WO2019219938A1 - Fmoc protected (2s)-2-amino-8-[(1,1-dimethylethoxy)amino]-8-oxo-octanoic acid, (s)-2-amino-8-oxononanoic acid and (s)-2-amino-8-oxodecanoic acid for peptide synthesis - Google Patents

Abstract

The invention discloses Fmoc protected (2S)-2-amino-8-[(1,1- dimethylethoxy)amino]-8-oxo-octanoic acid, (S)-2-amino-8- oxononanoic acid and (S)-2-amino-8-oxodecanoic acid for use in peptide synthesis, such as solid phase synthesis, as well as the peptide H3K27 (Ac-Lys-Ala-Ala-Arg-Aox-Ser-Ala-NH2) prepared from Fmoc protected (2S)-2-amino-8-[(1,1-dimethylethoxy)amino]-8-oxo-octanoic acid (Aox). These three exemplary compounds as well as their unprotected forms are claimed in the form of four generic formulae. The first of these four formulae is (formula (I)) where -NPro is a protected amino group, such as an amino group protected with a base-labile protecting group, -L- is alkylene, heteroalkylene, arylene or aralkylene, -X- is a covalent bond, -N(H) - or -N(R^N)-, where -R^N is alkyl, -R² is hydrogen or alkyl, -R³ is alkyl, such as C_2-10 alkyl, or heterocyclyl, and -L^AA- and -R¹ are as defined in the claims.

Description

FMOC PROTECTED (2S)-2-AMINO-8-[(1,1 -DIMETHYLETHOXY)AMINO]-8-OXO-OCTANOIC ACID, (S)-2-AMINO-8-OXONONANOIC ACID AND (S)-2-AMINO-8-OXODECANOIC ACID

FOR PEPTIDE SYNTHESIS

Related Application

The present case claims the benefit and priority of GB 1808149.7 filed on 18 May 2018 (18.05.2018), the contents of which are hereby incorporated by reference in their entirety.

Field of the Invention

The present invention provides protected amino acids for use in peptide synthesis, such as solid-phase peptide synthesis. Also provided are methods for preparing the protected amino acids, and methods for preparing peptides using the protected amino acids.

Background

Peptides possessing amino acid residues with zinc-binding side chains are of interest owing to their useful biological proprieties. In particular, there is interest in amino acids having hydroxamic acid and hydroxyurea side chains.

For example, Komatsu et al. describe the cyclic peptides CHAP31 and CHAP50, with each peptide containing an amino acid residue with a hydroxamic acid side chain. These cyclic peptides have the ability to inhibit histone deacetlylase (HDAC).

The preparation of CHAP31 and CHAP50 was achieved in the solution phase (see the Supplementary Information for Furumai et al. cited by Komatsu et al.). Here, the cyclic peptides were prepared having an Asu amino acid residue contained with the ring. The acid functionality within the side chain of this amino acid was benzyl-protected. After cyclisation the benzyl group was removed under hydrogenating conditions. The free acid was then reacted with hydroxylamine to give a hydroxamic acid side chain.

Clearly, this solution phase synthesis is appropriate only for small peptides (both CHAP31 and CHAP50 contain only 4 amino acid residues). Further, the use of a protecting group that requires removal by heterogeneous catalysis is not particularly well-suited for a solid- phase synthesis.

The hydroxamic acid amino acid residue is accessible only in a late stage amide-coupling reaction that involves the reaction of a free carboxylic residue of the Asu amino acid. Such techniques are not generally applicable, for example where a peptide contains other amino acid residues with carboxylic acid functionality, such as Asp and Glu, as these may also react under these conditions. The use of an Asu amino acid as a building block only allows the synthesis of hydroxamic acid-containing products, and hydroxyurea-containing products cannot be accessed in this way.

Watson et al. have shown that the peptide H4K16Hx is able to mimic substrate binding to HDAC1. Here, H4K16Hx comprises residues 12-18 of histone H4, with K16 replaced with a hydroxamic acid-containing amino acid residue (Hx).

The preparation of the H4K16Hx peptide is unusual, as it requires the hydroxamic acid- containing amino acid, Hx, to be connected to a solid-phase via the hydroxamic acid group. The H4K16Hx product is generated by modification of the Hx C terminal to provide the amino acids for the C terminal portion of the product, followed by modification of the Hx N terminal to provide the amino acid residues for the N terminal portion of the product. The resin-bound peptide is then cleaved from the solid phase with TFA.

Given that the hydroxamic acid-containing amino acid is the point of connection to the solid phase, there is no opportunity to incorporate additional hydroxamic acid-containing residues into the peptide generated on the solid phase.

The need to perform an amide coupling reaction using a resin-bound carboxylic acid is also a limitation as standard solid-phase amide forming reactions are optimized for resin-bound amines instead.

It is clear that the methods described by Watson et al. do not have applicability to the preparation of peptide products having hydroxyurea-containing amino acid residues.

There is a need to develop methods for the ready incorporation of hydroxamic acid- and hydroxyurea-containing amino acids into peptides. Accordingly, the present inventors have developed protected amino acids for use in the preparation of peptides having hydroxamic acid- and hydroxyurea-containing amino acid residues.

Summary of the Invention

The present invention generally provides a protected hydroxamic acid-containing amino acid, a protected hydroxyurea-containing amino acid and a ketone-containing amino acid. The amino acids are suitable for use in solid-phase peptide synthesis, and generally the amino acids are Fmoc-protected on the amino group, and generally have a free carboxyl group.

The amino acids of the invention allow the introduction of hydroxamic acid, hydroxyurea or ketone functionality into a peptide at an early stage, and it is not necessary to perform a late stage chemical transformation to introduce these groups. The amino acids may be used in standard amide-coupling reactions, without requiring any adaptations. The amino acids may be used on the solid-phase with requiring the side chain group to be linked to the

solid-phase through the side chain functionality, such as the hydroxamic acid or hydroxyurea functionality. Moreover, the methods of the invention allow multiple amino acids having hydroxamic acid and hydroxyurea functionality to be introduced into a peptide, whilst the prior art methods, such as those described by Watson et al., only permit a single hydroxamic acid amino acid to be incorporated into a peptide.

In a first aspect of the invention there is provided a protected amino acid of formula (I):

wherein:

-IX- is a covalent bond or alkylene, such as -CH₂-,

-R¹ is hydrogen or alkyl,

-NPro is a protected amino group, such as an amino group protected with a base-labile protecting group, such as -NHFmoc,

-L- is alkylene, heteroalkylene, arylene or aralkylene, such as alkylene,

-X- is a covalent bond, -N(H)- or -N(R^N)-, where -R^N is alkyl,

-R² is hydrogen or alkyl, and

-R³ is alkyl, such as C_2-10 alkyl, such as butyl, or heterocyclyl, such as tetrahydropyranyl,

and salts, solvates and activated forms thereof.

Preferably, the compound is:

^R3°YX'^lY O H

I o ' R

R FmocHN

wherein, -L-, -R¹, -X-, -R² and -R³, and salts, solvates and activated forms thereof. This is a compound of formula (I) where -IX- is a covalent bond and -NPro is -NHFmoc. The compound may be an L- or D-amino acid, or it may be an enantiomeric mixture.

When -X- is a covalent bond, the compound is a hydroxamic acid-containing amino acid. When -X- is -N(H)- or -N(R¹)-, the compound is a hydroxyurea-containing amino acid.

The compound may be selected from:

O NHFmoc O NHFmoc

and salts, solvates and activated forms thereof.

The D-isomer forms, as shown below, are particularly preferred:

In a second aspect of the invention there is provided a peptide incorporating an amino acid residue obtained or obtainable from the compound of formula (I), such as a peptide held on the solid phase.

In a third aspect of the invention there is provided a method of preparing an amide, such as a peptide according to the second aspect of the invention, the method comprising the step of reacting a compound of formula (I) with an amine, optionally in the presence of one or more amide coupling agents and base.

In a fourth aspect of the invention, there is provided a compound of formula (II):

where -L⁴⁴-, -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I), and salts and solvates thereof.

The compound of formula (II) may be used as an intermediate in the preparation of the compounds of formula (I).

In a fifth aspect of the invention, there is provided a method of preparing a compound according to formula (I), the method comprising the step of protecting the amino group of a compound of formula (II) with a protecting group, thereby to yield the compound of formula (I).

In a sixth aspect of the invention, there is provided a compound of formula (IV):

where -L⁴⁴-, -L-, -X-, -R¹, -R², -R³ and -NPro have the same meanings as the compound of formula (I), and -C(0)OPro is a protected carboxylic acid group, such as -C(0)0Bn, and salts and solvates thereof.

In a seventh aspect of the invention, there is provided a method of preparing a compound according to formula (I), the method comprising the step of deprotecting the carboxylic acid group of a compound of formula (IV), thereby to yield the compound of formula (I).

In further aspect of the invention, there is provided a peptide synthesizer having a protected amino acid of formula (I), optionally together with additional protected amino acids, amide coupling reagents, and base.

The present inventors have also found that the methods of synthesis described herein may be usefully adapted for the preparation of alternative protected amino acids having side chains with functional groups suitable for zinc binding. In particular, the inventors have prepared in a stereoselective manner protected amino acids containing a ketone group within the side chain.

Thus, in a yet a further aspect there is provided an amino acid of formula (VIII):

wherein:

-L⁴⁴- is a covalent bond or alkylene, such as -CH2-,

-R¹ is hydrogen or alkyl,

-NPro is a protected amino group, such as an amino group protected with a base-labile protecting group, such as -NHFmoc,

-L- is alkylene, heteroalkylene, arylene or aralkylene, such as alkylene, -L^T is alkyl,

and salts, solvates and activated forms thereof. Preferably, the compound is:

L

O

Fmoc

where -L-, -L^T, and -R¹ have the same meanings as the compound of formula (VII), and salts, solvates and activated forms thereof. This is a compound of formula (VIII) where -L⁴⁴- is a covalent bond and -NPro is -NHFmoc.

These and other aspects and embodiments of the invention are discussed in detail below.

Detailed Description of the Invention

The present invention provides a protected hydroxamic acid-containing amino acid, a protected hydroxyurea-containing amino acid and a ketone-containing amino acid for use in peptide synthesis.

The amino acids of the invention may be readily incorporated into standard solid-phase peptide synthesis routines without the need for specialized coupling reagents for amide synthesis, without the need for specialized cleavage agents for release from the solid phase, and without specialized deprotecting agents for deprotection. Thus, the coupling, cleavage and deprotecting agents for use with the amino acids of the invention are commonplace.

The amino acids of the invention therefore allow solid-phase techniques to be used in the preparation of hydroxamic acid-, hydroxyurea- and ketone-containing peptides. This provides an advantage over the solution-phase work described by Furumai et al.

Nevertheless, the amino acids of the invention may be used in the solution-phase, if desired.

The amino acids of the invention have a free carboxylic acid group and a protected amino group, typically an Fmoc-protected amino group. Accordingly, the amino acids may be used in a standard Fmoc-based solid-phase organic synthesis.

The side chain of certain amino acids of the invention has hydroxamic acid or hydroxyurea functionality and this is in a protected from, for example as an optionally substituted alkyl hydroxamic acid ester. In the preferred embodiments of the invention this protected form is cleavable under acidic conditions, such as the acidic conditions typically used to remove amino acid side chain protecting groups in solid-phase organic synthesis. Thus, the protected from may be cleavable under TFA-mediated cleavage conditions. In a solid phase synthesis, the amino acid compounds of the invention do not require the side chain functionality, such as hydroxamic acid or hydroxyurea functionality, to act as a linker between the amino acid and a solid phase. Instead, the amino acids of the invention may be anchored via the carboxy functionality, as is standard in solid-phase synthesis, such as solid-phase peptide synthesis. This provides an advantage over the synthetic strategy described by Watson et al., where it is necessary to link a hydroxamic acid-containing amino acid through the hydroxamic acid functionality.

The amino acids of the invention may be obtained in L- and D-forms by stereoselective synthesis. These methods are adaptable to allow for a variety of different hydroxamic acid, hydroxyurea or ketone side chains to be incorporated into an amino acid.

The inventors have also established alternative methods for preparing the amino acids of the invention, which increases the possibilities for preparing a range of different amino acids.

WO 2015/106200 describes an oamino acid having a hydroxamic ester-containing side chain: this a hydroxamic acid that is protected with tBu (see Example 33). This exemplified compound has a short (Ci) alkylene spacer between the a-carbon and the hydroxamic ester. The oamino group is protected with the acid labile Boc protecting group. The amino acids described in this disclosure are for use in the solution phase synthesis of a dipeptide.

The work in WO 2015/106200 does not look to prepare peptides having a hydroxamic acid functional group. Instead, the dipeptides retain the hydroxamic ester functionality (as shown in claim 7 and Table 3).

In contrast, the preferred compounds of the present invention have a base-labile nitrogen protecting group, such as Fmoc. Furthermore, the preferred compounds have a linker -L- that has more than one carbon atom, such as C₄ or Cs alkylene linkers. The amino acids for use in the present case find use in the solid phase synthesis of peptides, including those having three or more, such as seven, continuous amino acid residues.

WO 2015/106200 does not describe any compound having a hydroxyurea-protected side chain.

WO 2009/105824 discloses an amino acid having a hydroxamic acid-containing side chain in the form of a Weinreb amide: see the compound of Example 19. The amino acid also have Cbz protection of the amino functionality, and there is a short (Ci) alkylene spacer between the a-carbon and the hydroxamic acid.

The Weinreb amide is used a mask for an aldehyde that is later revealed in a downstream synthesis step. Thus, the compound of Example 19 is used to make the aldehyde- containing diazepane of Example 24. The aldehyde is used within a reductive amination to introduce amine-functionality into the final product (see Example 24). Thus there is no interest in preparing peptides having an amino acid with a hydroxamic acid-containing side chain.

It is noted that Chem. Abs. Acc. No. 2009:1081533 apparently incorrectly records the structure of an amino acid compound described in WO 2007/047608. The abstract indicates that the Fmoc-protected version of the amino acid describe above is also described, however there is no disclosure of such a compound.

As noted above, the preferred compounds in the present case have a base-labile nitrogen protecting group, and they also possess a longer alkylene spacer between the a-carbon and the hydroxamic acid.

WO 2009/105824 does not describe any amino acid having a hydroxyurea-protected side chain.

US 7550602 discloses as an intermediate amino acid having a hydroxamic acid-containing side chain in the form of a Weinreb amide (see Scheme 6, and the intermediate which is produced after step 2) in the first stage of the synthesis). The amino acid has Boc protection of the amino functionality, and there is a short (C₂) alkylene spacer between the a-carbon and the hydroxamic acid.

The Weinreb amide is used a mask for an aldehyde that is later revealed in a downstream synthesis step: here reduction with LAH provides the aldehyde which is reacted in a cyclisation reaction. Thus, thus there is no interest in preparing peptides having an amino acid with a hydroxamic acid-containing side chain.

In contrast to the amino acid of US 7550602, the preferred compounds of the present invention have a base-labile nitrogen protecting group, such as Fmoc, for the amino group. Furthermore, the preferred compounds have a linker -L- that has more than two carbon atoms, such as C4 or C5 alkylene linkers. The hydroxamic acid is also typically protected with an alkyl group having more than one carbon atoms, such as butyl, or is protected with heterocyclyl.

US 7550602 does not describe any amino acid having a hydroxyurea-protected side chain.

WO 2007/047608 discloses protected amino acid compounds for use in an automated synthesiser. Suitable amino acids are listed in Example 1 and these include Fmoc-protected glutamine (Gin) and Fmoc-protected glutamic acid (Glu). The side chain groups are protected with trityl (Trt) and -OTBu respectively. These amino acids do not contain a protected hydroxamic acid or a protected hydroxyurea. It is noted that Chem. Abs. Acc.

No. 2007:463352 apparently incorrectly records the structure of the amino acid compounds described in WO 2007/047608. WO 2006/086600 contains an example of a Dap amino acid where the side chain has an amino group protected with Fmoc. This is example compound G.1 1 .2. This amino acid is used to make a boron ester-containing product for use as a protease inhibitor (see compound B.9.2). This amino acid does not contain a protected hydroxamic acid or a protected hydroxyurea. It is noted that Chem. Abs. Acc. No. 2006:817369 apparently incorrectly records the structure of compound G.1 1.2.

Amino Acids

The present invention provides a protected amino acid as a compound of formula (I):

wherein:

-L⁴⁴- is a covalent bond or alkylene, such as -CH2-,

-R¹ is hydrogen or alkyl,

-NPro is a protected amino group, such as an amino group protected with a base-labile protecting group, such as -NHFmoc,

-L- is alkylene,

-X- is a covalent bond, -N(H)- or -N(R^N)-, where -R^N is alkyl,

-R² is hydrogen or alkyl, and

-R³ is alkyl, such as C2-10 alkyl, such as butyl, or heterocyclyl, such as tetrahydropyranyl,

and salts, solvates and activated forms thereof.

The group -L⁴⁴- is a covalent bond or alkylene. For example, -L⁴⁴- is a covalent bond or

-CH2-.

Where -L⁴⁴- is a covalent bond, the compound may be referred to as an oamino acid.

Where -L⁴⁴- is alkylene, this may be C1-6 alkylene, such as Ci_-4 alkylene, such as

Ci_-3 alkylene, such as C1 alkylene (methylene). The alkylene may be linear or branched.

The group -L⁴⁴- may be -CH2-, CH(CH₃)- or -C(CH₃)2-. Here, the compound may be referred to as a b-amino acid.

Preferably, -L⁴⁴- is a covalent bond.

Where -R¹ is alkyl, this may be C_1-6 alkyl, such as C_1-4 alkyl. For example the alkyl group may be selected from -CH₃, -CH₂CH₃, -CH(CH₃)₂, -CH₂CH(CH₃)₂, and -CH(CH₃)CH₂CH₃. The group -R¹ is preferably hydrogen or methyl (-CH₃), and most preferably hydrogen. The amino group of the amino acid of formula (I) is protected. Preferably the amino protecting group is orthogonal to the group -R³. That is, the conditions for the removal of the amino protecting group, -NPro, do not cause the loss of the group -R³. Similarly, the conditions for the removal of -R³, for example to generate the hydroxamic acid or the hydroxyurea, do not cause the loss of the amino protecting group.

The amine may be protected as a carbamate, such as -NHFmoc or -NHBoc.

Preferably, the amino protecting group is removable by base, such as organic base. The amino protecting group may be removable with piperidine or DBU

(1 ,8-diazabicyclo[5.4.0]undec-7-ene), such as piperidine.

In a preferred embodiment, -NPro is -NHFmoc, where Fmoc is 9-fluorenylmethoxycarbonyl. This is a base-labile protecting group.

The amino protecting group may be removable by acid, such as organic acid, such as TFA.

In one embodiment, -NPro is -NHBoc, where Boc is tert- butoxycarbonyl. This is an acid- labile protecting group.

The amino protecting group may be removable under hydrogenating conditions, such as hydrogenation in the presence of a Pd catalyst.

In one embodiment, -NPro is -NBn2, where each -Bn is benzyl.

It is preferred that the amino protecting group is base-labile.

It is most preferred that the amino protecting group is base-labile, and the group -R³ is acid labile.

The group -L- is a linker connecting the carbon bearing the amino group (-NPro) of the amino acid to the side chain functional group, -X-C(0)-N(R²)0R³. The linker may be selected from alkylene, heteroalkylene, arylene and aralkylene.

The group -L- may be an alkylene linker. This is preferred.

The alkylene linker may be a linear or branched alkylene, and is preferably a linear alkylene. The alkylene group may be C-MO alkylene, such as C1-6 alkylene. Preferably the alkylene is C5-6 alkylene.

In another embodiment, the alkylene is C1-4 alkylene, such as C1-2 alkylene.

Alternatively, the alkylene is C2-10 alkylene, C3-10 alkylene, such as C2-6 alkylene, such as C3-6 alkylene.

Typically -L- is C5 alkylene, when -X- is -N(H)- or -N(R^N)-. Typically -L- is C6 alkylene, when -X- is a covalent bond.

The group -L- may be a heteroalkylene linker. A heteroalkylene group is an alkylene group where one or two carbon atoms is each replaced with a heteroatom selected from the group consisting of O, S and N(H). The heteroalkylene linker may be a linear or branched heteroalkylene, and is preferably linear heteroalkylene.

Preferably the heteroalkylene group contains only one heteroatom, and preferably the heteroatom is O or S, and most preferably O.

The heteroatoms present in the heteroalkylene group are not present at the terminals of the heteroalkylene group, and therefore the heteroatoms are not bonded to -X- or

-C(NPro)(R¹)-L^AA-COOH.

The heteroalkylene group may be C_3-10 heteroalkylene, such as C_3-6 heteroalkylene.

Preferably the alkylene is C_5-6 heteroalkylene. Here, the value of C includes the number of both carbon and heteroatoms present.

Examples of heteroalkylene groups include C₅ heteroalkylene, such as -CH₂CH₂OCH₂CH₂-^*, -CH2CH2SCH2CH2-^* and -CH2CH2NHCH2CH2-^*, C₆ heteroalkylene, such as

-CH₂CH₂OCH(CH3)CH2-^* and -CH2CH2NHCH2CH2CH2-^*, where ^* indicates the point of attachment to the group -C(NPro)(R¹)-L^AA-COOH.

The group -L- may be an arylene linker. Here, the linker is an arylene group connecting -X- and -C(NPro)(R¹)-L^AA-COOH. Example arylene groups are described below, and these include carboarylene and heteroarylene groups.

Where the arylene linker is a heteroarylene linker, the heteroarylene group is preferably connected to -X- and -C(NPro)(R¹)-L^AA-COOH via carbon ring atoms of the heteroarylene group.

The group -L- may be an aralkylene linker. An aralkylene group is an alkylene group where a carbon atom is replaced with an arylene group, such as an arylene group described below.

An arylene group may be a carboarylene or a heteroarylene.

Examples of carboarylene include phenylene {Ce carboarylene) and naphthylene (C₁₀ carboarylene).

Examples of heteroarylene include C₅ heteroarylene, such as pyrrolylene, O_Q heteroarylene, such as pyrindylene, C₉ heteroarylene, such as indolylene, and C₁₀ heteroarylene, such as quinolinylene.

Examples of aralkylene groups include C₇ aralkylene, such as -Ph-CH₂-^*, Cs aralkylene, such as -CH2-Ph-CH2-^*, C10 aralkylene such as -ln-CH2-^*, where Ph is phenylene, In is indolylene and ^* indicates the point of attachment to the group -C(NPro)(R¹)-L^AA-COOH. The group -R² may be hydrogen or alkyl. Where an alkyl group is present at this position it may be CMO alkyl, such as C1-6 alkyl. The alkyl group may be C alkyl, such as methyl or ethyl.

The group -R² is preferably hydrogen.

The group -X- is a covalent bond, -N(H)- or -N(R^N)-, and preferably -X- is a covalent bond or -N(H)-.

The group -X- may be a covalent bond. Here, the amino acid is a hydroxamic acid- containing amino acid.

The group -X- may be -N(H)- or -N(R^N)-. Here, the amino acid is a hydroxyurea-containing amino acid.

Where -R^N is present, the alkyl group may be CMO alkyl, such as Ci-₆ alkyl. The alkyl group may be Ci_-4 alkyl, such as methyl or ethyl.

The group -R³ may be referred to as the protecting group for the hydroxamic acid and hydroxyurea functionality of the amino acid.

Preferably, the group -R³ is removable by acid, such as organic acid. The group may be removed with TFA (trifluoroacetic acid).

However, it is not always necessary for the group -R³ to be removable. The group -R³ may be retained as part of a finished product, for example where a hydroxamic acid ester is a desirable structural feature for that product.

Preferably, the carbon attached to the oxygen atom to which -R³ is bonded is a quaternary carbon. That is to say, the carbon attached to the oxygen atom is also attached to three other non-hydrogen atoms, such as three carbon atoms.

The group -R³ is preferably alkyl, such as CM_O alkyl, such as CM_O alkyl. Alternatively, the group -R³ is preferably C2-10 alkyl, such as C3-10 alkyl. The alkyl group may be branched, and typically contains at least one quaternary carbon atom, and as noted above a quaternary carbon atom may be attached to the oxygen atom with which -R³ is bonded.

The group -R³ is most preferably butyl, such as tert- butyl.

The alkyl group is unsubstituted.

The group -R³ may be heterocyclyl, which heterocyclyl group contains one or two, such as one, ring heteroatoms selected from O, S and N(H). The heterocyclyl may be a single ring, or a plurality of fused rings, and is preferably a single ring. The heterocyclyl group is not unsaturated.

The heterocyclyl may be a C₅ heterocyclyl, such as tetrahydrofuranyl, C₆ heterocyclyl, such as tetrahydropyranyl. Preferably the heterocyclyl is as a O_Q heterocyclyl. Where -R³ is heterocyclyl, the heterocyclyl is attached to the oxygen atom to which -R³ is bonded via a ring carbon atom. This ring carbon atom is adjacent to a ring heteroatom within the heterocyclyl. Thus, the heterocyclyl together with the oxygen atom to which -R³ is bonded forms an acetal, or acetal-like group, such as a hemiaminal or an aminal.

The group -R³ may be tetrahydropyranyl, and more preferably tetrahydropyran-2-yl.

The amino acids described herein typically have one or more, such as one, stereocentres. An amino acid may be provided as an enantiomer, or as a mixture of enantiomers, or a mixture of diastereomers, for example where there are two or more stereocentres.

The amino acid is preferably an oamino acid, which may be provided in the L- or D- form, and preferably the D-form.

The methods of the present case, such as described in further detail below, permit the synthesis of amino acids in a desired stereoform. Alternatively, an amino acid may be prepared and provided initially as a mixture of stereoforms, such as a racemic or diastereomeric mixture, and the stereoforms may be separated by conventional methods, such as by HPLC. The amino acid may be provided as a mixture of stereoforms, such as a racemic or diastereomeric mixture.

The compound of formula (I) may be selected from:

o NFIFmoc o NFIFmoc and salts, solvates and activated forms thereof.

The compound of formula (I) may be selected from:

o NHFmoc

and salts, solvates and activated forms thereof. Also provided by the present invention are activated forms of the amino acids of formula (I). Such amino acids are typically those where the carboxylic acid group is in activated form for reaction in an amide-forming reaction.

The activated form may be generated in situ through the appropriate choice of coupling agents in the amide bond-forming reaction, optionally in the presence of base.

The protected amino acid is preferably for use in Fmoc-based peptide synthesis.

Thus, the amino acids of the invention are typically active for nucleophilic attack by an amino group on the solid-phase such as an amino group of a solid-phase bound amino acid residue, or an amino group of an amino-functionalized resin, such as a Rink Amide resin.

The carboxylic acid may be activated by reaction of a standard activating agent, such as a carbodiimide, such as DIC (L/,L/'-diisopropylcarbodiimide) or EDCI (water soluble

carbodiimide; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). The activated form of the acid is an O-acylisourea.

The carboxylic acid may be activated by a hydroxybenzotriazole or a

hydroxyazabenzotriazole, such as HOBt (1-hydroxy-benzotriazole) or HOAt (1-hydroxy-7- aza-benzotriazole). The activated form of the acid is an ester.

The ester may be formed via the carbodiimide-activated form, or it may be formed from the carboxylic acid directly, for example using an appropriate reagent, such as HATU, HBTU, PyBOP, PyBROP, or TBTU.

An organic base may be present in the formation of the activated form, such as DIPEA or TEA.

Also provided by the present invention is a compound of formula (II), which is an

intermediate compound for use in the preparation of a compound of formula (I):

where -L⁴⁴-, -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I), and salts and solvates thereof.

The preferences of the compounds of formula (I) apply also to the compounds of formula (II). The compound of formula (I) may be formed from the compound of formula (II) by protection of the amino group in the compounds of formula (II). A preferred compound of formula (II) is:

where -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I), and salts and solvates thereof. This is a compound of formula (II) where -L⁴⁴- is a covalent bond.

Also provided by the present invention is a compound of formula (III), which is an

intermediate compound for use in the preparation of a compound of formula (I), for example via the compound of formula (II), for example, where -L⁴⁴- is covalent bond:

where -R¹ is as defined for the compounds of formula (I);

-R^s is a group -L-X-C(0)N(R²)-0(R³), where -L-, -X-, -R² and -R³ have the same meanings as the compounds of formula (I);

M is a metal ion, such as Ni(ll); and

each of Ar¹ and Ar³ is an aryl group that is optionally substituted, and Ar² is an arylene group that is optionally substituted.

The group Ar¹ is preferably phenyl. Here, the phenyl is preferably unsubstituted.

The group Ar² is preferably phenylene. This group is phenyl-1 ,2-ene. The phenylene is preferably unsubstituted.

The group Ar³ is preferably optionally substituted phenyl, such as substituted phenyl. The phenyl group may be substituted with halo such as fluoro, such as 2-subsituted with halo, such as fluoro.

M may be selected from Ni(ll) and Fe(ll), and M is preferably Ni(ll).

Stereoselective control is provided by appropriate choice of the proline amino acid residue in the compound of formula (III). L- or D-proline may be used to prepare the L- and D-forms of an amino acid of the invention. In the worked examples of the present case, L-Pro is used to form an L-amino acid. Where stereoselective control is not required, a racemic mixture of the L- and D-forms of proline may be used to prepare the compound of formula (II).

Also provided by the present invention is a compound of formula (IV):

where -L⁴⁴-, -L-, -X-, -R¹, -R², -R³ and -NPro have the same meanings as the compound of formula (I), and -C(0)OPro is a protected carboxylic acid group, such as -C(0)0Bn, and salts and solvates thereof.

A preferred compound of formula (IV) is:

o

R FmocHN

where -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I), and -C(0)OPro is a protected carboxylic acid group, such as -C(0)0Bn, and salts and solvates thereof. This is a compound of formula (IV) where -L⁴⁴- is a covalent bond and -NPro is -NHFmoc.

The compound of formula (IV) may be used as an intermediate in the preparation of a compound of formula (I). For example, a compound according to formula (I) may be prepared by deprotecting the carboxylic acid group of a compound of formula (IV), thereby to yield the compound of formula (I).

The invention also provides a compound of formula (V):

where -L⁴⁴-, -X-, -R¹, -R², -R³, -NPro and -C(0)OPro have the same meanings as the compound of formula (IV), and -L^M- is alkenylene, and salts and solvates thereof.

A preferred compound of formula (V) is:

o

R FmocHN

where -X-, -R¹, -R², -R³, and -C(0)OPro have the same meanings as the compound of formula (IV), and -L^M- is alkenylene, and salts and solvates thereof. This is a compound of formula (V) where -L⁴⁴- is a covalent bond and -NPro is -NHFmoc. The compound of formula (V) may be an intermediate in the preparation of the compound of formula (IV). Thus, the alkenylene group in the compound of formula (V) may be reduced to yield the compound of formula (IV). Alternatively, the compound of formula (V) may be used an intermediate in the preparation of the compound of formula (I). Here, the alkenylene group in the compound of formula (V) may be reduced with concomitant removal of the protecting group for the carboxylic acid, -C(0)OPro. The reduction and removal of the protecting group may be achieved under hydrogenating conditions, for example where the carboxylic ester is -C(0)0Bn.

The group -L^M- is an alkenylene group, such as C2-10 alkenylene. Thus, a double bond is present in this group. This double bond may be formed in a cross-metathesis reaction.

The alkenylene linker may be a linear or branched alkenylene, and is preferably linear alkenylene.

The alkenylene group may be C2-10 alkenylene, such as C2-6 alkenylene. Preferably the alkylene is C5-6 alkenylene. In another embodiment, the alkylene is C2 alkenylene.

Typically -L- is C5 alkenylene, when -X- is -N(H)- or -N(R^N)-.

Typically -L- is C₆ alkenylene, when -X- is a covalent bond.

In an additional aspect of the invention there is provided a compound of formula (VIII):

where the groups -L⁴⁴-, -L-, -R¹ and -NPro have the same definitions and

preferences as given above for the compounds of formula (I), and -L^T is alkyl, and salts, solvates and activated forms thereof.

Preferably, the compound is:

where -L⁴⁴-, -L-, and -L^T have the same meanings as the compound of formula (VIII), and salts, solvates and activated forms thereof.

The group -L^T is a terminal group. It is an alkyl group, which may be linear or branched. The alkyl group may be CM_O alkyl, such as Ci-₆ alkyl, such as Ci_-4 alkyl, such as C1-2 alkyl. The group -L^T is preferably methyl or ethyl. The amino acid is preferably an oamino acid, which may be provided in the L- or D- form, and preferably the D-form.

The amino acid may also be provided in activated form, such as the activated forms described above in relation to the compounds of formula (I).

The compounds of formula (VIII) may be prepared from a compound of formula (IX), the structure of which is given below:

where -L⁴⁴-, -L-, -L^T and -R¹ have the same meanings as the compound of formula (VII), and salts and solvates thereof. Typically, the method of preparation comprises the step of protecting the amino group of a compound of formula (IX) with a protecting group, thereby to yield the compound of formula (VIII).

The compounds of formula (IX) may be prepared from a complex of formula (X), for example where -L⁴⁴- is covalent bond:

where -R¹ is as defined for the compounds of formula (VIII);

-R^s is a group -L-C(0)-L^T, where -L- and -L^T have the same meanings as the compounds of formula (VII);

M is a metal ion, such as Ni(ll); and

each of Ar¹ and Ar³ is an aryl group that is optionally substituted, and Ar² is an arylene group that is optionally substituted.

The definitions and preferences for each of M, Ar¹, Ar² and Ar³ are the same as those for the compounds of formula (III). Methods of Preparation

The amino acids of the invention may be prepared from readily available starting materials using well-known techniques.

The amino acids of formula (I) may be obtained from the amino acid of formula (II), by protection of the amino group of the compounds of formula (II) with a protecting group.

Similarly, the amino acids of formula (VIII) may be obtained from the amino acid of formula (IX), by protection of the amino group of the compounds of formula (IX) with a protecting group.

For example, a compound of formula (II) of formula (IX) may be reacted with Fmoc-OSu, optionally in the presence of base, to yield a compound of formula (I) or formula (VIII), where -NPro is -NHFmoc.

The formation of the protected amino acid form will be well known and well understood to those of skill in the art.

A compound of formula (II) or formula (VIII), for example where -L⁴⁴- is a covalent bond, is obtainable by derivatisation of an amino acid such as glycine or an amino acid having a side chain group, such as alanine. Here, the amino acid starting material is chosen for the nature of its side chain group as this side chain group will ultimately become the group -R¹ in the amino acids of the invention. Thus, for example, glycine is chosen where -R¹ is hydrogen, and alanine is chose where -R¹ is methyl.

Here, the method of the invention is an adaptation of the stereoselective synthesis described by one of the present inventors in Aillard et al.

The method involves the formation of a precursor complex of an amino acid with a metal ion, such as Ni(ll), and a chiral auxiliary, such as 2-FBPB. Such a complex has a structure as shown for the compound of formula (III) and formula (X), except that the group -R^s is hydrogen in the precursor complex.

The complex is then reacted in the presence of base with an electrophilic reagent that contains the side chain for the amino acids of the present invention, for example the reagent contains the group -L-X-C(0)N(R²)-0(R³) (where compounds of formula (I) are the intended end products) or the reagent contains the group -L-C(0)-L^T (where compounds of formula (VIII) are the intended end products). The side chain is then added to the a-position of the amino acid in the complex in a stereospecific manner. This reaction gives a complex of formula (III), as described herein, or a compound of formula (X), as described herein. The complex is subsequently decomposed to give a compound of formula (II) or formula (IX) as appropriate.

The compound of formula (III) may be converted to a compound of formula (II) using methods known in the art. The complex may be treated with hydroxyquinoline or acid. Typically, however, strong acid conditions are avoided where the group -R³ is acid labile.

The inventors have found that where -R³ is an acid-labile group such as tert- butyl, such a group is not removed when the complex is removed under mild acid conditions.

The compound of formula (X) may be converted to a compound of formula (IX) using these same methods.

In an alternative method, an amino acid of the invention may be prepared by a cross metathesis method, from an amino acid precursor having alkene functionality. This method is less preferred owing to the need for expensive reagents and catalyst. Generally, the yields are not as high as those reported for the complex-based method described above.

In this method, a protected amino acid having an alkenyl-containing side chain is reacted in the presence of a metathesis catalyst and a reagent comprising an alkenyl group that is linked to protected hydroxamic acid and hydroxyurea functionality, or an alkenyl group linked to a ketone-containing group. The cross-metathesis product, which is a compound of formula (V) or formula (XII) is obtained and purified from homo-metathesis products.

Thus, the protected amino acid is a compound of formula (VI):

wherein -R¹, -NPro, -L⁴⁴-, and -C(0)OPro have the same meanings as the compound of formula (IV), and -R^M1 is an alkenyl group, and this may be reacted in the presence of a reagent comprising an alkenyl group of formula (VII):

where -X-, -R², and -R³, have the same meanings as the compound of formula (IV), and -R^M2 is an alkenyl group, to yield a protected amino acid of formula (V). The compound of formula (VI) is preferably:

wherein -R¹, -NPro, and -C(0)OPro have the same meanings as the compound of formula (IV).

Each of -R^M1 and -R^M2 is an alkenyl group, and the alkenyl may be a linear or branched alkenyl, and is preferably linear alkenyl.

Each alkenyl group may be C2-10 alkenyl, such as C2-6 alkenyl. Preferably each alkylene is C2-6 alkenyl, such as C2, C3 or C₄alkenyl.

Preferably, the double bond in the alkenyl group is provided at a terminal of the alkenyl.

The catalyst in this reaction may be a standard metathesis catalyst, such as a Grubbs’ catalyst, and more preferably a Grubbs’ II catalyst.

In a similar way, a compound of formula (VIII) may be prepared in a process that uses a method of metathesis, although this is less preferred, as the metathesis methods are not high yielding.

Also provided is a method of preparing a hydroxyl urea-containing amino acid of formula (IV), which is a compound where -X- is -N(H)- or -N(R^N)-, the method comprising the step of reacting a compound of formula (XX) with a hydroxylamine of formula (XXI), optionally in the presence of base, to give the compound of formula (IV), where the compound of formula (XX) is:

where -L⁴⁴-, -L-, -R¹, and -NPro have the same meanings as the compound of formula (I), -X- is -N(H)- or -N(R^N)-, where -R^N is alkyl, and -C(0)OPro is a protected carboxylic acid group, such as -C(0)0Bn, and salts and solvates thereof,

and the compound of formula (XXI) is:

where -R² and -R³ have the same meanings as the compound of formula (I), and -G is a leaving group, such as a tosyl hydroxylamine, and salts and solvates thereof.

The group -G is preferably -NHOS(0)₂Tol, where Tol is toluenyl. The methods of the present case may also make use of an intermediate of formula (XI):

)Pro

where -L⁴⁴-, -L-, -L^T and -NPro have the same meanings as the compound of formula (VIII), and -C(0)OPro is a protected carboxylic acid group, such as -C(0)0Bn, and salts and solvates thereof.

The metathesis product is an amino acid of formula (XII):

)Pro

where -L⁴⁴-, -L^T, -NPro and -C(0)OPro have the same meanings as the compound of formula (X), and -L^M- is alkenylene, and salts and solvates thereof.

A compound of formula (XII) may be converted to a compound of formula (XI) by reduction of the alkenylene group in the compound of formula (XII).

A protected amino acid of formula (VI), as previously defined, may be reacted in the presence of a reagent comprising an alkenyl group of formula (XII):

where -L^T has the same meanings as the compound of formula (VIII), and -R^M2 is an alkenyl group.

The definitions and preferences for each of -L^M-, -R^M1 and -R^M2 are the same as those for the compounds of formula (V) to (VII).

A compound of formula (V) or (XII) may be converted directly to a compound of formula (I) or formula (VIII) by removal of the carboxylic acid protecting group, and reduction of the double bond within the group -L^M-.

Thus, -C(0)OPro may be converted to -COOH, using methods know to those of skill in the art. The double bond may be reduced using methods know to those of skill in the art.

Typically, -C(0)OPro is -0C(0)Bn, where Bn is benzyl. The removal of the protecting group and the reduction of the double bond may be achieved in one step by hydrogenation in the presence of a catalyst. Where two steps are required, the hydrogenation step may be performed first, to give the compound of formula (XI), followed by the removal of the carboxylic acid protecting group, to give the compound of formula (VIII).

The methods of preparation described above are exemplified in the present case for the preparation of a series of oamino acid products (that is, compounds where -L⁴⁴- is a covalent bond). These methods are adaptable for the preparation of other amino acids.

The present case also provides compounds where the amino and carboxy functionality are not provided on contiguous carbon atoms. These are the compounds of the invention where -L⁴⁴- is alkylene, and most preferably these are b-amino acids, where the group -L⁴⁴- is -CH2-, for example.

Such compounds may be prepared by the metathesis routes described herein, with appropriate choice of coupling partner, such as the compound of formula (VI), in the cross- coupling reaction. Other methods for the preparation of b-amino acids of formula (I) and (VIII) may be used, such as adaptations of the methods reviewed by Ma relating to asymmetric carbon-carbon bond-forming reactions, asymmetric carbon-nitrogen bond- forming reactions, and asymmetric hydrogenation reactions.

Use of Amino Acids

The present invention also provides a peptide incorporating one or more amino acid residues derived from the amino acids according to the invention, such as the amino acids of formula (I) and (VIII) (or the amino acids of formula (II) and (IX)). Here, at least one of the amino and carboxyl functionality in the amino acid of the invention is part of an amide bond to another amino acid within the peptide. The peptide incorporates the amino acid of the invention as an amino acid residue having a protected hydroxamic acid or a protected hydroxyurea in its side chain. Additionally or alternatively, the peptide incorporates the amino acid of the invention as an amino acid residue having a ketone group in its side chain.

Here, a peptide comprises two or more contiguous amino acid residues linked by amide bonds, where at least one of those amino acid residues is derived from an amino acid of the present invention, such as an amino acid residue derived from the compounds of formula (I) or (VIII). Typically the peptide comprises five or more, such as ten or more, such as twenty or more amino acid residues.

The peptide may be held on the solid-phase. Thus, an amino acid residue in the peptide may be connected to a resin, preferably via the carboxy group of that residue. The peptide of the present invention contains an amino residue derived from the amino acid of the invention. Thus, the amino and/or the carboxy functionality of the amino acid are incorporated into amide bonds. Thus, the group -NPro in the compound of formula (I) or (VIII) may be provided as -NH- which participates in an amide bond with a carboxylic acid group of a neighboring amino acid residue. The group -COOH in the compound of formula (I) or (VIII) may be provided as -C(O)- which participates in an amide bond with an amino group of a neighboring amino acid residue.

An amino acid residue of the invention may be provided at the N terminal of the peptide. Here the carboxy functionality of the amino acid is incorporated into an amide bond. The N terminal may be protected in the form of -NPro, or the N terminal may be a free amine, for example as might be obtained by removal of the amine protecting group, such as prior to the reaction of the amino acid residue in an amide coupling reaction.

Accordingly, in a further aspect of the invention there is provided a peptide of formula (XIII):

where -L⁴⁴-, -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I),

-S^c is -OH, -OMe, -OEt or one or more contiguous amino acid residues;

-S^N is hydrogen, -Me, -Et, -C(0)Me, -C(0)Et or one or more contiguous amino acid residues;

where at least one of -S^c and -S^N is one or more contiguous amino acid residues, and optionally one of -S^c and -S^N, such as -S^c is connected to the solid phase,

and salts and solvates and protected forms thereof.

In a further aspect of the invention there is provided a peptide of formula (XIV):

where -R¹, -L⁴⁴-, -L- and -L^T have the same meanings as the compound of formula (I), and -S^c and -S^N have the same meanings as the compound of formula (XIII), and salts and solvates and protected forms thereof. The present invention also provides an amide containing an amino acid residue derived from the amino acid of the invention. An amide of the invention may be prepared by reaction of an amino acid of formula (I) or (VIII) with an amine, such as an amine of a second amino acid, optionally in the presence of base and one of more coupling agents. The amine may be present on the solid-phase. Thus, the reaction of the amine with the amino acid of formula (I) or (VIII) adds the amino acid to the solid-phase.

After the amide bond-forming reaction is complete, the product may be at least partially purified. Thus, the product may be separated from unreacted amino acid of formula (I), as well as base and coupling reagents, and any solubilised reaction by-products. In a solid- phase synthesis, the purification may be achieved by simple filtration and washing, where solid-bound material is separated from the solution-phase material.

Where a product is prepared on the solid-phase, such as a peptide, it may be removed from the solid-phase with concomitant removal of the protecting group of the hydroxamic acid and hydroxyurea functionality. Thus, typically the protecting group -R³ is acid labile and the standard strategy for removal of the peptide from the solid-phase is acid cleavage, for example with TFA, optionally in the presence of a scavenger.

In the worked examples of the present case a peptide product is formed on the solid phase using an amino acid of the present invention in combination with standard Fmoc-based chemistries.

The methods for preparing the peptide typically use standard Fmoc-based chemistry.

Thus, a method of preparing a peptide product may include the steps of reacting a protected amino acid, which may be an Fmoc-protected amino acid of the invention or another Fmoc-protected amino acid, with a resin, such as Rink-Amide resin, thereby to load the resin with the amino acid residue.

Subsequent extension steps in the synthesis involve the removal of the amino protecting group, for example using piperidine to remove Fmoc, and the subsequent coupling of the amino functionality of the resin-bound amino acid residue with a further protected amino acid, which may or may not be a protected amino acid according to the invention.

At least one step in the synthesis, including the loading and extension steps, makes use of an amino acid of formula (I) and/or (VIII).

The amino acids of formula (I) and (VIII) may be used as building blocks for standard peptide synthesis on the solid-phase. The amino acid may be used in an automated synthesis using a peptide synthesizer as is well known in the art. Accordingly, the invention also provides a peptide synthesizer having an amino acid of formula (I) or formula (VIII), optionally together with additional protected amino acids, amide coupling reagents, and base.

Also provided by the present invention is a kit comprising an amino acid of formula (I) or formula (VIII) held in a container, together with one or more protected amino acids, with each amino acid provided in a separate container. Here each of the other amino acids may be an Fmoc-protected amino acid.

Other Forms

Compounds of the invention may be provided in salt form.

Compounds having a carboxyl group, such as the compounds of formula (I), (II), (VIII) and (IX), may be provided as salts, for example base addition salts of strong mineral bases and base addition salts of strong organic bases.

Compounds having an amine group, such as the compounds of formula (II) and (IX) may be provided as salts, for example acid addition salts of strong mineral acids such as HCI and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulfates and acetates such as trifluoroacetate or trichloroacetate.

In one embodiment the compounds such as those of formula (II) and formula (IX) may be provided as a sulfate salt or a trifluoroacetic acid (TFA) salt.

A reference to a compound of the invention or any other compound described herein, is also a reference to a solvate of that compound. Examples of solvates include hydrates.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and

chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

One aspect of the present invention pertains to compounds in substantially purified form and/or in a form substantially free from contaminants.

In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. Unless specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.

Other Preferences

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example.

Experimental and Results

The preparation of the Fmoc-protected amino acid (S)-2-({[(9H-fluoren-9- yl)methoxy)carbonyl]amino}-8-(fe/f-butoxyamino)-8-oxooctanoic acid (7a) via a Schiff base is shown schematically below. Scheme 1 - Synthesis of Schiff base complex (4)

4 Scheme 2 - (S)-2-({[(9H-fluoren-9-yl)methoxy)carbonyl]amino}-8-(fe/f-butoxyamino)-8- oxooctanoic acid (7a)

FmocHN CO,H

7a (S)-1-(2-Fluorobenzyl)pyrrolidine-2-carboxylic acid (2)

L-Proline (1 ) (10.0 g, 86.8 mmol) was added to a solution of potassium hydroxide (14.6 g, 260.2 mmol, 3.0 equiv.) dissolved in isopropyl alcohol (100 ml) at 40°C. 2-Fluorobenzyl bromide (10.5 ml_, 86.8 mmol, 1.0 equiv.) was added to the solution drop wise. The solution was allowed to stir at 40°C for 18 hours and progress was monitored by TLC (MeOH-CH₂CI₂, 1 :4). Aqueous hydrochloric acid (37%) was added dropwise to the mixture until the solution reached pH 6-5 (as determined using a pH probe). The suspension was then cooled in an ice bath, filtered and the filtrate concentrated in vacuo to give

(S)-1-(2-fluorobenzyl)pyrrolidine-2-carboxylic acid (2) as a yellow solid (18.4 g, 82%); m.p: 78-80°C.

¹H NMR (400 MHz, CDCI3) d: 8.15 (s, 1H), 7.54 (tt, J = 7.4, 1.6 Hz, 1H), 7.41-7.32 (m, 1H), 7.19-7.07 (m,2H), 4.38 (d, J= 13.1 Hz, 1H), 4.22 (d, J= 13.1 Hz, 1H), 3.74 (dd, J = 9.4,

6.0 Hz, 1 H), 3.61 (ddd, J= 10.8, 7.0, 3.7 Hz, 1H), 2.86 (td, J = 10.0, 7.7 Hz, 1H), 2.40-2.26 (m, 1 H), 2.26-2.18 (m, 1H), 1.95 (qt, J= 12.7, 4.5 Hz, 2H); ¹³C NMR (101 MHz, CDCI3) d: 171.5, 163.2, 132.4, 131.4, 124.1, 119.1, 115.2, 67.6, 53.1, 50.1, 29.6, 23.0; ¹⁹F NMR (376 MHz; CDCI3) d: -115.9; IR (FTIR) cm¹: 3464, 3014, 2965, 1732, 1625, 1445, 1371, 1230, 1116, 875; HRMS (m/z) [M+H]⁺ calcd for CI₂HI₅N0₂F [M+H]⁺ 224.1087, found

224.1092.

(S)-/V-(2-Benzoylphenyl)-1 -(2-fluorobenzyl)pyrrolidine-2-carboxamide {(2S)-FBPB) (3)}

Methanesulfonyl chloride (1.7 mL, 22.4 mmol, 1.0 equiv.) was added to a solution of

(S)-1-(2-fluorobenzyl)pyrrolidine-2-carboxylic acid (2) (5.0 g, 22.4 mmol) and

/V-methylimidazole (3.7 mL, 49.3 mmol, 2.2 equiv.) in CH2CI2 (45.0 mL) at 0°C. After 5 min. 2-aminobenzophenone (3.9 g, 20.2 mmol, 0.9 equiv.) was added. The reaction mixture was heated to 50 °C for 12 hours, cooled and then saturated aqueous sodium hydrogen carbonate solution (30.0 mL) was added. The two layers were separated, and the aqueous layer extracted with CH2CI2 (3 x 30.0 mL). The combined organic layers were dried (MgS0₄) and concentrated in vacuo. Purification by flash column chromatography (15% ethyl acetate- petroleum ether) followed by a recrystallisation with hexane and few drops of EtOAc gave the title compound (3) as a pale yellow crystals (6.9 g, 77%); m.p: 86-88°C.

¹H NMR (400 MHz, CDCI3) d: 11.40 (s, 1H), 8.54 (d, J= 8.4 Hz, 1H), 7.81-7.71 (m, 2H),

7.59 (td, J= 7.5, 1.4 Hz, 1H), 7.53-7.45 (m, 4H), 7.09 (q, J= 7.7, 7.1 Hz, 2H), 6.92 (td,

J= 7.5, 1.4 Hz, 1 H), 6.78 (dd, J = 9.9, 8.4 Hz, 1H), 3.89 (d, J= 13.3 Hz, 1H), 3.73 (d,

J= 13.4 Hz, 1 H), 3.35 (dd, J = 10.2, 4.5 Hz, 1H), 3.22 (ddd, J = 9.4, 6.3, 2.1 Hz, 1H),

2.46 (td, J= 9.3, 6.8 Hz, 1H), 2.29-2.18 (m, 1H), 1.94 (ddt, J= 12.4, 7.9, 4.2 Hz, 1H), 1.87-1.72 (m, 2H); ¹³C NMR (101 MHz, CDCI₃) d: 197.9, 174.4, 162.3, 159.8, 139.0, 138.6,

133.3, 132.5, 131.7, 131.7, 130.1, 128.9, 128.8, 128.3, 125.6, 124.9, 124.8, 123.9, 123.9,

122.3, 121.5, 115.2, 115.0, 67.9, 53.8, 52.0, 52.0, 31.1, 24.3; ¹⁹F NMR (376 MHz; CDCI3) d: -117.2; IR (FTIR) cm¹: 3269, 1686, 1637, 1561, 1515, 1448, 1292, 1121, 912, 697; HRMS (m/z) [M+H]⁺ calcd for C25H24FN2O2 [M+H]⁺ 403.1822, found 403.1826. (S)-({2-[1 -(2-Fluorobenzyl)-pyrrolidine-2-carboxamide]phenyl}phenylmethylene)-glycinato- N,N',N',O} nickel(ll) {Gly-Ni-2-FBPB complex, (4)}

(S)-/V-(2-Benzoylphenyl)-1 -(2-fluorobenzyl)pyrrolidine-2-carboxamide (2-FBPB) (3) (2.0 g,

4.9 mmol, 1.0 equiv.), Ni(NC>₃)₂-6H₂0 (2.8 g, 9.9 mmol, 2.0 equiv.) and glycine (0.9 g,

9.9 mmol, 2.0 equiv.) were dissolved in methanol (50.0 ml_, 0.1 M) at 50 °C. Potassium hydroxide (1 .9 g, 34.8 mmol, 7.0 equiv.) was added and the mixture was heated to 70°C for 1 hour. The reaction mixture was cooled and then concentrated. The resulting residue was taken up in water (40.0 ml.) and extracted with EtOAc (3 x 40.0 ml_). The combined organic layers were washed with saturated brine solution (3 ^c 30.0 ml_), dried (MgS0₄) and concentrated in vacuo to give the title compound (4) as a red crystalline solid (1.7 g, 70%); m.p: 125-127°C.

¹H NMR (400 MHz, CDCIs) d: 8.40-8.26 (m, 2H), 7.56-7.47 (m, 3H), 7.32 (td, J = 7.6, 5.5 Hz, 1 H), 7.28-7.20 (m, 3H), 7.15-7.07 (m, 2H), 7.02-6.94 (m, 1 H), 6.85-6.79 (m, 1 H), 6.72 (ddt, J = 9.0, 7.8, 1 .7 Hz, 1 H), 4.48 (d, J = 12.9 Hz, 1 H), 3.94 (d, J = 13.0 Hz, 1 H), 3.77-3.60 (m, 3H), 3.46 (dd, J = 10.8, 5.7 Hz, 1 H), 3.40-3.28 (m, 1 H), 2.65 (q, J = 6.9 Hz, 1 H), 2.47 (dq,

J = 12.3, 8.9 Hz, 1 H), 2.20-2.01 (m, 2H); ¹³C NMR (101 MHz, CDCIs) d: 181 .6, 179.3, 172.3, 163.2, 160.7, 141 .6, 134.4, 133.9, 133.9, 132.8, 131.5, 131 .3, 131 .2, 129.8, 129.5, 129.1 , 126.5, 125.8, 125.7, 124.6, 124.5, 124.2, 121.9, 121 .7, 121 .1 , 1 15.8, 1 15.5, 71 .2, 57.6, 56.5, 48.3, 30.4, 23.2; ¹⁹F NMR (376 MHz; CDCIs) d: -1 13.7; IR (FTIR) cm ¹: 2931 , 2362, 1671 , 1628, 1576, 1491 , 1445, 1356, 1243, 1 162, 1 109, 1068, 968, 726; HRMS (m/z) [M+H]⁺ calcd for C27H25FN3N1O3 [M+H]⁺ 516.1233, found 516.1235.

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]-phenyl}phenylmethylene)-(S)- (N-(tert- butoxy)hexanamide) glycinato-L/,L/',L/'',O} nickel(ll) (5a)

Freshly ground sodium hydroxide (0.24 g, 5.8 mmol, 5.0 equiv.) was taken up in DMF (4.0 ml.) with stirring at 0°C under an atmosphere of nitrogen. (S)-({2-[1 -(2-Fluorobenzyl)- pyrrolidine-2-carboxamide]phenyl}phenylmethylene)-glycinato- N,N',N",0} nickel(ll) (4)

(0.6 g, 1.1 mmol, 1.0 equiv.) was added and stirred for 2 minutes; the solution darkened in colour and the ice bath was removed. A solution of 6-bromo-/V-(ferf-butoxy)hexanamide (9a) (1 .2 g, 4.5 mmol 4.0 equiv.) was added to reaction mixture. The solution was left to stir for 25 minutes at room temperature, then quenched with the addition of water. The mixture was concentrated in vacuo, taken up in water (10.0 ml.) and extracted with dichloromethane (3 x 15.0 ml_). The combined organic extracts were washed with aqueous lithium chloride solution (5% v/v) (3 x 20.0 ml_), brine (3 x 20.0 ml_), dried (MgS0₄) and concentrated in vacuo. Purification by flash column chromatography (10% methanol-dichloromethane) gave the title compound (5a) as a deep red-orange solid (0.57 g, 74%); m.p: 182-184°C. ¹H NMR (400 MHz, CD₃OD) d: 8.34 (td, J = 7.2, 3.1 Hz, 1 H), 7.94 (dd, J = 8.6, 1.1 Hz, 1 H), 7.62-7.50 (m, 3H), 7.37 (dt, J = 7.3, 1.8 Hz, 1 H), 7.23 (dp, J = 7.3, 4.7, 4.1 Hz, 2H),

7.17-7.01 (m, 3H), 6.76-6.64 (m, 2H), 4.23-4.16 (m, 1 H), 3.88 (dd, J = 8.1 , 3.5 Hz, 1 H), 3.73 (d, J = 12.8 Hz, 1 H), 3.60 (q, J = 7.0 Hz, 4H), 3.36-3.28 (m, 2H), 2.78-2.64 (m, 2H), 2.24 (tdd, J = 17.5, 7.5, 5.2 Hz, 2H), 2.07 (q, J = 8.4, 7.9 Hz, 3H), 1 .67-1.52 (m, 4H), 1 .21 (s, 9H), 1 .16 ((t, J = 7.0 Hz, 2H); ¹³C NMR (101 MHz, CD₃OD) d: 180.9, 179.8, 172.1 , 163.6, 160.9, 141 .2, 134.1 , 133.3, 133.2, 132.1 , 131.5, 131.3, 131.8, 129.9, 129.1 , 129.0, 126.1 , 125.4, 124.6, 124.9, 124.2, 121.9, 1 15.2, 1 15.0, 88.9, 71.2, 56.1 , 48.2, 32.8, 30.1 , 28.3, 26.9, 25.4, 23.7 23.2; ¹⁹F NMR (376 MHz; CD₃OD) d: -1 13.8; IR (FTIR) cm ¹: 3287, 3091 , 2966, 2356, 1670, 1625, 1571 , 1487, 1442, 1351 , 1237, 1 160, 1076, 963, 722; HRMS (m/z) [M+H]⁺ calcd for C₃₇H₄₄FN₄Ni0₅ [M+H]⁺ 701 .2649, found 701 .2643.

(S)-2-amino-8-(fe/f-butoxyamino)-8-oxooctanoic acid (6a)

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]-phenyl}phenylmethylene)-(S)- (N-(tert-butoxy)hexanamide) glycinato-N,N’,N”,0} nickel(ll) (5a) (500 mg, 0.72 mmol,

1 equiv.) and 8-hydroxyquinoline (259 mg, 1 .79 mmol, 2.5 equiv.) were taken up in acetonitrile (10 ml_). Water (1 .5 ml.) was added and the mixture heated to 30°C for 18 h.

The mixture was filtered and the precipitate washed with water. The filtrate was extracted with ethyl acetate (3 x 20 ml_). The aqueous layer was lyophilised to give (S)-2-amino-8-(fe/f- butoxy)-8-oxooctanoic acid (6a) as a white fluffy powder (182 mg, 94%); m.p: 208-210°C.

¹H NMR (400 MHz, D₂0) d: 3.64 (t, J = 6.1 Hz, 1 H), 2.13 (t, J = 7.3 Hz, 2H), 1 .77-1 .75 (m, 2H), 1 .59-1 .50 (m, 2H), 1 .36-1.26 (m, 4H), 1.23 (s, 9H); ¹³C NMR (101 MHz, D₂0) d: 174.9, 169.9, 88.9, 55.8, 31.3, 28.3, 26.9, 23.2; IR (FTIR) cm ¹: 3432, 3261 , 2953, 1680, 1 156; HRMS (m/z) [M+H]⁺ calcd for Ci₂H₂₅N₂0₄ [M+H]+ 261 .1814, found 261.1823.

(S)-2-({[(9H-fluoren-9-yl)methoxy)carbonyl]amino}-8-(fe/f-butoxyamino)-8-oxooctanoic acid (7a)

(S)-2-amino-8-(fe/f-butoxy)-8-oxooctanoic acid (6a) (100 mg, 0.38 mmol, 1 equiv.) and potassium carbonate (85 mg, 0.77 mmol, 2 equiv.) were taken up in water (1.5 ml.) and cooled to 0°C. Fmoc succinimide (155 mg, 0.58 mmol, 1.5 equiv.) was dissolved in dioxane (3 ml.) and added dropwise to the aqueous solution over 20 mins. The reaction was warmed to room temperature and left to stir for 24 h. Excess water was added to the mixture then extracted with ethyl acetate (3 x 10 ml_). The combined organic layers were back extracted with sodium bicarbonate (3 x 20 ml.) then the aqueous acidified to pH 1 with 3 M

hydrochloric acid. The aqueous fractions were then extracted with ethyl acetate (3 x 30 ml_). The combined organic layers were washed with brine (3 x 30 ml_), dried (MgS0₄) and concentrated in vacuo. Purification was carried out by flash column chromatography (10% methanol-dichloromethane). Fractions containing the product were combined and the solvent removed in vacuo to give (S)-2-({[(9/-/-fluoren-9-yl)methoxy)carbonyl]amino}-8-(ferf- butoxyamino)-8-oxooctanoic acid (7a) as an off white powder (178 mg, 97%); m.p: 1 12- 1 14°C.

¹H NMR (400 MHz, CD₃OD) d: 7.78 (dt, J = 7.6, 1.0 Hz, 2H), 7.65 (dd, J = 7.6, 2.4 Hz, 2H), 7.40-7.35 (m, 2H), 7.29 (td, J = 7.2, 1 .0 Hz, 2H), 4.39 (dd, J = 10.6, 7.0 Hz, 1 H), 4.36-4.25 (m, 1 H), 4.19 (t, J = 6.8 Hz, 1 H), 4.02 (dd, J = 8.5, 4.7 Hz, 1 H), 2.68 (s, 1 H), 2.13 (t, J = 7.4 Hz, 2H), 1 .82 (p, J = 7.6, 7.1 Hz, 1 H), 1 .63 (h, J = 7.7, 6.8 Hz, 3H), 1 .42-1 .33 (m, 3H), 1.26 (s, 9H); ¹³C NMR (101 MHz, CD₃OD) d: 174.8, 169.8, 155.6, 143.7, 141 .2, 126.5, 126.3, 126.0, 125.9, 125.1 , 88.9, 54.9, 32.8, 28.3, 26.9, 23.2; IR (FTIR) cm ¹: 3331 , 2932, 2858, 1715, 1526, 1449, 121 1 , 1 151 ; HRMS (m/z) [M+H]⁺ calcd for C₂₇H₃₅N₂0₆ [M+H]⁺ 483.2495, found 483.2497; [a]_D ²⁰ = 10.3 (c = 0.5, CHCI₃).

6-Bromo-/V-(ferf-butoxy)hexanamide (9a)

Scheme 3 - Synthesis of 6-bromo-/V-(ferf-butoxy)hexanamide (9a)

6-Bromohexanoic acid (200 mg, 1.0 mmol, 1 equiv.) was dissolved in dry dichloromethane (10 ml.) at 0°C under a nitrogen atmosphere. O-(ferf-butyl) hydroxyl amine hydrochloride (192.2 mg, 1.5 mmol, 1.5 equiv.) was added to the solution followed by DMAP (25 mg, 0.25 mmol, 0.2 equiv.) were added and allowed to stir for 5 mins at 0°C. DCC (316 mg, 1 .5 mmol, 1.5 equiv.) was added and the mixture warmed to room temperature, then stirred for a further 20h at room temperature. The mixture was filtered and the filtrate washed with water (10 ml_). The two layers were separated and the organic layer was dried (MgS0₄), then the solvent removed in vacuo. Purification was carried out by flash column chromatography (20% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give 6-bromo-/V-(ferf-butoxy)hexanamide (9a) (145.8 mg, 55%) as a colourless oil.

¹H NMR (400 MHz, CDCIs) d: 7.60 (s, 1 H), 3.41 (t, J = 6.7 Hz, 2H), 2.36 (t, J = 7.4 Hz, 1 H), 2.14 (s, 1 H), 1 .92-1 .84 (m, 2H), 1.68 (q, J = 7.5 Hz, 2H), 1 .53-1.46 (m, 2H), 1 .27 (s, 9H); ¹³C NMR (101 MHz, CDCIs) d: 178.4, 77.3, 77.2, 77.0, 76.7, 33.7, 33.6, 33.4, 32.5, 32.4, 27.8, 27.6, 26.2, 24.6, 23.9; IR (FTIR) cm ¹: 3326, 3104, 1680, 1412, 784; HRMS (m/z) [M+H]⁺ calcd for CioH₂oBrN0₂ [M+H]⁺ 266.0755, found 266.0758. Synthesis of (S)-2-({[(9H-fluoren-9-yl)methoxy)carbonyl]amino}-8-(tert-butoxyamino)-8- oxooctanoic acid (7a) - Metathesis Approach

The preparation of the Fmoc-protected amino acid (S)-2-({[(9H-fluoren-9- yl)methoxy)carbonyl]amino}-8-(fe/f-butoxyamino)-8-oxooctanoic acid (7a) by metathesis is shown schematically below.

Scheme 4 - Synthesis of (S)-2-({[(9H-fluoren-9-yl)methoxy)carbonyl]amino}-8-(fe/f- butoxyamino)-8-oxooctanoic acid (7a)

H₂, Pd/C, eth

FmocHN

Fmoc-allylglycine-OBn (11 )

Fmoc-allylglycine-OH (1.0 g, 2.96 mmol, 1 equiv.) and sodium hydrogen carbonate (1.24 g, 14.82 mmol, 5 equiv.) were taken up in /V,/V-dimethylformamide (6 ml_, 0.5 M). Benzyl bromide (0.7 ml_, 5.92 mmol, 2 equiv.) was added and the reaction allowed to stir at room temperature for 20 h. Water (10 ml.) was added to crash out the product. The resulting solution was extracted with dichloromethane (3 x 10 ml_). The combined organic layers were washed with 5% aqueous lithium chloride solution (3 c 30 ml.) and brine (3 c 30 ml_), dried (MgS0₄) and concentrated to give a colourless oil. The oil was dissolved in chloroform and left to slowly crystallise, yielding Fmoc-allylglycine-OBn (11 ) as white crystals (1.1 g, 86%); m.p: 76-78°C. ¹H NMR (400 MHz, CDCI₃) d: 7.74 (d, J = 7.4 Hz, 2H), 7.60 (d, J = 7.1 Hz, 2H), 7.45-7.31 (m, 9H), 5.70-5.61 (m, 1 H), 5.37 (d, J = 7.7 Hz, 1 H), 5.28-5.09 (m, 4H), 4.51 -4.50 (m, 1 H), 4.41 (d, J = 7.4 Hz, 2H), 4.24 (t, J = 6.8 Hz, 1 H), 2.70-2.49 (m, 2H); ¹³C NMR (100MHz, CDCI₃) d : 171 .6, 155.7, 143.8, 141 .3, 135.2, 131.9, 128.6, 128.5, 128.4, 127.7, 127.0, 125.1 , 120.0, 1 19.5, 67.3, 67.1 , 53.3, 47.1 , 36.7; I R (FTIR) cm ¹: 3328, 28 3036, 2952, 1728, 1691 , 1536, 1450, 1259, 1 182; HRMS (m/z) [M+H]⁺ calcd for C₂₇H₂₆N0₄, 428.1862; found

428.1864.

(S)-2-({[(9H-fluoren-9-yl)methoxy)carbonyl]amino}-8-(fe/f-butoxyamino)-8-oxooctanoic acid (7a)

To a solution of Fmoc-allylglycine-OBn (11 ) (500 mg, 1.2 mmol, 1 equiv.) and N-(tert- butoxy)pent-4-enamide, (1 .4 g, 8.4 mmol, 7equiv.) in dichloromethane (20 ml_, 0.1 M) was added Grubbs second generation catalyst (51 mg, 0.06 mmol, 5 mol%). The solution was heated to reflux for 2 h., cooled to room temperature and the solvent evaporated in vacuo. The residue was taken up in ethanol (12 ml_, 0.1 M) followed by the addition of Pd/C (18 mg, 10% loading). The flask was flushed with hydrogen then left under a positive hydrogen pressure for 24 h. at room temperature. The solution was filtered through Celite and the solution concentrated in vacuo. Purification was carried out using flash column

chromatography (10% methanol-dichloromethane) to give the title compound (7a) as an off white powder.

¹H NMR (400 MHz, CD₃OD) d: 7.78 (dt, J = 7.6, 1.0 Hz, 2H), 7.65 (dd, J = 7.6, 2.4 Hz, 2H), 7.40-7.35 (m, 2H), 7.29 (td, J = 7.2, 1 .0 Hz, 2H), 4.39 (dd, J = 10.6, 7.0 Hz, 1 H), 4.36-4.25 (m, 1 H), 4.19 (t, J = 6.8 Hz, 1 H), 4.02 (dd, J = 8.5, 4.7 Hz, 1 H), 2.68 (s, 1 H), 2.13 (t, J = 7.4 Hz, 2H), 1 .82 (p, J = 7.6, 7.1 Hz, 1 H), 1 .63 (h, J = 7.7, 6.8 Hz, 3H), 1 .42-1 .33 (m, 3H), 1.26 (s, 9H); ¹³C NMR (101 MHz, CD₃OD) d: 174.8, 169.8, 155.6, 143.7, 141.2, 126.5, 126.3, 126.0, 125.9, 125.1 , 88.9, 54.9, 32.8, 28.3, 26.9, 23.2; IR (FTIR) cm ¹: 3331 , 2932, 2858, 1715, 1526, 1449, 121 1 , 1 151 ; HRMS (m/z) [M+H]⁺ calcd for C₂₇H₃₅N₂0₆ [M+H]⁺ 483.2495, found 483.2497; [a]_D ²⁰ = 10.3 (c = 0.5, CHCI₃).

Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b) - Schiff Base Approach

The preparation of the Fmoc-protected amino acid (S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b) is shown schematically below. Scheme 5 - (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b)

FmocHN CO,H

7b

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]-phenyl}phenylmethylene)-(S)- (heptan-2-one) glycinato-N,N’,N”,0} nickel(ll) (5b)

Freshly ground sodium hydroxide (0.24 g, 5.8 mmol, 5.0 equiv.) was taken up in DMF (4.0 ml.) with stirring at 0°C under an atmosphere of nitrogen. (S)-({2-[1 -(2-Fluorobenzyl)- pyrrolidine-2-carboxamide]phenyl}phenylmethylene)-glycinato- N,N',N",0} nickel(ll) (4)

(0.6 g, 1.1 mmol, 1.0 equiv.) was added and stirred for 2 minutes; the solution darkened in colour and the ice bath was removed. A solution of 7-bromoheptan-2-one (9b) (0.85 g,

4.5 mmol 4.0 equiv.) was added to reaction mixture. The solution was left to stir for 25 min. at room temperature, then quenched with the addition of water. The mixture was

concentrated in vacuo, taken up in water (10.0 ml.) and extracted with dichloromethane (3 x 20.0 ml_). The combined organic extracts were washed with aqueous lithium chloride solution (5% v/v) (3 x 25.0 ml_), brine (3 x 25.0 ml_), dried (MgS0₄) and concentrated in vacuo. Purification by flash column chromatography (7% methanol-dichloromethane) gave the title compound (5b) as a deep red-orange solid (0.6 g, 89%); m.p: 174-176°C.

¹H NMR (400 MHz, CD₃OD) d: 8.41 -8.30 (m, 1 H), 7.92 (dd, J = 8.5, 1 .3 Hz, 1 H), 7.64-7.50 (m, 3H), 7.38 (dt, J = 7.2, 1.8 Hz, 1 H), 7.21 (ddd, J = 7.0, 3.9, 2.0 Hz, 2H), 7.14 (ddd, J =

8.7, 6.9, 1.8 Hz, 1 H), 7.07 (ddt, J = 8.9, 7.2, 2.0 Hz, 2H), 6.74 (ddd, J = 8.1 , 6.9, 1 .2 Hz, 1 H), 6.67 (dd, J = 8.2, 1 .8 Hz, 1 H), 4.20 {dd, J = 12.9, 1.1 Hz, 1 H), 3.89 (dd, J = 8.0, 3.5 Hz, 1 H), 3.75 (dd , J = 12.9, 1 .1 Hz, 1 H), 3.67-3.51 (m, 2H), 3.35 (d, J = 6.2 Hz, 1 H), 2.79-2.62 (m, 2H), 2.49-2.37 (m, 2H), 2.34-2.20 (m, 2H), 2.20 (s, 3H), 2.16-1 .93 (m, 2H), 1 .69-1.56 (m, 2H), 1 .55-1 .43 (m, 2H), 1 .25-1 .06 (m, 2H);¹³C NMR (101 MHz, CD₃OD) d: 214.0, 182.2, 172.1 , 162.0, 143.1 , 135.0, 134.3, 133.0, 132.6, 132.5, 131 .1 , 130.2, 130.0, 129.1 , 128.5,

128.1 , 126.0, 124.3, 123.1 , 122.4, 1 17.0, 1 16.7, 72.3, 71 .5, 58.6, 57.3, 42.2, 36.4, 35.4,

29.8, 29.2, 26.0, 24.2, 24.0; ¹⁹F NMR (376 MHz; CD₃OD) d: -1 14.9.0; IR (FTIR) cm ¹: 2930, 2369, 1705, 1660, 1625, 1569, 1543, 1409, 1258, 1 155, 1 1 10, 1068, 1026, 970, 840, 750, 710, 650; HRMS (m/z) [M+Na]⁺ calcd for C35H38FN3N1O4 [M+Na]⁺ 650.1941 , found 650.1943.

(S)-2-amino-8-oxononanoic acid (6b)

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]-phenyl}phenylmethylene)-(S)- (heptan-2-one) glycinato -N,N’,N”,0} nickel(ll) (5b) (500 mg, 0.80 mmol, 1 equiv.) and 8-hydroxyquinoline (289 mg, 1.99 mmol, 2.5 equiv.) were taken up in acetonitrile (10 ml_). Water (1.5 ml.) was added and the mixture heated to 30°C for 18 h. The mixture was filtered and the precipitate washed with water. The filtrate was extracted with ethyl acetate (3 x 20 ml_). The aqueous layer was lyophilised to give (S)-2-amino-8-oxononanoic acid (6b) as a white fluffy powder (141 mg, 94%); m.p: 190-192°C.

¹H NMR (400 MHz, D₂0) d: 3.72 (t, J = 6.1 Hz, 1 H), 2.55 (t, J = 7.4 Hz, 2H), 2.10 (s, 3H),

1 .92-1 .74 (m, 2H), 1 .62-1.52 (m, 2H), 1 .45-1 .29 (m, 4H); ¹³C NMR (101 MHz, D₂0) d: 220.4, 174.4, 54.3, 41 .4, 35.6, 30.0, 27.9, 24.1 , 23.0; IR (FTIR) cm ¹: 2971 , 2934, 2865, 1705,

1579, 1510, 1462, 1441 , 1414, 1 184, 1 1 17, 1076, 972, 855, 810, 660, 552; HRMS (m/z) [M+Na]⁺ calcd for C1₀H1₉NO₃ [M+Na]⁺ 210.1 106, found 210.1 109.

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b)

(S)-2-amino-8-oxononanoic acid (6b) (200 mg, 1.1 mmol, 1 equiv.) and potassium carbonate (295 mg, 2.1 mmol, 2 equiv.) were taken up in water (1.5 ml.) and cooled to 0°C.

Fmoc-succinimide (557 mg, 1 .65 mmol, 1.5 equiv.) was dissolved in dioxane (3 ml.) and added dropwise to the aqueous solution over 20 mins. The reaction was warmed to room temperature and left to stir for 24 h. Excess water was added to the mixture then extracted with ethyl acetate (3 x 10 ml_). The combined organic layers were back extracted with sodium bicarbonate (3 c 20 ml.) then the aqueous acidified to pH 1 with 3 M hydrochloric acid. The aqueous fractions were then extracted with ethyl acetate (3 c 30 ml_). The combined organic layers were washed with brine (3 c 30 ml_), dried (MgS0₄) and

concentrated in vacuo. Purification was carried out by flash column chromatography (10% methanol-dichloromethane). Fractions containing the product were combined and the solvent removed in vacuo to give (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8- oxononanoic acid (7b) as an off white powder (432 mg, 96%); m.p: 104-106°C.

¹H NMR (400 MHz, CDCI₃) d: 7.74 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 6.6 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.4 Hz, 2H), 5.48 (s, 1 H), 4.37 (t, J = 7.7 Hz, 2H), 4.20 (t, J = 7.0 Hz, 1 H), 2.39 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.86 (d, J = 9.6 Hz, 1 H), 1 .75-1 .65 (m, 1 H), 1.59- 1 .51 (m, 2H), 1 .40-1 .24 (m, 4H); ¹³C NMR (101 MHz, CDCIs) d: 209.6, 176.8, 156.2, 143.9, 143.7, 141 .3, 127.7, 127.1 , 125.1 , 124.8, 120.0, 67.1 , 53.8, 47.2, 43.6, 43.5, 32.1 , 29.9, 28.6, 25.1 , 23.4; IR (FTIR) cm ¹: 3331 , 2932, 2858, 1715, 1526, 1449, 121 1 , 1 151 ; HRMS (m/z) [M+H]⁺ calcd for C₂₄H₂₈N0₅, 410.1967; found 410.1966; [a]_D ²⁰ = 17.3 (c = 0.5, CHC ).

Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b) - Metathesis Approach

The preparation of the (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b) by a metathesis approach is shown schematically below.

Scheme 6 - Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-8-oxononanoic acid (7b)

(S)-2-{[(9/-/-fluoren-9-yl)methoxy)carbonyl]amino}-8-oxononanoic acid (7b)

To a solution of Fmoc-allylglycine-OBn (11 ) (500 mg, 1.2 mmol, 1 equiv.) and hex-5-ene-2- one, (971 mg, 8.4 mmol, 7equiv.) in dichloromethane (20 ml_, 0.1 M) was added Grubbs second generation catalyst (51 mg, 0.06 mmol, 5 mol%). The solution was heated to reflux for 2 h, cooled to room temperature and the solvent evaporated in vacuo. The residue was taken up in ethanol (8 ml_, 0.1 M) followed by the addition of Pd/C (16 mg, 10% loading). The flask was flushed with hydrogen then left under a positive hydrogen pressure for 24 hours at room temperature. The solution was filtered through celite and the solution concentrated in vacuo. Purification was carried out using flash column chromatography (5% methanol-dichloromethane) to give the title compound (7b) as an off white powder m.p: 104- 106°C.

¹H NMR (400 MHz, CDCI₃) d: 7.74 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 6.6 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (t, J = 7.4 Hz, 2H), 5.48 (s, 1 H), 4.37 (t, J = 7.7 Hz, 2H), 4.20 (t, J = 7.0 Hz, 1 H), 2.39 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.86 (d, J = 9.6 Hz, 1 H), 1 .75-1 .65 (m, 1 H), 1 .59- 1 .51 (m, 2H), 1 .40-1 .24 (m, 4H); ¹³C NMR (101 MHz, CDCI₃) d: 209.6, 176.8, 156.2, 143.9, 143.7, 141.3, 127.7, 127.1 , 125.1 , 124.8, 120.0, 67.1 , 53.8, 47.2, 43.6, 43.5, 32.1 , 29.9,

28.6, 25.1 , 23.4; IR (FTIR) cm ¹: 3331 , 2932, 2858, 1715, 1526, 1449, 121 1 , 1 151 ; HRMS (m/z) [M+H]⁺ calcd for C₂₄H₂₈N0₅, 410.1967; found 410.1966; [a]_D ²⁰ = 17.3 (c = 0.5, CHCI₃).

Synthesis of (S)-2((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-8-oxodecanoic acid (7c)

The preparation of the Fmoc-protected amino acid (S)-2((((9/-/-Fluoren-9- yl)methoxy)carbonyl)amino)-8-oxodecanoic acid (7c) is shown schematically below.

Scheme 7 - Synthesis of (S)-2((((9/-/-Fluoren-9-yl)methoxy)carbonyl)amino)-8-oxodecanoic acid (7c)

FmocHN CO,H

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]-phenyl}phenylmethylene)-(S)- (octan-3-one) glycinato-/V,/V',/V” 0} nickel(ll) (5c)

Freshly ground sodium hydroxide (0.39 g, 7.8 mmol, 5.0 equiv.) was taken up in DMF (8.0 ml.) with stirring at 0°C under an atmosphere of nitrogen. (S)-({2-[1 -(2-Fluorobenzyl)- pyrrolidine-2-carboxamide]phenyl}phenylmethylene)-glycinato-/V,/V',/V",0} nickel(ll) (4) (1 .0 g, 1 .9 mmol, 1.0 equiv.) was added and stirred for 2 min, the solution darkened in colour and the ice bath was removed. A solution of 8-bromooctan-3-one (9c) (1 .6 g, 7.8 mmol,

4.0 equiv.) in dry DMF (10 ml.) was added to reaction mixture. The solution was stirred for 25 min. at room temperature, then quenched with the addition of water. The mixture was concentrated in vacuo, taken up in water (10.0 ml.) and extracted with dichloromethane (3 x 25.0 ml_). The combined organic extracts were washed with aqueous lithium chloride solution (5% v/v) (3 x 30.0 ml_), brine (3 x 30.0 ml_), dried (MgS0₄) and concentrated in vacuo. Purification by flash column chromatography (8% methanol-dichloromethane) gave the title compound (5c) as a deep red solid (1.1 g, 88%); m.p: 183-185°C.

¹H NMR (400 MHz, CD₃OD) d: 8.40-8.32 (m, 1 H), 7.93 (dd, J = 8.7, 1.1 Hz, 1 H), 7.65-7.50 (m, 3H), 7.38 (dt, J = 7.3, 1.8 Hz, 1 H), 7.24 (ddd, J = 7.2, 3.9, 2.0 Hz, 2H), 7.14 (ddd, J =

8.7, 6.9, 1.8 Hz, 1 H), 7.07 (ddt, J = 8.9, 7.2, 2.0 Hz, 2H), 6.74 (ddd, J = 8.1 , 6.9, 1.2 Hz, 1 H), 6.67 (dd, J = 8.2, 1 .8 Hz, 1 H), 4.20 {dd, J = 12.9, 1.1 Hz, 1 H), 3.89 (dd, J = 8.0, 3.5 Hz, 1 H), 3.75 (dd , J = 12.8, 1 .0 Hz, 1 H), 3.67-3.51 (m, 2H), 3.34 (d, J = 6.1 Hz, 1 H), 2.79-2.62 (m, 2H), 2.49-2.37 (m, 4H), 2.35-2.17 (m, 2H), 2.17-1 .94 (m, 2H), 1 .69-1 .56 (m, 2H), 1 .56-1 .42 (m, 2H), 1 .26-1 .06 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) d: 214.1 ,

182.3, 172.7, 162.0, 143.0, 135.1 , 134.5, 133.1 , 132.5, 132.5, 131 .1 , 130.3, 130.2, 129.2,

128.4, 128.2, 126.0, 124.9, 123.2,122.4, 1 17.1 , 1 16.9, 72.9, 71.5, 58.8, 57.8, 42.7, 36.6, 35.9, 31 .8, 29.6, 26.2, 24.8, 24.5, 8.1 ; ¹⁹F NMR (376 MHz; CD₃OD) d: -1 15.0; IR (FTIR) cm ¹: 2932, 2361 , 1705, 1667, 1636, 1589, 1543, 1489, 1443, 1335, 1258, 1 165, 1 1 1 1 ,

1065, 1026, 972, 849, 756, 710, 625, 563, 532, 501 , 417; HRMS (m/z) [M+Na]⁺ calcd for C H FN N O [M+Na]⁺ 664.2092, found 664.2084.

('S)-2-Amino-8-oxodecanoic acid (6c)

(S)-({2-[1 -(2-Fluorobenzyl)benzyl)pyrrolidine-2-carboxamide]phenyl}phenylmethylene)-(S)-8- oxooctylglycinato-/V,/V’,/V”,0}nickel(ll) (5c) (1.0 g, 1 .63 mmol, 1.0 equiv.) and

8-hydroxyquinoline (591 mg, 4.1 mmol, 2.5 equiv.) were taken up in acetonitrile (25 ml_). Water (3.5 ml.) was added and the mixture heated to 30°C for 18 h. The mixture was filtered and the precipitate washed with water. The filtrate was extracted with ethyl acetate

(3 x 40 ml_). The aqueous layer was lyophilised to give (S)-2-amino-8-oxodecanoic acid (6c) as a white powder (302 mg, 92%); m.p: 201 -203°C.

¹H NMR (400 MHz, D₂0) d: 3.72 (t, J = 6.1 Hz, 1 H), 2.56 (q, J = 7.4 Hz, 2H), 2.55 (t, J = 7.4 Hz, 2H), 1 .93-1 .76 (m, 2H), 1 .64-1 .49 (m, 2H), 1 .46-1 .25 (m, 4H), 1 .00 (t, J = 7.4 Hz, 3H);

¹³C NMR (101 MHz, D₂0) d: 220.1 , 174.5, 54.4, 41.7, 35.7, 30.1 , 27.9, 24.0, 23.0, 7.2; IR (FTIR) cm ¹: 2972, 2936, 2860, 1707, 1578, 1512, 1462, 1441 , 1414, 1375, 1348, 1317,

1 184, 1 1 17, 1076, 972, 852, 804, 660, 552; HRMS (m/z) [M+Na]⁺ calcd for C₁₀H₁₉NO₃

[M+Na]⁺ 224.1257, found 224.1255. (S)-2((((9/-/-Fluoren-9-yl)methoxy)carbonyl)amino)-8-oxodecanoic acid (7c)

(S)-2-amino-8-oxodecanoic acid (6c) (197 mg, 0.98 mmol, 1.0 equiv.) and potassium carbonate (271 mg, 2.0 mmol, 2.0 equiv.) were dissolved in water (4.5 mL) and the solution was cooled to 0°C. Fmoc succinimide (496 mg, 1 .5 mmol, 1 .5 equiv.) was dissolved in dioxane (9 mL) and added dropwise to the aqueous solution over 20 min. The reaction was warmed to room temperature and stirred for 24 h. Excess water was added to the mixture then extracted with ethyl acetate (3 x 10 mL). The combined organic phases were back extracted with sodium bicarbonate (3 c 20 mL) and the aqueous phases were acidified to pH 1 with 3.0 M aqueous HCI. The aqueous fractions were then extracted with ethyl acetate (3 c 30 mL). The combined organic layers were washed with brine (3 c 30 mL), dried (MgS0₄) and concentrated in vacuo. Purification was carried out by flash column chromatography (12% methanol-dichloromethane). Fractions containing the product were combined and the solvent removed in vacuo to give afford (S)-2((((9/-/-fluoren-9-yl)methoxy)carbonyl)amino)-8- oxodecanoic acid (7c) as an off-white powder (406 mg, 98%); m.p: 109-1 1 1 °C.

¹H NMR (400 MHz, CD₃OD) d: 7.80 (d, J = 7.5 Hz, 2H), 7.68 (t, J = 7.2 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 7.5, 1.1 Hz, 2H), 4.36 (d, J = 7.0 Hz, 2H), 4.23 (t, J = 7.0 Hz, 1 H), 4.13 (dd, J = 9.2, 4.8 Hz, 1 H), 2.45 (q, J = 7.3 Hz, 2H), 2.44 (t, 6.9 Hz, 2H), 1 .90-1 .76 (m,

1 H), 1 .74-1 .61 (m, 1 H), 1.63-1 .49 (m, 2H), 1 .47-1 .26 (m, 4H), 1 .00 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) d: 214.5, 176.0, 158.7, 145.4, 145.2, 142.6, 128.8, 128.2, 126.3, 120.9, 67.9, 55.2, 48.4, 42.9, 36.5, 32.5, 29.7, 26.7, 24.7, 8.1 ; IR (FTIR): 3341 , 3063, 2986, 2947, 2862, 1744, 1690, 1520, 1450, 1404.18, 1335, 1281 , 1227, 1 196, 1 173, 1088, 1042, 841 , 733, 617, 540, 424; HRMS (m/z) [M+Na]⁺ calcd for C₂₅H₂₉N0₅ [M+Na]⁺ 446.1938, found 446.1935. [a]_D ²⁴ = 1.9 (c = 1 .0, MeOH).

Scheme 8 - Synthesis of 7-bromoheptan-2-one (9b)

6-Bromo-/V-methoxy-/V-methylhexanamide (8a)

To a solution of 6-bromohexanoic acid (8) (2.50 g, 12.8 mmol, 1.0 equiv.) in dry CH2CI2 (25 ml.) at 25°C under nitrogen was added dropwise a solution of 2.0 M oxalyl chloride in dry CH2CI2 (1 .63 mL in 9.6 ml_, 19.2 mmol, 1.5 equiv.). After dropwise addition of 1.4 ml_, dry DMF was added (6.25 pl_) and gas evolution was initiated. Addition of the remaining oxalyl chloride solution was added. The reaction was stirred at 25°C for 3 h, then concentrated in vacuo. The residue was dissolved in dry CH₂Cl₂ (6.25 mL) and added dropwise over 5 min to a solution of N, O-dimethyl hydroxylamine hydrochloride (1.53 g, 15.7 mmol, 1 .23 equiv.) and Et₃N (5.35 mL) in CH2CI2 (50 mL), and the reaction was stirred for 22 h. The reaction mixture was then washed with water (30 mL), 1.0 M aqueous HCI (30 mL), 1 .0 M aqueous NaOH (30 mL) and brine (30 mL), dried with MgS0₄ and concentrated in vacuo to afford 6-bromo-/V-methoxy-/V-methylhexanamide (8a) (2.07 g, 68%) as a yellow oil.

¹H NMR (400MHz, CDCI₃) d: 3.67 (s, 3H), 3.40 (t, J = 6.8 Hz, 2H), 3.16 (s, 3H), 2.42 (t, J = 7.4 Hz, 2H), 1 .92-1 .83 (m, 2H), 1 .70-1 .59 (m, 2H), 1 .52-1.41 (m, 2H); ¹³C NMR (101 MHz, CDCI₃) d: 174.4, 61.3, 33.8, 33.7, 32.3, 31 .7, 28.0, 23.8; IR (FTIR): 2932, 2014, 1659, 1412, 1381 , 1319, 1265, 1 173, 1 1 1 1 , 995, 640, 563; HRMS (m/z) [M+Na]⁺ calcd for C₈Hi₆BrN0₂ [M+Na]⁺ 260.0257, found 260.0255.

7-bromoheptan-2-one (9b)

To a solution of 6-bromo-/V-methoxy-/V-methylhexanamide (8a) (1.0 g, 4.22 mmol,

1 .0 equiv.) in dry THF (12.5 mL) at 0°C under nitrogen was added a solution of 3.0 M methyl magnesium bromide in Et2<D (2.25 mL, 6.32 mmol, 1.5 equiv.). The reaction mixture was stirred at 0°C for 4.5 h. and a white precipitate formed. Then 1 .0 M aqueous HCI (12.5 mL) was cooled to 0°C and added slowly to the reaction mixture, which was then stirred for a further 0.5 h. THF was removed in vacuo and the aqueous phase was extracted with EtOAc (3 x 25 mL). The combined organic phases were then washed with brine (25 mL), dried with MgS0₄ and concentrated in vacuo. Purification was carried out by flash column

chromatography (30% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give 7-bromoheptan-2-one (9b) (640 mg, 79%) as a yellow oil.

¹H NMR (400MHz, CDCI₃) d: 3.42 (t, J = 6.6 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.21 (s, 3H), 1 .90-1.82 (m, 2H), 1.63-1.56 (m, 2H), 1.46-1 .36 (m, 2H); ¹³C NMR (101 MHz, CDCI₃) d: 21 1 .2, 42.0, 36.1 , 33.5, 32.6, 27.4, 23.1 ; IR (FTIR): 2930, 1715, 1458, 1360, 1255, 1 1 10, 980, 733, 640, 565; HRMS (m/z) [M+Na]⁺ calcd for C₇Hi₃BrO [M+Na]⁺ 215.0048, found 215.0045.

Scheme 9 - Synthesis of 8-bromooctan-3-one (9c)

8-Bromooctan-3-one (9c)

To a solution of 6-bromo-/V-methoxy-/V-methylhexanamide (8a) (1 .10 g, 4.62 mmol,

1 .0 equiv.) in dry THF (12.5 ml) at 0 °C under nitrogen was added a solution of 3.0 M ethyl magnesium bromide in Et₂0 (2.31 ml_, 6.93 mmol, 1.5 equiv.). The reaction mixture was stirred at 0°C for 4.5 h and a white precipitate formed. Then 1 .0 M aqueous HCI (12.5 ml.) was cooled to 0°C and added slowly to the reaction mixture, which was then stirred for a further 0.5 h. THF was removed in vacuo and the aqueous phase was extracted with EtOAc (3 x 25 ml_). The combined organic phases were then washed with brine (25 ml_), dried with MgS0₄ and concentrated in vacuo. Purification was carried out by flash column

chromatography (35% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give 8-bromooctan-3-one (9c) (905 mg, 75%) as a yellow oil.

¹H NMR (400MHz, CDCIs) d: 3.40 (t, J = 6.8 Hz, 2H), 2.43 (t, J = 7.3 Hz, 2H), 2.42 (q, J = 7.3

Hz, 2H), 1 .91 -1 .82 (m, 2H), 1 .65-1 .56 (m, 2H), 1 .50-1 .36 (m, 2H), 1.05 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCI3) d: 21 1 .4, 42.1 , 36.1 , 33.7, 32.7, 27.9, 23.0, 7.94; IR (FTIR): 2932, 1713, 1450, 1412, 1366, 1250, 1 1 1 1 , 1018, 980, 733, 640, 563; HRMS (m/z) [M+Na]⁺ calcd for C₈Hi₅BrO [M+Na]⁺ 229.0195, found 229.0198.

Synthesis of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert.-butoxycarbamoyl)-L-lysine

(23)

The preparation of the /V2-(((9H-fluoren-9-yl)methoxy)carbonyl)-/V6-(ferf-butoxycarbamoyl)- L-lysine (23) is shown schematically below.

Scheme 10 - Synthesis of /V-Boc-O-tosyl hydroxylamine (18)

16 90% 17 85% 18 Scheme 1 1 - Synthesis of /V2-(((9H-fluoren-9-yl)methoxy)carbonyl)-/V6-(ferf- butoxycarbamoyl)-L-lysine (23)

/V-Boc-O-tosyl hydroxylamin

Hydroxylamine hydrochloride (1.0 g, 14.4 mmol, 1 .0 equiv.) and sodium hydrogen carbonate (3.8 g, 46.0 mmol, 3.2 equiv.) were taken up in methanol (20 ml.) at room temperature.

Di-fe/f-butyl dicarbonate (3.4 g, 15.8 mmol, 1.1 equiv.) was added dropwise over a period of 30 min. After stirring the reaction mixture at 25°C for 5 h, methanol was removed under vacuum and the residue was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine and dried over Mg₂S0₄. The crude N- Boc- hydroxylamine (17) was obtained in (1 .7 g, 90%) as colourless oil.

The crude /V-Boc-hydroxylamine (1.6 g, 1 1.8 mmol, 1 equiv.) was dissolved in

dichloromethane (15 ml.) and 4-toluenesulfonyl chloride (2.5 g, 13.0 mmol, 1.1 equiv.) in dichloromethane (15 ml.) was added to the solution. After 5 mins, 4-methylmorpholine (1 .3 ml_, 1 1.8 mmol, 1 equiv.) was added to the solution. The reaction mixture was stirred at room temperature for 3h, then diluted with water, washed the organic layer with water and 5% citric acid solution in water, concentrated the mixture. Purification by flash column chromatography (40% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give /V-Boc-O-tosyl hydroxylamine (18) as white solid (2.9 g, 85%); m.p: 96-98°C. ¹H NMR (400 MHz, CDCI3) d: 7.88 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.46 (s, 3H), 1 .30 (s, 9H); ¹³C NMR (101 MHz, CDCI3) d: 154.2, 145.9, 130.6, 129.6, 128.1 , 83.8, 27.7, 21 .7; IR (FTIR) cm ¹: 3300, 2914, 1680, 1490, 1080; HRMS (m/z) [M+H]⁺ calcd for

C5H12NO3, 134.0817; found 134.0819.

Fmoc-Lys(Boc)-OBn (20)

Fmoc-Lys(Boc)-OH (1.0 g, 2.1 mmol, 1 equiv.) was dissolved in DMF (5 ml.) in a dried 100 ml. round bottom flask and sodium hydrogen carbonate (882.3 mg, 10.5 mmol, 5 equiv.) was added. Benzyl bromide (0.5 ml_, 4.3 mmol, 2 equiv.) was added to the reaction mixture. The reaction was left to stir overnight and then concentrated to remove DMF. Purification by flash column chromatography (50% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give Fmoc-Lys(Boc)-OBn (20) as white solid (1 .0 g, 87%); m.p: 70-72°C.

¹H NMR (400 MHz, CDCIs) d: 7.76 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.4 Hz, 2H), 7.45 -7.28 (m, 9H), 5.18 (q, J = 12.2 Hz, 2H), 4.51 (s, 1 H), 4.40 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.0 Hz,

1 H), 4.12 (q, J = 7.1 Hz, 2H), 3.05 (m, 2H), 1 .87-1 .71 (m,2H), 1.43 (m, 1 1 H), 1 .25 (t, J = 7.1 Hz); ¹³C NMR (101 MHz, CDCIs) d: 171 .1 , 156.0, 143.9, 143.7, 141.3, 135.3, 128.6, 128.5, 128.3, 127.7, 127.1 , 125.1 , 1 19.9, 79.2, 67.0, 60.4, 53.8, 47.2, 40.1 , 32.2, 28.4, 22.3, 21.0; IR (FTIR) cm ¹: 3333, 3061 , 2947, 1751 , 1685, 1517, 1477, 1388, 1 176, 1 101 , 883, 846,

800, 754, 707, 696, 621 ; HRMS (m/z) [[M+H]⁺ calcd for C₃₃H₃₉N₂O₆, 559.2808; found 559.2801 .

Fmoc-Lys-OBn-TFA (21 )

Fmoc-Lys(Boc)-OBn (20) (600 mg, 1.1 mmol, 1 equiv.) was dissolved in a solution of TFA-DCM (5 ml_, 1 :1 ). After stirring the reaction mixture at room temperature for 2h, the solvent was removed in vacuo to give Fmoc-Lys-OBn.TFA (20) (724 mg, 98%) as a colourless oil.

¹H NMR (400 MHz, CD₃OD) d: 7.79 (d, J = 7.5 Hz), 7.64 (d, J = 1 1 .2 Hz), 7.42-7.23 (m, 9H), 5.16 (q, J = 12.3 Hz, 2H), 4.41 (s, 1 H), 4.26 (dd, J = 15.3 Hz, 2H), 4.18 (m, J = 7.0 Hz, 1 H, CH), 2.86 (m, J = 7.5 Hz, 2H), 1 .79 (m, 2H), 1 .64 (m, J = 7.2 Hz, 2H), 1.43 (m, J = 7.1 Hz, 2H); ¹³C NMR (101 MHz, CD₃OD) d: 172.2, 157.3, 143.9, 143.7, 141 .2, 135.8, 128.1 , 127.9, 127.4, 126.8, 124.8, 124.7, 1 19.5, 66.5, 65.6, 53.8, 46.9, 39.1 , 30.5, 26.5, 22.4; IR (FTIR) cm ¹: 3417, 3063, 2965, 1685, 1517, 1477, 1 176, 1 101 , 784, 707, 696; HRMS (m/z) [M+H]⁺ calcd for C30H32F3N2O6, 573.2212; found 573.2213. Benzyl /V2-(((9H-fluoren-9-yl)methoxy)carbonyl)-/V6-(fe/f-butoxycarbamoyl)-L-lysinate (22)

To a stirred solution of Fmoc-Lys-OBn.TFA (21 ) (600.0 mg, 1.0 mmol, 1 equiv.) in DMF (5 ml.) was added potassium carbonate (361 .8 mg, 2.6 mmol, 2.5 equiv.). To this mixture was added /V-boc-O-tosyl hydroxylamine (18) (301 .4 mg, 1.1 mmol, 1 equiv.). After stirring at room temperature for 3h, another lot of /V-boc-O-tosyl hydroxylamine (301 .4 mg,

1 .1 mmol, 1 equiv.) added and stirred at room temperature for 3 h. Purification by flash column chromatography (65% ethyl acetate-petroleum ether). Fractions containing the product were combined and the solvent removed in vacuo to give the title compound (22) as a colourless oil (487 mg, 85%).

¹H NMR (400 MHz, CD₃OD) d: 7.79 (d, J = 7.6 Hz, 2H), 7.66 (d, J = 9.7 Hz, 2H), 7.42-7.24 (m, 9H), 5.15 (d, J = 9.1 Hz, 2H), 4.38 (s, 1 H), 4.22 (dd, J = 8.7 Hz, 2H), 4.13-4.05 (m, 1 H), 3.16 (m, J = 6.5 Hz, 2H), 1.78 (m, J = 38.2 Hz, 2H), 1 .52(m, 2H), 1.46 (m, J = 8.3 Hz, 2H), 1 .21 (s, 9H); ¹³C NMR (101 MHz, CD₃OD) d: 172.6, 162.5, 157.3, 143.9, 141 .2, 135.9,

128.2, 127.8, 127.4, 126.8, 124.9, 124.8, 1 19.5, 79.9, 66.6, 66.4, 54.2, 47.0, 38.8, 30.8, 29.4, 25.2, 22.8; IR (FTIR) cm ¹: 3390, 2869, 2512, 2225, 2071 , 1388, 1370, 1258, 1 122, 989, 858, 702; HRMS (m/z) [M+H]⁺ calcd for C₃₃H₄oN₃0₆, 574.2917; observed 574.2916.

/V2-(((9H-fluoren-9-yl)methoxy)carbonyl)-/V6-(ferf-butoxycarbamoyl)-L-lysine (23)

To a solution of compound 22 (450 mg, 0.78 mmol) in ethanol (4 ml.) was added Pd/C (8.3 mg, 0.08 mmol). The flask was flushed with hydrogen, then left under a positive hydrogen pressure for 24 hours at room temperature. The solution was then filtered through celite and the solution concentrated in vacuo. Purification was carried out using flash column chromatography (5% methanol-dichloromethane) to give the title compound (23) as white powder (247 mg, 65%); m.p: 104-106°C.

¹H NMR (400 MHz, CD₃OD) d: 7.67 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 1 1.7 Hz, 2H), 7.27 (t, J = 7.3 Hz, 2H), 7.19 (t, J = 7.1 Hz, 2H), 4.23 (dd, J = 6.7 Hz, 2H), 4.09 (m, 1 H), 3.95 (dd, J = 8.2, 4.8 Hz, 1 H), 3.09 (t, 2H), 1.67 (d, J = 58.5 Hz, 2H), 1 .42 (dd, J = 12.9, 6.4 Hz, 2H), 1.29 (dd, J = 15.3, 8.0 Hz, 2H), 1.1 1 (s, 9H); ¹³C NMR (101 MHz, CD₃OD) d: 162.4, 157.1 , 156.5, 143.8, 141 .2, 127.4, 126.8, 124.8, 1 19.5, 80.0, 66.5, 55.4, 47.1 , 38.7, 31.5, 29.5, 25.2, 22.6; IR (FTIR) cm ¹: 3392, 3022, 2880, 2526, 2232, 2078, 1710, 1392, 1368, 1265, 993, 698; HRMS (m/z) [M+H]⁺ calcd for C₂₆H₃₄N₃0₆, 484.2447; observed 484.2445; [a]_D ²⁰ = 19.8 (c = 0.5, CHCIs). Synthesis of Peptide Incorporating an Amino Acid Residue

The preparation of a peptide incorporating an amino acid residue having hydroxamic acid functionality within its side chain is shown below for a representative synthesis of peptide (H3K27).

Scheme 12 - Synthesis of peptide (H3K27)

General Comments

Peptides were synthesized on an automated microwave-assisted solid-phase peptide synthesizer (Biotage Initiator-·- Alstra machine) using a 15 mL Teflon reactor vessel at 100 pmol scale using Rink Amide Chemmatrix resin (substitution: 0.47 mmol/g). The resin was swollen for 20 min at ambient temperature in DMF (9 mL) prior to synthesis. Coupling Reactions

/V-oFmoc protected amino acids (0.2 M / DMF, 4 equivalents (eq.) relative to resin loading) were coupled to resin bound peptide amines using HCTU (0.5 M / DMF, 4.0 eq.) and DIPEA (2.0 M / NMP, 8 eq.) at 75°C for 5 min.

Fmoc-L-Arg(Pbf)-OH was coupled for 45 min at ambient temperature, followed by coupling at 75°C for 5 min. Fresh reagents were added and arginine was double coupled at 75°C for 5 min.

Once the coupling reagents had been drained, the peptide resin was then washed four times with DMF (4 x 9 ml.) for 45 s at ambient temperature.

Fmoc Deprotection Reactions

/V-oFmoc resin bound peptides were deprotected using two aliquots of 20% piperidine /

DMF (v:v) and 0.1 M Oxyma pure (9 ml.) for 3 min, followed by 10 min at ambient temperature. The peptide resin was then washed four times with DMF (4 x 9 ml.) for 45 s at ambient temperature.

Capping Reactions

N-Terminal acetyl capping was achieved using a mixture of DIPEA (2.0 M / NMP, 50 eq.) and acetic anhydride (AC2O, 5.0 M / DMF, 50 eq.) at ambient temperature for 10 min. Once capping reagents had been drained, the peptide resin was then washed four times with DMF (4 x 9 ml.) for 45 s at ambient temperature.

Peptide Resin Cleavage

Resin bound peptide was washed with DCM (4 x 9 ml.) for 45 s at ambient temperature. Synthesized peptides were cleaved from the resin using a cleavage cocktail of

TFA/TIPS/DCM (90:5:5) for 24-36 hours before being drained and the TFA blown off with a stream of nitrogen. The peptide was precipitated and washed three times in cold diethyl ether and spun down to a pellet before the diethyl ether removed and the peptide dried under a steady stream of nitrogen. Peptides were then purified by reverse phase high pressure liquid chromatography (RP-HPLC).

Peptide (H3K27)

The peptide H3K27 was prepared according to the general protocol described above, and as shown in Scheme 12. The collected peptide product was analysed. Ac-Lys-Ala-Ala-Arg-Aox-Ser-Ala-NH₂ (H3K27): 1.3 mg, (97% purity); HRMS (m/z) [M+H]⁺ calcd for C H N O , 830.4849; found 830.4815; analytical HPLC 5-50% acetonitrile/H20 (0.1% trifluoroacetic acid), 20 min gradient, TR = 10.88 min; 5-50% acetonitrile/water (0.1% trifluoroacetic acid), 25 min gradient, TR = 11.87 min.

The peptide was tested against the HDAC co-repressor complex MTA1 :RBBP4 complex. The I C50 was determined to be 390 nM.

References

All documents mentioned in this specification are incorporated herein by reference in their entirety.

Aillard et al. Org. Biomol. Chem. 2014, 12, 8775

Furumai et al. PNAS 2001 , 98, 87

Komatsu et al. Cancer Research 2001 , 61, 4459

Ma Angew. Chem. Int. Ed. 2003, 42, 4290

Watson et al. Nature Communications 2016, 1 1262

US 7550602

WO 2006/086600

WO 2007/047608

WO 2009/105824

WO 2015/106200

Claims

Claims:

1 . A compound of formula (I):

wherein:

-L⁴⁴- is a covalent bond or alkylene,

-R¹ is hydrogen or alkyl,

-NPro is an amino group protected with a base-labile protecting group,

-L- is alkylene, heteroalkylene, arylene or aralkylene

-X- is a covalent bond, -N(H)- or -N(R^N)-, where -R^N is alkyl,

-R² is hydrogen or alkyl, and

-R³ is C2-10 alkyl or heterocyclyl,

and salts, solvates and activated forms thereof.

2. The compound of claim 1 , wherein -L⁴⁴- is a covalent bond.

3. The compound of claim 1 or claim 2, wherein -R¹ is hydrogen.

4. The compound of any one the preceding claims, wherein -NPro is -NHFmoc.

5. The compound of any one the preceding claims, wherein -L- is alkylene, such as

C2-10 alkylene, such as C3-10 alkylene.

6. The compound of claim 5, wherein -L- is -(CH2)5- or -(CH2)4.

7. The compound of claim 5, wherein -L- is -(CH2)5- and -X- is a covalent bond.

8. The compound of claim 5, wherein -L- is -(CH2)4 and -X- is -N(H)- or -N(R^N)-, such as

-X- is -N(H)-.

9. The compound of any one the preceding claims, wherein -R² is hydrogen.

10. The compound of any one the preceding claims, wherein -R³ is C2-10 alkyl, such as

C4-10 alkyl.

1 1. The compound of any one the preceding claims, wherein -R³ is butyl (C4 alkyl), such as tert- butyl.

12. The compound of any one the preceding claims, which is a D-amino acid.

13. A compound of formula (II):

where -L⁴⁴-, -L-, -X-, -R¹, -R² and -R³ have the same meanings as the compound of formula (I) as set out in claims 1 to 12, and salts and solvates thereof.

14. A compound of formula (VIII):

wherein:

-L⁴⁴- is a covalent bond or alkylene,

-R¹ is hydrogen or alkyl,

-NPro is an amino group, protected with a base-labile protecting group,

-L- is alkylene, heteroalkylene, arylene or aralkylene,

-L^T is alkyl,

and salts, solvates and activated forms thereof.

15. The compound of claim 14, wherein -L⁴⁴- is a covalent bond.

16. The compound of claim 14 or claim 15, wherein -R¹ is hydrogen.

17. The compound of any one claims 14 to 16, wherein -NPro is -NHFmoc.

18. The compound of any one of claims 14 to 17, wherein -L- is alkylene, such as C2-10 alkylene, such as C3-10 alkylene.

19. The compound of claim 18, wherein -L- is -(Chh^- or -(Chh^.

20. The compound of any one of claims 14 to 19, wherein -L^T is methyl or ethyl.

21 . A compound of formula (IX):

where -L⁴⁴-, -L-, -L^T and -R¹ have the same meanings as the compound of formula (VII) as set out in claims 14 to 20, and salts and solvates thereof.

22. A peptide containing one or more amino acid residues obtainable from the compound of formula (I) according to any one of claims 1 to 12, and/or obtainable from the of formula (II) according to claim 13, and/or obtainable from the compound of formula (VIII) according to any one of claims 14 to 20, and/or obtainable from the compound of formula (IX) according to claim 21.

23. A method of forming an amide, the method comprising the step of reacting a compound of formula (I) according to any one of claims 1 to 12 or a compound of formula (VIII) according to any one of claims 15 to 20, with an amine, optionally in the presence of one or more amide coupling agents and base, thereby to yield the amide.

24. A method of:

(a) preparing a compound of formula (I), the method comprising the step of protecting the amino group of a compound of formula (II) according to claim 13 with a protecting group, thereby to yield the compound of formula (I); or

(b) preparing a compound of formula (VIII), the method comprising the step of protecting the amino group of a compound of formula (IX) according to claim 21 with a protecting group, thereby to yield the compound of formula (VIII).

25. A method of de-protecting a peptide, the method comprising the step of treating the peptide according to claim 22 with a deprotecting agent, such as acid, thereby to provide a peptide having one or more amino acid residues having a hydroxamic acid group or a hydroxyurea group.