 6 is H and the other is alkyl or aryl (e.g. unsubstituted or substituted phenylmethyl). R ₇ and R ₈ are independently selected from H, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; and more preferably one of R ₇ and R ₈ is H and the other is alkyl (e.g. 2-propyl, 2-methyl-2-propyl or 2-methyl-1-propyl) or arylmethyl (e.g. unsubstituted or substituted phenylmethyl or naphthylmethyl). Rg is a five-membered heterocycle having from one to three heteroatoms selected from ·· ·· ·· • · and 4 * *

4 4 4 4

4 4 4 4 4 • · 4 4 4

4444 44 44 of the group consisting of O, N and S, more preferably a five-membered heterocycle containing at least one nitrogen heteroatom and at least one oxygen heteroatom (for example, unsubstituted or substituted 1,2-oxazolyl (i.e. isoxazolyl), 1,3-oxazolyl (i.e. oxazolyl) or oxazolyl (1,2,3-oxazolyl, 1,2,4-oxazolyl or 1,2,5-oxazolyl). When R ₉ represents oxazolyl, unsubstituted and monomethyl-substituted 1,2,4-oxazolyl are preferred. In particularly preferred embodiments, R 9 represents 3-isoxazolyl or 5-isoxazolyl, either unsubstituted or substituted with one or two methyl groups and/or halogen atoms, with chlorine and fluorine being preferred halogen substituents.

In a preferred embodiment, the compounds, prodrugs, pharmaceutically acceptable salts, pharmaceutically active metabolites and solvates have antipicornaviral activity with an EC ₅₀ of less than or equal to 100 μΜ in the Hl-HeLa cell culture assay and more preferably antirhinoviral activity with an EC ₅₀ of less than or equal to 10 μΜ in the Hl-HeLa cell culture assay.

The present invention also relates to intermediates of general formula II, preferably of general formula II', which are useful for the synthesis of certain compounds:

where :

• p is an integer ranging from 0 to 5;

• Au represents CH or N, A _i2 and each A _i3 is independently selected from the group consisting of C(R _e i)(R ₆ i), N(R ₆ i), S, S(O), S(0) ₂ and 0 and A _X4 represents NH or NR _6X , where each R ₆ i independently represents H, alkyl, acyl or aryl, provided that no more than two heteroatoms occur consecutively in the above-mentioned ring in general formula II formed by Au, A _X2 , (A _X3 ) _n , A _X4 and C=0;

• each R _i4 i independently represents H or lower alkyl;

• R ₅ i represents H, alkyl, acyl or aryl;

• R52, R53 and R54 are independently selected from the group consisting of H, hydroxy, alkyl, acyl and aryl; or any two of the substituents _R52 , _R53 and _R54 together represent =O or =C( _R57 )( _R58 ), where _R57 and _R58 are independently selected from the group consisting of H, alkyl, _CO2 ( _Cx - _C6 )alkyl, CO2N(Cx _{- C6} )alkyl and _CO2 (aryl ₎ ; and • R55 and _R56 independently represent H or a suitable nitrogen protecting group.

The present invention also relates to pharmaceutically acceptable salts of the compounds of general formulae II and II'.

The present invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of formula I or a precursor thereof, a pharmaceutically acceptable salt, a pharmaceutically active metabolite or a solvate thereof. In addition, the present invention relates to methods of inhibiting picornaviral 3C protease by administering a therapeutically effective amount of at least one compound of formula I or a precursor thereof, a pharmaceutically acceptable salt, a pharmaceutically active metabolite or a solvate thereof.

Detailed description and advantageous designs • 44

4 4 • 4 _4 · * • · ♦♦ 4* *» «4 «494

4 4 4 4

4 4 4

4 4 4 4

4444 44 44

The present invention relates to compounds of general formula I:

wherein Y, Ri, R ₂ , R ₃ , R 5 , Rg, R ₇ , R ₈ , Rg, Z and Zχ are as defined above and pharmaceutically acceptable salts, precursors, active metabolites and solvates thereof. Preferably, such compounds, pharmaceutically acceptable salts, precursors, active metabolites and solvates have antipicornaviral activity, more preferably antirhinoviral activity, corresponding to an EC ₅₀ value of less than or equal to 100 μΜ in the Hl-HeLa cell culture assay, more preferably corresponding to a μΜ value in the Hl-HeLa cell culture assay.

The present invention further relates to general formulas I-A, I-B and I-C:

EC ₅₀ less than or equal to preferred compounds

(I-A) (I-B)

in which R _y (in general formula IA) represents H or lower alkyl and R _lř R ₂ , R ₃ , R ₅ , Rg, R ₇ , Rs, R9, Z and Zχ have the above • 4

4 4 • ·

-· 4 4 • 4

44 *· *4 »4 4 4 » 44 4

4 4 · 4 ·

4* 4 4 4 4

4 4 4 4 4

444 4444 4« 44 the meaning given and their pharmaceutically acceptable salts, prodrugs, active metabolites and solvates.

The compounds of the invention of general formulas I-A, hereinafter referred to as peptide-like compounds, I-B, hereinafter referred to as ketomethylene-type compounds, and I-C, hereinafter referred to as depsipeptide compounds, differ in their backbone, which may affect specific biodistribution or other physical properties; however, they all exhibit potent rhinoviral protease inhibitory activity.

In preferred embodiments of the compounds of general formulas I-A, I-B and I-C listed above:

R 1 represents H, F or an alkyl group;

R _y (in general formula IA) represents H or methyl;

R ₃ , R ₅ and R 6 all represent H;

R ₂ is selected from a set containing the following groups:

yA?

H

C.N.

nh ₉ one to including heteroatoms

R ₆ represents an alkyl group which preferably carries an aryl group as an optional substituent;

R ₇ represents an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group;

R ₉ represents a five-membered heterocycle having from three heteroatoms selected from the group consisting of O, N and S, preferably where at least one of them is nitrogen, which is unsubstituted or substituted, where the optional substituents are preferably a halogen atom or lower alkyl and more preferably a mono-chloro or -fluoro or methyl group; and

Z and Z _x independently represent H, F, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, -C(O)R ₂₁ , -CO2R 21 , -CN, -C(O)NR ₂ iR ₂₂ , -C(O)NR ₂ iOR ₂₂ , -C(S)R ₂ i, -C (S) NR ₂₁ R ₂₂ ,

-N0 ₂ , -SOR21, -SO2R21, SO2NR21R22, -SO(NR ₂ i) (OR ₂₂ ) , -SONR21,

SO3R21, -PO(OR ₂₁ ) ₂ , -PO (R ₂ i) (R ₂₂ ) , -PO(NR ₂ iR ₂₂ ) (OR23) ,

PO(NR ₂ iR ₂₂ ) (NR ₂₃ R ₂₄ ), -C (0) NR 21NR 22R 23 or ^-C (S) ÇR 21NR 22R 23 , where Z and Zi do not both represent H and where R _2X , R ₂₂ , R ₂₃ and R ₂₄ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or where any two of the substituents R _2i , R ₂₂ , R ₂₃ and R ₂₄ , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, or Z and Zi (both as defined above), together with the atoms to which they are attached, form a heterocycloalkyl group.

In preferred embodiments, the compounds of the present invention are of general formulae I-A', I-B' and I-C':

·· · ·· ·· it· • · · · it ····

in which:

R 1 , Z and Z 1 have the meanings given above; n is 1 or 2;

R _y (in general formula I-A') represents H or lower alkyl;

R ₆ represents alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

R ₇ represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -OR ₁₇ , -SR ₁₇ , NR ₁₇ R ₁₈ , -NR ₁₉ NR ₁₇ R ₁₈ or NR ₁₇ OR ₁₈ , where R _X7 , R ₁₈ and R _X9 independently represent H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or acyl;

R _g represents a five-membered heterocycle having one to three heteroatoms selected from the group consisting of O, N and S, which is unsubstituted or substituted, where the optional substituents are preferably one or two lower alkyl groups and/or halogen atoms.

• · · • · · • · · · • · · · <

The present invention also relates to prodrugs, pharmaceutically acceptable salts, pharmaceutically active metabolites and solvates of such compounds.

In preferred embodiments of the RVP-inhibiting agents of the present invention, the compounds of any stereospecific general formula I-A, I-B and I-C are:

OR ₇ Ry OR ₂ Z

H O Rr H (I-A)

R ₇

Λ o R ₂ of Rf

HOR ₆ HR ₁ (IB'

Oh R,

HOR ₆ HR ₁ :iC) in which R _y , R 1 , R ₂ , R 8 , R 7 , R 9 , Z and Z 1 have the above meanings and their pharmaceutically acceptable salts, prodrugs, active metabolites and solvates.

In preferred embodiments of compounds of general formula IA, I-B or I-C:

R 1 represents H, F or methyl;

R _y (in general formula I-A') represents H or methyl;

R ₂ is selected from a set containing the following groups:

H

CK^N.

η H -N ,x>

A^nh ₂ O , in

R ₆ represents arylmethyl or arylthiomethyl, wherein aryl is preferably an optionally substituted phenyl group;

R ₇ represents an alkyl group, more preferably selected from the group consisting of 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl and arylmethyl, wherein the aryl group is preferably phenyl or naphthyl;

R ₉ represents isoxazolyl, oxazolyl or oxazolyl optionally substituted with one or two lower alkyl groups and/or halogen atoms; and

Z represents H and Z _x represents -CO 2 R 21 , -CN or C(O)NR ₂ i R ₂ 2 , where R ₂ i and R 22 are as defined above or Z and Z 1 together represent a cyclic ester or amide.

Even more preferably, the RVP-inhibiting agents of the present invention are compounds of any of the general formulas I-A', I-B''' and I-C':

About • · • ·

wherein n, R _y , R 1 , R ₂₀ , R 7 /· R 9r Z and Z 1 have the meanings given above and pharmaceutically acceptable salts, prodrugs, active metabolites and solvates thereof.

In preferred compounds of general formula (I-A'''), (IB' ) or (I-C' ):

R 1 represents H, F or methyl;

R _y (in general formula I-A') represents H or methyl;

R20 represents a hydrogen atom;

R ₆ represents arylmethyl or arylthiomethyl, wherein aryl is preferably phenyl unsubstituted or substituted by halogen, lower alkyl and/or lower alkoxy;

R ₇ represents an alkyl group and is more preferably selected from the group consisting of 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl and arylmethyl, wherein the aryl group is preferably phenyl or naphthyl;

R ₉ represents isoxazolyl, oxazolyl or oxazolyl, each of which is optionally substituted with one or two lower alkyl groups and/or halogen atoms; and

Z represents H and Zχ represents -CO ₂ R 2z or -CN or C(O)NR ₂ iR ₂₂ , where R ₂ i and R ₂₂ are as defined above or Z and Z 1 together represent a cyclic ester or amide.

In particularly preferred compounds of the present invention of generic formula I (and derived formulae), R 1 represents H or F.

• ·

The present invention also relates to compounds of general formulae II and II':

concerns intermediate products

(II) ^R56

(11') in which variables (p, An, An, A ₁₃ , A _i4 , R51, R52, R53, R ₅₄ ,

R55, R56 and R141) are as defined above. These compounds are useful for the synthesis of pharmaceutically useful compounds of general formula I.

Preferred R ₅₅ and R ₅₆ groups are H and suitable nitrogen protecting groups, for example BOC (tert-butyloxycarbonyl), CBZ (benzyloxycarbonyl), FMOC (fluorene-9-methyloxycarbonyl), other alkyloxycarbonyls (for example methyloxycarbonyl) and trityl (triphenylmethyl). Other suitable nitrogen protecting groups can be readily selected by one skilled in the art (see, for example, Greene and Wutz, Protecting groups in Chemical Synthesis (2 ^nd ed.), John Wiley & Sons, NY (1991)). Preferred R ₅₂ , R ₅₃ and R ₅₄ groups are H, alkoxy, hydroxy and carbonyl.

Preferred compounds of formula II include the following, wherein P _N is a suitable nitrogen protecting group

HOOC

HOOC

_EtO2C

• · · · · • · · · · • · · · · * • · · · · · · · · · ·

MeO

Other preferred intermediates include the following compounds, where BOC is tert-butyloxycarbonyl:

• ·

Particularly preferred intermediates include the following compounds:

BOC

In accordance with conventions commonly used in the art, ^z is used herein in structural formulae to denote a bond that is the point of attachment of a group or substituent to the nucleus or backbone structure.

If chiral carbon atoms are included in chemical structures, then unless a specific orientation is given, the general formula includes both possible stereoisomeric forms.

As used herein, the term alkyl is used to mean a monovalent saturated and/or unsaturated straight-chain or branched radical consisting of carbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl, isopropyl, butyl, isobutyl, tert-butyl, ethenyl, pentenyl, butenyl, propenyl, ethynyl, butynyl, propynyl, pentynyl, hexynyl and the like, which may be unsubstituted (i.e. containing only carbon atoms and hydrogen atoms) or substituted with one or more suitable substituents as defined below (for example, one or more halogen atoms such as F, Cl, Br or I, with F and Cl being preferred). The term lower alkyl is used to mean an alkyl group having 1 to 4 carbon atoms in the chain.

The term cycloalkyl group is used to mean a non-aromatic monovalent monocyclic, bicyclic or tricyclic radical containing 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms in the ring, each of which may be saturated or unsaturated and may be unsubstituted or substituted with one or more suitable substituents as defined below and to each of which may be fused one or more heterocycloalkyl groups, aryl groups or heteroaryl groups, which themselves may be unsubstituted or substituted with one or more substituents. Illustrative examples of cycloalkyl groups include the following:

• » • 9 • 9 •

• • 99··

The term heterocycloalkyl group is used to mean a non-aromatic monovalent monocyclic, bicyclic or tricyclic radical, which is saturated or unsaturated, containing 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 ring atoms, which contains 1, 2, 3, 4 or 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, where the radical is unsubstituted or substituted with one or more suitable substituents as defined below and to which one or more cycloalkyl groups, aryl groups or heteroaryl groups, which themselves may be unsubstituted or substituted with one or more suitable substituents, may be fused. Illustrative examples of heterocycloalkyl groups include the following:

The term aryl group is used to mean an aromatic monovalent monocyclic, bicyclic or tricyclic radical containing 6, 10, 14 or 18 atoms.

• ·4 ·* »· ·· * « · · » * • « · · · · • **···· • · · · * · «·» *··« ·· ·· carbon in the ring, which may be unsubstituted or substituted with one or more suitable substituents, as defined below, and to which may be fused one or more cycloalkyl groups, heterocycloalkyl groups or heteroaryl groups, which themselves may be or more suitable aryl groups unsubstituted or substituted with one substituent. Illustrative examples include the following:

The term heteroaryl group is used to mean an aromatic monovalent monocyclic, bicyclic or tricyclic radical containing 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 ring atoms, including 1, 2, 3, 4 or 5 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, which may be unsubstituted or substituted with one or more suitable substituents as defined below and to which may be fused one or more cycloalkyl groups, heterocycloalkyl groups or aryl groups, which themselves may be unsubstituted or substituted with one or more suitable substituents. Illustrative examples of heteroaryl groups include the following:

• ·· · ·· **» <4* ·»·· ·« * · « · · · • · · ····· • · · · ·*·*·· ·· ·· ···· ···*· *·· ···· «« ··

The term heterocycle is used to mean a heteroaryl or heterocycloalkyl group (each of which, as defined above, is optionally substituted).

The term acyl group is used to mean the residue -C(O)-R, where R represents a substituent as defined below.

The term thioacyl group is used to mean the residue -C(S)-R, where R represents a substituent as defined below.

The term sulfonyl group is used to mean the radical -SO ₂ R, where R represents a substituent as defined below.

The term hydroxy group is used to mean the residue -OH.

The term amino group is used to mean the residue -NH ₂ .

The term alkylamino group is used to mean the radical -NHR _a , where R _a represents an alkyl group.

The term dialkylamino group is used to mean the radical -NR _a R _b , where R _a and R _b independently represent alkyl groups.

The term alkoxy group is used to mean the radical -OR _a , where R _a represents an alkyl group. Examples of alkoxy groups include methoxy, ethoxy, propoxy and the like.

The term alkoxycarbonyl group is used to mean the radical -C(O)OR _a , where R _a represents an alkyl group.

The term alkylsulfonyl group is used to mean the radical -SO ₂ R _a , where R _a represents an alkyl group.

The term alkylaminocarbonyl group is used to mean the radical -C(O)NHR _a , where R _a represents an alkyl group.

The term dialkylaminocarbonyl group is used

so that it represents the residue -C(O)NR _a R _b , where R _a and R _b represent independently of itself an alkyl group. The term mercapto group is used so, that means the residue -SH. The term alkylthio group is used so, that means the radical -SR _a , where R _a represents an alkyl group. The term carboxy group is used as follows: that means

the residue -C(O)OH.

• ·

The term carbamoyl group is used to mean the residue -C(O)NH ₂ .

The term aryloxy group is used to mean the radical -OR _c , where R _c represents an aryl group.

The term heteroaryloxy group is used to mean the radical -OR _d , where R _d represents a heteroaryl group.

The term arylthio group is used to mean the radical -SR _c , where R _c represents an aryl group.

The term heteroarylthio group is used to mean the radical -SR _d , where R _d represents a heteroaryl group.

The term suitable organic group is used to mean any organic group that can be verified by one skilled in the art, for example by routinely performed tests, to not adversely affect the inhibitory activity of the compounds of the invention. Illustrative examples of suitable organic groups include, but are not limited to, hydroxy groups, alkyl groups, oxo groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkoxy groups, carboxy groups, amino groups, alkylamino groups, dialkylamino groups, carbamoyl groups, arylthio groups, heteroarylthio groups, and the like.

The term substituent or suitable substituent is used to mean any suitable substituent that can be recognized or selected by one skilled in the art, for example based on routine tests. Illustrative examples of suitable substituents include hydroxy groups, halogen atoms, oxo groups, alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups,

alkoxy groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, carboxy groups, amino groups, alkylamino groups, dialkylamino groups, carbamoyl groups, aryloxy groups, heteroaryloxy groups, arylthio groups, heteroarylthio groups and the like.

The term optionally substituted is intended to expressly state that the specified group is unsubstituted or substituted with one or more suitable substituents unless the optional substituents are expressly stated; in such a case, the term means that the group is unsubstituted or substituted with the specified substituents. As defined above, various groups may be unsubstituted or substituted (i.e., optionally substituted) unless expressly stated otherwise herein (for example, by stating that the specified group is unsubstituted).

The term prodrug is used to mean a compound that is converted under physiological conditions or by solvolysis or metabolically into a specified compound that is pharmaceutically active.

The term pharmaceutically active metabolite is used to mean a pharmacologically active product of a specified compound, produced by metabolism in the body.

The term solvate is used to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological activity of such compound. Examples of solvates include compounds of the present invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

The term pharmaceutically acceptable salt is used to mean a salt which retains the biological activity of the free acids and bases of the specified compound and which is not

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfites, monohydrogenphosphates, caproates, succinates, biologically or otherwise undesirable hydrogen sulfates, sulfites, phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, heptanoates, propiolates, oxalates, malonates, corkanes, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates and almonds.

When the compound of the invention is a base, the desired salt may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid such as hydrochloric acid; hydrobromic acid; sulfuric acid; nitric acid; phosphoric acid and the like or an organic acid such as acetic acid; maleic acid; succinic acid; mandelic acid; fumaric acid; malonic acid; grape acid; oxalic acid; glycolic acid; salicylic acid; pyranosidic acid such as glucuronic acid or galacturonic acid; alpha-hydroxy acid such as citric acid or tartaric acid; amino acid such as aspartic acid or glutamic acid; aromatic acid such as benzoic acid or cinnamic acid; sulfonic acid such as

If the compound is a desired salt, it may be p-toluenesulfonic acid or ethanesulfonic acid and the like.

according to the invention, the acid is prepared by any suitable method known in the art, including treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary); an alkali metal or alkaline earth metal hydroxide, and the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine; ammonium; primary, secondary or tertiary and cyclic amines such as piperidine, morpholine and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminium and lithium.

In the case of compounds, salts or solvates that are solids, it will be apparent to those skilled in the art that the compounds, salts and solvates of the invention may exist in various crystalline forms, all of which are within the scope of the present invention and the specified general formulae.

The compounds of the invention may exist as single stereoisomers, racemates and/or mixtures of enantiomers and/or diastereomers.

All such single stereoisomers, racemates and mixtures thereof are within the scope of the present invention. Preferably, however, the compounds of the invention are used in optically pure form.

It is generally understood by those skilled in the art that an optically pure compound is one that is enantiomerically pure. As used herein, the term optically pure is used to mean a compound comprising at least a sufficient amount of the active agent. Preferably, the optical amount of a single enantiomer that gives a compound that is a pharmacologically pure compound of the present invention, • · • ·

29; -.· contains at least 90% of the single isomer (80% enantiomeric excess (e.e.), more preferably at least 95% (90% e.e.), even more preferably at least 97.5% (95% e.e.) and most preferably at least 99% (98% e.e.).

Preferably, in compounds of formula I (or any formula derived therefrom), R 1 represents H or F.

In compounds of the general formula IR ₉ preferably represents an unsubstituted or substituted isoxazolyl group, in which the optional substituents are preferably one or two methyl groups and/or halogen atoms.

Particularly preferred embodiments of the present invention are set forth below with reference to the following general formula I-A'':

OR ₇ Ry OR ₂ Z

W/b

H O Rg H R-| j )

Preferred compounds of the present invention include peptide compounds (A1) - (A-8) of the general formula I-A'' shown above, wherein R _x represents H, Z represents H, R _y represents H and R ₂ , R ₆ , R ₇ , Z _x and R ₉ are as defined below:

(Al) R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents

CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and

R ₉ represents ^H 3 ^C (A-2) R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents

CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and

R ₉ represents

·· ·♦ ·· » · · · · · • · · 4 · • · 4 · · * • · · 4 · h ₃ c' (A 3) R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , Rg represents χΎ'·' , R ₇ represents C(CH ₃ ) ₃ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ n- ·' aX represents ^Η 3θ (A-4) R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , Rg represents represents C(CH ₃ ) ₃ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ represents ^H 3 ^C aX η H

Xn

XU (A-5) R ₂ represents r ₆ represents F ^z

R ₇ represents CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉

-,-N

AX represents ^H 3C (A-6) R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , Rg represents

V , R ₇ represents CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ represents ^H 3° _n -N

AX η π vX>

_F x (A-7) R ₂ represents , R ₆ represents

R ₇ represents C(CH ₃ ) ₃ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉

-,-N represents ^H 3 ^C

AX (A-8)

R ₂ represents •p , R ₆ represents

R ₇ represents CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉

-,-N.

represents ^H 3 ^C

AX

• ·· to • «to to • ·· · • · to · ·· to·

Preferred peptide compounds of general formula IA further include compounds (A-9) - (A-13) below, wherein R _x represents H, Z represents H, Z _x represents CO ₂ CH ₂ CH ₃ , R _y represents CH ₃ and R ₂ , R 8 , R ₇ and R ₉ have the meanings given below:

(A-9) R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , R ₆ represents on . , . V &Ϊ, R ₇ represents ha R ₉ represents ^H s ^c (A-10) R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , Rg represents nN

CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ and R ₉ represents ^H ®c (A-ll) R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , Rg represents , R ₇ represents xr nN

Xha R ₉ represents ^H and ^c (A-12)

IN·

R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , R ₆ represents αχ ^F , í<7 represents CH ₂ CH(CH ₃ ) ₂ and R ₉ represents ^H and ^c (A-13) R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , R ₆ represents

Λ ί-Ν.

_R ,

-,-N represents 'Ό'ο' _{and Rg} represents h ₃ c'

Other preferred peptide compounds include the following:

(A-14)

00 0 00 00 00 0000 00 00 0 0 0 0

0 0 00000 0 00 0 000000 0~~0 00 0000

000 0000 000 0000 00 00

(Α-15) (Α-16)

0^νη ₂

_{CO2CH2CH3 ​ ​}

Ξ. Η

(Α-17)

NH ₂

(Α-18) ck.nh ₂

CO ₂ CH ₂ CH ₃ (Α-19) °^νη ₂

_{CO2CH2CH3 (} Α- ₂₀ )

»· ·· ♦ · · ♦ • · · · • · · · « · · · · · ·

(A-21) (A-22) (A-23)

H

CW ^N A o

'CO ₂ CH ₂ CH ₃ (Α-24)

(Α-25) (Α-26) ·· 99

9 B · • 9 B 9 • 9 9 ·

9 9 9

9 9 9

Η ^0; =/ ^ν α ° λ °Ύ

Ν y ^Ν Ν ^^CO ₂ CH ₂ CH ₃ (Α-27)

(Α-28)

Preferred ketomethylene-type compounds (B-1) - (B-4) of the present invention are listed below with reference to the following general formula I-B'':

or ₂ zor ₇ χ

RgN

N

HOR ₆ HR _x (I-B'') (Bl) /CT

R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , R ₆ represents , R ₇ represents CH(CH ₃ ) ₂ , Z represents H, Z _x

->-N represents CO ₂ CH ₂ CH ₃ and R _g represents ^Η 3θ η H (B-2) R ₂ represents R ₆ represents

R ₇ represents CH(CH ₃ ) ₂ , Z represents H, Z _x represents

CO ₂ CH ₂ CH ₃ and Rg represents vA

XX » » 4

F 4 4 «

B 4 4 4

B ♦ 4 I

B 4 4 4 «4 «4 (B-3) η Η

R ₂ represents γΆ*^, r ₆ represents _F xr,

K ₃ C

R ₇ represents CH(CH ₃ ) ₂ , Z' and Ζχ together form , where the carbonyl group is in the cis position with respect to the hydrogen corresponding to Rx in general formula I and R9 represents

Λ>ί _F xr ^r ar (B-4) R ₂ represents , r ₆ represents

R7 represents CH( _CH3 ) ₂ , Z represents H, Zχ represents on

CO ₂ CH ₂ CH ₃ and Rg represents ^H 3°

Preferred depsipeptide-type compounds (C-1) and (C-2) according to the present invention are listed below with reference to the following general formula I-C'', where Rx represents H:

d-c'')

Z represents H, R ₂ represents CH ₂ CH ₂ C (0) NH ₂ ,

R6 represents , r _? represents CH(CH ₃ ) ₂ , Ζχ _n -N

A^ represents CO ₂ CH ₂ CH ₃ and R ₉ represents (C-2) represents

Z represents H, R ₂ represents _f r ₆

XA ^v > ^r 7 represents CH(CH ₃ ) ₂ , Ζχ represents

AX

CO ₂ CH ₂ CH ₃ and Rg represents ^Η 3θ'

• 99 ·· 9 9 • 9 9 9 9 — 9

·· « 9 9 * * • 4 9 · * 9 9 ·9

9 9 9

9999 99 · ··

Other compounds can be prepared with reference to general formula I by selecting variables from the following substituents:

R _y = H or CH ₃ ;

R 1 - H or CH ₃ ;

H

_R2 or

// about

//

R ₆ = or phenylmethyl (ie benzyl), wherein the aryl group is optionally substituted with one, two or three substituents independently selected from the group consisting of halogen, methoxy and methyl;

R ₇ = 2-methyl-1-propyl, 2-propyl, 2-methyl-2-propyl, benzyl or

nr. ?'V ϋ- Λ-Λ ₌ nzh SC* qr>

AND

IN

The present invention provides a method for inhibiting picornaviral 3C protease activity, which comprises contacting the protease with an effective amount of a compound of formula I, or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite, or solvate thereof. For example, the picornaviral 3C protease activity may be

inhibited in mammalian tissue by administering a compound of formula I or a pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite or solvate thereof. More preferably, the method of the present invention is directed to inhibiting the activity of rhinoviral protease.

The term treatment or cure is used to mean at least alleviating a disease state in a mammal such as a human that is alleviated by inhibiting the activity of one or more picornaviral 3C proteases, for example, human rhinoviruses, human polioviruses, human coxsackieviruses, encephalomyocarditis viruses, meningitis viruses, and hepatitis viruses, and includes: (a) prophylactic treatment of a mammal, particularly where the mammal is found to be predisposed to acquiring the disease state but has not yet been diagnosed with it; (b) inhibiting the disease state; and/or (c) alleviating, in whole or in part, the disease state.

The activity of the compounds of the invention as inhibitors of picornaviral 3C protease activity can be measured by any suitable method known to those skilled in the art, including in vivo and in vitro assays. An example of a suitable assay for measuring activity is the antiviral activity assay in H1-HeLa cell culture, which will be described below.

The administration of the compounds of formula I and their pharmaceutically acceptable metabolites and solvates can be carried out by any of the accepted routes of administration, prodrugs, salts, active ingredients known in the art. Illustrative examples of suitable routes of administration include oral, nasal, parenteral, topical, transdermal and rectal administration. Intranasal administration is particularly preferred.

The compound of the invention of formula I or a pharmaceutically acceptable salt, prodrug, active metabolite or lyophilized or solvate thereof may be administered in any pharmaceutical formulation recognized by the skilled artisan. Suitable formulations include solid, semi-solid, liquid formulations such as tablets, powders, capsules, suppositories, suspensions, liposomes and aerosols. The pharmaceutical compositions of the present invention may also contain suitable carriers and agents, depending on the preferred embodiments.

For example, the process using excipients, diluents, vehicles may be further pharmaceutically active for the intended use. In the pharmaceutical compositions of the invention, they are administered intranasally in the form of suspensions.

Acceptable methods for preparing suitable pharmaceutical forms of pharmaceutical compositions are known or can be routinely determined by one skilled in the art. Pharmaceutical preparations are prepared by conventional pharmaceutical chemistry techniques, which include the steps of mixing, granulating, and compressing when a tablet form is being prepared, or mixing, filling, and dissolving the ingredients as needed to obtain the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.

Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles or excipients may be used in the pharmaceutical compositions. Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate and stearic acid. Illustrative liquid carriers include syrups, peanut oil, olive oil, saline solutions and water. The carrier or diluent may contain a suitable sustained release material such as glyceryl • · ····

monostearate or glyceryl distearate, alone or together with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or anhydrous or aqueous liquid suspension.

A dosage form of a pharmaceutical composition contains at least a therapeutically effective amount of the active compound (i.e., a compound of Formula I or a pharmaceutically acceptable salt, prodrug, active metabolite, or solvate thereof) and is preferably comprised of one or more pharmaceutical dosage units. The selected dosage form may be administered to a mammal, e.g., a human patient in need of treatment by inhibiting picornaviral 3C protease activity, by any known or suitable route of administration, including topical administration, e.g., as an ointment or cream; oral administration; rectal administration, e.g., as a suppository; parenteral administration by injection; or continuous administration by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion.

The term therapeutically effective amount is used to mean an amount of a compound of the invention which, when administered to a mammal in need thereof, is sufficient to treat disease states by achieving relief by inhibiting the activity of one or more picornaviral 3C proteases, such as human rhinoviruses, human polioviruses, human coxsackieviruses, encephalomyocarditis viruses, meningoviruses, and hepatitis A viruses. The amount of a compound of the present invention administered that is therapeutically effective will vary depending on factors such as the compound selected, the disease state and its severity, the identity of the mammal in need of treatment, and can be routinely determined by one of ordinary skill in the art.

• ·

By way of illustration, nasal formulations of the compounds of the invention for the treatment of rhinoviral infections may be prepared as follows, wherein all percentages are by weight and the suspension is prepared in purified water. The compound of formula I is micronized to reduce the particle size to achieve a D ₉₀ < 10 pm. The suspension is prepared to contain a final concentration of from about 0.01% to about 2% of the active compound, preferably from about 0.2% to 2%. A suitable preservative may be added from those known in the art, for example benzalkonium chloride/EDTA, in a suitable range of final concentrations, for example about 0.02%/0.01%. A suitable suspending agent may be added, such as a mixture of microcrystalline cellulose (final concentration of about 1% - 1.5%, preferably about 1.2%) and sodium carboxymethylcellulose (final concentration of about 0.1% - 0.2%, preferably about 0.13%). A suitable surfactant such as polysorbate 80 may be added at a final concentration of about 0.05% to 0.2%, preferably about 0.1%. A tonicity modifying agent such as dextrose may be added to give a final concentration of about 4% to 6%, preferably about 5%. The pH of the resulting solution is adjusted appropriately to a physiological range, for example 4-6, using a non-toxic acid and/or base such as HCl and/or NaOH.

Examples of formulations for nasal administration of the compound of the invention from Example 17 have the following composition, where all percentages are by weight and the suspension is prepared in purified water:

Effective compound (B-2) 0.2 - 2% Conservative Benzalkonium chloride/EDTA 0.02% / reagent 0.01% Suspension Microcrystalline cellulose/ 1.2% / reagent Na-carboxymethylcellulose 0.13% Surfactant Polysorbate 80 0.1% Tonicity modifier Dextrose 5% PH adjustment NaOH/HCl pH 4 - 6

General synthesis methods

The compounds of the invention of general formula (I) can be conveniently prepared by the methods of the present invention, including the general methods described below. In each of these general methods, R1, R2, R3, R5, R6, R7, R8, R9, R10, R11, Z and Zχ have the meanings given above and R4 is used (in abbreviated form) as:

OR ₇

wherein R7, R8 and R9 have the meanings given above.

• ·

I, useful for the synthesis of general formula I-A, is a suitable protecting group for amido-forming reaction with

In a general method for peptide compounds, amino acid A, where Ρχ is a nitrogen atom, is treated with amino alcohol (or its salt) B to form amide C. Compound C is then deprotected to give free amine (or its salt) D. Amine D and amino acid E, which may contain either an R ₄ group or a nitrogen protecting group (P ₂ ), are subjected to a bond-forming reaction to give compound F. Compound F is oxidized to intermediate G, which is then converted to unsaturated product H. If protecting groups have been used on amino acid E or on any R group (R1-R9) and/or on R _y and/or on Z and/or Ζχ, product H is deprotected and/or further modified to give deprotected or modified H.

G

H deprotected or modified

H • ·

An alternative method for preparing intermediate F is as follows. Amino acid E and amino acid (or its salt)

I, where P3 is a suitable oxygen protecting group, undergoes a bond forming reaction to form the intermediate

J. Molecule J is deprotected to yield the free carboxylic acid K, which is then subjected to an amide-forming reaction with amino alcohol (or its salt) B to form intermediate F.

In general method II, which is also applicable to the synthesis of peptide-like compounds of general formula IA, the amino acid L, where Ρχ is a suitable nitrogen protecting group, is converted to a carbonyl derivative M, where Lv is a leaving group. Compound M is subjected to a reaction in which Lv is replaced by Rx to give derivative N. Derivative N is then converted to an unsaturated product O. The unsaturated compound O is deprotected to give the free amine (or salt thereof) P or modified at Z or Ζχ first to give O' and then deprotected to P. Intermediate P is subsequently subjected to an amide-forming reaction with a carboxylic acid K to give the final product H. If protecting groups have been used on any of the R groups (R1-R9) and/or on R _y and/or on Z and/or Ζχ, product H is deprotected and/or further modified to give a deprotected or modified H.

• ·

Pi

OH

R ₂

L

H

N

Level ₂

M

- 44 -:

*« ···

♦ · ··

r ₂

N

An alternative method for preparing intermediate N is as follows. Compound M is subjected to a reaction where Lv is reduced to the protected amino alcohol Q. Intermediate Q is subsequently oxidized to derivative N.

R ₂

Q

In general method III, useful for the synthesis of peptide compounds of general formula I-A, an amino acid L, where Pj is a suitable nitrogen protecting group, is converted to a carbonyl derivative M, where Lv is a leaving group. Compound M is deprotected to give the free amine (or salt thereof) R, which is subsequently subjected to an amide-forming reaction with a carboxylic acid K to give intermediate S. Compound S is then either directly converted to a carbonyl intermediate G or is first reduced stepwise to an alcohol F, which is oxidized to G. Compound G is reacted to give the unsaturated

9 · » »*

999« of the resulting product H. If protecting groups were used on any R group (R1-R9) and/or on R _y and/or on Z and/or Z 1 , the product H is deprotected and/or further modified to obtain a deprotected or modified H.

r ₂

_____deprotected or modified

H

F

In general method IV, applicable to the synthesis of peptide-like compounds of general formula IA, the free amine (or salt thereof) P, prepared from intermediate O as described in general method II, is converted to amide T by reaction with amino acid A, where Pi is a suitable nitrogen protecting group. Compound T is further deprotected to free amine (or salt thereof) U, which is subsequently converted to H by treatment with amino acid E. If protecting groups have been used on any of the R groups (R1-R9) and/or on R _y and/or on Z and/or Zq, product H is deprotected and/or further modified to obtain the deprotected or modified

O

P

A • · φ φφ·· ► · »φ φφ··

__|_| ______deprotected or modified

H

In general method V, useful for the synthesis of ketomethylene compounds of general formula I-B, the optically active lactone AA, where P4 is a suitable nitrogen protecting group and R5 and R6 are H (which can be prepared as described below and by various methods known in the literature, including: (a) Herold et al., J. Org.

Chem. 1989, 54, 1178; (b) Bradbury et al., Tetrahedron Lett.

1989, 30, 3845; (c) Bradbury et al., J. Med. Chem. 1990, 33,

2335; (d) Wuts et al., J. Org. Chem. 1992, 57, 6696; (E)

Jones et al., J. Org. Chem. 1993, 58, 2286; (f) Pégorier et al., Tetrahedron Lett. 1995, 36, 2753; and (g) Dondoni et al., J. Org. Chem. 1995, 60, 7927) is converted in a two-step procedure (basic hydrolysis followed by oxidation) to the carboxylic acid BB. This material is not isolated, but is subjected to an amide-forming reaction with an amine (or salt thereof) P to give the resulting product CC. The protecting group P ₄ , together with any protecting groups that were used on any R group (R 1 , R 2 , R 3 , R 6 and/or R 7 ) and/or on Z and/or on Zi, is then removed and/or the product is further modified to give a deprotected or modified CC.

·: · :

• 0

0

0 0

0 0

Lactone AA can be prepared in optically active form by the following general method VI (see: Herold et al., J.

Org. Chem.

1989, 54, 1178; Bradbury et al.,

Lett. 1989, 30, 3845; and Bradbury et al., J. Med. Chem. 1990, 33, 2335). The γ,δ-unsaturated carboxylic acid DD, which contains R7, is converted to the corresponding acid chloride (not shown). This acid chloride is subjected to an amide-forming reaction with a chiral amine or chiral oxazolidone to give the derivative EE (wherein Xχ is a chiral amine or chiral oxazolidone). The compound EE is then deprotonated and the resulting enolate is diastereoselectively alkylated with an electrophile of the R6 substituent to give the compound H. This material is subjected to a halolactonization reaction to give the halolactone wherein R5 and R6 are H and hal is Br or I. The halolactone GG is then converted to the azide HH and this material is then

Tetrahedron with corresponding

FF, then where R5 subjects

GG, in converts to the lactone AA, where P4 is the nitrogen atom.

is a suitable protecting group

AA HH GG The γ,δ-unsaturated carboxylic acid DD can be prepared by the following general method VII (see Herold et al., J. Org. Chem. 1989, 54, 1178). The aldehyde II, which contains R7, is coupled with vinylmagnesium bromide to give the alcohol JJ. The alcohol JJ is then converted to the γ,δ-unsaturated carboxylic acid DD in the following three-step procedure: (i) treatment with diethyl malonate and catalytic Ti(OEt)4 at 160°C for 1 hour, (ii) heating at 190°C for 4 hours, and (iii) hydrolysis with ethanolic KOH at reflux.

Carboxylic General Method 1997, 53, 7119).

BB acid can also be prepared by Vlil (see Hoffman et al., Tetrahedron, Amino acid KK containing R7 and in

999 M·· • 1·1·

9

9 ·

9 · «9 ·

9 ·

9 in which Ρ4 is a suitable nitrogen protecting group, is converted to the β-ketoester LL. Compound LL is deprotonated and the resulting anion is condensed with the triflate MM, which contains R6. The coupling product obtained in this manner is treated with trifluoroacetic acid to give the ketoesters NN, and this material is subsequently hydrolyzed to give the carboxylic acid BB. If basic hydrolysis leads to epimerization, the ketoester NN can be transesterified (allyl alcohol, Ti(Oi-Pr)4) and subsequently deprotected under neutral conditions (Pd(PPh3)4, morpholine) to give the carboxylic acid BB. The triflate MM can be prepared from the corresponding alcohol by treatment with trifluoromethanesulfonic anhydride and 2,6-lutidine.

Lactone AA can be prepared by general method IX (see Askin et al., J. Org. Chem. 1992, 57, 2771; and McWilliams et al., J. Am. Chem. Soc. 1996, 118, 11970). Amino acid KK, which contains R7 and in which P4 is a suitable nitrogen protecting group, is converted to epoxide 00 (a single diastereomer) by the method described by Luly et al., J. Org. Chem. 1987, 52, 1487. Epoxide 00 is condensed with an anion derived from compound PP, which contains R6 and in which X2 is a chiral auxiliary (containing acetonide (1S,2R)-1τ· »· » · • · · · · and » · < <

aminoindan-2-ol) to give the coupling product QQ. This material is then cyclized under acidic conditions to give the lactone AA. Compound PP can be prepared from the corresponding carboxylic acid by the method described by Askin et al. (not J. Org.

shown)

Chem. 1992,

57, 2771.

Ry

P4x

P ₄ .

O

K.K.

Ry'o

Re x ₂ oo

PP

• H»Rg r ₇ o

OH R ₆

AA QQ

General Method X, useful for the preparation of depsipeptide compounds of Formula I-C, illustrates a method for preparing intermediate TT. Amino acid E and alcohol RR, where P5 is a suitable oxygen protecting group, are subjected to an ester bond forming reaction to form intermediate SS. The SS molecule is deprotected to provide the free carboxylic acid TT, in place of the carboxylic acid K of the methods described above.

which can be used in any of the general ^R 5N

HO^X Rr

OP _B (P ₂ )R ₉ ·

Ry

N

HR ₈

O.

Rr

OP ₅ (P ₂ )R ₉ N

HR _g

R ⁰ °,h

OH

RR

SS

TT

4* • 4 ··· ···· ·« • · • ·«·· • · • 4 • · • 4 ·· ·· »·

Specific syntheses

The following specific methods can also be used to prepare various compounds of the present invention.

Specific Method I describes the preparation of specific intermediate O1, which can be used as intermediate O in the general methods described above. Thus, ester A1 (prepared as described in Chida et al., J. Chem. Soc., Chem. Commun. 1992, 1064) is hydrolyzed to give acid B1, which is itself converted to oxazolidinone C1. Compound C1 is subsequently deprotonated and the resulting enolate is diastereoselectively alkylated to give allylic intermediate D1. This compound is oxidized by ozonolysis and the resulting aldehyde (not shown) is subjected to a reductive amination reaction to form lactam E1. Acid-catalyzed methanolysis of E1 then gives alcohol F1. This intermediate is oxidized by the Swern method (or under other suitable oxidizing conditions) to the resulting aldehyde (not shown), which is subsequently subjected to an olefin-forming reaction to give specific intermediate O1.

• ·

Specific to intermediate O2, method II describes the preparation of a specific intermediate which can be used as intermediate O in the general methods described above. The allylic intermediate D1 is subjected to a sequence of hydroboration and oxidation to give the primary alcohol (not shown). This compound is oxidized by the Swern method (or other suitable oxidizing conditions) and the resulting aldehyde (not shown) is subjected to a reductive amination reaction to form the lactam G1. Acid-catalyzed methanolysis of G1 then gives the alcohol H1. This intermediate is oxidized by the Swern method (or other suitable oxidizing conditions) to the resulting aldehyde

(not shown), the olefin to obtain which is subsequently subjected to the reaction of a specific intermediate 02.

creating

The following intermediates P1, P2 and P3 can be used in the above general methods in place of intermediate O to vary the substituent group at position _R2 .

P3 • 9

9

9 ·

9 9

9 9

9 9 9

9 9

9

The synthesis of intermediate P1 is described below. Intermediate C1 (described above) is deprotonated and the resulting enolate is trapped with a suitable disulfide (symmetrical or mixed) to give sulfide p1 (P is a suitable oxygen protecting group). The oxygen protecting group is then removed to give alcohol p2. This intermediate is oxidized by the Swern method (or under other suitable oxidizing conditions) and the resulting aldehyde amination reaction (not shown) is subjected to reductive yielding lactam p3. Catalyzed methanolysis of p3 then gives alcohol p4. This intermediate is oxidized by the Swern method (or under other suitable oxidizing conditions) to the resulting aldehyde (also not shown), which is then subjected to an olefin-forming reaction to give intermediate p5. This compound can be used in place of intermediate O in the above synthetic routes; alternatively, the lactam protecting group can be removed to give intermediate P1.

Acid

p5

p4

electrophilic oxygen of the alcohol p6. This functionalized ether P7 atom). The resulting amine nitrogen atom is

For the synthesis of intermediate P2, intermediate C1 is deprotonated and the resulting enolate is trapped with a suitable source (e.g., oxaziridine) to afford the intermediate alkylated with a suitable alkyl halide or triflate for (P is a suitable nitrogen protecting group) the protecting group is then removed and (not shown) subjected to cyclization to afford the lactam p8. Acid-catalyzed methanolysis of p8 then affords the alcohol p9.

oxidized by the Swern method (or oxidation conditions) to the resulting product shown), which is then subjected to an olefin-forming reaction to obtain intermediate P2.

This intermediate is converted to other suitable aldehydes (also not

P2

BoCx

p9

The synthesis of a specific intermediate P3 will now be described. Intermediate D1 (described above) is ozonized and the resulting aldehyde (not shown) is reduced to the corresponding alcohol • · (also not shown). This intermediate is then protected to give compound p10 (Pχ is a suitable oxygen protecting group). The imide functionality present on p10 is hydrolyzed to give the carboxylic acid p11 and this intermediate is coupled with a suitably protected hydroxyamine derivative to give amide p12 (P2 is a suitable oxygen protecting group that is stable under the conditions under which Pχ is removed). The Pχ protecting group is then removed and the resulting alcohol (p13) is converted to a suitable leaving group (halide or triflate, not shown). The P2 protecting group is then removed and the resulting hydroxamic acid is cyclized to give intermediate p14. Acid catalyzed methanolysis of p14 then gives alcohol p15. This intermediate is oxidized by the Swern method (or other suitable oxidation conditions) to the resulting aldehyde (not shown), which is then subjected to an olefin-forming reaction to obtain intermediate P3.

• · · · · ♦ • · · · ♦ • · · · · 9 • 9 9 9 9 9 • 999 99 99

(Ikuta et al. Independently, the silyl ether ester p15 p3

Specific Method III describes the preparation of intermediates Q1, Q2 and Q3 which can be used in the general methods described above. The known compound II is converted to the molecule J1 known from the literature by modifying the published procedure J. Med. Chem. 1987, vol. 30, p. 1995). The K1 amino acid is protected to give the Ether is reduced with DIBAL (or using other suitable reducing conditions) and the resulting aldehyde (not shown) is subjected to an olefin-forming reaction with intermediate J1 to form M1. Silyl deprotection of M1 then gives the alcohol N1. This intermediate is subjected to a variety of hydrogenation conditions to give intermediates Q1, Q2 and Q3. These intermediates can be

IF.

converted into intermediates analogous to intermediate Ol (see

• * · ♦ • · · · • · fe · • · · ·

Specific olefination.

method I above) by oxidation and subsequent

Boc

_NH2

Ph ₃ P

OH

J1

Q3

It will be apparent to one skilled in the art that the various compounds of the present invention can be prepared by the above-mentioned general and specific methods as well as by prior art methods, including the above-mentioned references, which are incorporated herein by reference. In addition, one skilled in the art can prepare the various compounds of the present invention by the following examples or by routine modifications of the synthetic methods described herein.

Examples of embodiments of the invention

Examples of various preferred compounds of formula I are given below. The structures of the compounds of the following examples have been confirmed by one or more of the following methods: proton magnetic resonance spectroscopy, ·· to ·· ·* ·· ♦<· · ·♦ · · f ·· to < to to 9 9 9 9 9 to ·· · tototo··· _·· ·· ·· ··· infrared spectroscopy, elemental microanalysis, mass spectrometry, thin layer chromatography, melting point determination, and boiling point determination. In the event of a discrepancy between the structural formula shown for a compound and its chemical name, the structural formula shall prevail.

Proton magnetic resonance (1H NMR) spectra were determined using a Varian UNITYplus 300 spectrometer operating at a field frequency of 300 megahertz (MHz). Chemical shifts are reported in parts per million (ppm, δ) relative to an internal tetramethylsilane standard. Alternatively, 1H NMR spectra were referenced to residual protic solvent signals as follows: CHCl3 = 7.26 ppm; DMSO =

2.49 ppm; CgHDs = 7.15 ppm. Peak multiplicities are indicated as follows: s = singlet; d = doublet; dd = doublet of doublets; t = triplet; q = quartet; br = broad resonance; and m = multiplet. Coupling constants are given in Hertz. Infrared absorption (IR) spectra were obtained using a Perkin-Elmer 1600 series FTIR spectrometer. Elemental microanalyses were performed by Atlantic Microlab Inc. (Norcross, GA) and the results for the indicated elements were within ±0.4% of theoretical values. Flash column chromatography was performed on Silica gel 60 (Merck Art 9385). Analytical thin layer chromatography (TLC) was performed on Silica 60 F254 (Merck Art 5719) plates. Melting points were determined on a Mel-Temp apparatus and are uncorrected. All reactions were carried out in sealed bottles under a slight overpressure of argon unless otherwise stated. All commercial reagents were used as received from the manufacturer with the following exceptions: tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl before use;

·· fefe • · · 1 • · · 1 • · · · ι • fe · ι • fe fefe Dichloromethane (CH2Cl2) was distilled from calcium hydride before use; anhydrous lithium chloride was prepared by heating at 110°C under vacuum overnight.

The following abbreviations are used: Et2O refers to diethyl ether; DMF refers to N,N-dimethylformamide; DMSO refers to dimethyl sulfoxide; and MTBE refers to tert-butyl methyl ether. Other abbreviations include: CH3OH (methanol), EtOH (ethylene glycol Ph (phenyl), Tr (ethanol), EtOAc (ethyl acetate), DME dimethyl ether), Ac (acetyl), Me (methyl), (triphenylmethyl), Cbz (benzyloxycarbonyl), Boc (tert-butoxycarbonyl), TFA (trifluoroacetic acid), DIEA (N,Ndiisopropylethylamine), DBU (1,8-diazabicyclo[5,4.0]undec-7ene), HOBt (1-hydroxybenzotriazole hydrate), PyBOP (benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), HATU (O-(7-azabenzotriazol-1-yl)Ν,Ν,Ν',W'-tetramethyluronium hexafluorophosphate), EDC (1—(3— dimethylaminopropyl)-3-ethylcarbamodiimide hydrochloride), DCC (dicyclohexylcarbodiimide), DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), DMAP (4-dimethylaminopyridine), Gln (glutamine), Leu (leucine), Phe (phenylalanine), Val (valine), His (histidine), 1-naphth (1-naphthylalanine), 2-naphth (2naphthylalanine), α-tert-butyl-Gly (tert-butyl glycine), (S)pyrrole-Ala ((2S,3'S)-2-amino-3-(2'-oxopyrrolidin-3'-yl)-propionic acid) and (S)-piper-Ala ((2S,3'S)-2amino-3-(2'-oxo-piperidin-3'-yl)-propionic acid). In addition, L represents naturally occurring amino acids.

A simplified nomenclature system using amino acid abbreviations is used to designate some intermediates and final products. In naming compounds, the amino acid abbreviation in italics indicates a modification at the C-terminus of that residue for which the following applies: (1) esters ·· 99

9 9

9 9

9 9

9 9

99

acrylic acids are referred to as E (trans) propenoates; (2) substituted 3-methylene-dihydrofuran-2-ones are referred to as E (trans) 2-(α-vinyl-γbutyrolactones); and (3) 5-vinylisoxazoles are referred to as E (trans) propenisoxazoles. In addition, the terminology

The term AA1-NCH3AA2 denotes that for any peptide sequence the amide bond that usually connects two amino acids (AAi and AA2) is replaced by an N-methylamide bond. The term AAχ-ΟAA2 denotes that for any peptide sequence the amide bond that usually connects two amino acids (AAi and AA2) is replaced by an ester bond.

Examples of embodiments of the present invention are given below.

Example 1 - Preparation of Comparative Compound #1: 5-(3'-(Cbz-LLeu-L-Phe-L-Gln)-E-propene)-isoxazole

(Comparative compound

Preparation of the intermediate Cbz-L-(Tr-Gln)-OMe

Cbz-L-(Tr-Gln) (0.26 g, 0.50 mmol, 1 equivalent) was added to a solution of acetyl chloride (0.25 mL, 3.52 mmol, 7.0 equivalents) in CH3OH (5 mL) and stirring was continued at

Φ ··

Φ· ♦ φ • φ • ♦ • · ·· • · · · · · φ φ φ φ φ φ · · · · · ·Φ at 23°C for 1 hour. The solvent was removed under reduced pressure and the residue was dissolved in CH2Cl2 (100 mL) and washed with water (100 mL), saturated NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography (20% EtOAc in hexanes) to give Cbz-L-(Tr-Gln)-OMe (0.23 g, 84% yield) as a white solid: mp = 139-140°C; IR (cm ^-1 ) 1742, 1207; 1h

NMR (DMSO-dg) δ 1.16 (t, IH, J = 7.0), 1.77 (m, IH), 1.97 (m, IH), 3.61 (s, 3H), 4.99 (m, IH), 5.03 (s, 2H), 7.02-7.55 (m, 20H), 7.69 (d, IH, J = 7.7), 8.59 (s, 1H); Anal. (C33H32N2O5) C, Η, N.

Preparation of the intermediate Cbz-L-(Tr-glutaminol)

Lithium chloride (0.24 g, 5.66 mmol, 2.0 equiv) was added to a solution of Cbz-L-(Tr-Gln)-OMe (1.50 g, 2.79 mmol, 1 equiv) in 2:1 THF:EtOH (30 mL) and the mixture was stirred at 23°C until all solids dissolved (10 min). Sodium borohydride (0.21 g, 5.55 mmol, 2.0 equiv) was added and the reaction mixture was stirred overnight at 23°C. The solvents were removed under reduced pressure, the residue was taken up in water (50 mL) and the pH was adjusted to 2-3 with 10% HCl. The product was extracted with EtOAc (50 mL) and the organic layer was washed with water (50 mL) and brine (50 mL) before drying over MgSO4. The organic layer was concentrated and the residue was purified by flash column chromatography (gradient elution, 20->50% EtOAc in benzene) to afford Cbz-L-(Tr-glutaminol) (1.02 g, 72% yield) as a white glass: mp = 66-70°C; IR (cm ^-1 ) 3318, 1699, 1510, 1240; !h NMR (DMS0-d6) δ 1.40 (m, 1H), 1.72 (m, 1H), 2.26 (m, 2H), 3.173.50 (m, 3H), 4.64 (t, 1H, J= 5.0), 5.00 (s, 2H), 7.00-7.40 (m, 20H), 6.96 (d, 1H, J = 8.5), 8.54 (s, 1H) ; Anal.

(C32H32N2O4) C, H, N.

• ···· • ···· » 9 9 4 ' · · I » 9 9 4 * 9 9 I

99

Preparation of the intermediate L-(Tr-glutaminol)

A suspension of Cbz-L-(Tr-glutaminol) (1.93 g, 3.79 mmol) in

CH3OH (25 ml) and Pd/C (10% hydrogen atmosphere (balloon) filtered through a pad of Celite. The filtrate was concentrated under reduced pressure to give L-(Tr-glutaminol) as a white amorphous solid (1.38 g, 98% yield): Melting point = 191193°C; IR (cm ^-1 ) 3255 (br) , 1642, 1527; 1h NMR (DMSO-Cl6) δ

1.29 (m, 1H), 1.53 (m, 1H), 2.29 (m, 2H), 3.08 (m, 1H), 3.18 (m, 2H), 3.38 (s, br, 2H), 4.43 (s, br, IH), 7.14-7.28 (m, 15H), 8.62 (s, IH).

0.19 g) was stirred under for 4 hours, then

Preparation of the intermediate Cbz-L-Leu-L-Phe-L-(Tr-glutaminol)

Carbonyldiimidazole (0.17 g, 1.05 mmol, 1.0 equivalents) was added to a solution of Cbz-L-Leu-L-Phe-OH (0.41 g, 1.0 mmol, 0.95 equivalents) in THF (10 mL) and the reaction mixture was stirred at 23°C for one hour. L-(Trglutaminol) (0.39 g, 1.05 mmol, 1 equivalent) was then added and the resulting solution was stirred overnight. Volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (gradient elution, 2->4% CH3OH in CHCl3) to afford Cbz-L-Leu-L-Phe-L-(Tr-glutaminol) (0.47 g, 62% yield) as a white amorphous solid: mp = 92-95°C; IR (cm ¹ ) 3302, 1657, 1520, 1238; !h NMR (DMSO-c/6) δ 0.79 (t, 6H, J = 7.0), 1.30 (m, 2H) , 1.44 (m, 2H) , 1.75 (m, · * · · * ·

9 9 9 9 9 9 9 9

9 9 · · · • 9 9·· 9999 99 99

1H), 2.22 (m, 2H), 2.82 (m, 1H), 2.97 (m, 1H), 3.14 (m, 1H),

3.25 (m, 1H), 3.63 (m, 1H), 3.95 (m, 1H), 4.48 (m, 1H), 4.65 (t, 1H, J= 5.0), 4.96 (d, 1H, J= 13.0), 5.02 (d, 1H, J = 13.0), 7.07-7.33 (m, 25H), 7.42 (d, 1H, J = 8.0), 7.66 (d, 1H, J = 8.5), 7.86 (d, 1H, J = 8.0), 8.52 (s, 1H); Anal.

(C47H52N4O6Ό,5H2O) C, H, N.

Preparation of the intermediate Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H o-Iodoxybenzoic acid (0.63 g, 2.25 mmol, 3.0 equiv) was added to a solution of Cbz-L-Leu-L-Phe-L-(Trglutaminol) (0.58 g, 0.75 mmol, 1 equiv) in DMSO (7.5 mL) at 23°C. After stirring for 2 h, the DMSO was removed under reduced pressure. The residue was diluted twice with CH2Cl2 and the solvent was evaporated to remove any remaining DMSO. The residue was diluted with EtOAc (30 mL) and filtered, and the filtrate was washed with 5% Na2S2O3/5% NaHCO3 solution (30 mL), water (30 mL), and brine (30 mL), then dried over Na2SO4. The solvent was removed under reduced pressure to give Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H (0.53 g, 92% yield) as a white glass which was used immediately without further purification: NMR (DMSO-d6)

0.79 (m, 6H) , .1.00-1.98 (m, 5H) , 2.27 (m, 2H) , 2.84 (m,

1H), 3.02 (m, 1H), 3.98 (m, 2H), 4.58 (m, 1H), 4.99 (s, 2H), 7.14-7.32 (m, 25H), 7.39 (d, 1H, J= 8.1), 7.97 (d, 1H, J= 8.5), 8.38 (d, 1H, J = 8.0), 8.60 (s, 1H), 9.20 (s, 1H).

Preparation of the intermediate 5-{3'-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-Epropene}-isoxazole

KN((CH3)3S1)2 solution (0.95 ml of a 0.5 M solution in THF,

0.477 mmol, 1.0 equivalents) was added to the suspension ·· • 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

00 isoxazol-5-ylmethyl-triphenylphosphonium bromide (0.222 g, 0.525 mmol, 1.1 equivalents) in THF (20 mL) at 0°C and the reaction mixture was stirred at 0°C for 30 min. A solution of Cbz-L-Leu-L-Phe-L-(Tr-Gln)-H (0.366 g, 0.477 mmol, 1 equivalents) in THF (10 mL) was added and the reaction mixture was then stirred overnight at 23°C. The solvent was removed in vacuo and the residue was diluted with EtOAc (30 mL), washed with water (30 mL) and then dried over MgSO 2 . The solvent was removed under reduced pressure and the residue was purified by flash silica gel chromatography (gradient elution, 0-»1% CH3OH in CHCl3) to give 5{3'-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-E-propene}-isoxazole (0.307 g, 70% yield) as an amorphous solid: IR (cm“l)

3423, 1678, 1568, 1265, 1043, , 711; 3-H NMR (DMSO- cr6) δ 0.77- 0.81 (m, 6H) , 1.21-1.36 (m, 2H) , 1.40-1.55 (m, 1H) , 1.60- 1.80 (m, 2H) , 2.34-2.45 (m, 2H) , 2.82-2.87 (m, 1H) , 2.91- 3.04 (m, 1H) , 3.95-4.00 (m, 1H), 4.41-4.50 (m, 1H), 4.53- 4.60 (m, 1H), 4.99 (q, 2H, J = 6.0), 6.19 (d, 1H, J = 15.0), 6.36 (dd, 1H, J = 15.0, 6.0 ), 6.46 (s, 1H), 7, 15-7.33 (m, 20H) , 7.42 (d, 1H, J = 9, , 0) , 7.56-7.63 (m, 5H) , 7 .96 (d, 1H, J = 9 ,0) , 8.08 (d, 1H, J = 9, 0), 8.51 (s, 1H), 8, 58 (s, 1H);

HRMS calcd for C51H53N5O6+CS 964.3050 (M+Cs), found 964.3018.

Preparation of the product 5-(3(Cbz-L-Leu-L-Phe-L-Gln)-E-propene)isoxazole

Trifluoroacetic acid (1 mL) was added to a solution of 5-{3’-(Cbz-L-Leu-L-Phe-L-(Tr-Gln))-E-propene}-isoxazole (0.214 g, 0.257 mmol) in CH2Cl2 (10 mL) and the reaction mixture was stirred at 23°C overnight. The solvent was removed in vacuo and the residue was purified by flash silica gel chromatography (gradient elution, 0->1% CH3OH in CHCl3) to give 5-(3'-(Cbz-L-Leu-L-Phe-L-Gln)-Epropene)-isoxazole as a white solid (0.054 g, 36% yield): 1-H NMR (DMSO-d6) δ 0.77-0.83 (m, 6H), 1.26-1.46

(m, 2H), 1.47-1.62 (m, 1H), 1.69-1, 79 (m, 2H), 2.04-2.29 (m, 2H) , 2.83-2.88 (m, 1H) , 2.97-3.10 (m, 1H), 3.99-4.12 (m, 1H) , 4.37-4.43 (m, 1H) , 4.48-4.57 ( :m, 1H), 5.01 (q, 2H, J = 6.0) , 6.20 (d, 1H, J = 15.0), 6.36 (dd, 1H, J = 15.0, 6 ,0) , 6.45 (d, 1H, J = 3.0) , 6.75 (with, 1H), 7.14-7.29 (m, 6H) , 7.31 -7.40 (m, 5H), 7, 45 (d, 1H, J = 9.0), 8.04 (d, 1H, J = 9.0) , 8.07 (d, 1H, J = 9.0), 8.51 (d, 1H, J = 3.0) ; A: return

(C32H39N5O6) C, H, N.

Example 2 - Preparation of Compound A-1: Ethyl 3-((5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate

(A-1)

44 44 • 4 · » 4 • · · · ·

I »4 4 · 4 • · 4 4

H 44 «·

Preparation of the intermediate Boc-L-(Tr-Gln)-N(OMe)Me

Isobutyl chloroformate (4.77 mL, 36.8 mmol, 1.0 equiv) was added to a solution of Boc-L-(Tr-Gln)-OH (18.7 g, 36.7 mmol, 1 equiv) and 4-methylmorpholine (8.08 mL, 73.5 mmol, 2.0 equiv) in CH2Cl2 (250 mL) at 0°C. The reaction mixture was stirred at 0°C for 20 min, then N,O-dimethylhydroxyamine hydrochloride (3.60 g, 36.7 mmol, 1.0 equiv) was added. The resulting solution was stirred at 0°C for 20 min and at 23°C for 2 h, and then partitioned between water (150 mL) and CH2Cl2 (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification by chromatography (gradient flash column elution, 40—>20% hexanes in

EtOAc;

gave Boc-L-(Tr-Gln)N(OMe)Me (16.1 g, 82% yield) as a white foam: IR (cm“l) 3411, 3329, 3062, 1701, 1659; 1h NMR (CDCl3) δ 1.42 (s, 9H), 1.63-1.77 (m, 1H), 2.06-2.17 (m, 1H), 2.29-2.43 (m, 2H),

3.17 (s, 3H), 3.64 (s, 3H), 4.73 (s, br, 1H), 5.38-5.41 (m,

1H), 7.20-7.31 (m, 15H); Anal. (C31H37N3O5) C, Η, N.

Preparation of the intermediate Boc-L-(Tr-Gln)-H

Diisobutylaluminum hydride (50.5 mL of a 1.5 M solution in toluene, 75.8 mmol, 2.5 equiv) was added to a solution of Boc-L-(Tr-Gln)-N(OMe)Me (16.1 g, 30.3 mmol, 1 equiv) in THF at -78°C and the reaction mixture was stirred at -78°C for 4 h. Ethanol (4 mL) and 1.0 M HCl (10 mL) were added sequentially and the mixture was warmed to 23°C. The resulting suspension was diluted with Et2O (150 mL) and washed with 1.0 M HCl (3 x 100 mL), half-saturated NaHCO3 (100 mL) and water (100 mL). The organic layer was dried over MgSO4, filtered and concentrated to give crude Boc-L-(TrGln)-H (13.8 g, 97% yield) as a white solid: mp = 114-116°C; IR (cm ^-1 ) 3313, 1697, 1494; 1h NMR (CDCl ₃ ) δ 1.44 (s, 9H), 1.65-1.75 (m, 1H), 2.17-2.23 (m,

IH), 2.31-2.54 (m, 2H), 4.11 (s, br, IH), 5.38-5.40 (m, IH),

7.11 (s, 1H), 7.16-7.36 (m, 15H), 9.45 (s, 1H).

Preparation of the intermediate ethyl 3-(Boc-L-(Tr-Gln))-E-propenoate

Sodium bis(trimethylsilyl)amide (22.9 mL of a 1.0 M solution in THF, 22.9 mmol, 1.0 equiv) was added to a solution of triethylphosphonoacetate (5.59 g, 22.9 mmol, 1.0 equiv) in THF (200 mL) at -78°C and the resulting solution was stirred for 20 min at this temperature. Crude Boc-L-(TrGln)-H (10.8 g, 22.9 mmol, 1 equiv) in THF (50 mL) was added via cannula and the reaction mixture was stirred for 2 h at -78°C, warmed to 0°C for 10 min, and then partitioned between 0.5 M HCl (150 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (40% EtOAc in hexanes) gave ethyl 3-[Boc-L-(Tr-Gln)]-E-propenoate (10.9 g, 88% yield) as a white foam: IR (cm ^-1 ) 3321, 1710; !h NMR (CDCl 3 ) δ 1.27 (t, 3H, J= 7.2), 1.42 (s, 9H), 1.70-1.78 (m, 1H), 1.80-1.96 (m, 1H), 2.35 (t, 2H, J= 7.0), 4.18 (q, 2H, J= 7.2), 4.29 (s, br, IH) , 4.82-4.84 (m, IH) , 5.88 (dd, IH, J = 15.7,

1.6), 6.79 (dd, 1H, J = 15.7, 5.3), 6.92 (s, 1H), 7.19-7.34 (m, 15H); Anal. (C33H38N2O5) C, Η, N.

Preparation of the intermediate ethyl-3-(Boc-L-Phe-L-(Tr-Gln) )-Epropenoate • ·· ·· · · • ·

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was added to a solution of ethyl 3-(Boc-L-(Tr-Gln))-E-propenoate (3.26 g, 6.01 mmol, 1 equiv) in the same solvent (15 mL) at 23°C. After 2 h, the volatiles were removed under reduced pressure to give ethyl 3-(H2N-L(Tr-Gln))-E-propenoate«HCl. This material was dissolved in CH2Cl2 (60 mL) and Boc-L-Phe-OH (1.59 g, 6.01 mmol, 1.0 equiv), HOBt (1.22 g, 9.02 mmol, 1.5 equiv), 4-methylmorpholine (1.98 mL, 18.03 mmol, 3 equiv) and EDC (1.73 g, 9.02 mmol, 1.5 equiv) were added sequentially. The reaction mixture was stirred at 23°C overnight and then partitioned between water (100 mL) and CH2Cl2 (2 x 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was purified by flash column chromatography (40% EtOAc in hexane) to afford ethyl 3(Boc-L-Phe-L-(Tr-Gln))-E-propenoate (3.55 g, 85%) as a white foam: IR (cm“ ¹ ) 3306, 1706, 1661; 1h NMR (CDCl3) δ 1.29 (t, 3H, J= 7.2), 1.38 (s, 9H), 1.65-1.76 (m, 1H), 1.87-1.99 (m, 1H), 2.25-2.27 (m, 2H), 2.94-3.01 (m, 2H), 4.14-4.26 (m,

3H), 4.48-4.53 (m, 1H), 4.95 (s, br, 1H), 5.64 (d, 1H, J =

15.8), 6.29 (d, 1H, J = 8.1), 6.64 (dd, 1H, J = 15.8, 5.4),

6.80 (s, br, 1H), 7.14-7.32 (m, 20H); Anal. (C42H47N3O6)C,

H, N.

Preparation of the intermediate ethyl 3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) was added to a solution of ethyl 3-(Boc-L-Phe-L-(Tr-Gln))-E-propenoate (6.40 g, 9.28 mmol, 1 equiv) in the same solvent (15 mL) at 23°C. After 2 h, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (100 mL) and Boc-L-Leu-OH (2.58 g, 11.1 mmol, 1.2 equiv), HOBt (1.88 g, 13.9 mmol, 1.5 equiv), 4-methylmorpholine (3.06 mL, 27.8 mmol, 3 equiv) and EDC (2.67 g, 13.92 mmol, 1.5 equiv) were added sequentially. The reaction mixture was stirred at 23°C overnight and then partitioned between water (100 mL) and CH2Cl2 (2 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated, and the residue was purified by flash column chromatography (2% CH3OH in CH2Cl2) to give ethyl 3-(BocL-Leu-L-Phe-L-(Tr-Gln))-E-propenoate (6.46 g, 87% yield) as a white foam: IR (cm ^-1 ) 3284, 1651, 1515; A NMR

(CDC 13) δ 0.86 (d, 3H, J = 6.0), 0.89 (d, 3H, J = 6.0) , 1.29 (t, 3H, J = 7, 2) , 1.34 (s, 9H), 1.38-1.60 (m, 3H), 1.62-1.89 (m, IH) , ¹ .,95- 1, 97 (m, IH) , 2.28-2.30 (m, 2H) , 3.06-3.08 (m, 2H) , 3, 92- 3, 94 (m, IH), 4, 17 (q, 2H, J = 7 ,2) , 4.48-4.51 (m, 2H) , 4, 67 (m, ΪΗ) , 5, 66 (d, IH, J = 15, 9), 6.51-6.57 (m, 2H) , 6, 69 (dd, IH, J = 15, 6, 5,1), 7,10-7, 33 (m, 21H); Anal.

(C48H58N4O7*0.33H2O) C, Η, N.

Preparation of the intermediate ethyl 3-((5'-methylisoxazole-3'carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 3 mL) was added to a solution of ethyl 3-(Boc-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate (0.216 g, 0.27 mmol, 1 equiv) in the same solvent (3 mL) at 23°C. After 2 h, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (15 mL), cooled to 0°C, and triethylamine (0.112 mL, 0.81 mmol, 3.0 equiv) and 5-methylisoxazole-3-carbonyl chloride (0.058 g, 0.40 mmol, • · · · · ·

1.5 equivalents). The reaction mixture was stirred at 0°C for 30 min and then partitioned between water (50 mL) and CH2Cl2 (2 x 50 mL). The combined organic layers were dried over Na2SO4 and concentrated and the residue was purified by flash column chromatography (2% CH3OH in CH2Cl2) to give ethyl 3-((5'-methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-(TrGln) )-E-propenoate (0.199 g, 91% yield) as a white solid.

foams: IR (cm' Ί) 3286, 1650, 1541; ¹ h nm: R (CDCl3) δ 0.8 6 (d 3H, J = 5 ,4), 0.89 (d, 3H, J = 5.7) , 1, 28 (t, 3H, J = 7.2) 1.43-1.59 (m , 2H), 1.67-1.75 (m, IH) , 1.95-1.99 (m, 2H) 2.28 (t, 2H, J = 7.2 ), 2.41 (s, 3H) , 2.97-3.04 (m, IH) 3.06-3.13 (m, 1H), 4.17 (q, 2H, J = 7.2) , 4.31-4.33 (m, IH) 4.48-4.52 (m, , 2H), 5.72 (d, 1H, J = 1 5.9), 6.19 (with, IH) 6.41 (d, IH, J = 7.5), 6.59 (d, 1H, J = 8.1), 6.71 (dd , IH J = 15.3, 6, 0) , 6.95 (d, 1H, J = 6 ,6) , 7.09-7.21 (m, 21H)

Anal. (C48H53N5O7-H2O) C, Η, N.

Preparation of the product ethyl 3-((5'-methylisoxazole-3'-carbonyl)L-Leu-L-Phe-L-Gyn)-E-propenoate

Triisopropylsilane (0.077 mL, 0.376 mmol, 1.8 equiv) and trifluoroacetic acid (3 mL) were added sequentially to a solution of ethyl 3-((5'-methylisoxazole-3'-carbonyl)L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate (0.185 g, 0.21 mmol, 1 equiv) in CH2Cl2 (3 mL) at 23°C to form a bright yellow solution. The reaction mixture was stirred for 30 min at 23°C, during which time it became colorless. The volatiles were removed under reduced pressure and the resulting white solid was triturated with Et2O (10 mL), filtered and air dried to give ethyl 3-((5'-methylisoxazole3'-carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate (0.87 g, 81% yield).

• · · ·

yield) in the form of a white solid: Melting point = 223

225°C; IR (cm"1) 3298, 1662, 1544, 1457, 1278; 3-H NMR (DMSO-

cr6) δ 0.81 (d, 3H, J = 6.0 ), (t, 3H, J = 6.9), 1.38-1 .42 (m, (t, 2H, J = 7.2), 2.46 ( s, 3H), (m, 1H), 4.11 (q, 2H, J = 7.2), IH, J = 15.6), 6.56 (s, IH), 6, 6.76 (s, br, IH), 7.19 ( m, 6H) , (d, IH, J = 7.8), 8.58 1 (d, IH,

C, H, N.

0.85 (d, 3H, J = 6.3), 1.23

IH), 1.48-1.77 (m, 4H), 2.04 2.78-2.86 (m, IH), 2.93-3.00 4.36-4.54 (m, 3H), 5.63 (d, 68 (dd, IH, J = 15, 9, 5.4) , 8.09 (d, IH, J = 8.1), 8.14 J = 7.5); Anal. (C29H39N5O7)

Example 3 - Preparation of Compound A-2: Ethyl 3-((isoxazole-5'carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate

what ₂ ch ₂ ch ₃

(A-2)

Preparation of the intermediate ethyl 3-((isoxazole-5'-carbonyl)-L-LeuL-Phe-L-(Tr-Gln))-E-propenoate

This compound was prepared from ethyl 3-(Boc-L-Leu-LPhe-L-(Tr-Gln))-E-propenoate and isoxazole-5-carbonyl chloride using the procedure described above (Example 2) for the preparation of ethyl 3-((5'-methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-(TrGln) )-E-propenoate: IR (cm 3-) 3282, 1643, 1530; 3.p nmr (CDCl 3 ) δ 0.87 (t, 6H, J = 6.6), 1.29 (t, 3H, J = 7.2), 1.49-1.64 (m, 3H) , 1.69-1.80 (m, 1H) , 1.90-1.96 (m, 1H) ,

2.30 (t, 2H, J= 7.2), 2.92-2.96 (m, 1H), 3.02-3.09 (m, 1H),

4.17 (q, 2H, J = 7.2), 4.42-4.48 (m, 3H), 5.69 (d, 1H, J =

15.3), 6.65 (s, br, IH), 6.66 (dd, IH, J = 15.9, 5.4), 6.76 *

Β· ΒΒ • · « · · Β • · · · W

Β Β Β Β Β Β Β Β Β Β • ΒΒΒ · · ΒΒ

6.79 (m, 2Η), 7.00-7.31 (m, 22Η), 8.24 (s, 1H); Anal.

(C47H51N5O7·0.75 H2O) C, Η, Ν.

Preparation of ethyl 3-((isoxazole-5'-carbonyl)-L-Leu-L-Phe-L-GIn) E-propenoate

The title compound was prepared from ethyl 3-((isoxazole-5'-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-E-propenoate using a procedure analogous to that described above (Example 2) for the preparation of ethyl 3-((5'-methylisoxazole-3'carbonyl)-L-Leu-L-Phe-L-Gln)-E-propenoate: Melting point = 217-220°C; IR (cm ¹ ) 3302, 1655, 1541; NMR (DMSO-d6) δ

0.81 (d, 3H, J= 6.0), 0.86 (d, 3H, J= 6.0), 1.21 (t, 3H, J = 7.2), 1.42-1.75 (m, 5H), 2.04 (t, 2H, J = 7.2), 2.78-2.87 (m, ÍH), 2.94-3.01 (m, ÍH) , 4.11 (q, 2H, J = 7.2), 4.37 (m, ÍH) , 4.41-4.52 (m, 2H) , 5.64 (d, ÍH, J = 15.6), 6.68 (dd, ÍH, J = 15.9, 5.4), 6.76 (s, br, ÍH) , 7.12-7.19 (m, 7H), 8.02 (d, ÍH, J = 8.1), 8.20 (d, ÍH, J = 8.1), 8.74 (d, ÍH, J = 1.8), 8.94 (d, ÍH, J = 7.8); Anal. (C28H37N5O7) C, Η, N. Example 4 - Preparation of compound A-3: ethyl 3-((5'methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-MePhe)-L-Gln)-E-propenoate

(A-3)

Preparation of the intermediate ethyl 3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))E-propenoate

Ethyl 3-(H2N-L-(Tr-Gln))-E-propenoate»HCl (prepared as described in Example 2 above, 1.37 g, 3.10 mmol) • to • · • to • to

9· ·· was dissolved in DMF (10 mL) at 23°C. Diisopropylethylamine (1.08 mL, 6.20 mmol) was added, followed by BocL-(4-Me-Phe)-OH (0.87 g, 3.10 mmol). The reaction mixture was cooled to 0°C. HATU (1.18 g, 3.10 mmol) was added and the reaction mixture was allowed to warm to ambient temperature. DMF was removed in vacuo. The residue was dissolved in EtOAc (30 mL) and the organic phase was washed sequentially with 10% HCl solution (25 mL), saturated NaHCO3 solution (25 mL), H2O (25 mL) and brine (25 mL). The solvent was dried (MgSO4) and filtered and the residue was purified by flash column chromatography (gradient elution, 0—>0.75% CH3OH in CHCl3) to give ethyl 3-(Boc-L-(4-Me-Phe)-L-(TrGln) )-E-propenoate (1.48 g, 68% yield) as a white amorphous solid: IR (cm ^-1 ) 1713, 1655, 1491, 1175; ^H NMR (DMSO-d6) δ 1.20 (t, 3H, J = 7.0), 1.30 (s, 9H), 1.62-1.66 (m, 2H), 2.23 (s, 3H), 2.32 (m, 2H), 2.72 (m, 1H), 2.84 (m,

1H), 4.07-4.09 (m, 1H), 4.10 (q, 2H, J= 7.0), 4.38 (m, 1H), 5.64 (d, 1H, J = 15.5), 6.72 (dd, 1H, J = 15.5, 5.5), 6.88 (d, 1H, J = 8.0), 7.04 (d, 2H, J = 7.7), 7.10 (d, 2H, J = Ί,Ί), 7.14-7.28 (m, 15H), 8.02 (d, 1H, J = 8.0), 8.53 (s,

1H); Anal. (C43H49N3O5) C, Η, N.

Preparation of the intermediate ethyl 3-(Boc-L-α-(tert-butyl-Gly)-L(4-Me-Phe)-L-(Tr-Gln))-E-propenoate

Ethyl 3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate (1.45 g, 2.06 mmol) was dissolved in 1,4-dioxane (27 mL) and a solution of HCl in 1,4-dioxane (4.0 M, 14 mL) was added. The reaction mixture was stirred at ambient temperature for 4 h. The solvent was removed by evaporation and the residue was taken up in EtOAc (50 mL). The organic phase was washed with saturated NaHCO3 solution (50 mL) and then brine (50 mL), dried (MgSO4) and the solvent was removed under reduced pressure to give

1.23 g of an off-white amorphous solid. This material was coupled with Boc-L-α-(tert-butyl-Gly)-OH using the procedure described for the synthesis of ethyl 3-(Boc-L-(4-Me-Phe)-L-(Tr-Gln))-Epropenoate and given above to afford ethyl 3-(Boc-La(tert-butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate (49% yield) as a white amorphous solid: IR (cm ^-1 ) 1655, 1507,

1248, 1171; ^Τ Η NMR (DMSO-d 6 ) δ 0.81 (with, 9H), 1.21 (t, 3H, J = 7.0), 1.37 (s, 9H), 1.52-1.70 (m, 2H) , 2.22 (s, 3H), 2.26- 2.28 (m, 2H) , 2.73-2.91 (m, 2H) , 3.86 (d, 1H, J = 9.6), 4.05-4.14 (m, 2H), 4.31-4.36 (m, IH) , 4.47-4.55 (m, 1H), 5.54 (d, IH, J= 15.4), 6.37 (d, IH, J = 9.6), 6.65 (dd, IH, J = 15.8, 5.5), 7.01 (d, 2H, J = 8.1) , 7.07 (d, 2H, J = 7.7), 7.11-7.32 (m, 15H), 8.03 (d, IH, J = 8.1), 8.10 (d,

1H, J = 7.7), 8.49 (s, 1H); Anal. (C49H6ON4O7·0.4H2O) C, H,

N.

Preparation of the intermediate ethyl-3-((5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-Epropenoate

Ethyl 3-(Boc-L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-(TrGln))-E-propenoate was deprotected using the procedure described for the deprotection of ethyl 3-(Boc-L-(4-Me-Phe)-L-(TrGln) )-E-propenoate and given above and the resulting amine (0.22 g, 0.30 mmol) was dissolved in CH2Cl2 (3 mL). Pyridine (0.025 mL, 0.32 mmol) was added and the reaction mixture was cooled to 0°C. 5-Methylisoxazole-3-carbonyl chloride (0.046 g, 0.32 mmol) was added. The reaction mixture was allowed to warm to ambient temperature and stirred for one hour.

• ·

The solvent was removed in vacuo and the residue was subjected to flash column chromatography (gradient elution, 0->1% CH3OH in CH2Cl2) to give ethyl 3-((5'methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-MePhe)-L-(Tr-Gln))-E-propenoate (0.19 g, 77% yield) as

white amorphous substances: IR ( cm 1) 1651, 1518; 1 h NMR (DMSO-Cl 6 ) δ 0.88 (s, 9H), 1.20 (t, 3H, J = 7.0), 1.55-1.67 (m, 2H), 2.14 (s, 3H), 2.18-2.28 (m, 2H) , 2 .45 (s, 3H), 2.70-2.77 (m, 1H), 2.86-2.93 (m, 1H), 4.07-4.14 (m, 2H) , 4.46 (d, 1H, J = 9.6), 4.50-4.55 (m, 1H) , 5.54 (d, 1H, J = 15.8), 6.59 (s, 1H) , 6.65 (dd, 1H, J = 15.8, 5.5, 15.8), 6.95 (d, 2H, J = 8.1),

7.05 (d, 2H, 8.1), 7.13-7.28 (m, 15H), 7.60 (d, 1H, J =

9.6), 8.13 (d, 1H, J = 8.1), 8.41 (d, 1H, J = 8.1), 8.51 (s, 1H); Anal. (C49H55N5O7) C, Η, N.

Preparation of the product ethyl 3-((5'-methylisoxazole-3'-carbonyl)L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-propenoate

Ethyl 3-((5'-methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate (0.17 g, 0.20 mmol) was dissolved in CH2Cl2 (4 mL) at 23°C. Trifluoroacetic acid (0.4 mL) was added and the reaction mixture was stirred at ambient temperature for six hours. The solvents were removed in vacuo and the residue was subjected to flash column chromatography (gradient elution, 0->2% CH3OH in CH2Cl2) to give ethyl 3-((5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-Gln)-Epropenoate (0.085 g, 73% yield) as a white amorphous solid: IR (cm ^-1 ) 1661, 1541, 1206; !h NMR (DMSO-c/6) δ 0.88 (s, 9H), 1.21 (t, 3H, J= 7.0), 1.60-1.73 (m, 2H), 2.01-2.06 • · · (m, 2H), 2.14 (s, 3H), 2.50 (s, 3H), 2.70-2.77 (m, 1H) ,

2.86-2.93 (m, 1H) , 4.07-4.14 (m, 2H) , 4.34-4.37 (m, 1H) ,

4.45 (d, 1H, J= 9, 6), 4.50-4.55 (m, 1H), 5.57 (d, 1H, J =

15.8), 6.60 (s, 1H), 6.66 (dd, 1H, J = 15.8, 5.5), 6.75 (s, br, 1H), 6.96 (d, 2H, J= 8.1), 7.06 (d, 2H, J= 7.7), 7.17 (s, br, 1H) , 7.65 (d, 1H, J = 9.6), 8.14 (d, 1H, J = 8.1),

8.40 (d, 1H, J = 7.7); Anal. (C30H4iN5O7*0.5TFA*0.5H2O) C, H, N.

Example 5

Preparation of compound A-4: ethyl-3-((5'methylisoxazole-3'-carbonyl)-L-ct

Phe)-L-Gln)-E-propenoate

-(tert-butyl-Gly)-L-(4-F-

The solvent was subjected to flash (A-4)

Preparation of the intermediate ethyl 3-(Boc-L-(4-F-Phe)-L-(Tr-Gln))E-propenoate

Boc-L-(4-F-Phe)-OH (1.41 g, 5.0 mmol) was dissolved in THF (50 mL). Ethyl 3-(H2N-L-(Tr-Gln))-Epropenoate*HCl (prepared as described in Example 2 above, 1.0 g, 5.0 mmol) was added, followed by Et3N (0.70 mL, 5.0 mmol). Carbonyldiimidazole (0.81 g, 5.0 mmol) was added and the reaction mixture was stirred at ambient temperature for 20 h.

removed in vacuo and the residue was column chromatographed (gradient elution, 0—»1% CH3OH in CH2Cl2) to give ethyl 3-(Boc-L(4-F-Phe)-L-(Tr-Gln))-E-propenoate (1.13 g, 32% yield) as a white amorphous solid: IR ( ^cm_7 ) 1712, 1666, 1510, 1169;

• ·

1h NMR (DMSO-d6) δ 1.20 (t, 3H, J = 7.0), 1.29 (with, 9H) , 1.61-1.70 (m, 2H), 2.27-2.34 (m, 2H), 2.74-2.78 (m, 1H) , 2.86-2.90 (m, 1H), 4.06-4.13 (m, 3H), 4.36-4.40 (m, 1H) , 5.58 (d, 1H, J = 15.6), 6.71 (dd, 1H, J = 15.6, 5, 5), 6, 98 (d, 1H, J = 8.1), 7.03-7.09 (m, 2H), 7.14-7.28 (m, 17H) , 8.06 (d, 1H, J = 8.1), 8.53 (s, 1H) ; LRMS (M+Na) 730 • Preparation intermediate ethyl-3 -(Boc-L-α-(tert-butyl-Gly)-L-

of (4-F-Phe)-L-(Tr-Gln))-E-propenoate

Ethyl 3-(Boc-L-(4-F-Phe)-L-(Tr-Gln))-E-propenoate was deprotected and coupled with Boc-L-α-(tert-butyl-Gly)-OH using the procedures described above for the preparation of ethyl 3-(BocL-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-E-propenoate to afford ethyl 3-(Boc-L-α-(tert-butyl-Gly)-L-(4-F-Phe)L-(Tr-Gln))-E-propenoate (54% yield) as a white amorphous solid: IR (cm ^-1 ) 1720, 1651, 1506, 1168; ^H NMR (DMSO-c?6) δ

0.80 (s, 9H), 1.20 (t, 3H, J = 7.0), 1.36 (s, 9H), 1.53-1.67 (m, 2H), 2.23-2.28 (m, 2H), 2.79-2.94 (m, 2H), 3.85 (d, 1H, J= 9.9), 4.09 (q, 2H, J= 7.0), 4.31-4.35 (m, 1H), 4.534.55 (m, 1H), 5.46 (d, 1H, J = 15.8), 6.36 (d, 1H, J = 9.2), 6.64 (dd, 1H, J= 15.8, 5.5), 6.97-7.03 (m, 2H), 7.13-7.28 (m, 17H), 8.08 (d, 1H, J = 8.1), 8.14 (d, 1H, J = 8.1), 8.49 (s, 1H); Anal. (C48H57N4O7F) C, Η, N.

Preparation of the intermediate ethyl-3-((5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-Epropenoate

Ethyl 3-(Boc-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-(TrGln))-E-propenoate was deprotected and coupled with 5-methylisoxazole-3-carbonyl chloride using the procedures described above for the preparation of ethyl 3-((5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-Me-Phe)-L-(Tr-Gln))-Epropenoate to afford ethyl 3-((5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln))-Epropenoate (74% yield) as a white amorphous solid: IR (cm ^-1 ) 1659, 1535, 1510; NMR (DMSO-d6) δ 0.88 (s, 9H) ,

1.20 (t, 3H, J = 7.4), 1.52-1.67 (m, 2H), 2.23-2.28 (m, 2H), 2.45 (with, 3H) , 2.75-2.82 (m, IH) , 2.89-2.96 (m, IH), 4.09 (q, 2H, J = 7.0) , 4.32-4.36 (m, 1H) , 4.45 (d, 1H, J = 9.6), 4.50· -4.55 (m, IH) , 5.44 (d, IH, J = 15.6), 6.58 (s, IH) , 6, 63 (dd, Hello, J = 15.6, 5.5, 15.6), 6.93-6.99 (m, 2H), 7.13- 7.28 (m, 17H) , 7, 64 (d, 1H, J = 9.6) , 8.16 (d, 1H, J = 8.5), 8.46 (d, !H, J = 8.1), 8.51 (s, IH) ; Anal. (C48H52N5O7F) C,

H, N.

Preparation of ethyl 3-((5'-methylisoxazole-3'-carbonyl)-L-α(tert-butyl-Gly)-L-(4-F-Phe)-L-Gln)-E-propenoate

Ethyl 3-[(5'-methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-(Tr-Gln)]-E-propenoate was deprotected using a procedure analogous to that described above for the preparation of ethyl 3-((5'-methylisoxazole-3'-carbonyl)-La(tert-butyl-Gly)-L-(4-Me-Phe)-L-Gln)-E-propenoate to afford ethyl 3-((5'-methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-Gln)-E-propenoate (80% yield) as a white amorphous solid: IR (cm ^-1 ) 1653, 1543, 1223; !h NMR (DMSO-c? 6 ) δ 0.88 (s, 9H), 1.21 (t, 3H, J = 7.0), 1.59-1.75 (m, 2H), 2.01-2.06 (m, 2H), 2.46 (s, 3H), 2.75-2.82 (m, 1H), 2.89-2.96 (m, 1H), 4.09 (q, 2H, J = 7.0), 4.33-4.36 (m, 1H),

0

4.44 (d, ÍH, J= 9.6), 4.50-4.58 (m, ÍH), 5.47 (d, ÍH, J =

15.8), 6.59 (s, ÍH), 6.64 (dd, ÍH, J= 15.8, 5.5), 6.75 (s, br, ÍH), 6.94-7.00 (m, 2H), 7.16 (s, br, ÍH), 7.18-7.23 (m, 2H), 7.69 (d, ÍH, J = 9.6), 8.16 (d, 1H, J= 8.1), 8.44 (d, 1H, J= 8.1); Anal. (C29H38N5O7F) C, Η, N.

Example 6 - Preparation of Compound A-5: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-pyrroleAla)}-E-propenoate

Preparation of the intermediate tert-butyl ester of (4S)-4(2'-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid

Sodium hydroxide (27 mL of a 4.0 M solution in H2O, 108 mmol, 3.0 equiv) was added to a solution of (4S)-4-(2-methoxycarbonylethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (prepared as described in Chida et al., J. Chem. Soc., Chem. Commun. 1992, 1064) (10.5 g, 36.5 mmol, 1 equiv) in CH3OH (150 mL) and the resulting cloudy reaction mixture was stirred at 23°C for 3.5 h. The mixture was concentrated under reduced pressure to a volume of approximately 30 mL and then partitioned between 0.5 M HCl (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were dried over MgSO4 and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was dried under vacuum to give (4S)-4-(2'-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (10.0 g, 100% crude yield). This material was used without further purification: NMR (CDCl3, mixture of rotamers) δ 1.49 (s), 1.57 (s),

1.60 (s), 1.84-2.05 (m), 2.39-2.41 (m), 3.71-3.74 (m), 3.914.05 (m).

Preparation of the intermediate (4S,4'' S)-4-{3'-(4''-benzyl-2''-oxoxazolidin-3' '-yl)-3'-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester

Triethylamine (8.87 mL, 63.6 mmol, 3.0 equiv) and pivaloyl chloride (2.61 mL, 21.2 mmol, 1.0 equiv) were added sequentially to a solution of (4Sj-4-(2'-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (5.80 g, 21.2 mmol, 1 equiv) in THF (450 mL) at 0°C. The cloudy reaction mixture was stirred at 0°C for 3.5 h, then lithium chloride (0.988 g, 23.3 mmol, 1.1 equiv) and (S)(-)-4-benzyl-2-oxazolidinone (3.57 g, 20.1 mmol, 0.95 equiv) were added sequentially. After warming to 23°C and stirring for 19 h, the reaction mixture was The mixture was partitioned between 0.5 M HCl (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were washed with half-saturated Na2CO3 (150 mL), dried over MgSO4, and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography (30% EtOAc in hexanes) to give (4S, 4''S)-4-{3'-(4''-benzyl-2''-oxo-oxazolidin-3' 'yl)-3'-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (7.17 g, 83%) as a colorless oil: IR (cm“l) 2978, 1783,

1694; 1-H NMR (CDCl3, mixture of rotamers) δ 1.49 (s), 1.59 (s),

1.63 (s), 2.01-2.10 (m), 2.76 (dd, J = 13.5, 9.8), 2.82-3.13 (m), 3.30-3.41 (m), 3.76-3.82 (m), 3.90 (s, br), 3.97 (dd, J

= 9.0, 5.6), 4.10-4.19 (m), 4.63-4.71 (m), 7.22-7.36 (m);

Anal. (C23H32N2O6) C, H, N.

Preparation of the intermediate tert-butyl ester of (2'S,4S,4'' S)-4-{2'-(4''-benzyl-2''-oxo-oxazolidine-3''carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-carboxylic acid

A solution of (4S,4''S)-4-{3' (4''-benzyl-2''-oxo-oxazolidin-3''-yl)-3'-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (7.17 g, 16.6 mmol, 1 equivalent) in THF (50 mL) was added to a solution of sodium bis(trimethylsilyl)amide (16.6 mL of a 1.0 M solution in THF, 16.6 mmol, 1.0 equivalent) in the same solvent (150 mL) at -78°C. The reaction mixture was stirred for 20 min at -78°C and then allyl iodide (4.55 mL, 49.8 mmol, 3.0 equivalent) was added. After stirring for an additional 3 hours at -78°C, the reaction mixture was maintained at -45°C for 2 hours and then partitioned between a 2:1 mixture of half-saturated NH4Cl and 5% Na2S2Cl3 (300 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 200 mL). The combined organic layers were washed with H2O (200 mL), dried over MgSO4 and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography (15% EtOAc in hexanes) to give (2'S,4S,4''S)-4-{2'-(4''-benzyl-2''-oxo-oxazolidine3''-carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-

carboxylic (4.29 g, 55%) in in the form of a colorless oil: IR (cm 1) 2978, 1780, 1695; ^: H NMR (CDCl3, mixture of rotamers) δ 1.45 (s), 1.49 (s), 1.68-1.80 (m) , 2.13-2.47 (m), 2.49-2.67 (m) , 3.32 (dd, J = 13.4, 3, 1) , 3, 69-3, 97 (m) , 4,11-4,21 (m),

• · fe fe * · • fefe fefe fefe « fefe fefe • fefe · fefe fefe · • · · · • fefe fefe

4.66-4.74 (m) , 5.06-5.13 (m) , 5.74-5.88 (m) , 7.20-7.36 (m) i

Anal. ( ^C26H36N2C >6) C, Η, N.

Preparation of the intermediate tert-butyl ester of (1S,3S){3-(1'-(2'',4''-dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1hydroxymethylpropyl}-carbamic acid

Ozone was bubbled through a solution of (2' S, 4S, 4''S)-4-(2'-(4''-benzyl-2’'-oxo-oxazolidine-3''-carbonyl)-pent-4'-enyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (4.29 g, 9.08 mmol, 1 equiv) in CH2Cl2 (200 mL) and CH3OH (0.735 mL, 18.1 mmol, 2.0 equiv) at -78°C until the blue color persisted. The reaction mixture was then purged with argon until it became colorless. Methyl sulfide (6.67 mL, 90.8 mmol, 10 equiv) was added, the mixture was stirred at -78°C for 3.5 h, and maintained at 0°C for an additional 1 h. After partitioning the reaction mixture between H2O (200 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 200 mL), the combined organic layers were dried over MgSO4 and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was used immediately without further purification.

The above residue was dissolved in a 2:1 mixture of THF and EtOH (240 mL) at 23°C and 2,4-dimethoxybenzylamine hydrochloride (7.40 g, 36.3 mmol, 4.0 equiv), sodium acetate (2.98 g, 36.2 mmol, 4.0 equiv) and sodium cyanoborohydride (1.14 g, 18.1 mmol, 2.0 equiv) were added sequentially. The resulting suspension was stirred for 18 h at 23°C and then partitioned between 0.5 M HCl (400 mL) and EtOAc (2 x 200 mL). The combined organic layers were washed with half-saturated NaHCO3 (300 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was left • to • » • to · to · · · · · · · * · « • to · · ·

-· ·· to · · · • · «to ···· • to ···· ··· ··· ·* ·· pass through a short silica gel column (eluting with 50% EtOAc in hexanes) to obtain (3'S,4S)-4-{1' -(2'' ,4' '-dimethoxybenzyl)-2'-oxo-pyrrolidin3'-ylmethyl}-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester, contaminated with (S)-(-)-4-benzyl-2-oxazolidinone.

This material was dissolved in CH3OH (100 mL) and TsOH»H2O (0.345 g, 1.81 mmol, 0.20 equiv) was added. The reaction mixture was heated to 50°C and maintained at this temperature for 2.5 h. After cooling to 23°C, the reaction mixture was concentrated under reduced pressure to a volume of approximately 20 mL and partitioned between half-saturated NaHCO3 (150 mL) and a 9:1 mixture of CH2Cl2 and CH3OH (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification of the residue by flash column chromatography (3% CH3OH in CH2Cl2) gave (1S,3'S)-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-pyrrolidin-5'-yl)-1-

hydroxymethylethyl}-carbamic (1.62g, 44%) in form foams: IR (cm ^-1 ) 3328, 1669; 3-H NMR (CDCl3) δ 1.44 (with, 9H) , 1.50- 1.75 (m, 2H) , 1.90-2.00 (m, 1H) , 2.17-2.27 (m, 1H) , 2.52- 2.62 (m, 1H), 3.14-3.24 (m, 2H) , 3.51-3.65 (m, 3H) , 3.70- 3.78 (m, 1H), 3.80 (s, 6H), 4 , 35 (d, 1H, J = 14.3 ), 4, 48 (d, 1H, J = 14.3), 5.51-5.54 (m, 1H), 6.42-6.46 (m, 2H) , 7.09-

7.12 (m, 1H); Anal. (C21H32N2O6) C, Η, N.

Preparation of the intermediate ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala])-E-propenoate

DMSO (0.270 mL, 3.80 mmol, 3 equivalents) was added dropwise to a solution of oxalyl chloride (0.166 mL, 1.90 mmol, 1.5 equivalents) in CH2Cl2 (14 mL), cooled to -78°C.

·· ·· • · 9 9

9 9 9

9 9 9 • ·* 9 9

99

The reaction mixture was stirred for 20 min, then a solution of (1S, 3'S)-{2-(1'-(2'', 4''dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester (0.518 g, 1.27 mmol, 1 equiv) in CH2Cl2 (13 mL) was added via cannula to the side of the reaction vessel. After stirring for 20 min, triethylamine (1.06 mL, 7.60 mmol, 6 equiv) was added dropwise and the reaction mixture was stirred for 1.5 h. Acetic acid (0.479 mL, 8.37 mmol, 6.6 equiv) was added and the reaction mixture was warmed to 0°C for 5 min, then diluted with MTBE (200 mL) and washed with water, saturated NaHCO3 and brine (25 mL each). The organic phase was dried over Na2SO4 and concentrated to give the crude aldehyde as a foam (0.516 g, quant.), which was used without further purification.

Sodium bis(trimethylsilyl)amide (1.23 mL of a 1.0 M solution in THF, 1.23 mmol, 1 equiv) was added to a solution of triethylphosphonoacetate (0.244 mL, 1.23 mmol, 1 equiv) in THF (15 mL) at -78°C and the resulting solution was stirred for 20 min at this temperature. The crude aldehyde (prepared above, 0.500 g, 1.23 mmol, 1 equiv) in THF (13 mL) was added via cannula from the side of the reaction vessel and the reaction mixture was stirred for 45 min at -78°C, warmed to 0°C over 7 min and partitioned between 0.5 M HCl (20 mL) and MTBE (2 x 50 mL). The combined organic layers were dried over MgSO4 and concentrated. Purification of the residue by flash column chromatography (60% EtOAc in hexanes) gave ethyl 3-{Boc-L-({N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-

propenoate (0.356 g, 61%) in the form of white foam: Rf = 0.43 (60% EtOAc in hexanes); IR (cm ¹ ) 3307, 1708, 1678; !h NMR (CDCl3) δ 1.28 (t, 3H, J = 7.2), 1.43 (s, 9H) , 1.52-1.70 (m, 2H), 1.98-2.09 (m, 1H), 2.21-2.34 (m, 1H), 2.48-2.59 (m,

«4

IH), 3.16-3.24 (m, 2H), 3.80 (s, 6H), 4.18 (q, 2H, J= 7.2), 4.27-4.40 (m, IH), 4.41 (s, 2H), 5.40 (d, IH, J = 8.1), 5.95 (dd, IH, J = 15.6, 1.6). 6.41-6.48 (m, 2H), 6.86 (dd, 1H, J = 15.6, 5.3), 7.08-7.13 (m, 1H); Anal. (C25H36N2O7·0.25H2O)

C, H, N.

Preparation of the intermediate ethyl 3-{Boc-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate

This material was prepared from ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate and Boc-L-(4-FPhe)-OH using a procedure similar to that described for the preparation of ethyl 3-(Boc-L-(4-Me-Phe)-L-(Tr-G2n))E-propenoate (Example 4) above: Rf = 0.34 (60%

EtOAc in hexanes); IR (cm ^-1 ) 3258, 1705, 1666; 3-H NMR (CDCl 3 ) δ 1.28 (t, 3H, J = 7.2), 1.45 (s, 9H), 1.51-1.66 (m,

2H), 1.78-1.90 (m, 1H), 2.06-2.23 (m, 2H), 2.99 (dd, 1H, J =

13.7, 6.2), 3.11 (dd, 1H, J = 13.7, 5.3), 3.17-3.23 (m, 2H),

3.80 (s, 3H), 3.81 (s, 3H), 4.18 (q, 2H, J= 7.2), 4.35 (s,

2H), 4.38-4.51 (m, 2H), 5.29-5.37 (m, 1H), 5.76 (d, 1H, J =

15.8), 6.43-6.47 (m, 2H), 6.72 (dd, 1H, J = 15.8, 5.3),

6.83-6.91 (m, 2H), 7.09-7.17 (m, 3H), 7.92 (br, 1H); Anal. (C34H44FN3O8) C, Η, N.

Preparation of the intermediate ethyl 3-{Boc-L-Val-L-(4-F-Phe)-L-[(N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala]}-E-propenoate

This compound was prepared from ethyl 3-{Boc-L-(4-FPhe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-Epropenoate and Boc-L-Val-OH using a procedure similar to that described above for the preparation of ethyl 3-(Boc-L-Phe-L(Tr-Gln))-E-propenoate (Example 2): Rf = 0.24 (60% EtOAc in • ·» ·· «

4 ·· • »44· • ♦ · · * • · · · ϊ

44·· ··· ···· • · ¹ ·· ··

hexanes); IR (cm ) 3284 , 1713, 1678 br, 1643; lH (CDCl3) δ 0.91 (d, 3H, J = 6.8), 0.97 (d, 3H, J = 6.8) , (t, 3H, J = 7.2), 1 .45 (with, 9H), 1.50-1.62 (m, 2H), 1.66- (m, IH), 1.90-2.02 (m, IH) , 2.08-2.21 (m, 2H) , 2.94 (dd,

J = 13.5, 5.8), 3.17-3.27 (m, 3Η), 3.80 (s, 3Η), 3.82 (s,

3Η), 3.97-4.05 (m, 1Η), 4.17 (q, 2Η, J = 7.2), 4.27 (d, 1H,

J= 14.3), 4.29-4.38 (m, IH), 4.40 (d, IH, J= 14.3), 4.864.93 (m, IH), 5.10 (d, IH, J= 8.7), 5.76 (dd, IH, J = 15.6,

1.2), 6.45-6.52 (m, 2H), 6.70 (dd, IH, J = 15.6, 5.4), 6.796.88 (m, 3H), 7.12-7.22 (m, 3H), 8.30 (d, IH, J = 5.9);

Anal. (C39H53FN4O9) C, H, N.

Preparation of the intermediate ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-Val-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)pyrrole-Ala)}-E-propenoate

This compound was prepared from ethyl 3-{Boc-L-Val-L(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-Epropenoate and isoxazole-5-carbonyl chloride using the procedure described above (Example 2) for the preparation of ethyl 3-( (5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-(Tr-Gln))-Epropenoate: Rf = 0.36 (5% CH3OH in CH2Cl2); IR (cm ^-1 ) 3284,

1717, 1650; A NMR (CDCl3) δ 0.97 (d, 3H, J= 6.8), 1.01 (d, 3H, J = 6,8) , 1.28 (t, 3H, J = 7.2), 1.51-1.64 (m, 2H) , 1.72-1.84 (m, ÍH) , 1.95-2.05 (m, 1H), 2.11-2.33 (m, 2H) , 2.48 (s, 3H) , 2.98 (dd, IH, J = 13.7, 5, 6) , 3.16- -3.24 (m, 3H), 3.80 (with, 3H) , 3.81 (s, 3H), 4.17 (q, 2H, J = 7 ,2) , 4.23 (d, IH, J = 14.3) , 4.31-4.42 (m, IH) , 4.40 (d, !H, J =

14.3).

5.3) , 6.80-6.88 (m, 2H) , 6.94 (d, 1H, J = 9.3), 7.11-7.17

(m, 3H), 7.29 (d, 1H, J = 8.7), 8.33 (d, 1H, J = 6.2); Anal. (C39H48FN5O9O, 5H2O) C, Η, N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val-L-(4-F-Phe)-L-( (S)-pyrrole-Ala)}-E-propenoate

A suspension of ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-LVal-L-(4-F-Phe)-L-( (N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}E-propenoate (0.263 g, 0.351 mmol, 1 equivalent), water (2 drops) and DDQ (0.104 g, 0.458 mmol, 1.3 equivalents) was heated to reflux for 9 hours and then allowed to cool to ambient temperature over 8 hours. The reaction mixture was diluted with CH2Cl2 (200 mL) and washed with a 2:1 mixture of saturated NaHCO3 and 1 N NaOH (20 mL). The organic phase was dried over MgSO4 and evaporated. Purification of the residue by flash column chromatography (gradient elution 2—>3% CH3OH in CH2Cl2) gave ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-ValL-(4-F-Phe)-L-( (S)-pyrrole-Ala)}-E-propenoate (0.117 g, 56%) as a white solid: Melting point = 219-220°C; Rf = 0.23 (5% CH3OH in CH2Cl2); IR (cm ¹ ) 3401 br, 3295, 1655 br; ¹ H NMR (CDCl3) δ 0.94 (d, 3H, J = 6.8), 0.97 (d, 3H, J =

6.5), 1.29 (t, 3H, J = 7.2), 1.54-1.65 (m, 1H), 1.72-1.91 (m, 2H), 2.07-2.26 (m, 2H), 2.28-2.39 (m, 1H), 2.49 (d, 3H, J= 0.9), 3.01 (dd, 1H, J = 13.8, 6.1), 3.12 (dd, 1H, J =

13.8, 6.4), 3.26-3.38 (m, 2H), 4.18 (q, 2H, J = 7.2), 4.34 (dd, 1H, J = 8.7, 7.2), 4.43-4.54 (m, 1H), 4.90 (dt, 1H, J = 9.0, 6.2), 5.76 (dd, 1H, J = 15.6, 1.6), 6.00 (s, 1H), 6.42 (q, 1H, J = 0.9), 6.72 (dd, 1H, J = 15.6, 5.4), 6.86-6.94 (m, 2H), 7.01 (d, 1H, J = 9.0), 7.11-7.18 (m, 2H), 7.21 (d, 1H, J = 8.7), 7.76 (d, 1H, J = 7.2); Anal. (C30H38FN5O7)C,

H, N.

.4

Example 7 - Preparation of Compound A-6: Ethyl 3-[(5'methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)-L-Gln]-Epropenoate

Preparation of the intermediate ethyl 3-(Boc-L-(4-F)-Phe-L-(Tr-Gln)}E-propenoate

This compound was prepared from ethyl 3-(Boc-L-(TrGln))-E-propenoate and Boc-L-(4-F)-Phe-OH using a procedure similar to that described above (Example 2) for the preparation of ethyl 3-(Boc-L-Phe-L-(Tr-G_Zn) )-E-propenoate: IR (cm ^-1 ) 3328,

1707, 1506, 1168; !h NMR (CDCl 3 ) δ 1.29 (t, 3H, J = 7.2),

1.37 (s, 9H), 1.66- 1.78 (m, IH), 1.88-1.98 (m, IH), 2.32 (t, 2H, J = 6.6), 2.85- 2.92 (m, IH), 2.97-3.04 (m, IH), 4.18 (q, 2H, J = 7.2), 4.52 (m, IH) , 4.96 (m, 1H), 5.60 (d, IH, J = Í5,6) , 6.56 (d, IH, J = 8.1 ), 6.66 (dd, IH , J = 15.6, 5.1), 6, 80 (s, br, IH), 6, 92 -6.98 (m, 2H) , 7, 08-7.12 (m, 2H),

7.17-7.23 (m, 6H), 7.24-7.33 (m, 10H); Anal. (C42H45FN3O6)

C, H, N.

Preparation of the intermediate ethyl 3-(Boc-L-Val-L-(4-F)-Phe-L-(TrGln)}-E-propenoate

This compound was prepared from ethyl 3-{Boc-L-(4-F)Phe-L-(Tr-Gln)}-E-propenoate and Boc-L-Val-OH by the method described above (Example 2) for the preparation of ethyl 3-{Boc-L-Leu-LPhe-L-(Tr-Gln)}-E-propenoate: IR (cm ^-1 ) 3319, 1657, 1511,

1172; !h NMR (CDCl 3 ) δ 0.78 (d, 3H, J= 6.9), 0.87 (d, 3H, J

= 6, 9), 1.29 (t, 3H, J = 7, 2), 1.37 (s, 9H), 1.69-1.79 (m, 1H) , 1.93-2.06 (m, 2H), 2.33 (t, 2H, J = 7.2), 2.97-3.04 (m, 1H) , 3.73-3.77 (m, 1H), 4.18 (q, 2H, J = 7.2), 4.42-4.54 (m, 2H) , 4.80 (d, 1H, J = 6.9) , 5.61 (dd, 1H, J = 15.6, 1.5), 6.44 (d, 1H, J = 7.8) , 6, 69 (dd, 1H, J = 15, 9, 5.4), 6.71

(s, br, 1H), 6.92-6.98 (m, 2H), 7.07-7.13 (m, 2H), 7.18-7.31 (m, 16H), 8.02 (s, 1H); Anal. (C47H55FN4O7) C, Η, N.

Preparation of the intermediate ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-Val-L-(4-F)-Phe-L-(Tr-Gln)}-E-propenoate

This compound was prepared from ethyl 3-{Boc-L-Val-L(4-F)-Phe-L-(Tr-Gln)}-E-propenoate and 5-methylisoxazole-3-carbonyl chloride by the method described above (Example 2) for the preparation of ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Leu-LPhe-L-(Tr-Gln)}-E-propenoate: IR (cm~1) 3319, 1657, 1511,

1172; !h NMR (CDCl 3 ) δ 0.84 (d, 3H, J = 6.9), 0.89 (d, 3H, J = 6.9), 1.30 (t, 3H, J = 7.2), 1.68-1.80 (m, 1H), 1.95-2.06

(m, 1H), 2.08-2 ,17 (m, 1H), 2.34 (t, 2H, J = 7.2 ), 2.44 (s, 3H) , 2.87-2.94 (m, 1H) , 3.01-3.08 (m, 1H), 4.18 (q, 2H, J = 7.2) , 4.48-4.56 (m, 2H) , 5.68 (dd , 1H, J = 15.6, 1.8), 6.23 (with, 1H) , 6.39 (d, 1H, J = 7.8), 6.70 (dd, 1H, J = 15.9,

5.4), 6.84-6.90 (m, 3H), 7.04-7.08 (m, 4H), 7.17-7.30 (m,

16H); Anal. (C47H50N5O7) C, Η, N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val-L-(4-F)-Phe-L-Gln}-E-propenoate

Ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-L-(4-F)Phe-L-(Tr-Gln)}-E-propenoate was deprotected using the procedure described above (Example 2) to prepare ethyl 3-{(5'-92

methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate: IR (cm ^-1 ) 3284, 1652, 1542; !h NMR (DMSO-d6) δ 0.76 (d, 3H,

J = 6.9), 0.79 (d, 3H, J = 6.9), 1.20 (t, 3H, J = 7.2),

1.57-1.76 (m, 2H), 1.96-2.06 (3H), 2.46 (s, 3H), 2.75-2.83 (m, 1H), 2.89-2.96 (m, 1H), 4.09 (q, 2H, J= 7.2), 4.13-4.25 (m, 1H), 4.35 (m, 1H) , 4.49-4.56 (m, 1H) , 5.53 (d, 1H, J = 15.6), 6.57 (s, 1H) , 6.66 (dd, 1H, J = 15.6, 5.4), 6.75 (s, br, 1H), 6.97-7.03 (m, 2H), 7.17-7.24 (m, 3H), 8.15 (d, 1H, J = 7.8), 8.24 (d, 1H, J = 8.7), 8.32 (d, 1H, J = 8.1);

Anal. (C28H36N5O7) C, H, N.

Example 8 - Preparation of Compound A-7: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4-FPhe)-L-((S)-pyrrole-Ala)}-E-propenoate

Preparation of the intermediate Boc-L-(4-F-Phe)-Obn

DCC (0.765 g, 3.71 mmol, 1.05 equiv), benzyl alcohol (0.347 mL, 3.35 mmol, 0.95 equiv) and DMAP (0.022 g, 0.18 mmol, 0.05 equiv) were sequentially added to a solution of Boc-L-(4-F-Phe)-OH (1.0 g, 3.53 mmol, 1 equiv) in CH2Cl2 (15 mL). After stirring for 18 h, the precipitate was removed by filtration and the filtrate was diluted with MTBE (75 mL), washed with 10% KHSO4 and brine (10 mL each), dried over Na2SO4 and evaporated. Purification of the residue by flash column chromatography (12% EtOAc in hexanes) gave Boc-L-(4-F-Phe)OBn (0.992 g, 79%) as a white solid. 1h NMR

• · spectral data are consistent with the literature (see Jackson et al., J. Org. Chem. 1992, vol. 57, 3397).

Preparation of the intermediate Boc-L-α-(tert-butyl-Gly)-L-(4-F-Phe)OBn

Boc-L-(4-F-Phe)-OBn (2.0 g, 5.36 mmol, 1 equiv) was stirred for 1 h in a mixture of CH2Cl2 (20 mL) and TFA (10 mL), then more TFA (10 mL) was added and the reaction solution was stirred for another hour. The volatiles were evaporated and the residue was dissolved in DMF (30 mL). Boc-L-α(tert-butyl-Gly)-OH (1.24 g, 5.36 mmol, 1 equiv) was added and the solution was cooled to 0°C. N,N-diisopropylethylamine (2.80 mL, 16.1 mmol, 3 equiv) and HATU (2.04 g, 5.37 mmol, 1 equiv) were added sequentially. After stirring for 20 min, the reaction mixture was allowed to warm to ambient temperature over 1 h, then diluted with MTBE (500 mL) and washed with 5% KHSO4 (100 mL), saturated NaHCO3 (50 mL) and brine (50 mL). The organic phase was dried and evaporated. Purification of the residue by flash column chromatography (20% EtOAc in hexanes) gave Boc-L-α-(tert-butyl-Gly)-L-(4-F-

Phe)-OBn (2.04 g, 78%) in the form of white powder -íy : Rf = 0.49 (25% EtOAc in hexanes); IR (cm“1) 3307, 1737 r 1655 br; 1h NMR (CDCl 3 ) δ 0.94 (s, 9H) , 1.45 (s, 9H) , 3, 04 (dd, Hello, J = 14.2, 5.8), 3.11 (dd, ÍH, J= 14.2, 6.1) r 3.79 (d, Hello, J = 9.3), 4.88 (dt, ÍH, J = 7.8, 5.8), 5.08 (d ., IH, J = 12 ,0) , 5.16-5.23 (m, ÍH) , 5.19 (d, ÍH, J= 12.0) r 6.08 (d, 1H, J = 7.8), 6.83-6.97 (m, 4H), 7.28-7.40 (C27H35FN2O5) C, Η, N. (m, 5H) ; Anal.

• ·· · ·· ·· · ·· · · · · · • · · · · · · · · “* · · · ·

Preparation of the intermediate ethyl-3-{Boc-L-α-(tert-butyl-Gly)-L(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-Epropenoate

A solution of HCl in 1,4-dioxane (4.0 M, 8 mL) was added to a solution of ethyl 3-{Boc-L-( (N-2,4-dimethoxybenzyl)-(S)-pyrroleAla )}-E-propenoate (0.309 g, 0.648 mmol, 1 equiv) in 1,4-dioxane (8 mL). After stirring for 1.5 h, the volatiles were evaporated to give the crude amine salt as a foam.

Palladium on carbon (10%, 200 mg) was added to a solution of Boc-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-OBn (2.04 g, 4.19 mmol) in EtOAc (200 mL). The atmosphere was replaced with hydrogen via a balloon. After stirring for 3 h, the atmosphere was replaced with argon and the reaction mixture was filtered through Whatman #3 and #5 filter papers. The filtrate was evaporated to give a white foam.

This foam was combined with the crude amine salt (prepared above) in DMF (5 mL) and cooled to 0°C. N,N-diisopropylethylamine (0.339 mL,

1.95 mmol, 3 equiv) and HATU (0.247 g, 0.650 mmol, 1 equiv). After stirring for 20 min, the reaction mixture was allowed to warm to ambient temperature over 1 h, then diluted with MTBE (100 mL) and washed with 5% KHSO4, saturated NaHCO3 and brine (15 mL each). The organic phase was dried and evaporated. Purification of the residue by flash column chromatography (60% EtOAc in hexanes) gave ethyl 3-{Boc-L-α-(tert-butyl-Gly)-L-(4-FPhe)-L-( (N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate

(0.364g, 74%) in in the form of white foam: Ry = 0.34 (60% EtOAc in hexanes); IR (cm ^- !) 3284, 1713, 1655; !h NMR (CDCI3) δ 1, 01 (s, 9H) , 1.28 (t, 3H, J = 7.2), 1.46 (s, 9H) , 1.50-1 .63 ( m, 2H), 1.68-1.81 (m, 1H), 1.85-1.99 (m, 1H), 2.09 -2.20 ( m, 1H ) ,

fe · fe · • « fe « • ♦ fe ·

2.94 (dd, ÍH, J = 13.5, 5.4), 3.15-3.26 (m, 3H) , 3.80 (with, 3H) , 3.82 (s, 3H) , 3.97 (d, IH, J = 9.3) , 4.17 (q, 2H, J = 7.2) , 4.27-4.38 (m, H), 4.29 (d , 1H, J = 14.3), 4.42 (d, Hello, J = 14.3), 4.84-4, 92 (m, ÍH) , 5.22 (d, ÍH, J = 9.6), 5.74 (dd, ÍH, J = 15, 6, 1.6), 6.45-6, 53 (m, 2H) , 6.69 (dd, ÍH, J = 15.6, 5.4), 6.76-6 .87 (m, 3H) , 7.10-7.28 (m, 3H) , 8.26 (d,

1H, J = 5.9); Anal. (C40H55FN4O9) C, Η, N.

Preparation of the intermediate ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl) - (S) -pyrrole-Ala)}-E-propenoate

This compound was prepared from ethyl 3-{Boc-La(tert-butyl-Gly)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)(S)-pyrrole-Ala)}-E-propenoate and isoxazole-5-carbonyl chloride using the procedure described above (Example 2) for the preparation of ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-(TrGln)}-E-propenoate: Rf = 0.60 (10% CH3OH in CHCl3); IR ( ^cm_1 ) 3295, 1713, 1666, 1643; NMR (CDCl 3 ) δ 1.07 (s, 9H), 1.29 (t, 3H, J = 7.2), 1.51-1.64 (m, 2H), 1.71-1.83 (m, 1H),

1.96-2.07 (m, 1H) , 2.11-2.21 (m, 1H) , 2.49 (s, 3H) , 2.99 (dd, 1H, J = 13.7, 5.9), 3.13-3.26 (m, 3H) , 3.80 (s, 3H) ,

3.81 (s, 3H), 4.18 (q, 2H, J = 7.2), 4.23-4.48 (m, 4H),

4.85-4.93 (m, 1H), 5.76 (dd, 1H, J = 15.6, 1.4), 6.40-6.52 (m, 3H), 6.71 (dd, 1H, J = 15.6, 5.3), 6.79-6.88 (m, 2H),

6.92 (d, ÍH, J= 9.0), 7.09-7.22 (m, 3H), 7.37 (d, ÍH, J =

9.0), 8.27 (d, 1H, J = 6.2); Anal. (C40H50FN5O9·0.25H2O) C,

H, N.

• ·« o ••to · ··

·· ·· ·« • · · it · · • · · · >

• · · · · · • · · · · · ··· ·· ··

Preparation of the product ethyl-3-{(5'-methylisoxazole-3'-carbonyl)L-α-(tert-butyl-Gly)-L-(4-F-Phe)-L-((S)-pyrrole-Ala)}-Epropenoate

This compound was prepared from ethyl-3-{(5'methylisoxazole-3'-carbonyl)-L-α-(tert-butyl-Gly)-L-(4 — F—

Phe)-L-( (N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate using the procedure described above (Example 6) for the preparation of ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)-L((S)-pyrrole-Ala)}-E-propenoate: Rf = 0.30 (5% CH3OH in

CH 2 Cl 2 ); IR (cm ^-1 ) 3307 br, 1684, 1660; 1H NMR (CDCl 3 ) δ 1.02 (s, 9H), 1.29 (t, 3H, J = 7.2), 1.51-1.61 (m, 1H), 1.75-1.97 (m, 2H), 2.14-2.25 (m, 1H), 2.28-2.40 (m, 1H), 2.50 (s, 3H),

3.03 (d, 2H, J = 6.5), 3.27-3.42 (m, 2H), 4.18 (q, 2H, J =

7.2), 4.36 (d, 1H, J = 9, 8), 4.52-4.63 (m, 1H), 4.86-4.95 (m, 1H), 5.71 (dd, 1H, J = 15.6, 1.4), 6.44 (s, 1H), 6.57-6.64 (m, 1H), 6.73 (dd, 1H, J = 15.6, 5.3), 6.83-6.91 (m, 2H), 7.09-7.15 (m, 2H), 7.28-7.36 (m, 2H), 7.59 (d, 1H, J = 8.1);

Anal. (C31H40FN5O7) C, H, N.

Example 9 - Preparation of Compound A-8: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)-L-((S)-piperAla)}-E-propenoate

Preparation of the intermediate tert-butyl ester of (2'S,4S,4'' S) 4-(2' -(4'' -benzyl-2''-oxo-oxazolidine-3''-carbonyl)-5'hydroxypentyl}-2,2-dimethyloxazolidine-3-carboxylic acid • ·

A solution of borane-tetrahydrofuran complex (0.96 mL of a 1.0 M solution in THF, 0.96 mmol, 1 equiv.) was added to a solution of (2'S,4S,4''S)-4-{2'-(4''benzyl-2''-oxo-oxazolidine-3''-carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (prepared as described in Example 6, 0.455 g, 0.963 mmol, 1 equiv.) in THF (3 mL) at 0°C. After stirring for 30 min, water (3 mL) and sodium perborate tetrahydrate (0.148 g, 0.962 mmol, 1 equiv.) were added and the ice bath was removed. After another hour, the reaction mixture was diluted with MTBE (125 mL), washed with water (15 mL) and brine (2 x 15 mL), dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (50% EtOAc in hexanes) gave tert-butyl ester of {2' S, 4S, 4''S)-4-{2'-{4''-benzyl-2''-oxo-oxazolidine3''-carbonyl)-5'-hydroxypentyl}-2,2-dimethyloxazolidine-3-carboxylic acid (0.339 g, 72%) as a colorless gas: Rf = 0.41 (50% EtOAc in hexanes); IR ( ^cm_1 ) 3486, 1780, 1693; ^H NMR (CDCl 3 ) δ 1.42-1.85 (m, 21H), 2.13-2.24 (m, 1H), 2.70 (dd, 1H, J= 13.1, 10.0), 3.29-3.38 (m, 1H), 3.61-4.22 (m,

8H), 4.63-4.76 (m, 1H), 7.19-7.38 (m, 5H); Anal.

(C26H38N2O7'0.5H ₂ O) C, Η, N.

Preparation of the intermediate tert-butyl ester of (1S,3'S){2-(1'-(2'',4''-dimethoxybenzyl)-2'-oxo-piperidin-3'-yl)-1hydroxymethylethyl}-carbamic acid

(2'S,4S,4''S)-4-{2'-(4''benzyl-2''-oxo-oxazolidine-3 ^' '-carbonyl)-5'-hydroxypentyl}2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (3.88 g, 7.92 mmol, 1 equiv) was dissolved in Et3N (3.97 mL, 28.51 mmol, 3.6 equiv). The mixture was cooled to -12°C and a solution of sulfur trioxide-pyridine complex (5.04 g, 31.67 mmol, 4 equiv) in DMSO (150 mL) was added at a rate that maintained the temperature between 8-17°C. The solution was stirred at 23°C for 3 h. The reaction mixture was cooled in an ice bath and quenched by the addition of H2O (150 mL). The resulting solution was extracted with EtOAc (2 x 150 mL). The combined organic layers were washed with 5% citric acid (100 mL), brine (100 mL), dried over Na2SO4 and filtered. The solvent was removed under reduced pressure and the residue was dried under vacuum to give a white foam (3.53

g) ·

To a solution of this material (3.53 g, 7.22 mmol, 1 equiv) in a 2:1 mixture of THF and EtOH (120 mL) was added 2,4-dimethoxybenzylamine hydrochloride (5.88 g, 28.89 mmol, 4 equiv), NaOAc (2.37 g, 28.89 mmol, 4 equiv), and NaBH3CN (0.908 g, 14.45 mmol, 2 equiv). The reaction mixture was stirred overnight (20 h) and then diluted with MTBE (200 mL). The organic layer was washed with 10% KHSO4 (100 mL), saturated NaHCO3 (100 mL), and brine (100 mL), dried over Na2SO4, and concentrated to give a pale yellow foam.

To a solution of this foam (3.34 g, 7.22 mmol, 1 equiv) in CH3OH (50 mL) was added p-toluenesulfonic acid (0.275 g, 1.44 mmol, 0.2 equiv). The reaction mixture was stirred at 50°C for 2.5 h and then diluted with CH2Cl2 (100 mL). The organic layer was washed with saturated NaHCO3 (100 mL), dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography (3% CH3OH in CH2Cl2) to give (1S, 3'S) ~{2-(1'-(24''-dimethoxybenzyl)2'-oxo-piperidin-3'-yl)-1-hydroxymethyl-ethyl}-carbamic acid tert-butyl ester in • ·

- 99 as a white foam (1.33 g, 44% for three steps): NMR (CDCl3):

δ 1.44 (s, 9H) , 1.71-1.85 (m, 2H), 1.92-1.98 (m, 2H), 2.402.48 (m, IH) , 2.71-2.78 (m, IH) , 3.19-3.32 (m, 2H) , 3.453.69 (m, 4H), 4.11-4.20 (m, 2H), 4.68 (m, 1H), 5.47 (m, 1H), 6.44 (s, 1H), 7.20-7.33 (m, 2H).

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val-L-(4-F-Phe)-L-((S)-piper-Ala)}-E-propenoate

(1S,3'S)-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-piperidin-3'-yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester was converted to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)-L-( (S)-piperAla)}-E-propenoate in a manner analogous to the conversion of (1S,3'S)-{2-(1'-(2'',4''-dimethoxybenzyl)2'-oxo-piperidin-3'-yl]-1-hydroxymethylethyl)-carbamic acid tert-butyl ester to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-L-(4F-Phe)-L-((S)-pyrrole-Ala)}-E-propenoate described in Example 6 given above. above: Rf = 0.24 (5% CH3OH in CH2Cl2); IR (cm ^-1 ) 3284 br, 1713, 1655, 1637 br; ^Χ Η NMR (CDCl3) δ 0.94 (d, 3H, J =

6.8) , 0, 98 (d, 3H, J = 6.8) , 1.29 (t, 3H, J = 7.2), 1 ,43- 1.56 (m, 2H), 1.66- -1.78 (m, IH) , 1.83-2.05 (m, 4H), 2 ,16- 2.28 (m, 1H), 2.49 (s, 3H) , 3.00 (dd, IH, J = 13.7, 6 ,2) , 3.13 (dd , IH, J = 13.7, 5.9), 3.21-3.37 (m, 2H), 4.18 (q, 2H, J = 7.2), 4.36 -4.45 (m, 2H) , 4.80-4.88 (m, IH) , 5.76 (dd, IH, J = 15.6, 1.6), 5.96 (with, IH), 6.43 (with, IH), 6.70 (dd, IH, J = 15.6, 5.3), 6.81 (d, IH, J = 8 ,7), 6.86- 6, 98 (m, 2H) , 7.09-7.19 (m, 2H), 7.22-7, .29 (m, IH) , 8.07 (d, IH, J = 6.5)

·· 99 ► · · 4 • 9 9 4

100

Example 10

Preparation of Compound B-1: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val(COCH2)-L-(4-F-Phe)-LGln}-E-propenoate

(B-1)

Preparation of the intermediate trans-6-methyl-hept-4-enoic acid.

A solution of isobutyraldehyde (9.59 g, 133 mmol, 1 equivalents) in THF (50 mL) was added dropwise via an addition funnel to a solution of vinylmagnesium bromide (133 mL of a 1.0 M solution in THF, 133 mmol, 1.0 equivalents) in THF (300 mL) at 0°C. After the addition was complete, the reaction mixture was stirred for 30 min at 0°C and then ethylmalonyl chloride (17.0 mL, 133 mmol, 1.0 equivalents) was added. After stirring for 1 h at 0°C, the reaction mixture was partitioned between saturated NH4Cl (150 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 200 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by filtration through silica gel (eluting with 5% EtOAc in hexanes) afforded intermediate malonate ester (11.5 g, 40% yield). This material was not identified, but was combined (without solvent) with Ti(OEt)4 (1.13 mL, 5.39 mmol, 0.10 equiv) and heated to 190°C for 4 h and then cooled to 60°C. EtOH (50 mL) and 6.0 M KOH (50 mL) were added sequentially and the brown reaction mixture was heated to reflux for 4 h. After cooling to 23°C, the reaction mixture was filtered through a medium frit and the filtrate was partitioned between water (150 mL) and Et2O (2 x 150 mL). The aqueous layer was then acidified to pH = • ·

101

(as determined by pH paper) with concentrated HCl and extracted with a 1:1 mixture of EtOAc and hexanes (2 x 150 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was distilled under reduced pressure to give trans-6-methyl-hept-4-enoic acid (3.58 g, 47%) as a colorless liquid: bp = 107-112°C (1 Torr); IR (cm ^-1 ) 2960, 1711; 1h NMR (CDCl3) δ 0.96 (d, 6H, J = 6.5), 2.18-2.45 (m, 5H), 5.31-5.50 (m, 2H); Anal. (C8H14O2) C, H.

Preparation of the intermediate (2R-hydroxy-1R-methyl-2-phenyl-ethyl)methylamide of trans-6-methyl-hept-4-enoic acid.

Oxalyl chloride (2.25 mL, 25.8 mmol, 1.05 equiv) was added to a solution of trans-6-methyl-hept-4-enoic acid (3.50 g, 24.6 mmol, 1 equiv) and N,N-dimethylformamide (0.03 mL, 0.39 mmol, 0.016 equiv) in benzene (60 mL) at 23°C. The reaction mixture was stirred at 23°C for 2 h and then concentrated under reduced pressure. The resulting oil was dissolved in THF (20 mL) and added via cannula to a solution of (1R,2R)-(-)-pseudoephedrine (3.87 g, 23.4 mmol, 1 equiv) and triethylamine (3.92 mL, 28.1 mmol, 1.2 equiv) in THF (150 mL) at 0°C. The reaction mixture was stirred at 0°C for 30 min, then partitioned between half-saturated NH4Cl (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was purified by flash column chromatography (gradient elution 40-50% EtOAc in hexanes) to give trans-6-methyl-hept-4-enoic acid (2R-hydroxy-1R-methyl-2-phenylethyl)-methylamide (6.31 g, 93%) as a viscous oil: R_f = 0.35 (50%

EtOAc in hexanes); IR (cm !) 3382, 1622; !h NMR (CDCl3, mixture • · • · · · ·

102 • ····

rotamers) δ 0.96 (d, J = 6.8), 0.97 (d, J = 6.5), 1.11 (d, J = 6.9), 2.18-2.59 (m) , 2.82 (s), 2.92 (s) , 3.99-4.04 (m) ,

4.32-4.42 (m) , 4.44-4.49 (m) , 4.55-4.62 (m) , 5.32-5.49 (m) ,

7.24-7.42 (m); Anal. (C18H27NO2) C, Η, N.

Preparation of the intermediate product

(2E-hydroxy-1B-methyl-2trans-6-methyl-2S-(4fluorobenzyl)-hept-4-ene.

n-Butyllithium (32.5 mL of a 1.6 M solution in hexanes, 52.0 mmol, 3.1 equiv) was added to a suspension of anhydrous lithium chloride (7.18 g, 169 mmol, 10 equiv) and diisopropylamine (7.80 mL, 55.7 mmol, 3.3 equiv) in THF (250 mL) at -78°C. The reaction mixture was stirred for 30 min at -78°C, maintained at 0°C for 5 min, and then recooled to -78°C. (2R-hydroxy-1R-methyl-2-phenyl-ethyl)-trans-6-methyl-hept-4-enoic acid methylamide (4.91 g, 17.0 mmol, 1 equiv) in THF (50 mL) was added via cannula and the resulting solution was stirred at -78°C for 1.75 h, maintained at 0°C for 20 min, stirred at 23°C for 5 min, and then recooled to 0°C. A solution of 4-fluorobenzyl bromide (6.34 mL, 50.9 mmol, 3 equiv) in THF (15 mL) was added and the reaction mixture was stirred at 0°C for 30 min, then partitioned between half-saturated NH4Cl (230 mL) and a 1:1 mixture of EtOAc and hexanes (200 mL, 2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (gradient elution 20-»40% EtOAc in hexanes) gave trans-6-methyl-2S-(4fluorobenzyl)-hept-4-enoic acid (2R-hydroxy-1R-methyl-2phenylethyl)methylamide (6.33 g, 94%) as a viscous • · • · • ·

103 oil: Rf = 0.38 (40% EtOAc in hexanes); IR (cm 1) 3378,

1614; 1h NMR (CDCl3, mixture of rotamers) δ 0.85-0.95 (m) , 0.96 (d, J = 6.8), 2.10-2.32 (m) , 2.34-2.46 (m) , 2.58 (s), 2.672.79 (m) , 2.82-2.94 (m) , 3.00-3.18 (m), 3.94 (br), 4.37-4.52 (m), 5.24-5, 42 (m) , 5.44-5.56 (m), 6.89-7.01 (m), 7.08-7.14 (m) , 7.19-7.38 (m) ; Anal. (C25H32FNO2) C, Η, N.

Preparation of the intermediate 5S-(1R-bromo-2-methylpropyl)-3R-(4fluorobenzyl)dihydrofuran-2-one.

N-Bromosuccinimide (2.93 g, 16.5 mmol, 1.05 equivalents) was added in small portions over 10 minutes to a solution of trans-6methyl-2S-(4-fluorobenzyl)-hept-4-enoic acid (2R-hydroxy-1?-methyl-2-phenylethyl)methylamide (6.24 g, 15.7 mmol, 1 equivalent) and glacial acetic acid (4.49 mL, 78.4 mmol, 5 equivalents) in a 4:1 mixture of THF and H2O (165 mL) at 0°C. The resulting yellow solution was stirred for 15 min at 0°C and then warmed to 23°C and then refluxed for 45 min. After cooling to 23°C, the reaction mixture was partitioned between half-saturated NaHCO3 (200 mL) and a 1:1 mixture of EtOAc and hexanes (2 x

200 ml, 100 ml) Na2SO4 purification and were residues

The combined organic layers were dried and concentrated. Flash chromatography (gradient elution 4-10% EtOAc in hexanes) gave 5S-(1R-bromo-2-methylpropyl)-3H-(4fluorobenzyl)dihydrofuran-2-one (4.14 g, 80%) as a pale yellow oil (containing approximately 5-10% unidentified impurities by 1H NMR): Rf = 0.56 (25% EtOAc in hexanes); IR (cm- ¹ ) 1772; !h NMR (CDCl3, major isomer) δ 0.94 (d, 3H, J = 6.5), 1.00 (d, 3H, J = 6.8), 2.05-2.35 (m, 3H), 2.83 (dd, 1H, J = 13.6, 8.4), 2.92- 104 • ·

6, 4.7), 3.90 (dd, 1H, J

-7.06 (m, 2H), 7.14-7.20 • ····

3.03 (m, 1H), 3.11 (dd, 1H, J = 13, = 9.0, 3.7), 4.33-4.40 (m, 1H), 6.98 (m, 2H); Anal. (Ci5HisBrFO2)C,H.

Preparation of the intermediate 5S-(1S-azido-2-methylpropyl)-3R-(4fluorobenzyl)dihydrofuran-2-one.

A suspension of sodium azide (1.90 g, 29.2 mmol, 2.5 equiv) and 5S-(1R-bromo-2-methylpropyl)-3R-(4fluorobenzyl)dihydrofuran-2-one (3.85 g, 11.7 mmol, 1 equiv) in N,N-dimethylformamide (40 mL) was heated at 50°C for 67 h. The reaction mixture was cooled to 23°C and partitioned between half-saturated NaCl (200 mL) and a 1:1:1 mixture of EtOAc, hexanes, and acetone (2 x 200 mL, 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was purified by flash column chromatography (gradient elution 9—>17% EtOAc in hexanes) to afford 5S-(1S-azido-2-methylpropyl)-3R-(4fluorobenzyl)dihydrofuran-2-one (2.10 g, 62%) as a white solid (containing approximately 5-10% unidentified impurities by NMR): mp 91-96°C; Rf = 0.44 (25% EtOAc in hexanes); IR (cm ^-1 ) 2097, 1772; !h NMR (CDCl3, major isomer) δ 0.99 (d, 3H, J = 6.5), 1.02 (d, 3H, J = 6.8), 1.95-2.20 (m, 3H), 2.78-2.88 (m, 1H), 2.94 (dd, 1H, J = 7.0, 4.2), 3.03-3.17 (m, 2H), 4.37-4.43 (m, 1H), 6.97-7.09 (m, 2H), 7.14-7/21 (m, 2H).

Preparation of the intermediate tert-butyl ester of {2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}carbamic acid.

9 · • · 4

4 4

105

A suspension of 5S-(1S-azido-2-methylpropyl)-3R-(4fluorobenzyl)dihydrofuran-2-one (2.02 g, 6.93 mmol, 1 equivalent), di-tert-butyldicarbonate (2.12 g, 9.71 mmol, 1.4 equivalents) and Pd/C (10%, 0.20 g) in CH3OH (100 mL) was stirred under a hydrogen atmosphere (balloon) for 16 h. The reaction mixture was vacuum filtered through Whatman #3 paper and concentrated. Purification of the residue by flash column chromatography (15% EtOAc in hexanes) afforded {2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamic acid tert-butyl ester (1.58 g, 62%) as a white foam: Rf = 0.80 (5% MeOH in CH2Cl2); IR ( ^cm_l ) 3331, 1766, 1702; A NMR (CDCl3) δ 0.93 (d, 3H, J = 6.8),

0.95 (d, 3H, J = 6.5), 1.41 (s, 9H) , 1.71-1.83 (m, IH) , 1.95-2.06 (m, 1H), 2.16-2.27 (m, IH) , 2.80 (dd, IH, J = 13.5, 8.6) , 2.88-2.99 (m, 1H), 3.09 (dd, 1H, J = 13.5, 4.4) , 3.32-3.40 (m, 1H), 4.42-4.48 (m, 2H) , 6.95-7.03 (m, 2H) , 7.11-7.18 (m, 2H); Anal. (C20H28FNO4) C, Η, N. Preparation intermediate ethyl -3-{Boc-L-VaUF(COCH2)-L- (4-F- Phe)-L-(Tr -Gin)}-E-propenoate.

Lithium hydroxide (9.62 mL of 1 M aqueous solution, 9.62 mmol, 5 equiv) was added to a solution of {2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamic acid tert-butyl ester (0.703 g, 1.92 mmol, 1 equiv) in DME (25 mL) at 23°C. The resulting suspension was stirred at 23°C for 30 min and then partitioned between 10% KHSO4 (50 mL) and CH2Cl2 (3 x 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was dissolved in CH2Cl2 (30 mL). Powdered 4L molecular sieves were added sequentially.

106 (0.70 g), 4-methylmorpholine N-oxide (0.451 g, 3.85 mmol, 2 equivalents), and tetrapropylammonium perruthenate (0.068 g, 0.19 mmol, 0.10 equivalents). The resulting dark reaction mixture was stirred for 1.33 h at 23°C, then vacuum filtered through Whatman #3 paper and then Whatman #5 paper. The filtrate was concentrated under reduced pressure to give a dark residue, which was dissolved in CH2Cl2 (30 mL). Crude ethyl 3-(H2N-L-(Tr-Gln) ) E-propenoate*HCl (2.30 mmol, 1.2 equivalents, prepared as described in Example 2 for the preparation of ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln}-Epropenoate), 4-methylmorpholine (0.846 mL, 7.69 mmol, 4 equivalents), HOBt (0.390 g, 2.89 mmol, 1.5 equivalents) and EDC (0.553 g, 2.88 mmol, 1.5 equivalents) were added sequentially and the reaction mixture was stirred for 19 h at 23°C and then partitioned between brine (100 mL) and CH2Cl2 (3 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (gradient elution 35—>40% EtOAc in hexanes) afforded ethyl 3-{Boc-L-ValT (COCH2)-L-(4-F-Phe)-L(Tr-Gln)}-E-propenoate (0.820 g, 53%) as a brown foam: Rf = 0.50 (50% EtOAc in hexanes); IR (cm“A) 3307, 1708, 1666;

!h NMR (CDCl3) δ 0.67 (d, 3H, J = 6.8), 0.92 (d, 3H, J = 6.8), 1.28 (t, 3H, J = 7.2), 1.40 (s, 9H), 1.53-1.67 (m, 1H), 1.91-2.04 (m, 2H), 2.32-2.41 (m, 2H), 2.46-2.55 (m, 1H) , 2.63 (dd, 1H, J = 12.1, 5.9), 2.69-2.80 (m, 1H), 2.83 (dd, 1H, J = 12.1, 8.2), 3.03 (dd 1H, J = 17.7, 10 , 0) , 4.05-4.11 (m, 1H), 4.17 (q, 2H, J = 7.2), 4.40-4.50 (m, 1H) , 4.84 (d, 1H, J = 8.4), 5.38 (d, 1H, J = 15.7), 6.01 (d, 1H, J = 8.4), 6.60 (dd, 1H, J = 15.7, 5.0), 6.92-6.99 (m, 2H) ,

107 • ·· · ·· ·· ···«· ···♦ · » « · • · · »··· • · · · *···· “·· · · · · ·

7.03-7.12 (m, 3H), 7.17-7.30 (m, 15H); Anal. (C48H56FN3O7)

C, H, N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val(COCH2)-L-(4-F-Phe)-L-Gln)-E-propenoate

Ethyl 3-{Boc-L-ValT (COCH2)-L-(4-F-Phe)-L-(Tr-Gln)}-Epropenoate was converted to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-ValT (COCH2)-L-(4-F-Phe)-LGln}-E-propenoate in a manner similar to that described in Example 2 above for the conversion of ethyl 3-{Boc-L-Leu-LPhe-L-(Tr-Gln)}-E-propenoate to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate: Melting point = 220°C (decomposition); Rf = 0.35 (10% CH3OH in

CH 2 Cl 2 ); IR (cm ^-1 ) 3277, 1715, 1643; !h NMR (DMSO-d6) δ 0.81 (d, 3H, J = 6.2), 0.87 (d, 3H, J = 6.9), 1.21 (t, 3H, J =

6, 5), 1.59-1.67 (m, 2H) , 2.03 (s, br, 2H) , 2.21-2.24 (m,

ÍH) , 2.46 (s, 3H) , 2.57-2.68 (m, 3H) , 2.80-2.95 (m, 2H) , 4.09 (q, 2H, J= 6.5), 4.30-4.34 (m, 2H) , 5.41 (d, ÍH, J =

15.5), 6.55 (s, ÍH), 6.61 (dd, ÍH, J = 15.5, 5.5), 6.73 (s, ÍH), 6.99-7.16 (m, 5H), 8.01 (d, ÍH, J = 7.8), 8.69 (d, ÍH,

J = 8.7); Anal. (C29H37FN4O7)C,Η,N.

108

ΦΦ • φ • φ φ • φ φφ· φφφφ φφ • φ φ φ φ φ φφφφ • Φ φφ • φ · · • φ φ φ • * φ φ • φ φ φ φφ φφ

Example 11 - Preparation of Compound A-10: Ethyl 3-((5'methylisoxazole-3'-carbonyl)-L-Leu-NCH3~L-Phe-L-Gln}-Epropenoate

(A-10)

Preparation of the intermediate Boc-L-Leu-NCH3-L-Phe-OCH3

NCH3-L-Phe-OCH3«HCl (1.4 g) was dissolved in CH2Cl2 (50 mL) and poured into a combination of aqueous 1 N NaOH (7 mL) and saturated aqueous NaHCO3 (25 mL). After stirring, the organic phase was separated and the aqueous phase was washed with CH2Cl2 (3 x 50 mL). The combined organic phases were dried over Na2SO4 and evaporated to give the free amine as a clear colorless oil (1.14 g, 5.90 mmol). A solution of this amine and (iPr)2NEt (1.13 mL,

6.49 mmol) in DMF (10 mL) was added dropwise to a 0°C solution of Boc-L-Leu-OH (1.50 g, 6.49 mmol) and HOBt (0.877 g, 6.49 mmol) in DMF (10 mL). DCC (1.47 g, 7.12 mmol) was added. The reaction mixture was stirred at 0°C for 1 h and then stirred at 23°C for 48 h. The mixture was filtered to remove precipitate (ppt) and the filtrate was evaporated. The residue was dissolved in CH2Cl2 (200 mL), washed with saturated aqueous NaHCO3 (40 mL), dried over Na2SO4 and evaporated. The residue was purified by flash column chromatography (25% EtOAc in hexanes) to give Boc-L-Leu-NCH3~L-Phe-OCH3 as a white solid (2.04 g, 85%): mp = 126-127°C; IR (cm' ¹ ) 3401, 3319, 1743, 1708, 1649; NMR (CDCl3) (major isomer) δ 0.92 (d, 3H, J = 6.8), 0.95 (d, 3H, J = 6.5), 1.32-1.48 (m, 2H), 1.41 (s,

109

00 ·· « · • 0 0 0 0 0 •00 0000 ·· ·0 0· > 0 0 0 0 1 * 0 0 0 « • 0 0 0 0 «

3.04 (dd, 1H, J 5.5) , 3.72 (with, 3H) 5.20 (dd, 1H, J

9H) , 1.61-1.77 (m, 1H) , 2.90 (s, 3H) ,

14.5, 10.5), 3.37 (dd, 1H, J = 14.5,

4.48-4.57 (m, 1H) , 4.98-5.04 (m, 1H) ,

10.5, 5.5), 7.16-7.32 (m 5H); Anal. (C22H34N2O5) C, Η, N.

Preparation of the intermediate Boc-L-Leu-NCH3~L-Phe-OH

Boc-L-Leu-NCH3-L-Phe-OCH3 (0.625 g, 1.54 mmol) was dissolved in CH3OH (20 mL) and cooled to 0°C. Aqueous NaOH (6.15 mL of a 2N solution, 12.3 mmol) was added dropwise. The reaction mixture was stirred for 3 h at 23°C and then poured into 10% aqueous KHSO4 (150 mL). This mixture was extracted with CH2Cl2 (3 x 100 mL) and the combined organic phases were dried over Na2SO4 and evaporated to give Boc-LLeu-NCH3-L-Phe-OH as a white foam (0.617 g, quant.), which was used without purification.

Preparation of the intermediate ethyl-3-{Boc-L-Leu-NCH3~L-Phe-L-(TrGln)}-E-propenoate

This intermediate was prepared from Boc-L-Leu-NCH3~L-PheOH and ethyl 3-{H2N-L-(Tr-Gln)}-E-propenoate»HCl (prepared as described in Example 2) in a manner similar to that described for the preparation of ethyl 3-{Boc-L-Leu-L-Phe-L-(TrGln)}-E-propenoate in Example 2 above: IR (cm ^- !) 3295, 1713, 1672, 1649; 3-H NMR (CDCl3) (mixture of isomers) δ 0.65

(d, J = 6, 2), 0 .66 (d, J = 6.5) , 0.84 (d , J = 6.5) , 0.88 (d, J = 6.5), 1.02- •1.22 (m) , 1.23-1.38 (m) , 1.33 (s) , 1.41 (s) , 1.55-1.82 (m) , 1.89-2.07 (m), 2.23-2.30 (m), 2.90 (s) , 2.94 (s), 3.01 (dd, J = 14.6, 10.9), 3.03- 3.13 (m) , 3.26- 3.37 (m), 3.27 (dd, J = 14.6, 3.4), 3.42-3.54 (m) , 4.00- •4.22 (m) , 4.37-4.73 (m) , 4.82-4.89 (m), 5.63-5.70 (m) , 5.95 (dd, J =

*4

15.9, 1.2), 6.23-6.28 (m) , 6.66-6.75 (m) , 6.79-6.89 (m) ,

7.09-7.34 (m), 8.14 (d, J = 8.7); Anal. (C49H60N4O7) C, H,

N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Leu-NCH3~L-Phe-L-Gln}-E-propenoate

Ethyl 3-{Boc-L-Leu-NCH3-L-Phe-L-(Tr-Gln)}-E-propenoate was converted to the product ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-Leu-NCH3-L-Phe-L-G2n}-E-propenoate in a manner similar to that described in Example 2 above for the conversion of ethyl 3-{Boc-L-Leu-L-Phe-L-(Tr-Gln)}-Epropenoate to the product ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate: Rf = 0.23 (5%

CH3OH in CH2Cl2); IR (cm ^-1 ) 3295, 1713, 1666, 1637; !h NMR (CDCl3) (mixture of isomers) δ 0.65 (d, J = 6.5), 0.71 (d, J 6.5), 0.93 (d, J = 6.5), 0.94 (d, J = 6.5), 1.30 (t, J = 7.2), 1.24-1.73 (m) , 1.81-2.22 (m) , 2.45 (s), 2.48 (s),

2.86-2.93 (m), 2.96 (s), 2.97 (s), 3.03-3.14 (m) , 3.21-3.31 (m), 3.48 (dd, J= 14.0, 5.9), 4.19 (q, J = 7.2), 4.20 (q, J = 7.2), 4.38-4.45 (m) , 4.52-4.70 (m) , 4.74-4.81 (m) , 5.625.67 (m) , 5.73-5.79 (m), 5.81 (dd, J = 15.6, 1.6), 5.99 (dd, J = 15.6, 1.6), 6, 03-6.09 (m) , 6.35 (s), 6.39 (s) , 6.40-6.45 (m) , 6.77-6.94 (m), 7.42 (d, J = 7.2), 8.13 (d, J = 7.8).

Example 12 - Preparation of Compound C-1: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)-L-Gln}-Epropenoate

- 111 ·« ·« «* » * · · and Φ • · · · · • · · · · · • · · · >

>*·· 99 99

(C-1)

Preparation of the intermediate allyl-(S)-1-hydroxy-3-(4-fluorophenyl)propionate

In a flask equipped with a thermometer and a reflux condenser, methyl (S)-1-hydroxy-3-(4-fluorophenyl)propionate (prepared from L-H2N-(4-F-Phe)-OCH3 by the method described by Hoffman et al., Tetrahedron 1992, vol. 48, 3007) (0.99 g,

5.0 mmol;

allyl alcohol (50 ml). Titanium tetraisopropoxide (1.53 ml, 5.0 mmol) was added and the reaction mixture was heated to 90°C for 3.5 h. The reaction mixture was cooled to ambient temperature and poured into 250 ml of 1:1 EtOAc/saturated NH4Cl solution. The organic phase was separated and washed with water (100 ml), brine (100 ml), dried (MgSO4) and the solvent was removed. The residue was subjected to flash column chromatography eluting with a gradient of 5-10% EtOAc/hexanes to give 0.77 g (68%) of allyl-(S)-1-hydroxy-3-(4-fluorophenyl)propionate as a clear liquid: Rf = 0.21 (15% EtOAc/hexanes); IR (solvent-free) 3470 (broad), 1734, 1510, 1221 cm ^-1 ; NMR (DMSO-c/6) δ 1.24-1.27 (m, 1H), 2.92-2.99 (m, 1H), 3.09-3.15 (m, 1H), 4.43-4.47 (m, 1H), 4.65 (d, 2H, J = 5.9), 5.28-5.37 (m, 2H), 5.86-5.95 (m, 1H), 6.95-7.01 (m, 2H), 7.16-7.21 (m, 2H).

112 »· • · ···* t« »· • · Β • · Β • · · • · ·

B· *·

Preparation of the intermediate Boc-L-Val-O-L-(4-F-Phe)-OCH2CH=CH2

Allyl-(S)-1-hydroxy-3-(4-fluorophenyl)propionate (0.070 g, 0.31 mmol) was dissolved in CH2Cl2 (20 mL). Boc-LVal-OH (0.068 g, 0.31 mmol) was added, followed by DMAP (0.004 g, 0.03 mmol) and DCC (0.067 g, 0.33 mmol). The reaction mixture was stirred at ambient temperature overnight and the solvent was removed in vacuo. The residue was subjected to flash column chromatography eluting with a gradient of 3-5% EtOAc/hexanes. The Boc-L-Val-O-L-(4-F-Phe)-OCH2CH=CH2 product was

obtained as 0.12 g (90%) of pure oil: Rf = 0.18 (10% EtOAc/hexanes); IR (solvent free) 1752, 1717 1510 cm ^- 7; !h NMR (DMSO-c/6) δ 0.80-0.85 (m, 6H ), 1.36 (with, 9H) , 1.97- 2.04 (m, 1H) , 3.07-3.15 (m, 2H) , 3, 89-3.94 (m, ÍH) , 4.51- 4.55 (m, 2H) , 5.17-5.30 (m, 3H) , 5, 75-5.84 (m, ÍH) , 7.06- 7.17 (m, 3H), 7.27-7.32 (m, 2H); Anal • (C22H30NO6F) C, H, N.

Preparation of the intermediate ethyl 3-{Boc-L-Val-O-L-(4-F-Phe)-L(Tr-Gln)}-E-propenoate

Boc-L-Val-O-L-(4-F-Phe)-OCH2CH=CH2 (0.65 g, 1.52 mmol) was dissolved in THF (15 mL). Tetrakis(triphenylphosphine)palladium(0) (0.035 g, 0.03 mmol) was added and the reaction mixture was stirred for 5 min at 23°C. Morpholine (0.16 mL, 1.83 mmol) was added dropwise and the reaction mixture was stirred at ambient temperature for 2 h. The solvent was removed in vacuo and the residue was taken up in 50 mL of 4:1 hexanes/Et2O. The product was extracted in saturated NaHCO3 solution (50 mL) and the organic phase was removed. The aqueous phase was acidified to pH = 1-2 with solid KHSO4 and the product was re-extracted in EtOAc (50 mL). The organic phase was washed with brine (50 ml), dried

113 (MgSO4) and concentrated to give 0.50 g (86%) of the free acid as a clear oil. This material was dissolved in DMF (6 mL). Diisopropylethylamine (0.43 mL, 2.50 mmol) was added, followed by ethyl 3-{H2N-L-(Tr-Gln)}-Epropenoate•HCl (prepared as described in Example 2 above, 0.55 g, 1.25 mmol). The reaction mixture was cooled to 0°C. HATU (0.48 g, 1.25 mmol) was added and the reaction mixture was allowed to warm to ambient temperature. The DMF was removed in vacuo. The residue was dissolved in EtOAc (30 mL) and the organic phase was washed successively with 10% HCl solution (25 mL), saturated NaHCO3 solution (25 mL), H2O (25 mL) and brine (25 mL). The organic layer was dried (MgSO4), filtered and concentrated and the residue was purified by flash column chromatography (0—»1.0% MeOH/CH2Cl2) to give 0.40 g (39%) of ethyl 3-{Boc-L-Val-OL-(4-F-Phe)-L-(TrGln)}-E-propenoate as a white amorphous solid: Rf = 0.25 (3% MeOH/CHCl3); IR(KBr) 1691, 1512, 1159 cm ^-1 ; 1h NMR (DMSO-d6) δ 0.78-0.83 (m, 6H), 1.20 (t, 3H, J = 7.0), 1.35

(s, 9H), 1.58-1.66 (m, 2H), 1.96-2.02 (m, IH), 2 ,19-2,33 (m, 2H), 2.99-3.02 (m, 2H), 3.82-3.87 (m, IH) , 4.09 (q, 2H, J = 7.0), 4.33-4.37 (m, IH), 5.03-5.08 (m, IH), 5.60 (d, IH, J = 15.8), 6.67 (dd, IH, J = 15.8, 5.5) , 7.02-7, 08 (m, 2H) , 7.14-7.28 (m, 18H) , 8.12 (d, 1H, J = = 8.1), 8, 59 (with, IH) ;

Anal. (C47H54N3O8F) C, H, N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val-O-L-(4-F-Phe)-L-Gln}-E-propenoate

Ethyl 3-{Boc-L-Val-O-L-(4-F-Phe)-L-(Tr-Gln)}-Epropenoate was converted to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)-L-Gln}-E114 propenoate in a manner similar to that described in Example 2 above for the conversion of ethyl 3-{Boc-L-Leu-LPhe-L-(Tr-Gln)}-E-propenoate to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate:

Rf = 0.05 (3% MeOH/CHCl 3 ); IR (KBr) 1746, 1719, 1661, 1549 cm"¹; 3-H NMR (DMSO-dg) δ 0.87 (d, 3H, J = 6.6), 0.92 (d, 3H,

J = 6.6), 1.20 (t, 3H, J = 7.0), 1.61-1.74 (m, 2H), 1.962.01 (m, 2H), 2.15-2.22 (m, IH), 2.46 (s, 3H), 3.00-3.03 (m, 2H), 4.10 (q, 2H, J= 7.0), 4.27-4.32 (m, IH), 4.33-4.38 (m,

IH) , 5.06-5.11 (m, IH) , 5.63 (d, IH, J = 15.6), 6.54 (s,

IH) , 6.68 (dd, IH, J = 15.6, 5.5), 6.78 (s, br, IH) , 6.957.00 (m, 2H), 7.20-7.24 (m, 3H), 8.06 (d, IH, J= 8.1), 8.87 (d, IH, J= 7.7); Anal. (C28H35N4O8F) C, Η, N.

Example 13 - Preparation of Compound A-ll: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-(2-naphth)-NCH3-L-(4-F-Phe)-LGln}-E-propenoate

Preparation of the intermediate ethyl 3-{B0C-NCH3-L-(4-F-Phe)-L-(TrGln)}-E-propenoate

Boc-(4-F-Phe)-OH (4.46 g, 15.75 mmol, 1 equivalent) and CH3I (7.84 mL, 126 mmol, 8 equivalents) were dissolved in anhydrous THF (100 mL) and cooled to 0°C. NaH (1.89 g, 47.25 mmol, 3 equivalents) was added to this solution.

115 with vigorous stirring. After stirring at 23°C for 24 hours, EtOAc (3 mL) and H2O (3 mL) were carefully added to the mixture and the resulting suspension was evaporated to dryness. After dissolution in H2O (100 mL), the reaction mixture was washed with Et2O (2 x 100 mL). The aqueous layer was acidified to pH = 3 with 10% citric acid solution and then extracted with EtOAc (2 x 100 mL). The combined EtOAc extracts were washed sequentially with half-saturated NaHCO3 (150 mL), 5% Na2S2O3 (150 mL) and H2O (150 mL), dried over Na2SO4 and concentrated to give BocNCH3-(4-F-Phe)-OH as a pale yellow foam (4.37 g, 80%), which was used without further purification: 1H NMR (CDCl3, mixture of isomers) δ 1.35 (s), 1.40 (s), 2.69 (s), 2.75 (s), 2.96-3.13 (m), 3.23-3.33 (m), 4.54-4.58 (m), 4.76-4.81 (m), 6.96-7.01 (m), 7.15-7.17 (m).

A solution of HCl in 1,4-dioxane (4.0 M, 15 mL) of ethyl 3-(Boc-L-(Tr-Gln))-E-propenoate as described in Example 2 above, 3.57 g, 6.73 mmol, 1 equivalent) in the same solvent (15 mL) at 23° C. After 2 hours, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (50 mL) and BOC-NCH3-(4-F-Phe)-OH (prepared as in the previous paragraph, 2.0 g, 6.73 mmol, 1.0 equiv), HOBt (1.23 g, 9.09 mmol, 1.5 equiv), 4methylmorpholine (2.0 mL, 18.19 mmol, 3 equiv) and EDC (1.74 g, 9.09 mmol, 1.5 equiv) were added sequentially. The reaction mixture was stirred at 23°C overnight and then partitioned between water (100 mL) and CH2Cl2 (2 x 100 mL). The combined organic layers were dried over Na2SO4, concentrated, and the residue was purified by flash column chromatography (30% EtOAc in hexane) to give ethyl-3-{BOC-NCH3-L-(4-F-Phe)-L-(Trpyl)} prepared as

- 116 :Gln)}-E-propenoate (4.07 g, 84%) as a white foam: IR (cm !) 1666, 1510, 1167; A NMR (CDCl3, mixture of isomers) δ 1.29 (t,

J = 7.2), 1.37 (s), 1.65-1.75 (m), 1.95- 2.06 (m) , 2 .29-2.33 (m) , 2.66 (s), 2.91-2.99 (m) , 3.22-3.29 (m), 4.18 (q, J = 7.2) , 4.52-4.58 (m) , 5.68 (d, J = 15.9) , 6.45 (d, J = 8.4), 6, 74 (dd, J = 15.6, 5.4), 6.91-6.99 ( :m) , 7,11-7, 33 (m); Anal . (C43H48FN3O6) C, H, N.

Preparation of the intermediate ethyl 3-{Boc-L-(2-naphth)-NCH3-L-(4-FPhe)-L-(Tr-Gln)}-E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 3 mL) was added to a solution of ethyl 3-{BOC-NCH3-L-(4-F-Phe)-L-(Tr-Gln)}-Epropenoate (0.388 g, 0.54 mmol, 1 equivalent) in the same solvent (3 mL) at 23°C. After 2 h, the volatiles were removed under reduced pressure. The residue was dissolved in DMF (10 mL), cooled to 0°C, and

1.08 mmol, 2 0.54 mmol, 1.0 equivalents).

DIEA (0.188 ml, equivalents), Boc-L-(2-naphth)-OH (0.170 g, equivalents) and HATU (0.205 g, 0.54 mmol,

The reaction mixture was stirred at 23°C for 1 hour. The volatiles were removed under reduced pressure and the resulting residue was taken up in EtOAc (50 mL) and washed with 0.5 N HCl (50 mL), saturated NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, concentrated and the residue was purified by flash column chromatography (40% EtOAc in hexane) to give ethyl 3-{Boc-L-(2-naphth)NCH3-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (0.437 g, 88%) in

in the form of white foam: IR (cm !) 1656, 1509, 1162; A NMR 1 mixture of isomers) δ 0.88 (t, J = 7.2), 1.27 (s), 1.30 (s) , 1.58 (m) , 1.64-1.67 (m) , 1.97-2.11 (m), 2.23-2.28 (m) ,

··

- 117 ·· ··

2.50 (m) , 2.62-2.69 (m) , 2.80 (s), 2.90 (s) , 3.00- -3.07 (m), 3.15- 3.20 (m) , 3.25-3.32 (m) , 4.18 (q, J = 7.2) , 4.42-4.46 (m) , 4.53- 4.56 (m) , 4.61-4.66 (m), 4.72-4.82 (m), 5.94-5.00 (m) , 5, 63 (d, J = 15.6), 6.12 (d, J = 15.6), , 6, 60 (dd, J = 15.6, 5.4) , 6, 75-6, 89 (m) , 6 .70-7.08 (m), 7.19- 7.30 (m), 7.41- 7.50 (m), 7.70-7.82 (m), 8.80 (d, J = 8, 4) ; Anal.

(C ₅ 6H 59FN ₄ O7) C, H, N.

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-(2-naphth)-NCH3-L-(4-F-Phe)-L-G2n}-E-propenoate

Ethyl 3-{Boc-L-(2-naphth)-NCH3-L-(4-F-Phe)-L-(Tr-Gln)}-Epropenoate was converted to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-(2-naphth)-NCH3-L-(4-F-Phe)-LGln}-E-propenoate in a manner similar to that described in Example 2 above for the conversion of ethyl 3-{Boc-L-Leu-LPhe-L-(Tr-Gln)}-E-propenoate to the product ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Leu-L-Phe-L-Gln}-E-propenoate

IR ( cm 1) 3296, 1654, 151 .0; 1h NMR (DMSO -d.6, mixture isomer •ů) δ 1.17 -1.24 (m) , 1.62-1.78 (m), 2.04-2.15 (m) , 2.38 (s) , 2.42 (s) , 2.71- 2.79 (m), 2.84 (s), 2.87-2.92 (m) , 3.03 (s) , 3.15 (d, J = 7 ,5) , 3.98-4.06 (m), 4.08-4.12 (m) , 4.38 -4.42 (m) , 4.94 (m) , 5, 03- -5.07 (m), 5.09-5.18 (m), 5, 66 -5.82 (m) , 6, 42 (s) , 6, 43 (s) , 6.66-6.81 (m) , 6.88-6.94 (m) , 7.01 -7.06 (m) , 7.13 -7.17 (m) , 7.24-7.34 (m), 7.43-7.46 (m) , 7.56 (s), 7 ,76- 7.84 (m) , 8.08 (d, J = 7, 8) , 8.60 (d, J = 8.4 ), 9, 01 (d, J =

7.2); Anal. (C37H40FN5O7·0.75H2O) C, Η, N.

118

Example 14 - Preparation of Compound A-9: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-H1S-NCH3-L-(4-F-Phe)-L-Gln}-Epropenoate

Ethyl 3-{BOC-NCH3-L-(4-F-Phe)-L-(Tr-GIn)}-E-propenoate (described in Example 13 above) was converted to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-His-NCH3~ L-(4-F-Phe)-L-Gln}-E-propenoate in a manner analogous to the preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-(2naphth)-NCH3-L-(4-F-Phe)-L-Gln)-E-propenoate described in Example 13 above (using Boc-L-(Tr-His)-OH

instead of Boc-L-(2-naphth)-OH) : IR (cm 1) 3302, 1665, 1202; !h NMR (DMSO-d6< mixture of isomers) δ 1.21 (t, J = 7.2) , 1.70-1.78 (m) , 2.05-2.09 (m) , 2.41 (s) , 2.44 (s) , 2.69· -3, 26 (m), 4.11 (q, J= 7.2), 4.38-4.53 (m), 5.07-5.19 (m), 6, 51- -5.84 (m) , 6.44 (s), 6.48 (s), 6.63-6.86 (m), 6.89-7.01 (m), 7.09-7.19 (m) , 7.23-7.42 (m) , 8.02 (d, J = 8.7), 8.15 (d, J = 8.1), 8.59 (d, J= 8.7), 8.94 (s), 9.04 (s), 9.14 (d, J = 6.9) ; Anal. (C30H36FN7O7'TFA*H2O) C, Η, N.

- 119 ·· *·

Example 15 - Preparation of Compound A-12: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Leu-NCH3~L-(4-F-Phe)-L-Gln}-Epropenoate

(A-12)

Ethyl 3-{BOC-NCH3-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (described in Example 13 above) was converted to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Leu-NCH3L-(4-F-Phe)-L-Gln}-E-propenoate in a manner analogous to the preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-(2naphth)-NCH3-L-(4-F-Phe)-L-Gln}-E-propenoate described in

Example 13 above (using Boc-L-Leu-OH instead of 1h NMR

Boc-L-(2-naphth)-OH): IR (cm !) 3325, 1663, 1171 (DMSO-c/6, mixture of isomers) δ 0.64-0.67 (m), 0.86- 1.17-1.23 (m), 1.32 -1.40 (m), 1.59-1.75 (m), 1.98- 2.42 (s), 2.45 (s), 2.08 (s), 2.86-2.93 (m), 2.99 3.20 (m), 4.05-4.15 (m) , 4.41-4.50 (m), 4.82-5.07 (d, J = 15, 9) , 5.86 (d, J = 15.9), 6.51 (s), 6.53 6.73 (m) , 6.93-6.99 (m) , 7.09-7.21 (m), 7.26-7.31 (d, J = 7.8), 8.08 (d, J = 7.8) , 8.41 (d, J = 8.1) ,

J = 7.2); Anal. (C30H40FN5O7·1.25CH2C12) C, Η, N.

• 9 * «

- 120

Example 16 - Preparation of Compound A-13: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-(1-naphth)-NCH3-L-(4-F-Phe)-LGln}-E-propenoate

(A-13)

Ethyl 3-{BOC-NCH3-L-(4-F-Phe)-L-(Tr-Gln)}-E-propenoate (described in Example 13 above) was converted to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-(1-naphth)NCH3-L-(4-F-Phe)-L-Gln}-E-propenoate in a manner analogous to the preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-(2-naphth)-NCH3-L-(4-F-Phe)-L-Gln}-E-propenoate described in

Example 13 above, but instead of Boc-L-(2-naphth)-OH: IR NMR (DMSO-d6, mixture of isomers) δ using Boc-L-(1-naphth)-OH (cm ^-1 ) 3308, 1659, 1169; Ifí

1.16-1.23 (m) , 1.61-1.78 (m) ,

1.98-2.02 (m) , 2.07-2.12 (m) , 2.78 (s), 2.84-2.87 (m), 2.93- 3.38 (m), 4.00-4.15 (m), 4.27- 4.64 (m), 5.07-5.17 (m), 5.57- (m) , 6.71-6.88 (m), 6.89-6.91 (m) , 7.50-7.58 (m), 7.73-7.78 = 8, 1), 8.13 (d, J = 8.7), 8,

8.1); Anal. (C37H40FN5O7)C,H

2.41 (s), 2.43 (s) , 2.77 (s), 3.03 (m) , 3.08- 3.14 (m), 3,Šl- 4.32 (m), 4.40- 4.46 im), 4.58- 5.73 (m), 6.45 (s) , 6.57-6.61 (m) , 7.11-7.19 (m) , 7.31-7.38 (m) , 7.83-7.94 (m) , 8.08 (d, J 62 (d , J = 8,i; 9, 14 (d, J = , N.

121

Example 17 - Preparation of Compound B-2: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val(COCH2)-L-(4-F-Phe)-L((S)-pyrrole-Ala)}-E-propenoate

Preparation of the intermediate ethyl 3-{Boc-L-ValT(COCH2)-L-(4-FPhe)-L-{(N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala}-E-propenoate

This intermediate was prepared from {2-methyl-1S-(4R-(4-fluorobenzyl)-5-oxotetrahydrofuran-2S-yl)propyl}-carbamic acid tert-butyl ester (described in

Example 10) and ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(SJ pyrrole-Ala)}-E-propenoate (described in Example 6) in a manner analogous to the preparation of ethyl 3-{Boc-L-(4-Me-Phe)-L-(Tr-Gln)}E-propenoate (Example 4) above: Rf = 0.24 (60% EtOAc in hexanes); IR (cm ^-1 ) 3293, 1717, 1668; 1h NMR (CDCl3) δ 0.82 (d, 3H, J = 6.8), 1.01 (d, 3H, J = 6.8), 1.30 (t, 3H, J = 7.2), 1.51-1.65 (m, 2H), 1.84-1.96 (m, 1H), 2.16-2.37 (m,

2H) , 2.47 (s, 3H) , 2.49-2.55 (m, 3H) , 2.85-3.01 (m, 2H) ,

3.12-3.25 (m, 3H), 3.77 (s, 3H), 3.78 (s, 3H), 4.18 (q, 2H,

J = 7.2), 4.31-4.49 (m, 3H), 4.65-4.70 (m, 1H), 5.53 (dd,

1H, J = 15.7, 1, 4), 6.39-6.44 (m, 3H), 6.63 (dd, 1H, J =

15.7, 5.4), 6.93-7.01 (m, 2H), 7.05-7.10 (m, 1H), 7.12-7.18 (m, 2H), 7.24 (d, 1H, J= 8.7), 7.47 (d, 1H, J= 6.5); Anal. (C40H49FN4O9·0.5H2O) C, Η, N.

122 • ····

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-ValT(COCH2)-L-(4-F-Phe)-L-( (S)-pyrrole-Ala)}-E-propenoate

Ethyl 3-{Boc-L-ValT(COCH2)-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate was converted to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-Lν3ΐΨ(00<3Η2)-L-(4-F-Phe)-L-( (S)-pyrrole-Ala)}-E-propenoate in a manner analogous to the conversion of ethyl 3-{Boc-L-Val-L-(4-F-Phe)L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate to the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-L-(4F-Phe)-L-( (S)-pyrrole-Ala)}-E-propenoate described in Example 6 above: Melting point = 178-181°C; Rf = 0.49 (10%

CH3OH in CHCl3); IR (cm ^-1 ) 3295, 1678 br; NMR (CDCl 3 ) δ

0.85 (d, 3H, J= 6.8), 1.03 (d, 3H, J= 6.5), 1.30 (t, 3H, J

= 7.2), 1.51-1.62 ( m, ÍH), 1.71-1.93 (m, 2H) , 2.27-2.40 (m, 2H), 2.47 (s, 3H) , 2.51-2.75 (m, 3H) , 2.82- •2.98 (m, 2H) , 3.11-3.24 (m, 1H), 3.26-3.42 (m, 2H), 4.18 (q, 2H, J = 7.2), 4.41-4.53 (m, ÍH), 4.63-4.72 (m, ΙH), 5.50 (d, ÍH, J = 15.4), 5.88 (s, 1H) , 6.39 (s, IH) , 6.63 (dd, ÍH, J = 15.4, 5.3), 6.92-7.03 (m, 2H), 7.08-7.31 (m, 4H); Anal.

(C31H39FN4O7) C, H, N.

Example 18

Preparation of compound C-2: ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((S)pyrrole-Ala)}-E-propenoate

F (C-2)

123

Preparation of the intermediate (5'-methylisoxazole-3'-carbonyl)-L-Val0-L-(4-F-Phe)-OCH2CH=CH2

Boc-L-Val-O-L-(4-F-Phe)-OCH2CH=CH2 (prepared as described in Example 12 above, 0.91 g, 2.15 mmol) was dissolved in 1,4-dioxane (28 mL) and a solution of HCl in 1,4-dioxane (4.0 M, 14 mL) was added. The reaction mixture was stirred at ambient temperature for 14 h. The solvent was removed by evaporation and the residue was taken up in EtOAc (50 mL). The organic phase was washed with saturated NaHCO3 solution (50 mL) and

then brine (50 ml) 1 , dried (MgSO4 ) and solvent was removed for acquisition 0, 66 g (quant.) clear oil. This material was dissolved in CH2Cl2 (20 ml) . He was pyridine added (0.17 ml , 2.08 mmol) and the reaction mixture was

cooled to 0°C. 5-Methylisoxazole-3-carbonyl chloride (0.33 g, 2.27 mmol), dissolved in 2 mL CH2Cl2, was added and the reaction mixture was warmed to ambient temperature over 1 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography eluting with a gradient of 5—>10% EtOAc/hexanes. (5' — methylisoxazole-3'-carbonyl)-L-Val-OL-(4-F-Phe)-OCH2CH=CH2 product was obtained as 0.70 g (82%) of a white crystalline solid: Rf = 0.20 (30% EtOAc/hexanes); IR (KBr) 1745, 1661, 1553, 1186 cm- ¹ ; If NMR (DMSO-d6) δ 0.85-0.92 (m, 6H), 2.14-2.21 (m, 1H), 2.46 (s, 3H), 3.05-3.19 (m, 2H), 4.324.37 (m, 1H), 4.53-4.60 (m, 2H), 5.16-5.28 (m, 3H), 5.745.85 (m, 1H), 6.56 (s, 1H), 7.01-7.07 (m, 2H), 7.26-7.29 (m, 2H), 8.76 (d, 1H, J = 8.1); Anal. (C22H25N2O6F) C, Η, N.

124 » 4 <1

Preparation of the intermediate ethyl 3-{(5'-methylisoxazole-3'carbonyl)-L-Val-O-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)(S)-pyrrole-Ala)}-E-propenoate (5'-methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)OCH2CH=CH2 (0.67 g, 1.55 mmol) was dissolved in THF (15 ml). Tetrakis(triphenylphosphine)palladium(0) (0.036 g,

0.03 mmol) and the reaction mixture was stirred for 5 min. Morpholine (0.16 mL, 1.86 mmol) was added dropwise and the reaction mixture was stirred at ambient temperature for 6 h. The solvent was removed in vacuo and the residue was taken up in 50 mL Et2O. The product was extracted twice with saturated NaHCO3 solution (50 mL) and the organic phase was removed. The aqueous phase was acidified to pH = 1-2 with 10% HCl and the product was extracted twice with EtOAc (40 mL). The organic phase was washed with brine (50 mL), dried (MgSO4) and concentrated to give 0.57 g (95%) of an oil which crystallized on standing.

A portion of this material (0.19 g, 0.50 mmol) was dissolved in DMF (3 mL). Diisopropylethylamine (0.34 mL, 1.0 mmol) was added, followed by ethyl 3-{H2N-L-((17-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate*HCl (prepared as described in Examples 4 and 6 above, 0.19 g, 0.50 mmol). The reaction mixture was cooled to 0 °C. HATU (0.19 g, 0.50 mmol) was added and the reaction mixture was allowed to warm to ambient temperature. DMF was removed in vacuo. The residue was dissolved in EtOAc (30 mL) and the organic phase was washed sequentially with 10% HCl solution (25 mL), saturated NaHCO3 solution (25 mL), (25 mL). The solvent was dried

H2O (25 mL) and brine (MgSO4) and filtered and the residue was purified by flash column chromatography

125 • ·· «· ♦ * • * • · · • ♦ ♦ ·· ·· · · • · ·♦ #· • · · • · · • 9 9

9 9 (O—>1.0% MeOH/CH2Cl2) to give ethyl 3-{(5' methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate (0.27 g, 72%) as a white amorphous solid: Rf = 0.18 (3% MeOH/CHCl3);

IR(KBr) 1671, 1547, 1510, 1209 cm~3-; 3-H NMR (DMSO-Cl6) δ 0.88 (d, 3H, J = 7.0), 0.93 (d, 3H, J = 7.0), 1.20 (t, 3H, J =

7.0), 1.41-1.58 (m, 2H), 1.79-1.98 (m, 2H), 2.07-2.24 (m,

2H), 2.44 (s, 3H), 3.01-3.13 (m, 4H), 3.73 (s, 3H), 3.76 (s, 3H), 4.09 (q, 2H, J = 7.0), 4.24 (s, 2H), 4.30 (t, IH, J = 7.4), 4.45-4.48 (m, IH), 5.12 (t, IH, J= 6.3), 5.59 (d, IH, J= 15.8), 6.45 (dd, IH, J = 8.5, 2.2), 6.54-6.56 (m, 2H) ,

6.73 (dd, IH, J = 15.5, 4.8), 6.89-6.95 (m, 3H), 7.20 (d, IH, J= 8.5), 7.22 (d, IH, J= 8.5), 8.08 (d, IH, J= 8.8),

8.89 (d, 1H, J=7.4).

Preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)L-Val-O-L-(4-F-Phe)-L-((S)-pyrrole-Ala)}-E-propenoate

Ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-O-L-(4F-Phe)-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-Epropenoate (0.25 g, 0.33 mmol) was dissolved in CHCl3 (6 mL). Two drops of water were added, followed by DDQ (0.10 g, 0.43 mmol). The reaction mixture was heated to 50-55°C for 8 h. After cooling, the reaction mixture was poured into EtOAc (30 mL). The organic phase was washed with 30 mL of 2:1 NaHCO3/1N NaOH solution and then brine (30 mL), dried (MgSO4) and concentrated. The residue was subjected to flash column chromatography eluting with 0->2% MeOH/CH2Cl2. The ethyl 3{(5'-methylisoxazole-3'-carbonyl)-L-Val-O-L-(4-F-Phe)-L-((S)pyrrole-Ala)}-E-propenoate product was obtained as 0.18 g (90%) of a white amorphous solid: Rf = 0.09 (3% MeOH/CHCl3);

126

IR(KBr) 1680, 1549 cm ^-1 ; NMR (DMSO-O6) δ 0, 91 (d, 3H, J = 6.6) , 0.95 (d, 3H, J = 6.6), 1.20 (t, 3H h J = 7.0), 1 , 36- 1, 44 (m, 1H), 1.56-1.61 (m, 1H), 1.74-1, 82 (m, 1H) , 1 , 94- 1.99 (m, 2H), 2.18-2.25 (m, 1H), 2.45 (s, 3H) , 2.98-3.18 (m, 4H) , 4.09 (q, 2H, J= 7.0), 4.28-4.32 (m, 1H) , 4.43-4.46 (m, 1H) , 5.12 (m, 1H), 5.58 (d, 1H, J = 15, 8) , 6.56 (s, 1H) , 6, 72 (dd, 1H, J = 15.8, 4.8), 6.91-6.97 (m, 2H), 7.21 (d, 1H, J = 5.9), 7.23 (d, 1H, J = 5.9), 7.59 (with, 1H) , 8.06 (d, 1H, J = 8.8), 8.92 (d, 1H, J = 7.4); Anal - (C30H37N4O8F ) c,

H, N.

Example 19 - Preparation of Compound B-3: 2-{(5'-methylisoxazole3'-carbonyl)-L-Val*(COCHg)-L-(4-F-Phe)-L-((SJ-pyrrole-Ala)ΙΕ-(g-vinyl-y-butyrolactone)

Preparation of the intermediate Boc-L-{(N-2,4-dimethoxybenzyl)-(S)pyrrole-Ala}-E-(α-vinyl-γ-butyrolactone)

(15,3'S)-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester (0.360 g, 0.881 mmol, 1 equivalent) was oxidized to the corresponding aldehyde by the method described in the preparation of ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)(S)-pyrrole-Ala)}-E-propenoate (Example 6). This aldehyde was combined with 3-(triphenyl-X^-phosphanylidene)-dihydrofuran-2-one (prepared in a manner similar to that described

127

Φ φ φφ φ φφ ·· φφφ φ φφφ φ φ φφ φ φφφ —φ φ The residue was purified by flash column chromatography (2.5% CH3OH in CH2Cl2, then 67% EtOAc in CH2Cl2) to give Boc-L-{(N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala}-E-(α-vinyl-γ-butyrolactone) as an oil (0.250 g, 60%): Rf = 0.50 (67% EtOAc in

CH 2 Cl 2 ); IR (cm ^-1 ) 3307, 1754, 1678; ^-H NMR (CDCl 3 ) δ 1.42 (s, 9H), 1.46-1.68 (m, 2H), 2.02-2.13 (m, 1H), 2.18-2.30 (m, 1H), 2.44-2.56 (m, 1H), 2.91-3.04 (m, 1H), 3.15-3.27 (m, 3H), 3.80 (s, 6H), 4.34-4.43 (m, 5H), 5.63-5.69 (m, 1H), 6.42-6.47 (m, 2H), 6.48-6.53 (m, 1H), 7.09-7.13 (m, 1H).

Preparation of the product 2-{(5'-methylisoxazole-3'-carbonyl)-LValT(COCH2)-L-(4-F-Phe)-L-( (S)-pyrrole-Ala)}-E-(α-vinyl-ybutyrolactone)

Boc-L-{(N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala}-Ε-(avinyl-γ-butyrolactone) was converted to the product 2-{(5'methylisoxazole-3'-carbonyl)-L-ValT(COCH2)-L-(4-F-Phe)-L( (S)-pyrrole-Ala)}-E-(α-vinyl-γ-butyrolactone) in a manner similar to that described above for the preparation of the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-LValT(COCH2)-L-(4-F-Phe)-L-((S)-pyrrole-Ala)}-E-propenoate (Example 17): Rf = 0.28 (5% CH3OH in CH2Cl2); IR (cm ^-1 ) 3378 br, 1749, 1678 br; !h NMR (CDCl 3 ) δ 0.84 (d, 3H, J = 6.8),

- 128 • ·· ·· · » • ·

-9 9 9

9 • 99

9 9

9 • 9 9 ·

• 9 9 · • 9 9 9

9 9 9

9 9 9

99

1.03 (d, 3H, J= 6.8), 1.45-1.55 (m, IH), 1.75-2.00 (m, 2H), 2.25-2.43 (m, 2H), 2.46-2.59 (m, 2H), 2.48 (s, 3H), 2.632.72 (m, IH), 2.77-2.90 (m, 3H), 3.06-3.26 (m, 2H), 3.293.44 (m, 2H), 4.32-4.46 (m, 3H), 4.65-4.71 (m, IH), 5.72 (s, IH), 6.14-6.20 (m, IH), 6.40 (s, IH) , 6.94-7.02 (m, 2H),

7.03-7.11 (m, 2H), 7.24 (d, 1H, J = 9.0), 7.60 (d, 1H, J = 6.2); Anal. (C31H37FN4O7)C,Η,N.

Example 20 - Preparation of Compound B-4: Ethyl 3-{(5'methylisoxazole-3'-carbonyl)-L-ValT(COCH2)~ ^L ~(4-F-Phe)-L((S)-piper-Ala)}-E-propenoate

of ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-Val-L-(4-F-Phe)L-( (S)-piper-Ala)}-E-propenoate (Example 9) and the product ethyl 3-{(5'-methylisoxazole-3'-carbonyl)-L-ValT(COCH2)-L-(4F-Phe)-L-Gln}-E-propenoate (Example 10) described above: Melting point = 161-162°C; Rf = 0.30 (5% CH3OH in CH2Cl2); IR (cm ^-1 ) 3295, 1713, 1649; A NMR (CDCl 3 ) δ 0.84 (d, 3H, J = 6.8), 1.03 (d, 3H, J = 6.8), 1.30 (t, 3H, J = 7.2), 1.431.55 (m, 2H), 1.77-1.90 (m, 2H), 1.95-2.12 (m, 2H) . 4.39-4.49 (m, IH), 4.65-4.72 (m, IH), 5.50 (dd, IH, J

1H, •

that·· that

129 —· this • this this*· MM • this* ·· · · this this • this this · ·»· »··· ·· ·* « this « • · · · • this· · • ·· « ·« *· = 15.9, 1.6), J = 15.9, 5.3)

5.80 (s, 1H), 6.38-6.41 (m, 1H), 6.62 (dd, , 6.94-7.02 (m, 2H), 7.08-7.28 (m, 4H).

Examples 21 to 30

In Examples 21-30, the following compounds (A-14) to (A-23) were prepared using synthetic methods analogous to those described above for compounds of general formula I-A:

(A-14) (A-15)

NH, (A-16)

N

H

_CO2CH2CH3 ( _A - ₁₇ )

130 ♦ «9 9 ·

9 «99«

9 9 9 9

9 9 9 9 ·

9 9 9 9 *999 99 99

(A-18) (A-19)

(A-20)

(A-21) (A-22)

131 1 • 00 0

0 ··

CO2CH2CH3 (A-23)

Example 31 - Preparation of Comparative Compound #2: Ethyl-3(Cbz-L-Leu-L-Phe-Ii-Gln)-E-propenoate

•CO2CH2CH3 (Reference compound #2)

Preparation of the intermediate [Boc-L-(Tr-Gln)]-N(OMe)Me

Isobutyl chloroformate (4.77 mL, 36.8 mmol, 1.0 equiv) was added to a solution of Na-Boc-γ-trityl-L-glutamine (18.7 g, 36.7 mmol, 1 equiv) and 4-methylmorpholine (8.08 mL, 73.5 mmol, 2.0 equiv) in CH2Cl2 (250 mL) at 0°C. The reaction mixture was stirred at 0°C for 20 min and then N,O-dimethylhydroxyamine hydrochloride (3.60 g, 36.7 mmol, 1.0 equiv) was added. The resulting solution was stirred at 0°C for 20 min and at 23°C for 2 h and then partitioned between water (150 mL) and CH2Cl2 (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (gradient elution 40—>20% hexanes in EtOAc) gave {Boc-L-(Tr-Gln)}-N(OMe)Me (16.1 g, 82%) as a white foam: IR (cm ^-1 ) 3411, 3329, 3062, 1701, 1659; 3-H NMR (CDCl3) δ 1.42 (s, 9H), 1.63-1.77 (m, 1H), 2.06-2.17 (m, 1H), 2.29- 132 ί • · • · 99 99

9 9 9 9 9 9

9 9 9 9 9

9 9 9 9 9 9

9 9 9 9 9

999 9999 99 9 9

2.43 (m, 2H), 3.17 (s, 3H), 3.64 (s, 3H), 4.73 (s, br, 1H),

5.38-5.41 (m, 1H), 7.20-7.31 (m, 15H); Anal. (C31H37N3O5)C,

H, N.

Preparation of the intermediate {Boc-L-(Tr-GIn)}-H

Diisobutylaluminum hydride (50.5 mL of a 1.5 M solution in toluene, 75.8 mmol, 2.5 equiv) was added to a solution of {Boc-L-(Tr-GIn)}-N(OMe)Me (16.1 g, 30.3 mmol, 1 equiv) in THF at -78°C and the reaction mixture was stirred at -78°C for 4 h. Methanol (4 mL) and 1.0 M HCl (10 mL) were added sequentially and the mixture was warmed to 23°C. The resulting suspension was diluted with Et2O (150 mL) and washed with 1.0 M HCl (3 x 100 mL), half-saturated NaHCO3 (100 mL) and water (100 mL). The organic layer was dried over MgSO 4 , filtered and concentrated to give crude {Boc-L-(Tr-GIn)}-H (13.8 g, 97%) as a white solid: mp = 114-116°C; IR (cm ^-1 ) 3313, 1697, 1494; 1 H NMR (CDCl 3 ) δ 1.44 (s, 9H), 1.65-1.75 (m, ÍH), 2.17-2.23 (m, ÍH), 2.31-2.54 (m, 2H), 4.11 (s, br, ÍH), 5.38-5.40 (m, ÍH),

7.11 (s, 1H), 7.16-7.36 (m, 15H), 9.45 (s, 1H).

Preparation of the intermediate ethyl 3-{Boc-L-(Tr-GIn)}-E-propenoate

Sodium bis(trimethylsilyl)amide (22.9 mL of a 1.0 M solution in THF, 22.9 mmol, 1.0 equiv) was added to a solution of triethylphosphonoacetate (5.59 g, 22.9 mmol, 1.0 equiv) in THF (200 mL) at -78°C and the resulting solution was stirred for 20 min at this temperature. Crude {Boc-L-(TrGln)}-H (10.8 g, 22.9 mmol, 1 equiv) in THF (50 mL) was added via cannula and the reaction mixture was stirred for 2 h at -78°C, warmed to 0°C for 10 min and partitioned between 0.5 M HCl (150 mL) and a 1:1 mixture of EtOAc and

133

• ♦ it • this • this • • · • it ·· • · · • • that • that • · • • —-· • · • « ·· • that that * • • · • •

• to • to hexanes (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Purification of the residue by flash column chromatography (40% EtOAc in hexanes) gave ethyl 3-{Boc-L-(Tr-Gln)}-E-propenoate (10.9 g, 88%) as a

white foams: IR (cm ^-1 ) 3321, 1710; 1h NMR (CDCl3) δ 1.27 (t, 3H, 7.2), 1.42 (s, 9H), 1.70-1.78 (m, 1H), 1.80-1.96 (m, 1H) , 2.35 (t, 2H, J= 7.0), 4.18 (q, 2H, J = 7.2), 4.29 (with, br, 1H) , 4.82-4.84 (m, 1H) , 5.88 (dd, 1H, J = 15.7, 1, β) , 6.79 (dd, 1H, J = 15.7, 5.3), 6.92 (s, 1H), 7.19-7.34 (m,

15H); Anal. (C33H38N2O5) C, Η, N.

Preparation of the intermediate ethyl-3-{Cbz-L-Leu-L-Phe-L-(Tr-Gln)}E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 20 mL) was added to a solution of ethyl 3-{Boc-L-(Tr-Gln)}-E-propenoate (1.00 g, 1.84 mmol, 1 equiv) in the same solvent (20 mL) at 23°C. After 3 h, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (50 mL) and Cbz-L-Leu-L-Phe-OH (0.759 g, 1.84 mmol, 1.0 equiv), HOBt (0.373 g, 2.76 mmol, 1.5 equiv), 4-methylmorpholine (0.809 mL, 7.36 mmol, 4.0 equiv) and EDC (0.529 g, 2.76 mmol, 1.5 equiv) were added sequentially. The reaction mixture was stirred at 23°C for 18 h and then partitioned between water (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Flash chromatography of the residue (5% CH3OH in CH2Cl2) gave ethyl 3-{Cbz-L-LeuL-Phe-L-(Tr-Gln)}-E-propenoate (1.25 g, 83%) as a white solid: mp = 192-194°C; IR (cm ^-1 ) 3295, 1696, 1678, 1655, 1519; ^X H NMR (CDCl3) δ 0.84 (d, 3H, J = 6.5), *· • © • © ···©

- 134 ·· • · ·_ · · • © ··· ··©· ·· ·· ·· « · · · • · · © • © · · • © · · ·· ·©

0.86 (d, 3H, J = 6.5), 1.24-1.32 (m, 1H), 1.28 (t, 3H, J =

7.2), 1.43-1.75 (m, 3H) , 1.91-2.06 (m, 1H) , 2.20-2.38 (m, 2H) , 2.93-3.02 (m, 1H) , 3.07-3.18 (m, 1H) , 3.95-4.02 (m, 1H), 4.17 (q, 2H, J= 7.2), 4.43-4.55 (m, 2H), 4.82-4.95 (m,

2H), 5.69 (d, 1H, J = 15.7), 6.46 (d, 1H, J = 7.5), 6.60 (d,

1H, J = 8.1), 6.69 (dd, 1H, J = 15.7, 5.1), 7.09-7.38 (xn,

27H); Anal. (C51H56N4O7) C, Η, N.

Preparation of the product ethyl-3-(Cbz-L-Leu-L-Phe-L-G2n)-Epropenoate

Trifluoroacetic acid (20 ml) was added to a solution of ethyl 3-{Cbz-L-Leu-L-Phe-L-(Tr-Gln)}-E-propenoate (1.25 g,

1.49 mmol, 1 equiv) and triisopropylsilane (1.53 mL, 7.47 mmol, 5.0 equiv) in CH2Cl2 (20 mL) at 23°C to form a bright yellow solution. The reaction mixture was stirred for 20 min at 23°C, during which time it became colorless. Carbon tetrachloride (20 mL) was added and the volatiles were removed under reduced pressure. The residue was triturated with Et2O (20 mL) and the resulting white solid was collected by vacuum filtration, washed with Et2O (3 x 50 mL) and air dried to give ethyl 3-(Cbz-L-Leu-LPhe-LG-Zn)-E-propenoate (0.717 g, 81%): mp = 219-221°C; IR (cm ^-1 ) 3300, 1672, 1535; ¹ H NMR (DMSO-d6) δ 0.78 (d, 3H, J = 6.8), 0.82 (d, 3H, J = 6.5), 1.21 (t, 3H, J = 7.0), 1.25-1.37 (m, 2H), 1.42-1.54 (m, 1H), 1.58-1, 80 (m,

. 4.33-4.52 (m, 2H), 4.97 (d, 1H, J = 12.3), 5.04 (d, 1H, J = 12.3), 5.64 (d, 1H, J = 15.9), 6.69 (dd, 1H, J =

15.9, 5.4), 6.76 (s, 1H), 7.13-7.37 (m, 11H), 7.43 (d, 1H, J • *

- 135 _ · · = 7.8), 7.99 (d, 1H, J= 8.1), 8.04 (d, 1H, J= 8.1); Anal. (C32H42N4O7) C, Η, N.

Example 32 - Preparation of Comparative Compound #3: Ethyl 3(Cbz-L-Val-L-Phe-L-Gln)-E-propenoate

compound #3)

Preparation of the intermediate ethyl-3-{Boc-L-Phe-L-(Tr-Gln)}-Epropenoate

A solution of HCl in 1,4-dioxane (4.0 M, 10 mL) was added to a solution of ethyl 3-{Boc-L-(Tr-Gln)}-E-propenoate (prepared as described in Example 31, 3.05 g, 5.62 mmol, 1 equivalent) in the same solvent (20 mL) at 23° C. After 3 hours, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (50 mL) and Boc-L-Phe-OH (1.49 g, 5.62 mmol, 1.0 equiv), HOBt (0.911 g, 6.74 mmol, 1.2 equiv), 4-methylmorpholine (1.85 mL, 16.8 mmol, 3.0 equiv) and EDC (1.29 g, 6.73 mmol, 1.2 equiv) were added sequentially. The reaction mixture was stirred at 23°C for 18 h and then partitioned between water (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated. Flash chromatography purification of the residue (gradient elution, 40—»50% EtOAc in hexanes) afforded ethyl 3-{Boc-L-Phe-L-(TrGln)}-E-propenoate (2.77 g, 71%) as a white foam: IR (cm

!) 3306, 1706, 1661; !h NMR (CDCl 3 ) δ 1.29 (t, 3H, J= 7.2),

1.38 (s, 9H) , 1.65-1.76 (m, 1H) , 1.87-1.99 (m, IH) , 2.25- 2.27 (m, 2H), 2.94-3.01 (m, 2H), 4.14-4.26 (m, 3H) , 4.48- 4.53 (m, IH), 4.95 (s, br, IH), 5.64 (d, IH, J = 15.8) , 6.29 (d, IH, J= 8.1), 6.64 (dd, IH, J = 15.8, 5.4 ), 6.80 (s, br,

1H), 7.14-7.32 (m, 20H); Anal. (C42H47N3O6) C, Η, N.

Preparation of the intermediate ethyl-3-{Cbz-L-Val-L-Phe-L-(Tr-Gln)}E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 8 mL) was added to a solution of ethyl 3-{Boc-L-Phe-L-(Tr-Gln)}-E-propenoate (0.296 g, 0.429 mmol, 1 equivalent) in the same solvent (10 mL) at 23° C. After 3 hours, the volatiles were removed under reduced pressure. The residue was dissolved in CH2Cl2 (10 mL) and then Cbz-L-Val-OH (0.108 g, 0.430 mmol, 1.0 equiv), HOBt (0.070 g, 0.518 mmol, 1.2 equiv), 4-methylmorpholine (0.142 mL, 1.29 mmol, 3.0 equiv) and EDC (0.099 g, 0.516 mmol, 1.2 equiv) were added sequentially. The reaction mixture was stirred at 23°C for 4 h and then partitioned between water (100 mL) and EtOAc (2 x 100 mL). The combined organic layers were dried over Na2SO4 and concentrated. Flash chromatography purification of the residue (3% CH3OH in CH2Cl2) gave ethyl 3-{Cbz-LVal-L-Phe-L-(Tr-Gln)}-E-propenoate (0.220 g, 62%) as a white solid: mp = 195-198°C; IR 3284,

1689, 1646; A NMR (CDCl 3 ) δ 0.69 (d, 3H, J = 6.9), 0.82 (d, 3H, J = 6.5), 1.28 (t, 3H, J = 7.2), 1.63-1.74 (m, 1H), 1.96-2.02 (m, 2H), 2.22-2.35 (m, 2H) , 2.93 (dd, IH, J = 14.0, 7.6), 3.10 (dd, IH, G= 14.0, 6.7), 3.81-3.85 (m, IH),

4.17 (q, 2H, J = 7.2), 4.48-4.58 (m, 2H), 4.87 (d, 1H, J =

12.0), 4.94 (d, IH, J = 12.0), 5.06 (d, IH, J= 6.9), 5.67

(d, 1H, J= 15.6), 6.43 (d, 1H, J= 7.5), 6. 63-6, 72 (m, 2H) ,

7.10-7.40 (m, 26H); Anal. (C50H54N4O7) C, Η, N.

Preparation of the product ethyl-3-(Cbz-L-Val-L-Phe-L-Gln)-Epropenoate

Trifluoroacetic acid (5 mL) was added to a solution of ethyl 3-{Cbz-L-Val-L-Phe-L-(Tr-Gln)}-E-propenoate (0.188 g, 0.229 mmol, 1 equivalent) and triisopropylsilane (0.300 mL,

1.46 mmol, 6.4 equiv) in CH2Cl2 (10 mL) at 23°C to form a bright yellow solution. The reaction mixture was stirred for 20 min at 23°C, during which time it became colorless. Carbon tetrachloride (10 mL) was added and the volatiles were removed under reduced pressure. The residue was triturated with Et2O (20 mL) and the resulting white solid was collected by vacuum filtration, washed with Et2O (3 x 50 mL) and air dried to give ethyl 3-(Cbz-L-Val-LPhe-L-Gln)-E-propenoate (0.094 g, 71%): mp = 240°C (decomposition); IR (cm ^-1 ) 3263, 1686, 1640; !h NMR (DMSO-Cl 6 ) δ 0.73 (d, 6H, J = 6.9), 1.21 (t, 3H, J = 7.2), 1.60-1.75 (m, 2H), 1.83-1.90 (m, 1H), 2.03-2.08 (m, 2H), 2.83 (dd, 1H, J= 13.6, 8.6), 2.96 (dd, IH, J = 13, 6, 6.2), 3.79-3.84 (m, IH), 4.10 (q, 2H, J= 7.2), 4.37-4.49 (m, IH), 4.51-4.56 (m, IH), 4.99 (d, IH, J = 12.5), 5.06 (d, IH, J = 12.5), 5.61 (d, IH, J = 15.5), 6.67 (dd, IH, J = 15.5, 5.5), 6.76 (s,

1H), 7.13-7.36 (m, 12H), 8.06 (d, 2H, J = 8.1); Anal.

(C31H40N4O7) C, H, N.

Example 33 - Preparation of compound (A-24): ethyl 3-{Cbz-L-LeuL-Phe-L-((S)-pyrrole-Ala.)}-E-propenoate

Preparation of the intermediate ethyl-3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 4 mL) was added to a solution of ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrroleAla)}-E-propenoate (prepared as described in Example 6) (0.139 g, 0.292 mmol, 1 equivalent) in 1,4-dioxane (4 mL). After stirring for 1.5 h, the volatiles were evaporated to give the crude amine salt, which was used without purification.

This amine salt was dissolved in CH2Cl2 (7 mL) and then Cbz-L-Leu-L-Phe-OH (0.156 g, 0.378 mmol, 1.3 equiv), 4-methylmorpholine (0.128 mL, 1.16 mmol, 4 equiv), HOBt (0.067 g, 0.50 mmol, 1.7 equiv) and EDC (0.095 g, 0.50 mmol, 1.7 equiv) were added sequentially. After stirring for 20 h, the reaction mixture was poured into brine (15 mL) and extracted with 10% CH3OH in CH2Cl2 (3 x 25 mL). The combined organic phases were dried over Na2SO4 and evaporated. Purification of the residue by flash column chromatography (60% EtOAc in hexanes) afforded ethyl 3-{Cbz-L-Leu-L-Phe-L-( (N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate (0.158 g, 70%) as a foam: NMR (CDCl3) δ 0.87-0.92 (m, 6H), 1.28 (t,

3H, J = 7.2), 1.46-1.68 (m, 5H), 1.74-1.86 (m, 1H), 1.972.19 (m, 2H), 3.02 (dd, 1H, J = 13.7, 5.6), 3.11-3.24 (m,

3H) , 3.78 (s, 3H) , 3.79 (s, 3H) , 4.17 (q, 2H, J = 7.2),

139

4.20-4.30 (m, 2H) , 4.35-4.45 (m, 2H) , 4.82-4.90 (m, 1H) ,

5.07 (d, ÍH, J= 12.3), 5.13 (d, ÍH, J= 12.3), 5.36 (d, ÍH, J= 7.8), 5.82 (dd, ÍH, J= 15, 6, 1.2), 6, 42-6, 46 (m, 2H) ,

6.72 (dd, ÍH, J = 15.6, 5.3), 6.88 (d, ÍH, J = 8.7), 7.09 (d, ÍH, J= 9.0), 7.13-7.20 (m, 5H), 7.29-7.37 (m, 5H), 8.09 (d, ÍH, J = 6.5).

Preparation of the product ethyl-3-{Cbz-L-Leu-L-Phe-L-( (S)-pyrroleAla)}-E-propenoate

Ethyl 3-{Cbz-L-Leu-L-Phe-L-( (N-2,4-dimethoxybenzyl)(S)-pyrrole-Ala)}-E-propenoate (0.156 g, 0.202 mmol, 1 equiv) and ammonium cerium nitrate (0.277 g, 0.505 mmol, 2.5 equiv) were combined in 2:1 THF/water (3 mL) and stirred for 2 h. The reaction mixture was poured into brine (15 mL) and extracted with 10% CH3OH in CH2Cl2 (3 x 25 mL). The combined organic phases were dried over Na2SO4 and evaporated. Purification of the residue by flash column chromatography (gradient elution, 2—>5% CH3OH in CH2Cl2) afforded ethyl 3-{Cbz-L-Leu-L-Phe-L-( (S)-pyrrole-Ala)}-E-propenoate (0.059 g, 47%) as an off-white solid: NMR (CDCl3) δ 0.85-0.92 (m, 6H), 1.28 (t, 3H, J= 7.2), 1.39-1.65 (m, 4H), 1.68-1.93 (m, 2H), 2.08-2.20 (m, ÍH), 2.27-2.38 (m, ÍH), 3.02-3.13 (m, 2H), 3.24-3.32 (m, 2H) , 4.11-4.20 (m, ÍH), 4.18 (q, 2H, J = 7.2), 4.47-4.58 (m, ÍH), 4.81-4.89 (m, ÍH), 5.05 (d, ÍH, J = 12.1), 5.12 (d, ÍH, J= 12.1), 5.26 (d, ÍH, J = 8.1), 5.78 (dd, ÍH, J = 15.7, 1.2), 6.23 (s, 1H), 6.72 (dd, ÍH, J = 15.7, 5.3), 7.13-7.25 (m, 6H), 7.30-7.37 (m, 5H), 7.54 (d,

(H, J = 7.2).

Example 34 - Preparation of compound (A-25): ethyl 3-{Cbz-L-ValL-Phe-1·-((S)-pyrrole-Ala)}-E-propenoate

Preparation of the intermediate ethyl 3-{Cbz-L-Val-L-Phe-L-((N-2 _r 4dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate

In a manner analogous to that used for the conversion of ethyl 3-{Boc-L-(Tr-Gln)}-E-propenoate to ethyl 3-{Cbz-L-LeuL-Phe-L-(Tr-Gln)}-E-propenoate, described in Example 31, ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate (prepared as in Example 33) was coupled with Cbz-LVal-L-Phe-OH to give ethyl 3-{Cbz-L-Val-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate: IR (cm” ¹ )

3288, 1699, 1652; 3-H NMR (CDCl 3 ) δ 0.87 (d, 3H, J = 6.8),

0.95 (d, 3H, J = 6.5), 1.28 (t, 3H, J = 7.2), 1.48-1.60 (m, 2H), 1.70-1.84 (m, IH), 1.95-2.20 (m, 3H), 3.01 (dd, IH, J = 13.4, 5.6), 3.09-3.25 (m, 3H), 3.78 (s, 3H), 3.80 (s, 3H),

4.03-4.10 (m, IH), 4.17 (q, 2H, J = 7.2), 4.24 (d, IH, J = 14,2), 4, 33-4.44 (m, IH) 4.38 (d, IH, J = 14.2) >, 4, . 85-4.94 (m, IH) , 5.08 (d, IH, J = 12.1), 5.14 (d, IH, J = Í2,l) , 5.39 (d, IH, J = 8.1) , 5.80 (dd, IH, J = 15.6, í,2) , 6.42- 6.47 (m, 2H), 6.70 (dd, IH, J = 15 ,6, 5.3), 6.81 (d, 1H, J = 9.0), 7.1 1-7.20 (m, 6H), 7.31-7.39 (m, 5H), 8.11 (d, IH, J =

6.2); Anal. (C42H52N4O9) C, Η, N.

Preparation of the product ethyl-3-{Cbz-L-Val-L-Phe-L-((S)-pyrroleAla)}-E-propenoate

- ui-:.

A suspension of ethyl 3-{Cbz-L-Val-L-Phe-L-( {N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate (0.215 g, 0.284 mmol, 1 equiv), DDQ (0.071 g, 0.31 mmol, 1.1 equiv) and water (3 drops) in CHCl3 (4 ml) was stirred for 1 h at 23°C and then heated to reflux for 6 h. After cooling overnight, additional DDQ (0.019 g, 0.084 mmol, 0.3 equiv) was added and the mixture was heated at 67°C for 1 h and then evaporated. Purification of the residue by flash column chromatography (gradient elution, 2-5% CH3OH in CH2Cl2) gave slightly impure material which was dissolved in CH2Cl2 (70 mL) and washed with saturated NaHCO3 (2 x 30 mL) and brine (30 mL) and then dried over Na2SO4 and evaporated. The residue was stirred in Et2O (10 mL) for 20 min and the solid was collected by filtration and dried under vacuum to give ethyl 3-{Cbz-L-Val-LPhe-L-((S)-pyrrole-Ala)}-E-propenoate (0.060 g, 35%) as an off-white solid: mp = 215-217°C; IR (cm ^- !)

3413, 3295, 1696, 1649; 3-H NMR (CDCl 3 ) δ 0.83 (d, 3H, J =

6.5), 0.91 (d, 3H, J = 6.8), 1.28 (t, 3H, J = 7.2), 1.501.59 (m, 1H), 1.70-1.91 (m, 2H), 2.03-2.17 (m, 2H), 2.262.38 (m, 1H), 3.03 (dd, 1H, J = 13.5, 6.4), 3.12 (dd, 1H, J = 13.5, 6.4), 3.21-3.34 (m, 2H), 3.96 (dd, 1H, J = 8.3, 6.4), 4.17 (q, 2H, J = 7.2), 4.45-4.56 (m, 1H), 4.83-4.92 (m, 1H), 5.07 (d, 1H, J = 12.1), 5.13 (d, 1H, J = 12.1),

5.29 (d, 1H, J = 8.3), 5.77 (dd, 1H, J = 15.8, 1.2), 5.94 (s, 1H), 6.71 (dd, 1H, J = 15.8, 5.3), 6.95 (d, 1H, J = 9.0), 7.14-7.27 (m, 5H), 7.31-7.38 (m, 5H), 7.57 (d, 1H, J = 7.2); Anal. (C33H42N4O7) C, Η, N.

and * ·· *·

Example 35 - Preparation of compound (A-26): ethyl 3-{Cbz-L-LeuL-Phe-L-((S)-piper-Ala)}-E-propenoate

HN—v, i 0=/ \

AND

_{CO2CH2CH3 ​}

AND

H

(A-26)

Preparation of the product ethyl-3-{Cbz-L-Leu-L-Phe-L-((S)-piperAla)}-E-propenoate

(1S,3'S)-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-piperidin-3'-yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester (prepared as described in Example 8) was converted to the product ethyl 3-{Cbz-L-Leu-L-Phe-L-( (S)piper-Ala)}-E-propenoate in a manner analogous to the conversion of (1S,3'S)-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester to the product ethyl 3-{Cbz-L-Leu-L-Phe-L-( (S)pyrrolidin-Ala)}-E-propenoate as described in Example 34: IR (cm ^-1 ) 3422, 3307, 1713, 1649; A NMR (CDCl 3 ) δ 0.86-0.92

(m, 6H) , A 28 (t , 3H, J = 7.2) From 1.38 -1.75 (m, 6H) , 1.77-1.89 (m, ÍH) , 1, 96-2, 11 (m, 2H) , 3, 07 (d, 2H, J = 6.2), 3.20-3.27 (m, 2H) , 4, 13-4, 24 (m, H) , 4, 18 (q, 2H, J = 7.2), 4.41-4.53 (m, ÍH) , 4, 76-4, 85 (m, H) , 5, 06 (d, ÍH, J = 12.1), 5.12 (d, Hello, J = 12 ,D, 5.34 (d, Hello, J = 7 ,8), 5.78 (dd, ÍH, J = 15, f P 5, 4), 6.17 (s, 1 Η) , 6.70 ( dd, ÍH, J = 15.6, 5 ,4), 7.00 (d, Hello, J = 8.4) , 7.13 7.2 7 (m from 6H) , 7.30-7.37 (m, 5H), 7.83

(d, 1H, J = 6.8); Anal. (C35H46N4O7·0.5H2O) C, Η, N.

* «

143 • ·

Example 36 - Preparation of compound (A-27): ethyl 3-{Cbz-L-LeuL-Phe-L-((R)-pyrrole-Ala)}-E-propenoate

(A-27)

Preparation of the intermediate tert-butyl ester of (4S,4' ^r R)4-{3'-(4''-benzyl-2''-oxo-oxazolidin-3''-yl)-3'-oxopropyl}2,2-dimethyloxazolidine-3-carboxylic acid

Triethylamine (6.43 mL, 46.1 mmol, 3.0 equiv) and pivaloyl chloride (1.89 mL, 15.3 mmol, 1.0 equiv) were sequentially added to a solution of (4S)-4-(2'-carboxyethyl)-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (4.20 g, 15.3 mmol, 1 equiv) in THF (300 mL) at 0°C. The cloudy reaction mixture was stirred at 0°C for 3.5 h, and then lithium chloride (0.716 g, 16.9 mmol, 1.1 equiv) and (R)(+)-4-benzyl-2-oxazolidinone (2.59 g, 14.6 mmol, 0.95 equiv) were sequentially added. After warming to 23°C and stirring for 19 h, the reaction mixture was partitioned between 0.5 M HCl (150 mL) and EtOAc (2 x 150 mL). The combined organic layers were washed with half-saturated Na2CO3 (150 mL), dried over MgSO4, and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography (30% EtOAc in hexanes) to give (4S,4'' R)-4-{3'-(4''-benzyl-2''-oxoxazolidin-3' '-yl)-3'-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert.butyl ester (6.15 g, 97%) as a colorless oil: IR (cm

1) 2978, 1783, 1694; A NMR (CDCl3, mixture of isomers) δ 1.46

144 • to ·* « r · ·♦<» to · · · • · · · ♦ • * ♦ *

(s) , 1.58 (s) , 1.63 (s), 2.01-2.05 (m), 2.72-3.13 (m), 3.29 3.33 (m), 3.74-3.79 (m), 3.82-4.09 (m), 4.11-4.25 (m) , 4.67 4.70 (m) , 7.20-7.37 (m); Anal. (C23H32N2O6) C, H, N.

Preparation of the intermediate tert-butyl ester of (2'JR,4S,4 ^ř 'R)-4-{2'-(4-benzyl-2-oxo-oxazolidine-3-carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-carboxylic acid

A solution of (4S, 4''R)-4-{3'(4-benzyl-2''-oxo-oxazolidin-3''-yl)-3'-oxopropyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (6.15 g, 14.2 mmol, 1 equivalent) in THF (25 mL) was added to a solution of sodium bis(trimethylsilyl)amide (14.2 mL of a 1.0 M solution in THF, 14.2 mmol, 1.0 equivalent) in the same solvent (50 mL) at -78°C. The reaction mixture was stirred for 15 min at -78°C and then allyl iodide (3.90 mL, 42.6 mmol, 3.0 equivalent) was added. After stirring for an additional 2 hours at -78°C, the reaction mixture was kept at -45°C for 2 hours and then partitioned between a 2:1 mixture of half-saturated NH4Cl and 5% Na2S2O3 (200 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 150 mL). The combined organic layers were washed with H2O (100 mL), dried over MgSO4 and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was purified by flash column chromatography (15% EtOAc in hexanes) to give (2'R,4S,4R)-4-{2'-(4-benzyl-2''-oxo-oxazolidine-3''-carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (3.12 g, 46%) as a viscous foam: IR (cm ^-1 ) 2978, 1782, 1685; 1H NMR (CDCl3, mixture of isomers) δ 1.42 (s), 1.45 (s), 1.49 (s), 1.52 (s), 1.62-1.78 (m), 1.80-2.01 (m), 2.23-2.49 (m), 2.51-2.56 (m), 2.76 (dd, J = 13.3, 9.7), 3.26 (dd, J = 13.3, 3.6), 3.58-3.64 (m), 3.67 (d, J = 8.7), 3.903.98 (m), 4.02-4.15 (m), 4.16-4.30 (m), 4.75-4.82 (m), 5.065.11 (m), 5.74-5.88 (m), 7.22-7.36 (m); Anal. (C26H36N2O6) C, H,

N.

Preparation of the intermediate tert-butyl ester of (1S,3'R){2-(1'-(2'',4''-dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1hydroxymethylethyl}-carbamic acid

Ozone was bubbled through a solution of (2'R,4S,4''R)-4-{2'-(4''-benzyl-2''-oxo-oxazolidine-3''-carbonyl)-pent-4'-enyl}-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (3.12 g, 6.60 mmol, 1 equiv) in CH2Cl2 (200 mL) and CH3OH (0.535 mL, 13.2 mmol, 2.0 equiv) at -78°C until the blue color persisted. The reaction mixture was then purged with argon until it became colorless. Methyl sulfide (4.85 mL, 66.0 mmol, 10 equiv) was added and the mixture was stirred at -78°C for 3.5 h and then maintained at 0°C for an additional 1 h. After partitioning the reaction mixture between H2O (150 mL) and a 1:1 mixture of EtOAc and hexanes (2 x 150 mL), the combined organic layers were dried over MgSO4 and gravity filtered. The filtrate was concentrated under reduced pressure and the residue was used immediately without further purification.

The above residue was dissolved in a 2:1 mixture of THF and then EtOH (180 mL) at 23°C and 2,4-dimethoxybenzylamine hydrochloride (5.38 g, 26.4 mmol, 4.0 equiv), sodium acetate (2.17 g, 26.4 mmol, 4.0 equiv) and sodium cyanoborohydride (0.829 g, 13.2 mmol, 2.0 equiv) were added sequentially. The resulting suspension was stirred for 19 h at 23°C and then partitioned between 0.5 M HCl (150 mL) and EtOAc (2 x 100 mL). The combined organic layers were washed with half-saturated NaHCO3 (100 mL), dried over Na2SO4 and • *

• ·* • • • • • •

concentrated under reduced pressure. The residue was passed through a short silica gel column (eluting with 50% EtOAc in hexanes) to give (3' R,4S)-4-{1'-(2' ',4' '-dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-ylmethyl}-2,2-dimethyl-oxazolidine-3-carboxylic acid tert-butyl ester, contaminated with (R)-(+)-4-benzyl-2-oxazolidinone.

This material was dissolved in CH3OH (80 mL) and TsOH«H2O (0.251 g, 1.32 mmol, 0.20 equiv) was added. The reaction mixture was heated to 50°C and maintained at this temperature for 4 h. After cooling to 23°C, the reaction mixture was concentrated under reduced pressure to a volume of approximately 20 mL and partitioned between half-saturated NaHCO3 (150 mL) and a 9:1 mixture of CH2Cl2 and CH3OH (2 x 150 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Purification of the residue by flash column chromatography (3% CH3OH in CH2Cl2) gave (1S, ^3ZR )-{2-(1'-(2'',4''dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1hydroxymethylethyl}-carbamic acid tert-butyl ester (0.92 g, 34%) as a foam:

IR (cm-1) 3347 (br) , 2937, 1669; !h NMR (CDCl3) δ 1.44 (with, 9H) , 1.62-1.77 (m, 2H), 1.94-2.04 (m, IH) , 2.15-2.26 (m, IH) , 2.40-2.50 (m, IH) , 3.13-3.24 (m, 2H) , 3.56-3.77 (m, 3H) , 3.80 (with, 6H), 3.82-4.16 (m, IH) , 4.37 (d, IH, J = 14, 3), 4.45 (d, IH, J = 14.3), 5.49 (d, IH, J = 7.8), 6 ,42-

6.45 (m, 2H), 7.08-7.11 (m, 1H); Anal. (C21H32N2O6·0.25H2O)

C, H, N.

Preparation of the intermediate ethyl-3-{Boc-L-((ω-2,4-dimethoxybenzyl)-(R)-pyrrole-Ala)}-E-propenoate

- 147 • 0« « ♦ · _ ♦ · • · *«« 0000

By a method analogous to the conversion of (1S,3'S)-{2-(1'-(2'',4''-dimethoxybenzyl)-2'-oxo-pyrrolidin-3'yl)-1-hydroxymethylethyl}-carbamic acid tert-butyl ester to ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl) - (S) -pyrrole-Ala)}-E-propenoate, described in

Example 33, (1S,3'R){2-(1'-(2,4-dimethoxybenzyl)-2'-oxo-pyrrolidin-3'-yl)-1hydroxymethylethyl}-carbamic acid tert-butyl ester was converted to ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(R)-pyrrole-Ala)}-E-propenoate: IR (cm ^- !) 3307,

1713, 1674; 3-H NMR (CDCl3) δ 1.28 (t, 3H, J = 7.2), 1.45 (with , 9H), 1.57-1.82 (m, 2H), 2.02-2.19 (m, 2H) , 2.42-2.55 (m, ÍH), 3.11- 3.25 (m, 2H) , 3.79 (s, 3H), 3.80 (with, 3H), 4.19 (q, 2H, J = 7.2), 4.32-4.50 (m, 3H), 5.97 (dd, ÍH, J = 15.7, 1.4), 6.38 (d, H, J - 7.8), 6.42-6.47 (m, 2H), 6.86 (dd, ÍH, J = 15.7, 5.1) , 7.08- 7.13 (m, ÍH); Anal. (C25H36N2O7) C, H, N.

Preparation of the intermediate ethyl 3-{Cbz-L-Leu-L-Phe-L-({N-2,4-dimethoxybenzyl)-(R)-pyrrole-Ala)}-E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 6 mL) was added to a solution of ethyl 3-{Boc-L-((N-2,4-dimethoxybenzyl)-(R)-pyrroleAla)}-E-propenoate (0.233 g, 0.489 mmol, 1 equiv) in 1,4-dioxane (6 mL). After stirring for 1.5 h, the volatiles were evaporated to give the crude amine salt, which was used without purification.

This amine salt was dissolved in DMF (4 mL) and cooled to 0°C. Cbz-L-Leu-LPhe-OH (0.202 g, 0.490 mmol, 1 equivalent), DIEA (0.255 mL,

1.46 mmol, 3 equiv) and HATU (0.186 g, 0.489 mmol, 1 equiv). After stirring for 1.5 h, the reaction mixture was diluted with MTBE (100 mL), washed with 5% KHSO4, saturated NaHCO3 and brine (10 mL), dried over MgSO4 and evaporated. Purification

9*

9·

• 9 • · 9 «999 •

• 9 9 9 9 9 9 9 9 Flash column chromatography of the residue (gradient elution, 60-»75% EtOAc in hexanes) gave ethyl 3-{Cbz-LLeu-L-Phe-L- ( (N-2,4-dimethoxybenzyl)-(R)-pyrrole-Ala)}-Epropenoate (0.208 g, 55%) as a white solid (after evaporation from Et2<0): mp = 174-176°C; IR (cm ^-1 ) 3287, 1713, 1649; 1h NMR (CDCl3) δ 0.84-0.91 (m, 6H), 1.29 (t, 3H,

J = 7.2), 1.42-1.66 (m, 4H), 1.67-1.77 (m, 1H), 1.84-1.95 (m, 1H), 2.20-2.12 (m, 1H), 2.33-2.45 (m, 1H), 3.04-3.23 (m,

4H) , 3 .78 (s, 3H), 3, 79 (s, 3H), 4.15-4.29 (m, 1H), 4.17 (q, 2H, J = 7.2), 4.31 (d, 1H, J = 14.5) , 4.40 (d, 1H, j = 14, 5) , 4.67-4.84 (m, 2H) , 5.05 (d, 1H, J = 12.1) , 5.11 (d, 1H, J = 12.1), 5.35 (d, 1H, J = 8.1) , 5.76 (dd, 1H, J = 15, 6, 1.6), 6.42-6.46 (m, 2H) , 6.74-6.81 (m, 1H), 6, 75 (dd, 1H, J = 15.6, 5.0), 7.06-7.10 (m, 1H), 7.15-7.24 (m, 5H),

7.29-7.36 (m, 5H), 8.79 (d, 1H, J = 5.9); Anal. (C43H54N4O9)C,

H, N.

Preparation of the product ethyl-3-{Cbz-L-Leu-L-Phe-L-((R)-pyrroleAla)}-E-propenoate

Ethyl 3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)(R)-pyrrole-Ala)}-E-propenoate was converted to the product ethyl 3-{Cbz-L-Leu-L-Phe-L-( (R)-pyrrole-Ala)}-E-propenoate in a manner analogous to the conversion of ethyl 3-{Cbz-L-Leu-L-Phe-L-((N-2,4-dimethoxybenzyl)-(S)-pyrrole-Ala)}-E-propenoate to the product ethyl 3-{Cbz-L-Leu-L-Phe-L-((S)-pyrrole-Ala)}-E-propenoate

described in Example 34: IR (cm 1) 3290, 1702, 1642; !h NMR (CDCl3) δ 0.85-0.92 (m, 6H) , 1.30 (t, 3H, J = 7.2) , 1.35- 1.49 (m, 1H) , 1.52-1.71 (m, 3H) , 1.73-1.94 (m, 2H) , 2.15- 2.26 (m, 1H) , 2.32-2.43 (m, 1H) , 3.02-3.18 (m, 2H) , 3.19- 3.29 (m, 2H), 4.15-4.27 (m , 1H) , 4.18 (q, 2H, J = 7.2) ,

• 9

- 149

9999 ·9 · · « 9

9 «

9

9·· 9999 •

· 9 9 9 9 9 9

4.65-4.74 (m, IH) , 4.76-4.85 (m, IH) , 5.07 (d, IH, J =

12.3), 5.12 (d, 1H, J= 12.3), 5.18-5.25 (m, 1H), 5.76-5.84 (m, 2H), 6.64-6.78 (m, 2H), 7.15-7.40 (m, 10H), 7.91-7.98 (m, 1H); Anal. (C34H44N4O7) C, Η, N.

Example 37 - Preparation of Compound (A-28): Ethyl 3-{Cbz-L-LeuL-Phe-L-1-(2-imidazolidosi)Ala}-E-propenoate

Preparation of the intermediate ethyl 3-(Cbz-L-Leu-L-Phe-L-BocaminoAla)-E-propenoate (Carbethoxymethylene)triphenylphosphorane (1.20 g, 3.28 mmol, 1.2 equivalents) was added to a solution of Cbz-L-Leu-LPhe-L-N-Boc-aminoalaninal (prepared as described in Webber et al., J. Med. Chem. 1998, vol. 41, 2786) (1.60 g, 3.28 mmol, 1.2 equivalents).

2.73 mmol, 1 equiv) in THF (55 mL) and the reaction mixture was stirred at ambient temperature overnight. The volatiles were then removed in vacuo and the residue was purified by flash column chromatography eluting (gradient elution, 0-»1.5% CH3OH in CH2Cl2) to afford ethyl 3-{Cbz-L-Leu-L-PheL-N-Boc-aminoAla}-E-propenoate (0.968 g, contaminated with triphenylphosphine oxide). This material was used without further purification.

- 150 • *9 *: · :

• · » — · • ♦ · 9 · * <

1« «* ·♦ » · · · · • * * · · * ,· · · · ’ ·♦ ·· of the propenoate solution solvent

Preparation of the intermediate ethyl 3-{Cbz-L-Leu-L-Phe-L-(2-Boc-2azainoethyl)aminoAla}-E-propenoate

A solution of HCl in 1,4-dioxane (4.0 M, 10 mL) was added to ethyl-3-{Cbz-L-Leu-L-Phe-L-Boc-aminoAla}-E (0.95 g, 1.46 mmol, 1 equiv) in the same (20 mL) at 23°C. The reaction mixture was stirred at this temperature for 1.5 h and then additional HCl in 1,4-dioxane (4.0 M, 10 mL) was added. After stirring overnight, the volatiles were removed in vacuo and the residue was triturated with Et2O (20 mL). The resulting solids were filtered and washed with Et2O (3 x 10 mL) to give the crude amine salt (0.63 g 73%, 1.05 mmol) as a white solid.

This material was dissolved in CH3OH (10 mL) and then N-Boc-2-aminoethanal (prepared as described in Bischofberger et al., J. Org. Chem. 1988, vol. 53, 3457) (0.19 g, 1.16 mmol, 1.1 equiv) and NaBH3CN (0.069 g, 1.05 mmol, 1.0 equiv) were added sequentially. The reaction mixture was stirred at 23°C overnight and then the volatiles were removed under reduced pressure. The residue was dissolved in EtOAc (25 mL) and the organic layer was washed with water (25 mL) and brine (25 mL) and then dried over MgSO4 and concentrated. The residue was purified by flash column chromatography (gradient elution, 0->3% CH3OH in CH2Cl2) to give ethyl 3-{Cbz-L-Leu-L-Phe-L-(2-Boc-2aminoethyl)aminoAla}-E-propenoate (0.32 g, 44%) as a white amorphous solid: Rf = 0.20 (5% CH3OH in CHCl3); IR (cm ^-1 ) 1712, 1649, 1537, 1252, 1175; 1h NMR (DMSO-dg) δ 0.79 (d,

3H, J= 6.6), 0.82 (d, 3H, J= 6.6), 1.21 (t, 3H, J = 7.0), 1.26-1.37 (m, 13H), 1.46-1.54 (m, 1H), 2.56-2.60 (m, 2H),

• « • ·

8.1), 7.97 (d, 1H, J = 7.0), 8.07 (d, 1H, J = 7.0); Anal.

(C37H53N5O8* 0.50H2O) C, Η, N.

Preparation of the product ethyl-3-{Cbz-L-Leu-L-Phe-L-l-(2-izaidazolidone)Ala}-E-propenoate

TFA (0.8 mL) was added to a solution of ethyl 3-{Cbz-L-LeuL-Phe-L-(2-Boc-2-aminoethyl)aminoAla}-E-propenoate (0.29 g, 0.42 mmol, 1 equiv) in CH2Cl2 (8 mL) and the reaction mixture was stirred at 23°C for 2 h. The volatiles were removed in vacuo and the residue was dissolved in EtOAc (25 mL) and washed with saturated NaHCO3 solution (25 mL), water (25 mL) and brine (25 mL). The organic layer was dried over MgSO4 and concentrated to give the crude diamine (0.23 g, 92%, 0.39 mmol) as a tan amorphous solid.

This material was dissolved in THF (4 mL), carbonyldiimidazole (0.06 g, 0.36 mmol, 0.92 equiv) was added, and the reaction mixture was stirred at 23°C for 3.5 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography (gradient elution, 0->2% CH3OH in CH2Cl2) to give ethyl 3-{Cbz-LLeu-L-Phe-L-2-(2-imidazolidone)Ala}-E-propenoate (0.12 g, 54%) as a white amorphous solid: mp = 161-164°C; Rf = 0.21 (5% CH3OH in CHCl3); IR (cm ^-1 ) 1701, 1647, 1535, 1277; 1H NMR (DMSO-d6) δ 0.79 (d, 3H, J= 6.6), 0.82 (d, 3H,

J = 6.6), 1.21 (t, 3H, J = 7.0), 1.27-1.35 (m, 2H), 1.481.52 (m, 1H), 2.79-2.86 (m, 1H), 2.92-3.05 (m, 3H), 3.14-

- 152

9

9

9999 • 9 9 · 9

9 9

9 9

3.19 (m, 2H), 3.25-3.30 (m, 2H), 3.98-4.03 (m, 1H) , 4, 10 (q, 2H, J = 7.0), 4.47-4.49 (m, 1H) , 4.59-4.63 (m, 1H) , 4.97- 5, 02 (m, 2H), 5.72 (d, 1H, J = 15.8), 6.37 (s, 1H) , 6.71 (dd, 1H, J = 15, 8, 5.5), 7.15-7.39 (m, 10H), 7.42 (d, 1H, J = 8, 1) , 8.00 (d, 1H, J = ^: 8.1), 8.18 (d, 1H, J = 8 ,D ; Anal.

(C33H43N5O7) c, H, N.

Example 38 - Synthesis of intermediates ¢1, Q2 and Q3

Q1 Q2 Q3

Preparation of the intermediate l-acetyl-3-(triphenylphosphanylidino)pyrrolidin-2-one

2,4-Dibromobutyride (prepared according to the procedure of Ikuta et al., J. Med. Chem, 1987, vol. 30, 1995) (46.1 g, 188.2 mmol) in THF (1 L) was cooled to 0°C and treated with a solution of lithium bis(trimethylsilylamide) (40.9 g, 244.6 mmol) in THF (200 mL). The solution was kept at 0°C for 2.5 h and then poured into brine (800 mL), extracted with ethyl acetate (2 L) and dried (MgSO4). Evaporation gave 25.5 g of 3-bromo-pyrrolidin-2-one as a brown oil. This material was treated with Ac2O (76 mL) and heated to reflux for 5 h. Evaporation followed by purification (silica gel filtration, eluent EtOAc) gave 28 g of 1-acetyl-3-bromo-pyrrolidin-2-one as a dark oil. THF (272 mL) and triphenylphosphine (42.8 g, 163.3 mmol) were added and the resulting solution was heated to reflux for 8 h. After cooling to 23°C, a precipitate of 1-acetyl-3-(triphenylphosphanyl)* formed.

- 153 ·♦· ·· ·· * · • · « • · · · · * • · · • ♦ pyrrolidin-2-one bromide and was collected by filtration (27.1 g). Concentration of the mother liquor, followed by cooling to 0°C gave an additional 6.6 g. The combined materials in CH2Cl2 (1 L) were washed with IN NaOH (100 mL) and then brine (2 x 100 mL). Evaporation of the organic layer gave 26.9 g (37% overall) of l-acetyl-3-(triphenylphosphanylidine)-pyrrolidin-2-one as a brown oil. A NMR (CDCl3) δ 7.76-7.32 (15H, m), 3.90-3.85 (2H, m), 2.50 (3H, s), 2.56-2.30 (2H, m).

Preparation of the intermediate 2-t-butoxycarbonyl-3-(tert-butyldimethylsilanoxy)-propionic acid methyl ester

Boc-D-serine methyl ester (20.0 g, 91.2 mmol) in DMF (300 mL) was treated with imidazole (18.6 g, 273.7 mmol) followed by TBSC1 (13.0 g, 86.7 mmol). The solution was kept at ambient temperature for 8 h and then washed with saturated aqueous ammonium chloride (300 mL) and extracted with ethyl acetate (800 mL). The organic layer was washed with brine (300 mL) and then dried (MgSO 4 ) to give 30.2 g (100%) of 2-t-butoxycarbonyl-3-(tert-butyldimethylsilanoxy)propionic acid methyl ester as a colorless oil. A NMR (CDCl 3 ) δ 5.32 (IH, d, J = 8.3), 4.33 (IH, dt, J = 8.8, 2.7), 4.02 (IH, dd, J = 10.1, 2.6), 3.80 (IH, dd, J = 9.8, 3.1), 3.72 (3H, s), 1.43 (9H, s). 0.85 (9H, s), 0.0 (6H, s).

Preparation of the intermediate tert-butyl ester of {1-(1-acetyl-2-oxo-pyrrolidin-3-ylidenemethyl)-2-(tert-butyldimethylsilanyloxy)-ethyl}-carbamic acid

2-t-Butoxycarbonyl-3-(tert-butyldimethylsilanoxy)-propionic acid methyl ester (12.7 g, 38.0 mmol) in toluene (190 mL) was cooled to -78°C and worked up

- 154 y ·*>· • * ·· «· • * with a solution of diisobutylaluminumhydride (15.6 ml, 87.4 mmol) in toluene (175 ml). The internal temperature was maintained below -70°C. The solution was maintained at -78°C for a further 90 minutes and then methanol (7.7 ml, 190 mmol) was added. 1-Acetyl-3(triphenylphosphanylidino)-pyrrolidin-2-one (11.1 g, 28.6 mmol) in CH2Cl2 (50 ml) was added at -78°C and the resulting solution was allowed to warm to ambient temperature and maintained for 30 minutes. A solution of sodium potassium tartrate (150 g) in water (600 ml) was added and stirred vigorously for 30 minutes. The mixture was extracted with ethyl acetate (4 x 250 ml), dried (MgSO4) and evaporated. Purification by silica gel chromatography gave 7.04 g (60%) of {1-(1-acetyl-2-oxo-pyrrolidin-3-ylidenemethyl)-2-(tert-butyldimethylsilanyloxy)-ethyl}-carbamic acid tert-butyl ester as a colorless oil. A NMR (CDCl 3 ) δ 6.59 (IH, dt, J = 8.7, 2.9), 4.98 (IH, d, J = 6.8), 4.37-4.25 (IH, m), 3.77 (2H, t, J = 7.3), 3.70-3.58 (2H, m), 2.90-2.80 (IH, m), 2.75-2.60 (1H, m), 5.53 (3H, s), 1.41 (9H, s), 0.87 (9H, s), 0.04 (6H, s).

Preparation of the intermediate product {2-hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acid tert-butyl ester

{1-(1-Acetyl-2-oxopyrrolidin-3-ylidenemethyl)-2-(tert-butyldimethylsilanyloxy)ethyl}-carbamic acid tert-butyl ester (9.18 g, 22.2 mmol) in THF (150 mL) was treated with TBAF (22.2 mL of a 1M solution in THF, 22.2 mmol) at 0°C and kept for 1 hour. Saturated aqueous ammonium chloride was added and stirred for 10 minutes and then the solution was extracted with ethyl acetate (2 x 200 mL). The organic layer was dried (MgSO4) and then evaporated. Purification by silica gel

- 153 to <· · • to •

it · r

to to ·· *· _λ • ·· · • · · * • ·· * • »· ♦ ·· ·* chromatography gave 4.82 g (73%) of a colorless oil. This material was taken up in methanol (160 mL), treated with potassium carbonate (223 mg, 1.62 mmol) and refluxed for 1 h. The mixture was then treated with solid citric acid (311 mg, 1.62 mmol) and ethyl acetate (800 mL) was added. The solution was filtered through silica gel. Evaporation gave 4.30 g (73% overall) of {2-hydroxy-1-(2-oxo-pyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acid tert-butyl ester as a colorless oil. 3-H NMR (CDCl 3 ) δ 7.03 (1H, br s) , 6.35 (1H, dt, J = 8.6, 2.6), 5.37 (1H, d, J = 6.5), 4.40-4.20 (1H, m) , 3.66 (br s, 3H) ,

3.4 (2H, t, J = 6.7), 3.10-2.80 (1H, m), 2.80-2.70 (1H, m),

1.41 (9H, s).

Preparation of the intermediate tert-butyl ester of {2-hydroxy-1-(2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid (mixture of diastereomers) (Ql)

{2-Hydroxy-1-(2-oxopyrrolidin-3-ylidenemethyl)ethyl}-carbamic acid tert-butyl ester (4.30 g, 16.8 mmol) in ethyl acetate (168 mL) was treated with 5% palladium on carbon (1.78 g) and hydrogenated at ambient pressure for 1 hour. The mixture was filtered and then evaporated to give

3.92 g (91%) of tert-butyl ester of {2-hydroxy-1-(2oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid in the form of a colorless oil (1.5:1 mixture of diastereomers): 3-H NMR (CDCl3) δ 6.99 (1H, s), 5.49 (1H, d, J = 8.4), 3.70-3.50 (3H, m), 3.38-3.20 (2H, m), 2.60-2.20 (2H, m), 2.00-1.70 (2H, m),

1.65-1.45 (1H, m), 1.37 (9H, s).

156

*'3 • ·« ·· • · • · « • and * • • • · • • • • · • » * · • • • « • · • • • ·· «·

Preparation of the intermediate {2-hydroxy1-(R-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid tert-butyl ester (Q2)

{2-Hydroxy-1-(2-oxopyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acid tert-butyl ester (98 mg, 0.39 mmol) in methanol (5 mL) was treated with (R)-BINAP-RuCl (19 mg, 0.02 mmol) and then placed under a hydrogen atmosphere (1200 psi) at 50°C for 48 hours. The solution was evaporated and then filtered through silica gel (10% MeOH-EtOAc eluent). Evaporation gave 75 mg (75%) of {2-hydroxy-1-(R-2-oxy-pyrrolidin-3-ylmethyl)ethyl}-carbamic acid tert-butyl ester as a colorless oil (9:1 mixture of diastereomers): 1 H NMR (CDCl3) δ 6.32 (1H, br s), 5.40 (1H, d, J = 1.5), 3.82 (1H, br s), 3.71-3.63 (2H, m), 3.34-3.31 (2H, m), 2.45-2.30 (2H, m), 2.09-1.86 (2H, m), 1.70-1.63 (1H, m), 1.42 (9H, s).

Preparation of the intermediate {2-hydroxy1-(S-2-oxy-pyrrolidin-3-ylmethyl)-ethyl}-carbamic acid tert-butyl ester (Q3)

{2-Hydroxy-1-(2-oxopyrrolidin-3-ylidenemethyl)-ethyl}-carbamic acid tert-butyl ester (0.10 g, 0.39 mmol) in methanol (5 mL) was treated with (S)-BINAP-RuCl (19 mg, 0.02 mmol) and then placed under a hydrogen atmosphere (1200 psi) at 50°C for 48 hours. The solution was evaporated and then filtered through silica gel (10% MeOH-EtOAc eluent). Evaporation gave 74 mg (74%) of {2-hydroxy-1-(S-2-oxy-pyrrolidin-3-ylmethyl)ethyl}-carbamic acid tert-butyl ester as a colorless oil (9:1 mixture of diastereomers): 1h NMR (CDCl3) δ 6.66 (1H, br s), 5.51 (1H, d, J = 8.2), 3.72-3.57 (3H, m), 3.34-3.31 (2H, m), 2.52-2.33

157 (1H, m),

1.62-1.50 (1H, ·· ·· ·· ®· • · * · · · « · · · · • · · · · · and · · · · ··· ···· ···· ·* (2H, m) , 2.20-1.86 (1H, m),

m), 1.40 (9H, s).

1.86-1.70

The results of biochemical and biological tests performed using various compounds of the present invention are described below.

Biochemical and biological evaluation

Inhibition of rhinovirus 3C protease

Stock solutions (50 mM, in DMSO) of various compounds were prepared; dilutions were made in the same solvent. Recombinant rhinovirus 3C proteases (see Birch et al., Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli, Protein Expr. Pur. 1995, 6(5), 609-618) from serotypes 14, 16, and 2 were prepared by the following standard chromatographic procedures: (1) ion exchange using Q Sepharose Fast Flow from Pharmacia; (2) affinity chromatography using Affi-Gel Blue from Biorad; and (3) size separation using Sephadex G-100 from Pharmacia. Each test sample contained 2% DMSO, 50 mM Tris pH 7.6, 1 mM EDTA, the test compound at the indicated concentration, approximately ΙμΜ substrate, and 50-100 nM protease. The k _o bs/I values were obtained from reactions initiated by addition of enzyme rather than substrate. RVP activity was measured in a fluorescence resonance energy transfer assay. The substrate was (N-terminal) DABCYL-(Gly-Arg-Ala-Val-Phe-Gln-Gly-Pro-Val-Gly)-EDANS . In the uncleaved peptide, EDANS fluorescence was quenched by the proximal DABCYL group. If the peptide was cleaved, quenching was limited and activity was measured as an increase

158 fluorescence signal. The data were analyzed using standard nonlinear programs (Enzfit) and are shown in Tables 1 and 2 below. Table 1 shows all data for the 3C protease of rhinovirus HRV serotype-14 (generated from an infectious cDNA clone constructed by Dr. Robert Rueckert, Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin). Table 2 shows the protease inhibitory activity of the compounds against proteases of several rhinovirus serotypes other than RVP serotype-14. The data in the column labeled k _obs /[I] were measured from the progress curves in enzyme baseline experiments.

Antirhinoviral assay in Hl-HeLa cell culture

In this cell protection assay, the ability of compounds to protect cells against HRV infection was measured using the XTT dye reduction method described by Weislow et al., J. Natl. Cancer Inst. 1989, vol. 81, 577-586.

Hl-HeLa cells were infected with HRV-14 at a multiplicity of infection (m.o.i.) of 0.13 (number of virus particles/number of cells) or the infection was only simulated with medium. Infected or mock-infected cells were resuspended at 8 x 10^ cells per ml and incubated with appropriate concentrations of the compounds to be tested. Two days later, XTT/PMS was added to the assay plates and the amount of formazan formed was determined spectrophotometrically at wavelengths of 450/650 nm. The EC50 value was calculated as the concentration of compound that increased the percentage of formazan formation in compound-treated virus-infected cells to 50% of the formazan formation in compound-free mock-infected cells. The 50% cytotoxic dose (CC50) was calculated as the concentration of compound that reduced the percentage of formazan formed in compound-treated virus-infected cells.

9 formazan formation in compound-treated and mock-infected cells to 50% of the formazan formation in compound-free and mock-infected cells. The therapeutic index (TI) was calculated by dividing the CC50 by the EC50.

All human rhinovirus (HRV) strains used in this assay were purchased from the American Type Culture Collection (ATCC) with the exception of HRV serotype-14 (generated from an infectious cDNA clone constructed by Dr. Robert Rueckert, Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin). HRV stocks were amplified and viral assays were performed using HlHeLa cells (ATCC). Cells were grown in minimal essential medium with 10% fetal bovine serum, supplied by Life Technologies (Gaithersburg, MD).

The compounds were tested against the control compounds WIN 51711, WIN 52084 and WIN 54954 (obtained from Sterling-Winthrop Pharmaceuticals), Pirodavir (obtained from Janssen Pharmaceuticals) and Pleconaril (prepared as described in Diana et al., J. Med. Chem 1995, vol. 38, 1355). The antiviral data obtained for the test compounds are shown in Tables 1 and 3, where all data are for HRV serotype-14 unless otherwise stated in parentheses. The designation ND indicates that the value was not determined for the given compound.

• ·

• · • ·

- 160

Table 1. Activity against HRV Serotype-14 Compound Protease inhibition kobs/ ti] Cell protection EC50 Example 31 (Comparative Compound #2) 25,000 0.610 Example 35 (A-26) 239,000 0.030 Example 33 (A-24) 257,000 0.100 Example 36 (A-27) 18,000 1,600 Example 32 (Comparative Compound #3) 62,500 0.380 Example 34 (A-25) 500,000 0.030 Example 5 (A-4) 270,000 0.100 Example 8 (A-7) 980,000 0.004 Example 7 (A-6) 248,000 0.420 Example 9 (A-8) 900,000 ND Example 6 (A-5) 1,500.00 0.005 Example 12 (C-l) 68,400 0.100 Example 18 (C-2) 270,000 0.002 Example 10 (B-l) 240,000 0.100 Example 20 (B-4) 500,000 <0.030 Example 17 (B-2) 1,090.00 0.005 Example 1 (Comparative Compound #1) 573 >320,000 Example 2 (A-l) 260,000 0.250 Example 3 (A-2) 46,900 1,600 Example 4 (A-3) 310,000 0.050 Example 11 (A-10) 108,000 0.14 Example 13 (A-ll) 108,000 0.03

- 161

Table 1. Activity against HRV Serotype-14 Compound Inhibition of protease kobs/ [I] Cell protection EC50 Example 14 (A-9) 66,000 1.80 Example 15 (A-12) 59,300 0.40 Example 16 (A-13) 95,800 0.20 Example 19 (B-3) 465,000 0.18 Example 21 (A-14) 54,500 0.48 Example 22 (A-15) 237,100 0.22 Example 23 (A-16) 172,800 0.45 Example 24 (A-17) 167,000 0.06 Example 25 (A-18) 292,000 1.50 Example 26 (A-19) 27,750 25.10 Example 27 (A-20) 1,020 12.60 Example 28 (A-21) 17,800 2.50 Example 29 (A-22) 2,400 ND Example 30 (A-23) 26,000 ND

- 162

Table 2. Activity against other HRV serotypes Compound Rhinovirus serotype kobs/I (M-lsec-1) Comparative (16) 6,500 in (89) 3,400 in (2) 2,000 Comparative (2) 8,000 \\ (16) 16,900 (A-24) (2) 31,000

Table 3. Activity against rhinoviruses Number HRV EC50 (PM) CC50 (pM) YOU Comparative >320,000 >320 - (A-1) 0.250 >100 >400 In (2) 0.410 ND >243 (IA) 1,000 ND >100 (89) 0.220 ND >450 (A-2) 1,600 >100 >63 (A-3) 0.050 >10 >200 (A-4) 0.100 >100 >1000 (A-5) 0.005 >10 >2000 n (2) 0.010 ND >1000 in (16) 0.020 ND >500 in (39) 0.020 ND >500 \λ (89) 0.020 ND >500 (10) 0.050 ND >200

• 9

- 163

Table 3. Activity against rhinoviruses Number HRV EC50 (PM) CC50 (pM) YOU In (IA) 0.030 ND >333 (3) 0.050 ND >200 (9) 0.040 ND >250 In (12) 0.060 ND >166 (13) 0.020 ND >500 \\ (17) 0.020 ND >500 In (25) 0.180 ND >55 \\ (30) 0.060 ND >166 \\ (38) 0.130 ND >76 \\ (87) 0.210 ND >47 (A-6) 0.420 >100 >237 (A-7) 0.004 >10 >2500 \\ (2) 0.020 ND >500 w (16) 0.040 ND >250 w (39) 0.020 ND >500 at (89) 0.020 ND >500 5¾ (10) 0.060 ND >166 (IA) 0.030 ND >333 (3) 0.020 ND >500 At (9) 0.040 ND >250 At (12) 0.070 ND >143 In (13) 0.030 ND >333 >> (17) 0.020 ND >500 In (25) 0.200 ND >50

• · • · • ·

164 ··

1 Table 3. Activity against rhinoviruses Number HRV EC50 (µM) CC50 (pM) YOU (30) 0.090 ND >111 w (38) 0.170 ND >58 w (87) 0.590 ND >16 (A-8) ND ND ND (B-l) 0.100 >100 >1000 at (IA) 0.300 ND >333 at (10) 0.400 ND >250 (A-10) 0.140 >100 >714 (C-l) 0.100 >10 >100 (A-ll) 0.030 50 1667 (A-9) 1,800 >100 >55 (A-12) 0.400 >100 >250 (A-13) 0.200 >10 >50 (B-2) 0.005 >100 >20000 (2) 0.020 ND >5000 VI (16) 0.010 ND >10000 (39) 0.050 ND >2000 W (89) 0.009 ND >11111 ΛΛ (10) 0.020 ND >5000 In (IA) 0.020 ND >5000 \X (3) 0.020 ND >5000 At (9) 0.006 ND >16666 In (12) 0.050 ND >2000 (13) 0.010 ND >10000

165 fe fefefe· • fefe · • fefe ··

Table 3. Activity against rhinoviruses Number HRV EC50 (μM) CC50 (μM) YOU In (17) 0.010 ND >10000 in (25) 0.030 ND >3333 at (30) 0.040 ND >2500 w (38) 0.070 ND >1428 at (87) 0.060 ND >1666 (C-2) 0.002 >10 >5000 W (2) 0.004 ND >2500 In (16) 0.010 ND >1000 W (39) 0.010 ND >1000 In (89) 0.004 ND >2500 At (10) 0.020 ND >500 W (IA) 0.010 ND >1000 M (3) 0.020 ND >500 W (9) 0.010 ND >1000 In (12) 0.040 ND >250 In (13) 0.007 ND >1428 W (17) 0.007 ND >1428 W (25) 0.070 ND >142 \\ (30) 0.030 ND >333 w (38) 0.050 ND >200 \x (87) 0.020 ND >500 (B-3) 0.180 >100 >543 (B-4) <0.030 >100 >3333 (A-14) 0.480 >100 >208

- 166 • · • *«·♦

Table 3. Activity against rhinoviruses Number _EC50 (µM) CC ₅ 0 (pM) YOU (A-15) 0.220 >100 >454 In (IA) 7,100 ND >14 in (10) 2,700 ND >37 (A-16) 0.45 >100 >222 (IA) 4.80 ND >21 (10) 4.50 ND >22 (A-17) 0.06 >100 >1786 (IA) 1.80 ND >56 W (10) 3.30 ND >30 (A-18) 1.50 >100 >67 (A-19) 25.10 >100 >4 (A-20) 12.60 >100 >8 (A-21) 2.50 >100 >40 (A-22) ND ND ND (A-2 3) ND ND ND Comparative 0.61 >320 >524 (16) 2.30 >320 >139 (89) 6.30 >320 >50 In (10) 0.60 >320 >533 Comparative 0.38 >320 >842 (A-24) 0.10 >100 >1000 (A-25) 0.03 >100 >3333 (A-2 6) 0.03 >100 >3333 (A-2 7) 1.60 >100 >62

167

• · » » ι» · Φ · » · « · * * · · • · ♦ · ♦ · ··

I Table 3. Activity against rhinoviruses Number HRV EC50 (μM) CC50 (μM) YOU WIN 51711 0.78 >60 >77 WIN 52084 0.07 >10 >143 WIN 54954 2.13 >63 >30 Pirodavir 0.03 >10 >300 Pleconaril 0.01 >10 >1000

Coxsackievirus test in cell culture

Coxsackievirus types A-21 (CAV-21) and B3 (CVB-3) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Virus stocks were amplified and antiviral assays were performed on Hl-HeLa cells (ATCC). Cells were grown in minimal essential medium with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD).

The ability of the compounds to protect cells against CAV-21 or CVB-3 infection was measured by the XTT dye reduction method. This method is described by Weislow et al., J. Natl. Cancer Inst. 1989, vol. 81, 577-586. Hl-HeLa cells were infected with CAV-21 or CVB-3 at a multiplicity of infection (m.o.i.) of 0.025 and 0.075, respectively, or were mock infected with medium. HlHeLa cells were plated at 4 x 10^ cells per well in 96-well plates and incubated with appropriate concentrations of the test compound. One day (CVB-3) or two days (CAV21) later, XTT/PMS was added to the test plates and the amount of formazan formed was measured spectrophotometrically at wavelengths of 450/650 nm. The EC50 value was calculated as the concentration of compound that increased formazan production in compound-treated, virus-infected cells to 50% of that produced in untreated cells.

0«

0

168 » 0 0 « » 0 0 1 » 0 0 «

00 compound and uninfected. The 50% cytotoxic dose (CC50) was calculated as the concentration of compound that reduced formazan formation in uninfected cells to 50% of the value of compound and uninfected. Therapeutic index calculated by dividing CC50 by EC50.

The compounds were tested against the control compounds WIN 54954 (obtained from Sterling-Winthrop Pharmaceuticals), Pirodavir (obtained from Janssen Pharmaceuticals), and Pleconaril (prepared according to the procedure described by Diana et al., J. Med. Chem. 1995, 38, 1355). The antiviral data obtained for the test compounds against CAV21 and CVB-3 are shown in Table 4.

compound-treated and generated in cells without (TI) was

Table 4. Activity against coxsackieviruses Compound Tribe EC50 (µM) CC50 (pM) (A-5) CAV-21 0.23 >10 >43 \\ CVB-3 1.00 ND >10 (B-2) CAV-21 0.16 >100 >625 CVB-3 0.18 ND >555 WIN 54954 CAV-21 >100.00 >100 ND In CVB-3 >100.00 ND ND Pirodavir CAV-21 >100.00 >100 ND In CVB-3 >100.00 ND ND Pleconaril CAV-21 0.09 >10 >107 VI CVB-3 >10.00 ND ND

Cell culture assays against echoviruses and enteroviruses • ·

- 169

81, 577-586). MRC-5 with multiplicity was infection only

Echovirus type 11 (EV 11) and enterovirus type 70 (EV 70) were purchased from ATCC (Rockville, MD). Viral stocks were amplified and antiviral assays were performed on MRC-5 cells (ATCC). Cells were grown in minimal essential medium with 10% fetal bovine serum (Life Technologies, Gaithersburg, MD).

The ability of the compounds to protect cells against EV 11 or EV 70 infection was measured by the XTT dye reduction method (Weislow et al., J. Natl. Cancer Inst. 1989, vol.

cells were infected with EV 11 or EV infection 0.003 or 0.004, respectively, or mock medium. Infected or uninfected cells were plated at 1 x 10^ cells per well on plates and incubated with appropriate concentrations of compound. Four days later, XTT/PMS was added to the assay plates and the amount of formazan produced was measured spectrophotometrically at wavelengths of 450/650 nm. The EC50 value was calculated as the concentration of compound that increased formazan production in compound-treated, virus-infected cells to 50% of that produced in compound-free, uninfected cells. The 50% cytotoxic dose (CC50) was calculated as the concentration of compound that reduced formazan production in uninfected cells to 50% of that in compound-treated, uninfected cells. The therapeutic index was calculated by dividing the CC50 by the EC50 value.

The compounds were tested against the control compounds Pirodavir (obtained from Janssen Pharmaceuticals) and Pleconaril (prepared according to the procedure described by Diana et al., J. Med. Chem. 1995, vol. 38, 1355). Antiviral data obtained from compound-treated and non-treated cells (TI) were

- 170 • ·

for the tested compounds against strains EV 11 and EV 70 are listed below in Table 5.

Table 5. Activity against echoviruses and enteroviruses Tribe EC50 (µM) CC50 (pM) (A-5) EV-11 0.080 >10 >125 In EV-70 0.040 ND >250 (B-2) EV-11 0.010 >100 >10000 w EV-70 0.003 ND >33333 Pirodavir EV-11 3,700 >10 >3 At EV-70 0.060 ND >167 Pleconaril EV-11 0.160 >10 >62 In EV-70 ND ND ND

While the present invention has been described with reference to preferred embodiments and specific examples, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is not limited to the above detailed description, but is defined by the appended claims and their equivalents.

Represents:

Dr. O. Švorčík

171 fV yoo - 3W£

Claims (77)

PATENTED JUDr. Otakar Švorčík, attorney at law Halkova 2, 120 00 Prague 2 • · • · • ···· » · · · · · • · · · · · • · · · · I · · · · · ♦ · · · ·· ·· ·· 1. Compound of general formula I: OR ₇ About (I), in which: Y represents -N(R _y )-, -C(R _y )(R _y )- or -0-, where each R _y independently represents H or lower alkyl; R 1 represents H, F, an alkyl group, OH, SH or an O-alkyl group; R ₂ and R ₃ independently represent H; about> NH, \ ^A 2 ( ^R 4l)( ^R 4l) (A ₃ )n or ' ( ^R 4i)( ^R 4i) _r where n is an integer in the range from 0 to 5, A1 represents CH or N, _A2 and each _A3 is independently selected from the group consisting of C( _R4i )( _R41 ), N( _R41 ), S, S(O), S(O) ₂ and 0 and A ₄ represents NH or NR _4i , where each R _4I independently represents H or lower alkyl, provided that no more than 2 heteroatoms occur consecutively in the ring formed by A _x , A ₂ , (A ₃ ) n A ₄ and C=O; and provided that at least one of R ₂ and R ₃ (A ₃ ) n , ^A 1^ / A2 NH ₂ or ( ^R 4i)( ^R 4i) _r R 5 and R ₆ independently represent H, F, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group; introduce 7' • · - 172 R ₇ and R 8 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, -OR 17 , SR 17 , -NR 17 R 18 , -NR ₁₉ NR ₁₇ R 18 or -NR 19 OR 18 , where R 18 , R 19 and R 19 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group or an acyl group; Rg represents a five-membered heterocycle having from one to three heteroatoms selected from the group consisting of O, N and S; and Z and Z independently represent H, F, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, -C(O)R ₂₁ , -CO ₂ R ₂ i , -CN, -C(O)NR ₂ i R ₂₂ , -C(O)NR ₂₁ OR ₂₂ , -C(S)R ₂₁ , C(S)NR ₂ iR ₂₂ , -N0 ₂ , -SOR ₂₁ , -SO2R21, ~SO ₂ NR ₂ iR ₂₂ , -S0 (NR ₂ i) (OR ₂₂ ) , -SONR21, -SO ₃ R ₂ x, -PO(OR ₂ i) ₂ , -PO(R ₂₁ ) (R ₂₂ ) , PO (NR21R22) (OR23) , PO(NR ₂ iR ₂₂ ) (NR ₂ 3R ₂₄ ) , -C (0) NR ₂ xNR ₂₂ R ₂ 3 or -C (S) NR ₂ xNR ₂₂ R ₂ 3, where R 21, R ₂₂ of R 23 and R ₂₄ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or any two of the substituents R ₂ i, R ₂₂ r R 23 and R ₂₄ , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Ζχ do not both simultaneously represent H; or Zχ and Rx, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Ζχ, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; • · - 173· metabolite, or a precursor thereof, a pharmaceutically active pharmaceutically acceptable salt or solvate thereof. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 1, wherein R ₂ and R ₃ independently represent H; NH, (C(R4i)( _R4l ))n ( _R4l )( _R4l ) ( ^R4l )( ^R4l ) ¹² ; or ( _R4i )( _R4i ) ' , _{n is} a number ranging from 0 to 5, each _R41 independently represents H or lower alkyl and the stereochemistry at the carbon atom marked with an asterisk (*) may be R or S; provided that at least O X, R ₃ represents NH, or one R ₂ 3. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 1, wherein Y represents -N(R _y )-, wherein R _y represents H or lower alkyl. 4. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein Z and Ζχ are independently selected from the group consisting of H, F, lower alkyl, -CO ₂ R _2X and -C (0) NR _2X R ₂₂ , where • · - 174 R21 and R22 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or _R2X and _R22 , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and _Zx do not both represent H; or Z 1 and R _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Z _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 3, wherein R _x represents H, F or methyl. 6. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 3, wherein at least one of R ₂ or R ₃ represents η Η ~ H ^0<! r' ^N \ ° ^<5 ί ^χΝ 'Ί ⁰ yd-j, qdd _or λ>Λ _ΝΗ2 7. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 7, wherein one of R ₅ and R ₆ represents H and the other represents alkyl or aryl. 8. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein one of R5 and _R6 is H and the other is alkyl or aryl. • · 9. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein one of R ₅ and R ₆ is H and the other is unsubstituted or substituted phenylmethyl. 10. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein R ₇ and R ₈ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group. 11. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein one of R ₇ and R 8 represents H and the other represents alkyl or aryl. 12. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein one of R ₇ and R 8 is H and the other is 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl or naphthylmethyl. 13. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 3, wherein R 9 represents a five-membered heterocycle containing at least one nitrogen heteroatom and one oxygen heteroatom. 14. A compound, precursor, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to • · • ♦ • · 176 of claim 3, wherein Rg is selected from the group consisting of substituted and unsubstituted 1,2-oxazolyl, 1,3-oxazolyl and 1,2,4-oxazolyl. 15. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 3, wherein R ₉ represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from methyl and halogen atoms. 16. A compound according to claim 3 of general formula I-A'': OR ₇ Ry OR ₂ Z AVyVA HO Rg H Rj (I—A'' ) in which R _x , R ₂ , R 6 , R ₇ and R^ R _y , Z and Z ₃ have the same meaning as in claim 3, or a prodrug, a pharmaceutically active metabolite, a pharmaceutically acceptable salt or solvate thereof. 17. A pharmaceutically acceptable compound, precursor, metabolite of claim 16, wherein R ₂ represents a pharmaceutically active salt or solvate according to NH, 18. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 16, wherein: « · • · • · ··· ···· - 177 4444 R _y represents H or methyl; R 1 represents H, F or methyl; Z and Ζχ are independently selected from the group consisting of H, F, -CO ₂ R ₂₁ , -CN and ~C (0) NR ₂₁ R ₂₂ , where R ₂₁ and R ₂₂ independently represent a cycloalkyl group, H, an alkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or R ₂₁ and R ₂₂ , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Ζχ do not both simultaneously represent H; or Zχ and Rx, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Ζχ, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; .NH, o , unsubstituted R ₂ represents R ₆ represents phenylmethyl; R ₇ represents alkyl or aryl; and R ₉ represents 3-isoxazolyl unsubstituted or substituted by substituents selected from halogen atoms. or or oh. r substituted or one 5-isoxazolyl or two including methyl and 19. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 18, wherein R ₇ is selected from the group consisting of 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl and naphthylmethyl. • · 0 · • · 178 • · 0 0 0 0 · 0 0 · ·' · · · · · • · · · » • ·«·» ·’ ·· 20. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 16, wherein R _y , R _x and Z are all H and: R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R _{9 n} -N represents ^H 3° R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ represents No. R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents h ₃ c , _{R 2} represents C(CH ₃ ) ₃ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ represents nN h ₃ c' R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents C , R ₇ represents C(CH ₃ ) ₃ , Z 1 represents CO ₂ CH ₂ CH ₃ and R ₉ represents h ₃ c' _n -N A>*’ R ₂ represents y·^^ , r ₆ represents f , R ₇ represents -.-N CH(CH ₃ ) ₂ , Z 1 represents CO ₂ CH ₂ CH ₃ and R ₉ represents ^Η 3θ' An VH represents ^H 3° n- ^N AND?*' R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents < , R ₇ represents CH(CH ₃ ) ₂ , Z _x represents CO ₂ CH ₂ CH ₃ and R ₉ « * # ♦ • · • · » t · « • » · · · · · · · • ··· ♦♦ ·· 179 R7 π Η °^Ν νκ· R2 represents ΗRe represents f represents C(CH ₃ ) ₃ , Ζχ represents CO ₂ CH ₂ CH ₃ and Rg represents -i-N. H ₃ C aMH °n R ₂ represents 'W, r ₆ represents , _R? represents CH(CH ₃ ) ₂ , Ζχ represents CO ₂ CH ₂ CH ₃ and R ₉ represents ^H sC vy R ₂ represents _f r ₅ represents r ₇ represents C(CH ₃ ) ₃ , Ζχ represents CO ₂ CH ₂ CH ₃ and Rg represents L-N _H3C A/^ R ₂ represents -ř, R6 represents CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Ζχ represents CO ₂ CH ₂ CH ₃ and Rg CA ^Λ represents ; n h R ₂ represents A, R ₆ represents CH ₂ Ph, R ₇ represents CH ₂ CH(CH ₃ ) ₂ , Zχ represents CO ₂ CH ₂ CH ₃ and Rg represents o' NH represents CH ₂ CH(CH ₃ ) ₂ , Ζχ represents CO ₂ CH ₂ CH ₃ and R ₉ 'ο'Χ represents ; or R ₂ represents AA R6 represents _CH2Ph , _R7 99 ·· ·· 9 9 9 9 9 9 9 9 9 9 9 • 9 9 9 9 9 9 »999 9999 ·* ** 9 9 9 9 - 180 -, R ₂ represents represents CH(CH ₃ ) ₂ , Qp-X represents R6 _{represents CH2Ph} , _{R7 represents CO2CH2CH3 and R9} 21. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 16, wherein R _y represents CH ₃ , R _x and Z represent all H and: VR ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents f represents η , z _x represents CO ₂ CH ₂ CH ₃ and R ₉ represents H,C R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents CH ₂ Ph, R ₇ ... x represents CH ₂ CH(CH ₃ ) ₂ and R ₉ represents ^H and ^c ; R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents f ar· ¹ represents ar ₉ represents ^H 3° r ₇ R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents f^^ r ₇ -iN ; or represents CH ₂ CH(CH ₃ ) ₂ and R ₉ represents ^H 3 ^C ' ·♦ ·· ·· « « · · * * • · · · · • « · · · 181 — ··· ··· R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents f represents e ->-N. and R ₉ represents ^H with ^c ' AX 22. _A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 16 selected from the group consisting of ^the _following compounds: X . ^s - co ₂ ch ₂ ch ₃ CO ₂ CH ₂ CH ₃ y °^nh _{2 CO2CH2CH3 ​ ​} ΟγΝΗ; Ah OJ F °<sxNH ₂ co ₂ ch ₂ ch ₃ • 4 4 4 4 to · · * 182 • that 4 « * «··· co ₂ ch ₂ ch· ; and 'what ₂ ch ₂ ch _; 23. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 1, wherein Y represents -CH ₂ -. 24. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 23, wherein Z and Z _x are independently selected from the group consisting of H, F, lower alkyl, -CO ₂ R _2X and C(O)NR _2X R ₂ 2/ where R 21 and R 22 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or R _2X and R ₂ 2, together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Z _x do not both represent H; or Z _x and R _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Z _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group. • · 183 • · · 99 99 25. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 23, wherein R 1 represents H, F or methyl. 26. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 23, wherein at least one of R ₂ and R ₃ represents or O 27. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 26, wherein one of R ₅ and R ₆ is H and the other is alkyl or aryl. 28. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 23, wherein one of R ₅ and R ₆ is H and the other is alkyl or aryl. 29. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 23, wherein one of R ₅ and R 6 is H and the other is unsubstituted or substituted phenylmethyl. 30. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 23, wherein R ₇ and R ₈ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group. • · · • · ♦ 184 • · • · · · * ·· 31. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 23, wherein one of R ₇ and R ₈ is H and the other is alkyl or aryl. 32. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 23, wherein one of R ₇ and R ₈ is H and the other is 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl or naphthylmethyl. 33. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 23, wherein Rg represents a five-membered heterocycle containing at least one nitrogen heteroatom and one oxygen heteroatom. 34. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 33, wherein Rg represents unsubstituted or substituted 1,2-oxazolyl, 1,3-oxazolyl or 1,2,4-oxazolyl. 35. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 34, wherein Rg represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from methyl and halogen atoms. 36. A compound according to claim 23 of general formula I-B'': 185 O Ry OR ₂ Z n N z ₁ HO Rg H R-| (I-B'') in which Rx, _R2 , Rg, R7, R9, Z and Zχ have the same meaning as in claim 23, or a prodrug, a pharmaceutically active metabolite, a pharmaceutically acceptable salt or a solvate thereof. 37. metabolite A compound, prodrug, pharmaceutically acceptable pharmaceutically active salt or solvate according to claim 36, wherein R ₂ represents 38. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 36, wherein: R x represents H, F or methyl; Z and Ζχ are independently selected from the group consisting of H, F, -CO2R21, -CN and ~C(0) _NR21 , _R22 , where _R21 and _R22 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or _R21 and _R22 , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Ζχ do not both represent H; 186 or Z1 and R1, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Ζχ, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; O R ₂ is selected from the set including R ₆ represents unsubstituted or substituted phenylmethyl; R ₇ represents alkyl or aryl; and Rg represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from the group consisting of methyl and halogen atoms. 39. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 38, wherein R ₇ is selected from the group consisting of 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl and naphthylmethyl. 40. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 36, wherein Rx represents H and: R ₂ represents CH ₂ CH ₂ C (O) NH ₂ , R ₆ represents CH(CH ₃ ) ₂ , Z represents H, Zχ represents CO ₂ CH ₂ CH ₃ and Rg represents ^Η 3θ • ·· ·« · · • · • · ♦ ·»· ···· • · • 9 187 ν· « ·· • · « · φ · • * • · • ·· ···· π Η °^Ν Him R ₂ represents , r ₆ represents f ^z , r ₇ represents CH(CH ₃ ) ₂ , Z represents Η, Ζχ represents ι-N. CO ₂ CH ₂ CH ₃ and R ₉ represents ^Η 3θ η H °VN xX> R ₂ represents 'E' Ré represents , r ₇ represents CH(CH ₃ ) ₂ , Z and Ζχ together form where the carbonyl group is in the cis position with respect to the hydrogen of R _x and R ₉ in N. X>L represents ^H 3 ^C ; or „ H CU ,N R ₂ represents _f r ₆ represents , r ₇ represents CH(CH ₃ ) ₂ , Z represents Η, Z _x represents Λ Π CO ₂ CH ₂ CH ₃ and R ₉ represents ^Η 3θ 41. A compound according to claim 1 of the general formula: or a prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate thereof. 42. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 1, wherein Y represents -O-. 188 • • fe • fe • · • • • • • • • ·· ·· fefe • fe • · fe fefe • • • • fefe • • • • · · · • • fe fe fefe • fe fe • fe 43. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein Z and Ζχ are independently selected from the group consisting of H, F, lower alkyl, -CO ₂ R2x and C(0)NR ₂₁ , R ₂₂ , where R _2i and R ₂₂ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group, or R _2x and R ₂₂ , together with the atom or atoms to which they are attached, form a heterocycloalkyl group, provided that Z and Ζχ do not both represent H; or Zχ and Rx, together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; or Z and Ζχ, together with the atoms to which they are bonded, form a cycloalkyl or heterocycloalkyl group. 44. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 42, wherein Rx represents H, F or methyl. 45. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein at least one of R ₂ and R ₃ represents Η H ^N Ί ⁰ gJA _nsbo λΑΑ. 46. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 45, wherein one of R ₅ and R 6 is H and the other is alkyl or aryl. • · 189 47. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein one of R ₅ and R ₆ is H and the other is alkyl or aryl. 48. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein one of R ₅ and R ₆ is H and the other is unsubstituted or substituted phenylmethyl. 49. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein R ₇ and R ₈ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group. 50. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein one of R ₇ and R ₈ is H and the other is alkyl or aryl. 51. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein one of R ₇ and R ₈ is H and the other is 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl or naphthylmethyl. 52. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein R _g represents a five-membered heterocycle • · • · 190 containing at least one nitrogen heteroatom and one oxygen heteroatom. 53. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 42, wherein Rg is selected from the group consisting of substituted and unsubstituted 1,2-oxazolyl, 1,3-oxazolyl and 1,2,4-oxazolyl. 54. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 42, wherein Rg represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from methyl and halogen atoms. 55. A compound according to claim 42, of general formula I-C'': Rg N H OR <3 H Rj in which R ₁ , R ₂ , R 6, R 7, R ₉ , Z and Z _x have the same meaning as in claim 42, or a prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate thereof. 56. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of 191 of claim 55, wherein R2 represents or 57. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 55, wherein: R 1 represents H, F or methyl; Z and Z are independently selected from the group consisting of H, F, -CO2R21, -CN and “C(0) _NR2XR22 , where _R2X and _R22 independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or _{R2X and R22} together with the atom or atoms to which they are attached form a heterocycloalkyl group, provided that Z and _Zx do not both represent H; or Z _x and R _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group, or Z and Z _x , together with the atoms to which they are attached, form a cycloalkyl or heterocycloalkyl group; O is selected from a set including H Oh. .N. R ₆ represents unsubstituted or substituted phenylmethyl; • · 192 • · • · · • · · · • · · R ₇ represents alkyl or aryl; and R ₉ represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from the group consisting of methyl and halogen atoms. 58. The compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 57, wherein R ₇ is selected from the group consisting of 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, phenylmethyl and naphthylmethyl. 59. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 55, wherein Rx is H, Z is H and: R ₂ represents CH ₂ CH ₂ C (0) NH ₂ , R ₆ represents represents CH(CH ₃ ) ₂ , Ζχ represents CO ₂ CH ₂ CH ₃ and ar ₇ Rg r»- in represents ^H 3c' or R ₂ represents represents CH(CH ₃ ) ₂ , r ₆ represents f Ζχ represents CO ₂ CH ₂ CH ₃ r ₇ Rg represents ^H with ^c'n - ^N XXX 60. A compound, pharmaceutically acceptable salt, prodrug, pharmaceutically active metabolite or solvate according to claim 1, which has antipicornaviral activity corresponding to an EC ₅₀ value of less than or equal to 100 μΜ in an H1-HeLa cell culture assay. • · - 193 61. A compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 1, which has antirhinoviral activity corresponding to an EC ₅₀ value of less than or equal to 10 μΜ in an H1-HeLa cell culture assay. 62. A compound having the general formula selected from wherein: R 1 , R ₂ , Z and Z 1 have the same meaning as in claim 1, n is equal to 1 or 2; R _y represents H or lower alkyl, R ₆ represents alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; R ₇ represents alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -OR _i7 , -SR _X7 , -NR _i7 Ri ₈ , -NR _i9 NR _i7 R ₁₈ or NR _i7 OR _i8 , where R _X7 , Ri ₈ and R _X9 independently represent H, 194 alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or acyl; and R ₉ represents a five-membered heterocycle having one to three heteroatoms selected from the group consisting of O, N and S; or a prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate thereof. 63. A compound, prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate according to claim 62, wherein: R 1 represents H, F or alkyl; R _y represents H or methyl; R6 represents an alkyl group optionally substituted with one or more substituents selected from the group consisting of alkyl and aryl; R ₇ represents alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; Rg represents a five-membered heterocycle having from one to three heteroatoms selected from the group consisting of O, N and S, wherein at least one of the heteroatoms is nitrogen which is unsubstituted or substituted with one or two substituents selected from the group consisting of lower alkyl groups and halogen atoms; or a prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate thereof. 64. The compound, prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate of claim 62, wherein: R6 represents arylmethyl or arylthiomethyl; R ₇ represents an alkyl group; 195 • · Rg represents 3-isoxazolyl or 5-isoxazolyl unsubstituted or substituted with one or two substituents selected from methyl and halogen atoms; and Z represents H and Zχ represents -CO ₂ R ₂ i, -CN or C(O)NR ₂ iR ₂₂ , where R ₂ i and R ₂₂ independently represent H, an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, an acyl group or a thioacyl group or R ₂₁ and R ₂₂ together with the atom or atoms to which they are attached form a heterocycloalkyl group or Z and Zi together represent a cyclic ester or amide. 65. The compound, prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate of claim 64, wherein: R 1 represents H or F; R ₆ represents arylmethyl or arylthiomethyl; and R ₇ is selected from the group consisting of 2-propyl, 2methyl-2-propyl, 2-methyl-1-propyl and arylmethyl. 66. The compound, prodrug, pharmaceutically acceptable salt, pharmaceutically active metabolite or solvate of claim 65, wherein R ₆ represents phenylmethyl, wherein the phenyl group optionally has one to three substituents selected from the group consisting of halogen, lower alkyl and lower alkoxy. 67. A compound according to claim 62, comprising the following compounds selected from the group consisting of: 196 HN OSV V% ⁰ Va co ₂ ch ₂ ch ₃ o ,AND O. or a pharmaceutically active metabolite or solvate thereof. salt 68. A pharmaceutical composition comprising: that · • · 197 • that (a) a therapeutically effective amount of at least one antipicornaviral agent which is a compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate according to claim 1; and (b) a pharmaceutically acceptable carrier, diluent, vehicle or excipient. 69. A method of treating a disease state in a mammal mediated by picornaviral protease activity, comprising administering to a mammal in need thereof a therapeutically effective amount of at least one compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 1. 70. A method of inhibiting the activity of picornaviral 3C protease, comprising contacting the picornaviral 3C protease with an effective amount of at least one compound, prodrug, pharmaceutically active metabolite, pharmaceutically acceptable salt or solvate of claim 1. 71. The method of claim 70, wherein the picornaviral 3C protease is a rhinoviral protease. 72. Compound of general formula: O. (Al3)p NC(R ₅₂ )(R ₅₃ )(R ₅₄ ) in which: p is an integer ranging from 0 to 5; • · φ φ φ · 198 • φ An represents CH or N, A _i2 and each A _i3 is independently selected from the group consisting of C (Rďi) (Rďi) r N (Rďi) , S, S (O) , S(O) ₂ and O and A ₁₄ represents NH or NR ₆ i , where each R ₆₁ independently represents H, alkyl, acyl or aryl, provided that no more than two heteroatoms occur consecutively in the ring formed by Au, Aχ ₂ , (Ai ₃ ) _n , A ₁₄ and C=0; each R 14i independently represents H or lower alkyl; R ₅₁ represents H, alkyl, acyl or aryl; R ₅₂ , R53 and R54 are independently selected from the group consisting of H, hydroxy, alkyl, acyl and aryl or any two of the substituents R ₅₂ , R53 and R ₅₄ together represent =O or =C(R ₅ t) (R 5 t) r where R 57 and R 58 are independently selected from the group consisting of H, alkyl, C0 ₂ (C x ~ C g) alkyl, CO ₂ N (C 1 -C 8 ) alkyl and CO ₂ (aryl); and R55 and _R56 independently represent H or a suitable nitrogen protecting group; or a pharmaceutically acceptable salt of said compound. 73. Compound of the general formula: in which: p is an integer ranging from 0 to 5; R51 represents H, alkyl, acyl or aryl; R ₅₂ , R 53 and R ₅₄ are independently selected from the group consisting of H, hydroxy, alkyl, acyl and aryl or any two of the substituents R ₅₂ , R 53 and R ₅₄ together represent =0 or • φ • · • φ - 199 =C(R ₅₇ ) (R 58 ), where R 57 and R ₅₈ are independently selected from the group consisting of H, alkyl, CO ₂ (C 1 -C ₆ ) alkyl, CO ₂ N (C _x Οβ) alkyl and CO ₂ (aryl); and R ₅₅ and R ₅₆ independently represent H or a suitable nitrogen protecting group; or a pharmaceutically acceptable salt of said compound. 74. A compound or pharmaceutically acceptable salt according to claim 73, wherein n is equal to 1 or 2. 75. A compound or pharmaceutically acceptable salt according to claim 74, wherein R ₅₂ , R 53 and R ₅₄ are independently selected from the group consisting of H, alkoxy, hydroxy and carbonyl. 76. A compound according to claim 73, comprising the following compounds selected from the group, ^P N HIM. 200 ·· «· • 9 9 · • * « · MeO in which: Pn is a suitable nitrogen protecting group and q is equal to 1 or 2; or a pharmaceutically acceptable salt of said compound. 77. A compound according to claim 73, comprising the following compounds selected from the group, • · • · • · 201 wherein BOC is tert-butyloxycarbonyl; or a pharmaceutically acceptable salt of said compound.