WO2014108526A1 - Synthetic process for the manufacture of pipecolidepsin compounds - Google Patents

Synthetic process for the manufacture of pipecolidepsin compounds Download PDF

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Publication number
WO2014108526A1
WO2014108526A1 PCT/EP2014/050440 EP2014050440W WO2014108526A1 WO 2014108526 A1 WO2014108526 A1 WO 2014108526A1 EP 2014050440 W EP2014050440 W EP 2014050440W WO 2014108526 A1 WO2014108526 A1 WO 2014108526A1
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substituted
unsubstituted
formula
fmoc
independently selected
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PCT/EP2014/050440
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French (fr)
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Marta PELAY GIMENO
Yesica GARCíA-RAMOS
Judit Tulla Puche
Fernando Albericio Palomera
Mª Jesús MARTÍN LÓPEZ
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Pharma Mar, S.A.
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Publication of WO2014108526A1 publication Critical patent/WO2014108526A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • the present invention relates to synthetic processes, and in particular to the first synthetic process for the manufacture of Pipecolidepsins and analogues thereof, to intermediates useful for such syntheses, and to analogues of Pipecolidepsins.
  • Pipecolidepsins are a group of marine natural compounds which are disclosed to have cytotoxic properties. Examples of pipecolidepsins are provided by Pipecolidepsin A, B and C. Their isolation and cytotoxic properties against lung carcinoma (NSCLC), colorectal adenocarcinoma and breast adenocarcinoma human tumor cell lines have been described in WO 2010/070078. These natural compounds have been originally isolated from a sponge of the order Lithistida, family Neopeltidae, genus Homophymia, species Homophymia lamellosa Vacelet & Vasseur, 1971. However, the limited availability of natural material has resulted in the search for alternative synthetic methods being sought for the natural compounds and related analogs.
  • Pipecolidepsins A, B and C are represented in Figure I: Exocyclic linear region
  • Figure I the structure of pipecolidepsins is complex, comprising eight amino acids as a macrocyclic region (aal-aa8) and an exocyclic linear region of three amino acids (aa9-aal l) with a N-terminal polyketide-derived moiety.
  • the distinguishing structural characteristics of this family of cyclic depsipeptides include a preponderance of unusual amino acid residues, including non- proteinogenic amino acids, and an unique structural arrangement, a "head-to- side-chain" macrocyclic region through an ester bond, terminated in an exocyclic linear region with a N-terminal polyketide-derived moiety.
  • the inventors have developed for the first time a process for the synthesis of complex pipecolidepsin and related compounds, opening a new field of compounds with useful biological properties.
  • the present invention is directed to a process for the synthesis of pipecolidepsin compounds of general formula I:
  • Ri is selected from substituted or unsubstituted Ci-Cie alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each R2 and R3 is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl;
  • R 4 is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl
  • R11 is selected from hydrogen, COR a , COOR a , CONR a Rb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; n is 3 or 4;
  • Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
  • each Riy and R6 y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each F3 ⁇ 4y and R3 y is independently selected from hydrogen, COR a , COOR a , CONRaRb, S0 2 Ra, S0 3 Ra, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; each R 4y and Rs y is independently selected from hydrogen, OR c , NRaRb, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted
  • Rc is selected from hydrogen, COR a , COOR a , CONR a R b , S0 2 R a , S0 3 R a , substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; and each R a and Rb is independently selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the process comprises a macrolactamization step performed between positions 1 and 2 of an intermediate of formula Ila to give a compound of formula la in accord
  • R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3;
  • Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
  • each Riy, and R6 y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each R2y and R3 y is independently selected from hydrogen, COR a , COOR a , CONRaRb, S0 2 Ra, S0 3 Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or R2 y with R3 y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted 1 ,3- dioxolane; or each R2 y and R3 y is independently selected as
  • PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
  • X is selected from PG 1 and a group of formulae (f), (g) or (h):
  • Formula (f) Formula (g) Formula (h) Zi is selected from hydrogen and PG 1 ;
  • Z is selected from hydrogen, PG2 and a suitable insoluble support
  • Ri , R , R3, R4, Re, Rio and n are as defined for compounds of formula I;
  • R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3;
  • Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
  • PG2 is a a-carboxy protecting group
  • PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
  • the invention relates to the use of intermediates of formula II in the manufacture of compounds of formula I.
  • the invention relates to a process performed in solid phase for the synthesis of intermediates of formula II which comprise the step of forming an ester bond performed at the branching position 5 of a compound of formula III to provide an intermediate of formula lib in accordance with Scheme II:
  • Z is selected from PG2 and a suitable insoluble support; R3, R4, Re, Rio and n are as defined for compounds of formula I;
  • R5, and R11 are as defined for compounds of formula I or may be independently selected as PG3;
  • PG2 is a a-carboxy protecting group
  • PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
  • Alkyl groups may be branched or unbranched, and preferably have from 1 to about 18 carbon atoms.
  • One more preferred class of alkyl groups has from 1 to about 12 carbon atoms; and even more preferably from 1 to about 6 carbon atoms.
  • Methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl are particularly preferred alkyl groups in the compounds of the present invention.
  • Another preferred class of alkyl groups has from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms.
  • the term alkyl unless otherwise stated, refers to both cyclic and acyclic groups, although cyclic groups will comprise at least three carbon ring members.
  • Suitable aryl groups in the compounds of the present invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups.
  • Typical aryl groups contain from 1 to 3 separated and/ or fused rings and from 6 to about 18 carbon ring atoms.
  • Preferably aryl groups contain from 6 to about 10 carbon ring atoms.
  • Specially preferred aryl groups include substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted phenanthryl and substituted or unsubstituted anthryl.
  • Suitable heterocyclic groups include heteroaromatic and heteroalicyclic groups containing from 1 to 3 separated and/ or fused rings and from 5 to about 18 ring atoms. Preferably heteroaromatic and heteroalicyclic groups contain from 5 to about 10 ring atoms.
  • Suitable heteroaromatic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., coumarinyl including 8- coumarinyl, quinolyl including 8-quinolyl, isoquinolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benz
  • Suitable heteroalicyclic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., pyrrolidinyl, tetrahydrofuryl, dihydro furyl, tetrahydro thienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1 ,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3-
  • Suitable halogen groups or substituents in the compounds of the present invention include F, CI, Br, and I.
  • pharmaceutically acceptable salts refers to any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein.
  • non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts.
  • the preparation of salts can be carried out by methods known in the art. For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both.
  • nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred.
  • acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and -toluenesulfonate.
  • mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate
  • organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and -toluenesulfonate.
  • alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N- dialkylenethanolamine, triethanolamine and basic aminoacids salts.
  • Trifluoroacetate is one of the preferred pharmaceutically acceptable salts in the compounds of the invention.
  • any compound referred to herein is intended to represent such specific compound as well as certain variations or forms.
  • compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.
  • any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof.
  • the compounds of the present invention represented by the above described formulae I, II, and III may include enantiomers depending on their asymmetry or diastereoisomers.
  • Stereoisomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E) -isomer or (2)-isomer. If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same as, or different to, the stereoisomerism of the other double bonds of the molecule.
  • the single isomers and mixtures of isomers fall within the scope of the present invention.
  • any compound referred to herein may exist as tautomers.
  • the term tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily converted from one isomeric form to another. Common tautomeric pairs are amine-imine, amide- imidic acid, keto-enol, lactam-lactim, etc.
  • any compound referred to herein is intended to represent hydrates, solvates, and polymorphs, and mixtures thereof when such forms exist in the medium.
  • compounds referred to herein may exist in isotopically-labelled forms. All geometric isomers, tautomers, atropisomers, hydrates, solvates, polymorphs, and isotopically labelled forms of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.
  • R a refers to a specific monomer within the peptide chain. Therefore, “residue” refers to specific amino acids present in the peptide under construction which are numbered from aal to aal l for clarity purposes.
  • the term "moiety” refers to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures.
  • N-terminal fragment present in the linear region of compounds of general formula I is herein referred to as "N-terminal moiety”.
  • the processes according to the invention may make use of solid phase peptide synthesis (SPPS), i.e. where the peptide is bound to an insoluble support (R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149).
  • SPPS solid phase peptide synthesis
  • any un-reacted reagents left at the end of any synthetic step and soluble by-products can be removed by a simple wash procedure, greatly decreasing the time required for synthesis. Therefore, reagents can be used in excess allowing the reactions to proceed to completion in a minimum time resulting in faster synthesis of peptides with high purity.
  • the couplings typically proceed in a C ⁇ N terminal direction to allow the use of racemisation limiting a-amine protection for the activated species.
  • the insoluble support may be any support known in the art which is suitable for use in SPPS. See for example, James, I. W. Tetrahedron 1999, 55, 4855-4946; Guillier, F., Orain, D., Bradley, M. Chem. Rev. 2000, 100, 2091 - 2157 and Kates S.A. and Albericio, F. in Solid-Phase synthesis A practical guide; 1 st Ed. Boca Raton: CRC Press 2000. These references are incorporated here by reference in their entirety.
  • the insoluble support comprises a matrix polymer based resin, optionally with a linker group to which the growing peptide is attached during synthesis and which can be cleaved under desired conditions to release the target peptide from the support.
  • the matrix polymer based resin may be made from one or more polymers, copolymers or combinations of polymers, such as crosslinked polystyrene (PS)- , crosslinked polyamide (PA)-, crosslinked polyethylene glycol (PEG)-, crosslinked polylysine, composite PS-Polyethylene glycol (PEG)- or composite PA-Polyethylene glycol (PEG) -based resins .
  • Suitable linker groups may link the C-terminal function of the first amino acid residue to the insoluble support through an ester or an amide bond depending on the C-terminal functional group of the target peptide (respectively peptide-acid or peptide-amide).
  • Suitable linker groups may comprise trityl, amino or hydroxy moieties.
  • suitable insoluble supports for the syntyhesis of peptide-acids may include 4-hydroxymethylphenoxymethyl polystyrene Wang resin (Wang, S.S., J. Am. Chem. Soc. 1973, 95, 1328- 1333; J. Org. Chem. 1975, 40, 1235-1239; Albericio, F., Barany, G. Int. J. Pept. Protein Res. 1985, 26, 92-97), Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin (Atherton, E., et. al.
  • insoluble supports should not be interpreted as a limitation of the scope of the invention, since they have been mentioned as a mere illustration of suitable insoluble supports for peptide synthesis, but further insoluble supports having said function may be known by the skilled person in the art, and they are to be understood to be also encompassed by the present invention.
  • protecting groups may refer to side chain protecting groups (including protecting groups for hydroxyl, 1 ,2-diols, carboxyl, amino, amide and thiol groups), a-carboxy, or a-amino protecting groups for the amino acids used in the process according to the invention.
  • Suitable protecting groups are well known by the skilled person in the art. For instance, general reviews of protecting groups in organic chemistry are provided by Wuts, P. G. M. and Greene T. W. in Greene's Protective groups in Organic Synthesis, 4 th Ed. John Wiley & Sons Inc. 2006 and Kocienski, P. J.
  • An orthogonal protecting scheme is defined as one based on completely different classes of protecting groups such that each class of groups can be removed in any order and in presence of all other classes of protecting groups (Barany, G.; Albericio, F. J. Am. Chem. Soc. 1985, 107, 4936-4942). Therefore the a-amino and the side chain protecting groups are typically not the same. It also applies when a-carboxy protection is needed. In some cases, and depending on the type of reagents used in solid phase synthesis and other peptide processing, an amino acid may not require the presence of a side-chain protecting group.
  • a a-amino protecting group refers to a chemical moiety coupled to the alpha amino group of an amino acid. Typically, the a-amino protecting group is removed in a deprotection reaction prior to the coupling of the next amino acid to be incorporated to the growing peptide chain.
  • a-amino protecting groups are provided by 9-fluorenylmethoxycarbonyl (Fmoc), 2-(4- nitrophenylsulfonyl)ethoxycarbonyl (Nsc), ( 1 , l -dioxobenzo[fo]thiphene-2- yl)methyloxycarbonyl (Bsmoc), ( 1 , 1 -dioxonaphtol[ 1 ,2-b]thiophene-2- yl)mehyloxycarbonyl (a-Nsmoc), l -(4,4-dimethyl-2,6-dioxocyclohex- l -ylidine)- 3-methylbutyl (ivDde), 2,7-di-t-butyl-Fmoc (Fmoc*), 2-fluoro-Fmoc (Fmoc(2F)), 2-monoisooctyl-Fmoc (mi)
  • preferred a-amino protecting groups are selected from 9- fluorenylmethoxycarbonyl (Fmoc), 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc), ( 1 , l -dioxobenzo[fo]thiphene-2-yl)methyloxycarbonyl (Bsmoc), (1 , 1 - dioxonaphtol[ 1 ,2-b]thiophene-2-yl)mehyloxycarbonyl (a-Nsmoc), 1 -(4,4- dimethyl-2,6-dioxocyclohex- 1 -ylidine)-3-methylbutyl (ivDde), 2,7-di-t-butyl- Fmoc (Fmoc*), 2-fluoro-Fmoc (Fmoc(2F)), 2-monoisooctyl-Fmoc (mio-Fmo-Fm
  • More preferred a-amino protecting groups are selected from from 9-fluorenylmethoxycarbonyl (Fmoc), 2-(4- nitrophenylsulfonyl)ethoxycarbonyl (Nsc) and 2-fluoro-Fmoc (Fmoc(2F)). Most preferred a-amino protecting group is 9-fluorenylmethoxycarbonyl (Fmoc).
  • preferred a-amino protecting groups for pipecolic acid and pipecolic residue are selected from allyloxycarbonyl (Alloc), azidomethoxycarbonyl (Azoc) and -nitrobenzyloxycarbonyl (pNZ). More preferred a-amino protecting group is allyloxycarbonyl (Alloc).
  • a ⁇ -carboxy protecting group refers to a chemical moiety coupled to the alpha carboxy group of an amino acid.
  • the terminal a- carboxy group is usually linked to the insoluble support, and therefore, the linker/ handle acts as protecting group.
  • the linker/ handle acts as protecting group.
  • ⁇ -carboxy protection is needed.
  • Examples of a-carboxy protecting groups are provided by t-butyl (£Bu), benzyl (Bn), 2-chlorotrityl (2- Cl-Trt), 2,4-dimethoxybenzyl (Dmb), 2-phenylisopropyl (2-PhiPr), 5-phenyl-3,4- ethylenedioxythenyl derivatives (Phenyl-EDOT n ), 9-fluorenylmethyl (Fm), 4-(N- [ l -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl
  • a- carboxy protecting groups are selected from allyl and 1 , 1 -dimethylallyl (Dma) . More preferred a-carboxy protecting group is allyl.
  • a side chain protecting group refers to a chemical moiety coupled to the side chain of an amino acid that prevents a portion of the side chain from reacting with chemicals used in steps of peptide synthesis, processing etc.
  • a side chain protecting group may refer to a protecting group for hydroxyl (including 1 ,2-diols), carboxyl, amino, amide or thiol side chain groups.
  • hydroxyl side chain protecting groups are provided by benzyl (Bn), 2,6-dichlorobenzyl (Deb), 2-bromobenzyl (BrBn), o- nitrobenzyl (oNB), benzyloxycarbonyl (Z), 2-bromobenzyloxycarbonyl (BrZ), 4- (3,6,9-trioxadecyl)oxybenzyl (TEGBn), i-butyloxycarbonyl (Boc), Boc-N-methyl- N-[2-(methylamino)ethyl]carbamoyl (Boc-Nmec), 3-pentyl (Pen), cyclohexyl (cHx), i-butyl (iBu), allyl, triphenylmethyl (Trt), 2-chlorotriphenylmethyl (2-C1- Trt), t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TB
  • Preferred hydroxyl side chain protecting groups are selected from i-butyl (iBu), i- butyloxycarbonyl (Boc), triphenylmethyl (Trt), 2-chlorotriphenylmethyl (2-C1- Trt), and dimethyloxazolidines ( ⁇ ⁇ > ⁇ ⁇ ). More preferred are i-butyl (iBu), triphenylmethyl (Trt), and 2-chlorotriphenylmethyl (2-Cl-Trt). Most preferred is i-butyl (iBu).
  • Examples of protected 1 ,2-diol side chain groups include cyclic acetals and ketals, chiral ketones, cyclic ortho esters, silyl derivatives, cyclic carbonates and cyclic boronates.
  • the protecting group for the 1 ,2-diol can be selected from methylene, ethylidene, i- butylmethylidene, 1 -i-Butylethylidene, 1 -Phenylethylidene, 2-
  • the protecting group for the 1 ,2-diol can be selected from di-i-butylsilylene group, dialkylsilylene groups, 1 ,3-( 1 , 1 ,3,3- tetraisopropyldisiloxanylidene) derivative, 1 , 1 ,3,3-tetra-i- butoxydisiloxanylidene derivative, methylene-bis-(diisopropylsilanoxanylidene), l , l ,4,4-tetraphenyl- l ,4-disilanylidene, o-xylyl ether, 3,3 - oxybis(dimethoxytrityl) ether and l ,2-ethylene-3,3-bis(4 "4 -dimethoxytrityl) ether.
  • the protecting group for the 1 ,2-diol can be selected from methyl boronate, ethyl boronate, phenyl boronate and o- acetamidophenyl boronate.
  • Preferred 1 ,2-diol side chain protecting groups are selected from cyclic acetals and ketals.
  • More preferred are selected from ethylidene, i-butylmethylidene, 1 -i-Butylethylidene, 2,2,2-trichloroethylidene, acetonide, acetonide (isopropylidene), cyclopentylidene, cyclohexylidene and cycloheptylidene. Most preferred is acetonide.
  • amino side chain protecting groups are provided by formyl, benzyloxycarbonyl (Z) , 2- chlorobenzyloxycarbonyl (Cl-Z) , i-butyloxycarbonyl (Boc), cyclohexyloxycarbonyl (Hoc) , triphenylmethyl (Trt) , 4-methyltriphenylmethyl (Mtt) , monomethoxytriphenymethyl (Mmt) , dimethoxytriphenylmethyl (Dmt) , 9- fluorenylmethoxycarbonyl (Fmoc), 1 -(4,4-dimethyl-2,6-dioxoocyclohex- 1 - ylidene) -3 -methyl-butyl (ivDde), 2-(methylsulfonyl)ethoxycarbonyl (Msc), tetrachlorophthaloyl (TCP), allyloxycarbonyl (Alloc), 2-chlorobenzy
  • Preferred amino side chain protecting groups are selected from i-butyloxycarbonyl (Boc) and 4- methyltritryl (Mtt). More preferred is i-butyloxycarbonyl (Boc).
  • carboxy side chain protecting groups are provided by benzyl (Bn), cyclohexyl (cHx), i-butyl (iBu), -menthyl (Men), /3-3-methylpen-3-yl (Mpe), 2- phenylisopropyl (2-PhiPr), 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBn), 9- fluorenylmethyl (Fm), 4-(N-[ 1 -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3- methylbutyl]amino)benzyl (Dmab), allyl, -nitrobenzyl (pNB), 2- (trimethylsilyl) ethy
  • Preferred carboxy side chain protecting groups are selected from benzyl (Bn), cyclohexyl (cHx), i-butyl (iBu), -menthyl (Men), /3-3-methylpen-3-yl (Mpe), and 2-phenylisopropyl (2-PhiPr). More preferred is i-butyl (iBu).
  • amide side chain protecting groups are provided by 9-xanthenyl (Xan), triphenylmethyl (Trt), 4 -methyltriphenylmethyl (Mtt), cyclopropyldimethylcarbinyl (Cpd), 4, 4 '-dimethoxybenzhydryl (Mbh) and 2,4,6-trimethoxybenzyl (Tmob).
  • Preferred amide side chain protecting groups are selected from 9-xanthenyl (Xan), triphenylmethyl (Trt), and 4- methyltriphenylmethyl (Mtt). More preferred are triphenylmethyl (Trt) or 4- methyltriphenylmethyl (Mtt). Most preferred is triphenylmethyl (Trt).
  • thiol side chain protecting groups are provided by alkyl, acetamidomethyl (Acm), phenylacetamidomethyl (PhAcm), benzyl (Bn), -methylbenzyl (Meb), p- methoxybenzyl (Mob), 2,4,6,-trimethoxybenzyl (Tmob), o-nitrobenzyl (oNB), 9- fluorenylm ethyl (Fm), 9-xanthenyl (Xan), triphenylmethyl (Trityl, Trt), monomethoxytrityl (Mmt), S-alkyl disulfide 2,2,4,6,7-pentamethyl-5- dihydrobenzofuranylmethyl (Pmbf), 2-(2,4-dinitrophenyl)ethyl (Dnpe), 2- quinolyl, 4-picolyl, i-butyl ( £ Bu), 5-iert-butylmercapto (S'Bu), 1
  • Preferred thiol side chain protecting groups are selected from acetamidomethyl (Acm) , phenylacetamidomethyl (PhAcm) , benzyl (Bn) , -methylbenzyl (Meb) , triphenylmethyl (Trityl, Trt) and monomethoxytrityl (Mmt) . More preferred are selected from acetamidomethyl (Acm), phenylacetamidomethyl (PhAcm), triphenylmethyl (Trityl, Trt) and monomethoxytrityl (Mmt) . Most preferred is triphenylmethyl (Trityl, Trt) .
  • Suitable coupling reagents and additives for peptide synthesis are well known for the skilled person in the art. For instance a review for peptide coupling reagents is provided in Han, S-. Y. and Kim Y-. A. Tetrahedron 2004, 60, 2447-2467.
  • Examples of coupling reagents are ⁇ , ⁇ '- dicyclohexylcarbodiimide (DCC) , N-(3-dimethylaminopropyl)-N- ethylcarbodiimide (EDC) and its salts, l -[3-(dimethylamino)propyl] -3- ethylcarbodiimide methiodide (EDC methiodide), N,N'-diisopropylcarbodiimide (DIPCDI) , l -ieri-butyl-3 -ethyl carbodiimide, N-cyclohexyl-N'-(2- morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N'-di-tert- butylcarbodiimide, 1 ,3-Di-p-tolylcarbodiimide, ⁇ ,
  • HOTU hexafluorophosphate
  • TOTU N,N,N',N-tetramethyl- 0-(N-succinimidyl)u-ronium hexafluorophosphate
  • HSTU N,N,N',N-tetramethyl-0-(N-succinimidyl)u-ronium hexafluorophosphate
  • TSTU dipyrrolidino(N-succinimidyloxy)carbenium
  • HTPyU S-( 1 -oxido-2-pyridyl)- N,N,N',N'-tetramethylthiouronium tetrafluoroborate
  • TOTT l -[( l -(cyano-2- ethoxy-2-oxoeth
  • additives are 1 - hydroxybenzotriazole (HOBt), 1 -hydroxy-7-azabenzotriazole (HO At), 3,4- dihydro-3-hydroxy-4-oxo- l , 2, 3-benzotriazine (HODhbt), N-hydroxytetrazole, ethyl- 1 -hydroxy- l H- 1 , 2, 3-triazole-4-carboxylate (HOCt), ethyl 2-cyano-2- (hydroxyimino)acetate (Oxyma) and benzyltriphenylphosphonium dihydrogen trifluoride (PTF).
  • the compounds of formula I can be obtained synthetically from intermediates of formula Ila following a sequence of key reactions that comprise a macrolactamization step performed between positions 1 and 2 of the intermediate Ila to give a compound of formula la.
  • this process can further comprise the step of cleaving a compound of formula la from an insoluble support to which said compound may be attached through terminal R1 side chain group.
  • this process may further comprise the step of removing side chain protecting groups performed over a compound of formula la.
  • the macrolactamization step performed between positions 1 and 2 of the intermediate Ila to give a compound of formula la is typically effected using suitable coupling reagents.
  • suitable coupling reagents are as defined above, typically PyBOP/HOAt coupling system is used.
  • the macrolactamization step may be effected using solid or solution phase synthesis, being solid phase synthesis more preferred.
  • the step of cleaving a compound of formula la from a insoluble support to which said compound may be attached through terminal R12 side chain group is typically carried out following standard procedures known in solid phase peptide synthesis.
  • the precise conditions required to cleave said compound from the insoluble support may vary with the nature of the side chain functional group attached to the support and the linker group in the support and are similar to those known in the art.
  • the insoluble support is selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like; more preferably the insoluble support is selected from 4- hydroxymethylphenoxymethyl polystyrene Wang resin and Wang ChemMatrix resin; being 4 -hydroxymethylphenoxymethyl polystyrene Wang resin most preferred.
  • the cleavage step is typically effected by using acidic conditions; preferably by using TFA-H2O-TIS.
  • the step of removing side chain protecting groups performed over a compound of formula la is typically effected following standard procedures known in the art. The precise conditions may vary with the nature of the side chain protecting groups and are known in the art.
  • the side chain removal step may be effected before, at the same time or after the link between the insoluble support and the compound of formula la has been cleaved.
  • the step of cleaving said compound from the insoluble support and the step of removing side chain protecting groups are carried out by a single step.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein Ri is preferably selected from substituted or unsubstituted Ci-Cie alkyl and substituted or unsubstituted C2-C 18 alkenyl, which may be branched or unbranched. More preferred alkyl and alkenyl groups, which may be branched or unbranched, are those having from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12 , 13, 14, 15 or 16 carbon atoms.
  • Ri is a substituted alkyl group having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms wherein the substituent is OH. Even more preferably Ri is selected from 2 -hydroxy -pentyl, 2-hydroxy-hexyl, 2-hydroxy-heptyl, 2-hydroxy-octyl, 2-hydroxy-nonyl, 2- hydroxy-decyl, 2 -hydroxy -undecyl, 2-hydroxy-dodecyl, 2-hydroxy-tridecyl, 2- hydroxy-tetradecyl, 2-hydroxy-pentadecyl, 2-hydroxy-hexadecyl, 3-methyl-2- hydroxy-butyl, 4-methyl-2-hydroxy-pentyl, 5-methyl-2-hydroxy-hexyl, 6- methyl-2-hydroxy-heptyl, 7-methyl-2-hydroxy-octyl, 8 -methyl-2 -hydroxy - nonyl, 9 -methyl-2 -hydroxy-decyl, 10-
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein R2 and R3 are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • R2 is selected from hydrogen and a substituted alkyl having 1 carbon atom wherein substituents for the alkyl group are selected from OR', COOR', CONHR' and CON ⁇ wherein R' is as defined above.
  • R 2 is selected from hydrogen, -CH 2 COOR', -CH R ⁇ COOR', CH2CONHR', -CH 2 CON(R')2, -CHiOR ⁇ CONHR' and -CHiOR ⁇ CONiR ⁇ , wherein R' is as defined above.
  • R2 is selected from hydrogen, - CH2COOH, -CH(OH)COOH, -CH2CONH2, and -CH(OH)CONH 2 .
  • R3 is a substituted alkyl having 2, 3 or 4 carbon atoms, which may be branched or unbranched, wherein the substituents for the alkyl group are selected from -CONHR' and CONiR ⁇ wherein R' is as defined above.
  • R 3 is selected from -CH 2 CH 2 CONHR', -CHaCHaCONiR'Ja, - CH(CH 3 )CH 2 CONHR', -CHiCHaJCHaCONiR'Ja, -CH 2 CH(CH 3 )CONHR', CHaCHiCHaJCONiR'Ja, CH(CH 3 )CH(CH 3 )CONHR' and -CHiCHaJCHiCHaJCONiR'Ja wherein R' is as defined above.
  • R3 is selected from - CH2CH2CONH2, -CH(CH 3 )CH 2 CONH 2 , -CH 2 CH(CH 3 )CONH 2 and CH(CH 3 )CH(CH 3 )CONH 2 .
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein R 4 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferably R 4 is an alkyl group, which may be branched or unbranched, having 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • R 4 is selected from methyl, ethyl, n-propyl, iso-propyl, butyl, including n-butyl, tert- butyl, sec-butyl and iso-butyl, and pentyl, including n-pentyl, 1 -methyl-butyl, 2 -methyl-butyl, 3 -methyl-butyl, 1 , 1 -dimethyl-propyl, 1 ,2-dimethyl-propyl, 1 - ethyl-propyl, neopentyl and cyclohexyl; being methyl and 1 ,2-dimethyl-propyl the most preferred.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein R5 is independently selected from hydrogen, substituted or unsubstituted C 1-C6 alkyl, COR a , and COOR a , wherein R a is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl.
  • Particularly preferred R a is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably R5 is hydrogen.
  • R a and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n- propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso- butyl. More preferably R6 and R7 are hydrogen.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein Re and Rio are each independently selected from substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred alkyl groups, which may be branched or unbranched, are those having 1 , 2, 3, or 4 carbon atoms; being methyl and ethyl the most preferred. Preferably Re and Rio have different meaning in the compounds of the invention.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein Rn is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. More preferably Rn is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. Even more preferably Rn is independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl; being hydrogen the most preferred group.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein Rg and R12 are each independently selected from NR a Rb and OR c , wherein R c is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, COR a , and COOR a , and wherein R a and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl.
  • R a and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl. More preferably R c is hydrogen.
  • Rg and R12 are each independently selected from OH and NH2.
  • Particularly preferred processes according to invention are those that provide compounds of formula I wherein Y is selected from a group of formulae (a), (b), (c), (d) and (e); wherein particularly preferred Ri y and R6 y are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • each Riy and R6 y is independently selected from hydrogen, -CH2CONHR', - CHaCONiR'Ja -CHaCHaCONHR', -CHaCHaCONiR'Ja, - CH 2 (OR') and - CHfOROCHa.
  • R ly is selected from -CH 2 CONH 2 , -CH 2 CH 2 CONH 2 and -CH(OH)CH 3 .
  • R 6y is - CH 2 OH.
  • R 2y and R3 y are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, COR a and COOR a , wherein R a is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl.
  • Particularly preferred R a is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably F3 ⁇ 4y and R3 y are hydrogen.
  • each R 4y and Rs y is independently selected from hydrogen, substituted or unsubstituted C 1 -C6 alkyl, NR a Rb and OR c , wherein Rc is selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, COR a , and COORa, and wherein R a and Rb are each independently selected from hydrogen and substituted or unsubstituted C 1 -C 12 alkyl.
  • R a and Rb are each independently selected from hydrogen and substituted or unsubstituted C 1 -C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl.
  • Particularly preferred R c is hydrogen. More preferably each R 4y and Rs y is independently selected from hydrogen, NRaRb, OR c , and a substituted alkyl group having 1 or 2 carbon atoms wherein the substituent is OH. Most preferred each R 4y and Rs y is independently selected from hydrogen, N3 ⁇ 4 , OH and -CH2OH; or
  • More preferred processes according to the invention are those that provide compounds of formula I wherein Y is selected from a group of formulae (a), (c) and (e).
  • X is a group of formula (h): O R 2 o
  • particularly preferred Ri is selected from substituted or unsubstituted Ci-Cie alkyl and substituted or unsubstituted C2-C 18 alkenyl, which may be branched or unbranched. More preferred alkyl and alkenyl groups, which may be branched or unbranched, are those having from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms.
  • substituents for the above mentioned alkyl and alkenyl groups are selected from OR', OSO2R', OSO3R', halogen, OCOR', OCOOR', OCONHR', OCONiR'Ja, CONHR', and CONfROa, wherein each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; and even more preferred the substituent is OH.
  • Ri is a substituted alkyl group having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms wherein the substituent is OH. Even more preferably Ri is selected from 2 -hydroxy -pentyl, 2-hydroxy-hexyl, 2-hydroxy-heptyl, 2-hydroxy-octyl, 2-hydroxy-nonyl, 2- hydroxy-decyl, 2 -hydroxy -undecyl, 2-hydroxy-dodecyl, 2-hydroxy-tridecyl, 2- hydroxy-tetradecyl, 2-hydroxy-pentadecyl, 2-hydroxy-hexadecyl, 3 -methyl-2 - hydroxy-butyl, 4-methyl-2-hydroxy-pentyl, 5-methyl-2-hydroxy-hexyl, 6- methyl-2-hydroxy-heptyl, 7-methyl-2-hydroxy-octyl, 8 -methyl-2 -hydroxy - nonyl, 9-methyl-2-hydroxy-decyl,
  • R2 and R3 are each independently selected from hydrogen, and substituted or unsubstituted Ci- C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2 , 3 , 4, 5 or 6 carbon atoms.
  • R2 is selected from hydrogen, -CH 2 COOR', -CHfOROCOOR', -CH 2 CONHR', and - CHiOR'JCONHR', wherein R' is selected from hydrogen and a hydroxyl side chain protecting group when R' is linked to an O atom, a carboxy side chain protecting group when R' is linked to a COO- group or an amide side chain protecting group when R' is linked to a CONH- group.
  • R3 is selected from -CH 2 CH 2 CONHR', -CH(CH 3 )CH 2 CONHR', -CH 2 CH(CH 3 )CONHR' and -CH(CH 3 )CH(CH 3 )CONHR', wherein R' is selected from hydrogen and an amide side chain protecting group.
  • the different side chain protecting groups are selected following the guidance and preferences given above.
  • R 4 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred R 4 is an alkyl group, which may be branched or unbranched, having 1 , 2, 3, 4, 5 or 6 carbon atoms.
  • R 4 is selected from methyl, ethyl, n-propyl, iso-propyl, butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl, and pentyl, including n- pentyl, 1 -methyl-butyl, 2 -methyl-butyl, 3 -methyl-butyl, 1 , 1 -dimethyl-propyl, 1 ,2 -dimethyl-propyl, 1 -ethyl-propyl, neopentyl and cyclohexyl; being methyl and 1 ,2 -dimethyl-propyl the most preferred.
  • R5 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, COR a , and COORa, wherein R a is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl.
  • Particularly preferred R a is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl.
  • R5 is PG3.
  • PG3 is a side chain protecting group and is selected following the guidance and preferences given above.
  • Re and Rio are each independently selected from substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred alkyl groups, which may be branched or unbranched, are those having 1 , 2, 3, or 4 carbon atoms; being methyl and ethyl the most preferred. Preferably Re and Rio have different meaning in the compounds of the invention.
  • R9 is selected from NR a Rb and OR c , wherein R c is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, COR a , and COOR a , and wherein R a is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl and PG3.
  • More preferred alkyl groups are substituted or unsubstituted C1-C6 alkyl; and even more preferred alkyl groups are each independently selected from methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably each Rb and R c is independently selected from hydrogen and PG3. More preferably R9 is selected from OR c and NHRb wherein each Rb and R c is independently selected from hydrogen and PG3. PG3 is a side chain protecting group and is selected following guidance and preferences given above.
  • Rn is selected from hydrogen, PG3 and substituted or unsubstituted C1-C12 alkyl. More preferably Rn is independently selected from hydrogen, PG3 and substituted or unsubstituted C1-C6 alkyl. Even more preferably Rn is independently selected from hydrogen, PG3, methyl, ethyl, n-propyl, iso-propyl and butyl, including n- butyl, ieri-butyl, sec-butyl and iso-butyl; being PG3 the most preferred group. PG3 is a side chain protecting group and is selected following the guidance and preferences given above.
  • R12 is selected from NR a Rb and OR c , wherein R c is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, a suitable insoluble support, COR a , and COORa, and wherein Ra is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, PG3 and a suitable insoluble support.
  • More preferred alkyl groups are substituted or unsubstituted C1-C6 alkyl; and even more preferred alkyl groups are each independently selected from methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably each Rb and Rc is independently selected from PG3 and a suitable insoluble support. More preferably R12 is selected from OR c and NHRb wherein each Rb and R c is independently selected from PG3 and a suitable insoluble support. PG3 is a side chain protecting group and is selected following the guidance and preferences given above.
  • the suitable insoluble support is preferably selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like.; more preferably the insoluble support is selected from 4- hydroxymethylphenoxymethyl polystyrene Wang resin and Wang ChemMatrix resin; being 4-hydroxymethylphenoxymethyl polystyrene Wang resin most preferred.
  • particularly preferred Y is selected from formulae (a), (b) , (c), (d) and (e); wherein particularly preferred Ri y and R6 y are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2 , 3 , 4, 5 or 6 carbon atoms.
  • each Ri y and R6 y is independently selected from hydrogen, -CH 2 CONHR', -CH 2 CH 2 CONHR', - CH 2 (OR') and
  • R' is independently selected from hydrogen and a hydroxyl side chain protecting group when R' is linked to an O atom or an amide side chain protecting group when R' is linked to a CONH- group; most preferred R' is independently selected from a hydroxyl side chain protecting group and an amide side chain protecting group.
  • Most preferred Ri y is selected from - CH 2 CONHR', -CH 2 CH 2 CONHR' and -CHiOR CHa, wherein R' is selected from a hydroxyl side chain protecting group and an amide side chain protecting group.
  • R6y is - CH 2 (OR'), wherein R' is a hydroxyl side chain protecting group.
  • R 2y and R3y are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, COR a and COOR a , wherein R a is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl.
  • Particularly preferred R a is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl.
  • R 2y and R3y are each independently selected as PG3, even more preferred R 2y and R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted dioxolane; being 1 , 1 -dimethyl- 1 ,3 -dioxolane most preferred.
  • PG3 is a side chain protecting group and is selected following guidance and preferences given above.
  • each R 4y and Rs y is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, NRJ3 ⁇ 4 and OR c , wherein Rc is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, PG3, CORa, and COOR a , and wherein R a is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl and PG3. More preferably R a is selected from hydrogen and substituted or unsubstituted Ci-C-6 alkyl; and even more preferred is hydrogen.
  • each Rb and R c is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl and PG3; and even more preferred are each independently selected from hydrogen and PG3. More preferably each R 4y and Rs y is independently selected from hydrogen, NHRb, OR c and a substituted alkyl group having 1 or 2 carbon atoms wherein the substituent is OR c ; wherein each R c and Rb is independently selected from hydrogen and PG3.
  • PG3 is a side chain protecting group and is selected following guidance and preferences given above; or
  • particularly preferred Zi is hydrogen and particularly preferred Z2 are hydrogen and a suitable insoluble support. Most preferred Z is hydrogen.
  • More preferred compounds of formula II are those of formula Ila wherein X is a group of formula (h) and Y is a group selected from the group of formulae (a), (c) and (e).
  • Suitable starting materials for the synthesis of the intermediates of formula II include proteinogenic and non-pro teinogenic amino acids. Such starting materials may be either commercially available or synthetically prepared as previously described or as decribed herein. See for example, J. Org. Chem. 2003, 68, 7841 ; Tetrahedron 2001 , 57, 6353; Org. Lett. 2000, 2, 4157; J. Org. Chem 2006, 71 , 6351 and Amino Acid 2010, 39, 161 , which are incorporated herein by reference in their enterity.
  • intermediates of formula II are obtained by a process performed in solid phase following a sequence of key reactions that comprise a step of forming an ester bond performed at the branching position 5 of a compound of formula III to provide an intermediate of formula lib wherein X is PG 1.
  • this process can further comprise the step of removing terminal PG 1 group of the exocyclic chain and elongating said exocyclic chain performed over a compound of formula lib wherein X is PG 1 to provide a compound of formula lib wherein X is a group of formula (f) ; or performed over a compound of formula lib wherein X is a group of formula (f) to provide a compound of formula lib wherein X is a group of formula (g); or performed over a compound of formula lib wherein X is a group of formula (g) to provide a compound of formula lib wherein X is a group of formula (h).
  • this process further comprises the step of removing PG 1 group performed over an intermediate of formula lib wherein X is a group of formula (h) and Z2 is selected from PG2 and a suitable insoluble support.
  • this process can further comprise the step of removing PG2 group performed over an intermediate of formula lib wherein X is a group of formula (h) and Z 2 is PG2.
  • this process can further comprise the step of cleaving a compound of formula lib wherein X is a group of formula (h) and Z2 is a suitable insoluble support.
  • this process further comprises the step of elongating the peptidic chain performed over conveniently protected aal residue of the macrocyclic region which is attached to a suitable insoluble support to provide a compound of formula III.
  • this process further comprises the step of attaching conveniently protected aa 1 amino acid of the macrocyclic region to a suitable insoluble support to be used as starting point for elongating the peptidic chain to provide a compound of formula III .
  • the step of forming the ester bond is performed between secondary hydroxyl group at at branching position 5 of a compound of formula III and free carboxy group of a-amino protected pipecolic acid to provide an intermediate of formula lib wherein X is PG l .
  • This step is typically effected using suitable coupling reagents and may required to apply temperature depending on the esteric hindrance around the branching position 5.
  • this step is effected using a carbodiimide in the presence of catalytic amounts of a base as coupling reagents; being DIPCDI in the presence of catalytic amounts of DMAP more preferred.
  • this step is effected at a temperature from about 25 to about 65 °C; being a temperature from about 35 to about 55 °C more preferred.
  • Suitable a-amino protecting groups (PG l) to be used in pipecolic acid are selected following guidance and preferences given above.
  • the exocyclic chain elongation step performed over a compound of formula lib wherein X is selected from PG 1 and a group of formulae (f) or (g) is typically effected by cycles of a-amino deprotection and coupling reactions in a C ⁇ N terminal direction to finally provide a compound of formula lib wherein X is a group of formula (h).
  • the protecting groups for a-amino and for side chain functionalities that may be present are chosen following the guidance and preferences given above.
  • the coupling reactions are carried out using suitable coupling reagents. Suitable coupling reagents are selected following guidance given above, typically HATU/HOAt or PyBOP/HOAt coupling systems are independently used in each coupling reaction.
  • the last amino acid and N- terminal moiety of the exocyclic chain are incorporated as one unit. More preferably, the last amino acid and terminal moiety are incorporated as one unit using a phosphonium salt together with HOAt additive as coupling system; being PyBOP/HOAt coupling system most preferred.
  • PG 1 The step of removing PG 1 group performed over a compound of formula lib wherein X is a group of formula (h) and Z2 is selected from PG2 and a suitable insoluble support is typically effected by standard procedures known in the art. The precise conditions required may vary depending the nature of PG 1. PG 1 is selected following guidance and preferences given above.
  • the step of removing PG2 group performed over a compound of formula lib wherein X is a group of formula (h) and Z2 is PG2 is typically effected by standard procedures known in the art. The precise conditions required may vary depending the nature of PG2. PG2 is selected following guidance and preferences given above.
  • the step of cleaving a compound of formula lib wherein X is a group of formula (h) and Z2 is a suitable insoluble support from said insoluble support is typically carried out following standard procedures known in solid phase peptide synthesis.
  • the precise conditions required to cleave said compound from the insoluble support may vary with the nature of the support and the linker group in the support and are similar to those known in the art.
  • the insoluble support to be used in Z2 is selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like.; more preferably the insoluble support is selected from HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, benzhydryl bromide resins, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix; being benzhydryl bromide resins and 2-chlorotritylchloride (CTC) Barlos
  • the steps of removing PG 1 and PG2 groups performed over an intermediate of formula lib wherein X is a group of formula (h) and Z is PG2 to provide a compound of formula Ila are carried out by a single step.
  • this step is typically carried out by using neutral reductive conditions; preferably by using Pd(PPh3) 4 in the presence of a suitable scavenger such as PhSiH3, ⁇ - ⁇ or Me2NH-BH3; being PhSiH3 more preferred.
  • the step of elongating the peptidic chain is typically effected by repetitive cycles of a-amino deprotection and coupling reactions in a C ⁇ N terminal direction performed over conveniently protected aal residue of the macrocyclic region which is attached to a suitable insoluble support as starting point to provide a compound of formula III.
  • the coupling reactions are carried out using suitable coupling reagents that are known in the art and are selected following the guidance given above; typically HATU/HOAt or DIPCDI/HOBt coupling systems are independently used in each coupling reaction.
  • the protecting groups for a-amino, a-carboxy and side chain functionalities that may be present are chosen following the guidance and preferences given above.
  • the step of attaching conveniently protected aal amino acid of the macrocyclic region to a suitable insoluble support to be used as starting point for elongating the peptidic chain to provide a compound of formula III is performed by attaching said aal amino acid through terminal R1 side chain functional group or through its a-carboxy functional group to a suitable insoluble support.
  • the nature of the linkage formed may vary depending on the desired terminal R1 side chain functional group in the target peptide (acid or amide).
  • This step is typically effected using methods known in the art which are described in standard texts on solid phase peptide synthesis such as Kates, S. A. and Albericio, F.
  • the aal amino acid of the macrocyclic region is attached to a suitable insoluble support through terminal R1 side chain functional group forming an ester or amide linkage
  • the protecting groups for a-amino and ⁇ -carboxy functionalities are chosen following the guidance and preferences given above.
  • Suitable insoluble supports are selected following the guidance and preferences given above.
  • Particularly preferred processes according to invention are those that provide compounds of formula II according to the preferences previously described for intermediates of formula II.
  • This invention also relates to the use of intermediates of formula II the manufacture of compounds of formula I, and in particular in 1 manufacture of:
  • Analytical HPLC was performed on a Waters instrument comprising a separation module (Waters 2695), automatic injector, photodiode array detector (Waters 996 or Waters 2998), and system controller (Millenium 32 login).
  • the columns used were XbridgeTM C 18 reversed-phase analytical column 2.5 ⁇ x 4.6 mm x 75 mm and XbridgeTM BEH130 C 18 reversed- phase analytical column 3.5 ⁇ x 4.6 mm x 100 mm.
  • UV detection was at 220 and 254 nm, and linear gradients of ACN (+0.036% TFA) into H 2 0 (+0.045% TFA) were run at a 1 mL/min flow rate over 8 min.
  • HPLC-MS analysis was performed on a Waters instrument comprising a separation module (Waters 2695), automatic injector, photodiode array detector (Waters 2998), a Waters Micromass ZQ spectrometer and a system controller (Masslynx v4. 1).
  • the column used was a SunfireTM C 18 reversed- phase analytical column 3.5 mm x 2. 1 mm x 100 mm. UV detection was at 220 and 254 nm, and linear gradients of ACN (+0.07% Formic acid) into H 2 0 (+0.1% Formic acid) were run at a 0.3 mL/min flow rate over 8 min.
  • ⁇ and 13 C NMR spectra were recorded on a Varian MERCURY 400 (400 MHz for i H NMR, 100 MHz for NMR) spectrometer and a Bruker 600 Avance III Ultrashielded, provided with a cryoprobe TCI (600 MHz for ⁇ NMR, 150 MHz for 13 C NMR) spectrometer. Chemical shifts ( ⁇ ) are expressed in parts per million downfield from tetramethylsilyl chloride. Coupling constants are expressed in Hertz.
  • reaction mixture was acidified to pH 8 by the addition of 0.1 M aqueous HC1 and 2 eq of Fmoc-OSu (667.9 mg, 1.98 mmol) were added.
  • the product 19 ( 1.21 g, 78 %) was obtained as a colorless dense oil; ⁇ NMR (400 MHz, acetone-d6) ⁇ 0.85-0.92 (9H, m), 1.26 ( 1H, br m), 1.49 ( 12H, s), 1.56 (3H, s), 1.65 ( 1H, m), 3.76 (2H, m), 4.00 (2H, br m); NMR (100 MHz, acetone-d6) ⁇ 1 1.00, 21.55, 22.08, 25.65, 28.70, 33.08, 43.31 , 62.94, 66.63, 76.41 , 82.41 , 95.54, 150.7; HRMS (NanoESI) m/z calculated for C16H32NO4 [M+H] + 302.2331 , found 302.2329.
  • the methyl ester 21 (0.24 g, 1. 1 1 mmol) was refluxed with HC1 cone ( 15 mL) for 72 h. Then, the solution was concentrated to give the hydrochloride of 21 as a white solid (0.23 g, 100 %) which was used without further purification for the next step. A sample of the hydrochloride was dissolved in EtOH (6 mL) and propylen oxide (3 mL) and heated for 1 h to 90 °C.
  • HTMHA SYNTHESIS OF (2i?,3i?,4i?)-3-HYDROXY-2,4,6- TRIMETHYLHEPTANOIC ACID (HTMHA) 31 HTMHA was synthesized following a modified procedure reported in the literature. See J. Org. Chem. 2003, 68, 7841 -7844.
  • reaction mixture was cooled down to 0 °C and quenched by adding NaC10 2 (368.9 mg, 3.28 mmol, 4 equiv) and H 2 NS0 3 H (318.1 mg, 3.28 mmol, 4 equiv) to obtain a bright yellow suspension that was stirred for 2 h at room temperature.
  • 5% aqueous HC1 was added and the aqueous layer was extracted with DCM (x 3).
  • the combined organic extracts were washed with 5% aqueous HC1 (x 1) and brine, dried over MgS0 4 , filtrated and concentrated under vacuo to obtain crude HTMHA 31 as a yellowish oil that was used without further purification.
  • EXAMPLE 3 SYNTHESIS OF DIPEPTIDE BUILDING BLOCK: HTMHA-D- Asp( t BU)-OH 36
  • HTMHA 31 120.0 mg, 0.64 mmol
  • H-D-Asp(tBu)-OBzl 34 359.0 mg, 1.29 mmol
  • HOBt*H20 196.8 mg, 1.29 mmol
  • EDC*HC1 246.3 mg, 1.29 mmol
  • HTMHA-D-Asp(tBu)-OBzl 35 (68.9 mg, 0. 15 mmol) in dry MeOH (5 mL) was stirred with a catalytic amount of Pd-C (10%) under an atmosphere of H 2 (atmospheric pressure) for 12 h. EtOAc was added and the mixture was filtrated through celite and concentrated under vacuo to give 53.7 mg of crude HTMHA-D-Asp(tBu)-OH 36 (97%) as a yellow oil.
  • Non-proteinogenic amino acids appropriately protected Alloc-Pipecolic- OH 1, Fmoc-DADHOHA(Trt, Acetonide)-OH 10, and (Fmoc-D-ctZZo-AHDMHA- OH 23, were synthesized as described in Example 1.
  • the dipeptide building block (HTMHA-D-Asp(tBu)-OH) 36 was synthesized as described in Examples 2 and 3.
  • Fmoc-DiMe-Gln-OH 45 was synthesized by procedures known in the literature. See for example Tetrahedron 2001 , 57, 6353 and Org. Lett. 2000, 2, 4157. In addition, a-amino protection was carried out under standard procedures known in the literature.
  • Fmoc-NMe-Gln-OH 40 was synthesized by procedures known in the literature. See for example J. Org. Chem 2006, 71 , 6351.
  • Fmoc-ihreo-p-EtO-Asn(Trt)-OH 39 was synthesized as previously described by the inventors. See Amino Acid 2010, 39, 161. Experimental Protocol:
  • Aminomethyl resin (75. 1 mg, 0.36 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL).
  • Fmoc-D-Asp(OH)-OAllyl 37 (53.4 mg, 0. 135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0. 135 mmol, 5 equiv) were dissolved in DCM (0.2 mL) and DIPCDI (20.9 uL, 0. 135 mmol, 5 equiv) and DMAP ( 1.6 mg, 0.014 mmol, 0.5 equiv) in DMF (0. 15 mL) were added. The resulting mixture was added to the resin and stirred for 3 h (3 min of manual stirring and 177 min on a shaker) at 25 °C.
  • the solvents were removed and the resin subjected to the following washings/treatments: DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the loading as calculated by UV absorbance at 290 nm, was 0.36 mmol/g.
  • Fmoc-diMeGln-OH 45 48.2 mg, 0. 122 mmol, 4.5 equiv was coupled using DIPCDI ( 19.0 ⁇ L, 0. 122 mmol, 4.5 equiv) and HOBt*H 2 0 ( 18.6 mg, 0. 122 mmol, 4.5 equiv) in DMF (0.6 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the Fmoc group was kept in place until ester bond formation.
  • An aliquot of the resin was treated with a solution of TFA-H2O-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the octapeptide Fmoc-diMeGln-D-ctZZo- AHDMHA-D - aZZo-Thr-D -Lys-Leu-NMe-Gln- i reo- ⁇ -EtO -Asn-D -Asp-O Allyl was obtained with a purity of 87% as checked by HPLC-PDA.
  • the Fmoc group of diMeGln was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL).
  • the resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (48.0 mg, 0.068 mmol, 2.5 equiv) quickly added to the resin.
  • This aa was introduced using HATU (25.7 mg, 0.068 mmol, 2.5 equiv), HOAt (9.
  • the resin was then subjected to the following washings /treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • An aliquot of the resin was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • HTMHA-D-Asp( £ Bu)-OH 36 (60% purity, 80.9 mg, 0.135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0.135 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (47.0 ⁇ ⁇ , 0.270 mmol, 10 equiv) was added.
  • DMF 0.3 mL
  • DIEA 4.7.0 ⁇ ⁇ , 0.270 mmol, 10 equiv
  • the reaction mixture was added to the resin followed by the addition of solid PyBOP (70.3 mg, 0.135 mmol, 5 equiv) and it was allowed to react for 3.5 h (5 min of manual stirring and 205 min on a shaker) at 25 °C.
  • Acetonide - (3 SAR ⁇ -3 , 4 -diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ( 3 ⁇ 4u) -D - Lys(Boc)-Leu-i ⁇ e-Gln-t3 ⁇ 4reo-B-EtO ⁇
  • the peptide-resin 49 was treated with Pd(PPh 3 ) 4 (6.2 mg, 0.005 mmol, 0.2 equiv) and PhSiH 3 (66.6 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.3 mL) under N 2 . After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times.
  • the macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 ⁇ ⁇ , 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin followed by the addition of solid PyBOP (56.2 mg, 0.108 mmol, 4 equiv) and it was allowed to react for 3 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC- ESMS.
  • Non-proteinogenic amino acids appropriately protected Alloc-Pipecolic- OH 1, Fmoc-DADHOHA(Trt, Acetonide)-OH) 10, and (Fmoc-D-aZZo-AHDMHA- OH 23, were synthesized as described in Example 1.
  • HTMHA 31 was synthesized as described in Example 2.
  • Fmoc-DiMe-Gln-OH 45 was synthesized by procedures known in the literature. See for example Tetrahedron 2001 , 57, 6353 and Org. Lett. 2000, 2, 4157. In addition, a-amino protection was carried out under standard procedures known in the literature.
  • Fmoc-ihreo-P"EtO-Asn(Trt)-OH 39 was synthesized as previously described by the inventors. See Amino Acid 2010, 39, 161.
  • Aminomethyl resin 150 mg, 0.36 mmol/g was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). 3-(4- Hydroxymethylphenoxy)propionic acid (31.8 mg, 0. 162 mmol, 3 equiv) and HOBt*H 2 0 (24.8 mg, 0. 162 mmol, 3 equiv) were dissolved in DMF (0.5 mL) and DIEA (84.7 ⁇ L, 0.486 mmol, 9 equiv) first and HBTU (61.4 mg, 0.
  • Fmoc-D-Asp(OH)-OAllyl 37 ( 106.8 mg, 0.270 mmol, 5 equiv) and HOAt (38.8 mg, 0.270 mmol, 5 equiv) were dissolved in DCM (0.3 mL) and DIPCDI (41.8 ⁇ ., 0.270 mmol, 5 equiv) and DMAP (3.3 mg, 0.027 mmol, 0.5 equiv) in DMF (0.2 mL) were added. The resulting mixture was added to the resin and stirred for 3 h (3 min of manual stirring and 177 min on a shaker) at 25 °C.
  • the solvents were removed and the resin subjected to the following washings/treatments: DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the loading as calculated by UV absorbance at 290 nm, was 0.36 mmol/g.
  • Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the heptapeptide H-D-aZZo- AHDMHA-D-aZZo-Thr-D-Lys-Leu-NMe-Glu-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 100% as checked by HPLC-PDA.
  • Fmoc-diMeGln-OH 45 (96.3 mg, 0. 243 mmol, 4.5 equiv) was coupled using DIPCDI (37.6 ⁇ ., 0.243 mmol, 4.5 equiv) and HOBt*3 ⁇ 40 (37.2 mg, 0.243 mmol, 4.5 equiv) in DMF ( 1.0 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation.
  • the Fmoc group of diMeGln was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL).
  • the resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (96.0 mg, 0.135 mmol, 2.5 equiv) quickly added to the resin.
  • This aa was introduced using HATU (51.3 mg, 0.135 mmol, 2.5 equiv), HOAt ( 18.4 mg, 0.135 mmol, 2.5 equiv) and DIEA (47.0 ⁇ L, 0.270 mmol, 5 equiv) dissolved in DMF (0.5 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the resin was then subjected to the following washings /treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • An aliquot of the resin was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • HTMHA 31 (30.5 mg, 0.162 mmol, 3 equiv), DIPCDI (25.1 ⁇ L, 0.162 mmol, 3 equiv) and HOBt (8.3 mg, 0.162 mmol, 3 equiv) were dissolved in DMF (0.5 mL) and added to the resin without pre- activation. After 3.25 h of coupling (5 min of manual stirring and 190 min on a shaker) more reagents were added (HTMHA-DIPCDI-HOBt, 1:1:1) and the resulting mixture was stirred for 1 h more. Then, the solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • HTMHA-D-Asn-DADHOHA- diMeGln-D-a//o-AHDMHA(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Glu- i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 28% as checked by HPLC-PDA.
  • Conditions: linear gradient (30% to 60%) of ACN (0.036% TFA) into H 2 0 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (i R 6.27 min).
  • HPLC-ESMS(+) analysis showed: m/ z calculated for C81H136N16O29 1796.97; [M+H] + found, 1797.93.
  • the peptide-resin 58 was treated with Pd(PPh 3 ) 4 ( 12.5 mg, 0.01 1 mmol, 0.2 equiv) and PhSiH 3 ( 133.3 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.5 mL) under N 2 . After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times.
  • the macrolactamization step was carried out on solid phase. HO At (29.4 mg, 0.2 16 mmol, 4 equiv) was dissolved in DMF (0.5 mL) and DIEA (75.2 ⁇ ⁇ , 0.432 mmol, 8 equiv) was added. The reaction mixture was then added to the resin followed by the addition of solid PyBOP ( 1 12.4 mg, 0.2 16 mmol, 4 equiv) and it was allowed to react for 3 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5, 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC- ESMS.
  • 2-Chlorotrityl chloride resin (75 mg, 1.6 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). Fmoc-D- Asp( £ Bu)-OH (15.4 mg, 0.038 mmol) and DIEA (43.5 iL, 0.25 mmol) were dissolved in DCM (0.3 mL). The resulting mixture was added to the resin and manually stirred for 5 min.
  • the solvents were removed and the resin subjected to the following washings/ treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF (1:4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the loading as calculated by UV absorbance at 290 nm, was 0.40 mmol/g.
  • Fmoc-diMeGln-OH 45 (53.5 mg, 0. 135 mmol, 4.5 equiv) was coupled with resin 63 using DIPCDI (20.9 iL, 0. 135 mmol, 4.5 equiv) and HOBt*H 2 0 (20.7 mg, 0. 135 mmol, 4.5 equiv) in DMF (0.5 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the Fmoc group was kept in place until ester bond formation.
  • An aliquot of the resin 64 was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the octapeptide Fmoc- diMeGln-D-a//o-Thr-D-a//o-Thr-D-Lys-Leu-i ⁇ /Me-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 78% as checked by HPLC-PDA.
  • Fresh Alloc-pipecolic-OH 1 (51.2 mg, 0.240 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (37.2 ⁇ ., 0.240 mmol, 8 equiv) was added. The mixture was poured into the resin 64 followed by addition of DMAP ( 1.8 mg, 0.015 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the Fmoc group of diMeGln moiety in 65 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL) .
  • the resin was shortly washed with DMF (x 2 ; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-Ser( £ Bu)-OH 66 (40.3 mg, 0. 105 mmol, 3.5 equiv) quickly added to the resin.
  • This aa was introduced using HATU (39.9 mg, 0. 105 mmol, 3.5 equiv), HOAt ( 14.3 mg, 0.
  • the resin was then subjected to the following washings/ treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) .
  • An aliquot of the resin was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • HTMHA-D-Asp( £ Bu)-OH 36 (53.9 mg, 0. 150 mmol, 5 equiv) and HOAt (20.4 mg, 0. 150 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (52.3 ⁇ ., 0.300 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 68, and solid PyBOP (78.0 mg, 0.150 mmol, 5 equiv) was added.
  • the suitably protected linear precursor was cleaved from the resin 70 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL). All the filtrates were collected in a round-bottom flask provided with 20 mL of H2O in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 52.9 mg of fully protected linear precursor.
  • the macrolactamization step was then carried out in solution.
  • the peptide crude was dissolved in dry DCM (25 mL) and then a solution of HO At (5.5 mg, 0.040 mmol, 2 equiv) in DMF (0.05 mL) and DIEA ( 14 ⁇ L, 0.080 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP (20.8 mg, 0.040 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C.
  • the crude was partially purified by semi-analytical HPLC-PDA using a XBridgeTM Prep BEH130 C 18 5 ⁇ 10x 100 mm column and a linear gradient (0% to 30% over 5 min and 30% to 33% over 20 min) of ACN (0.036% TFA) into H 2 0 (0.045% TFA) with a flow rate of 3.0 mL/min.
  • 2-Chlorotrityl chloride resin (75 mg, 1.6 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL) . Fmoc-D- Asp('Bu)-OH ( 15.4 mg, 0.038 mmol) and DIEA (43.5 iL, 0.25 mmol) were dissolved in DCM (0.3 mL) . The resulting mixture was added to the resin and manually stirred for 5 min. Then, more DIEA (2 1 .8 ⁇ L, 0.
  • the solvents were removed and the resin subjected to the following washings/ treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the loading as calculated by UV absorbance at 290 nm, was 0.42 mmol/g.
  • the Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin 63 was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • Fmoc-Gln(Trt)-OH 67 (76.9 mg, 0. 126 mmol, 4 equiv) was coupled with resin 63 using HATU (47.9 mg, 0. 126 mmol, 4 equiv), HOAt ( 17.2 mg, 0. 126 mmol, 4 equiv) and DIEA (43.9 ⁇ ., 0.252 mmol, 8 equiv) in DMF (0.4 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C with 1 min of pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • Fresh Alloc-pipecolic-OH 1 (53.8 mg, 0.252 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (39. 1 ⁇ ., 0.252 mmol, 8 equiv) was added. The mixture was poured into the resin 72 followed by addition of DMAP ( 1.9 mg, 0.016 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the Fmoc group of Gin in resin 73 was then removed following a common deprotection treatment with piperidine-DMF ( 1 :4) (2 x 1 min, 2 > ⁇ 5 min, 1 x 10 min, 4 mL).
  • the resin was washed with DMF (3 x 1 min; 4 mL) and DCM ( 3 x 1 min; 4 mL) and the reaction mixture of the next residue Fmoc- DADHOHA(Trt, Acetonide)-OH 10 (55.9 mg, 0.079 mmol, 2.5 equiv) added to the resin.
  • This aa was introduced using HATU (29.9 mg, 0.079 mmol, 2.5 equiv), HOAt ( 10.7 mg, 0.079 mmol, 2.5 equiv) and DIEA (27.4 ⁇ iL, 0. 158 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the resin was then subjected to the following washings/ treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • An aliquot of the resin 74 was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • HTMHA-D-Asp( £ Bu)-OH 36 (56.6 mg, 0. 158 mmol, 5 equiv) and HOAt (21.4 mg, 0. 158 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (54.9 ⁇ ., 0.315 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 74, and solid PyBOP (81.9 mg, 0.158 mmol, 5 equiv) was added.
  • the peptide-resin 75 was treated with Pd(PPh 3 ) 4 (3.64 mg, 0.003 mmol, 0.1 equiv) and PhSiH 3 (38.8 ⁇ iL, 0.315 mmol, 10 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL) , DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times.
  • the suitably protected linear precursor was cleaved from the resin 76 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL) . All the filtrates were collected in a round-bottom flask provided with 20 mL of 3 ⁇ 40 in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 68. 1 mg of fully protected linear
  • the macrolactamization step was then carried out in solution.
  • the peptide crude was dissolved in dry DCM (52 mL) and then a solution of HOAt (7.1 mg, 0.052 mmol, 2 equiv) in DMF (0.05 mL) and DIEA ( 18 ⁇ L, 0. 103 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP (27.0 mg, 0.052 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C.
  • the reaction mixture was washed with 5% aqueous NaHCC solution (x 3), saturated aqueous NH 4 C1 solution (x 3) and brine (x 1), dried over Na2S0 4 , filtrated and concentrated under vacuo.
  • the protected cyclopeptide was treated with TFA- H 2 0-Tis (95:2.5:2.5) (1 x 1.5 h, 4 mL) at 25 °C.
  • the final deprotection step was monitored by analysis of the crude by HPLC-PDA and HPLC-ESMS. All organic solvents were eliminated under reduced pressure, the peptide crude was dissolved in H2O-ACN ( 1 : 1), and the resulting solution was lyophilized to provide 66.
  • EXAMPLE 7 SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 81
  • 2-Chlorotrityl chloride resin 50 mg, 1.6 mmol/g was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). Fmoc-D- Asp( £ Bu)-OH ( 10.3 mg, 0.025 mmol) and DIEA (29 ⁇ ., 0. 17 mmol) were dissolved in DCM (0.3 mL). The resulting mixture was added to the resin and manually stirred for 5 min.
  • the solvents were removed and the resin subjected to the following washings/treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the loading as calculated by UV absorbance at 290 nm, was 0.43 mmol/g.
  • Fmoc-diMeGln-OH 45 (38.4 mg, 0. 097 mmol, 4.5 equiv) was coupled with resin 63 using DIPCDI ( 15.0 ⁇ iL, 0.097 mmol, 4.5 equiv) and HOBt*3 ⁇ 40 ( 14.8 mg, 0.097 mmol, 4.5 equiv) in DMF (0.4 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C with 1 min of pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the Fmoc group was kept in place until ester bond formation.
  • An aliquot of the resin 64 was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the octapeptide Fmoc-diMeGln-D - ctZZo-Thr-D - ctZZo-Thr-D -Lys-Leu-NMe-Gln- ⁇ -EtO - Asn-D-Asp-OH was obtained with a purity of 85% as checked by HPLC-PDA.
  • Fresh Alloc-pipecolic-OH 1 (36.7 mg, 0. 172 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (26.6 ⁇ ., 0. 172 mmol, 8 equiv) was added. The mixture was poured into the resin 64 followed by addition of DMAP ( 1.3 mg, 0.01 1 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the Fmoc group of diMeGln moiety in resin 65 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL) .
  • the resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (38.2 mg, 0.054 mmol, 2.5 equiv) quickly added to the resin.
  • This aa was introduced using HATU (20.4 mg, 0.054 mmol, 2.5 equiv) , HOAt (7.3 mg, 0.054 mmol, 2.5 equiv) and DIEA ( 18.7 ⁇ ., 0. 108 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . The Kaiser's test proved quantitative coupling.
  • the resin was then subjected to the following washings/treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) .
  • An aliquot of the resin 78 was treated with a solution of TFA-H2O- TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the decapeptide H-DADHOHA-diMeGln-D-a//o-Thr(Alloc- pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 62% as checked by HPLC-PDA.
  • Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H 2 0 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> 2.589 min and 2.856 min, the second peak corresponding to our desired intermediate with the diol function protected as an acetal) .
  • HTMHA-D-Asp( £ Bu)-OH 36 38.6 mg, 0. 108 mmol, 5 equiv
  • HOAt 14.6 mg, 0. 108 mmol, 5 equiv
  • DMF 0.3 mL
  • DIEA 37.4 ⁇ ., 0.2 15 mmol, 10 equiv
  • the remaining mixture was poured into the resin 78, and solid PyBOP (55.9 mg, 0. 108 mmol, 5 equiv) was added.
  • HTMHA-D-Asp-DADHOHA- diMeGln-D-a//o-Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn- D-Asp-OH was obtained with a purity of 44% as checked by HPLC-PDA.
  • Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H 2 0 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (i R 4.42 min).
  • the suitably protected linear precursor was cleaved from the resin 80 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL). All the filtrates were collected in a round-bottom flask provided with 20 mL of H2O in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 31.6 mg of fully protected linear precursor.
  • the macrolactamization step was then carried out in solution.
  • the peptide crude was dissolved in dry DCM (26 mL) and then a solution of HO At (3.6 mg, 0.026 mmol, 2 equiv) in DMF (0.03 mL) and DIEA (9. 1 ⁇ ., 0.052 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP ( 13.6 mg, 0.026 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C.
  • the reaction mixture was washed with 5% aqueous NaHCOa solution (x 3), saturated aqueous NH 4 C1 solution (x 3) and brine (x 1), dried over Na2S0 4 , filtrated and concentrated under vacuo.
  • the protected cyclopeptide was treated with TFA- H 2 0-Tis (95:2.5:2.5) (1 x 1.5 h, 4 mL) at 25 °C.
  • the final deprotection step was monitored by analysis of the crude by HPLC-PDA and HPLC-ESMS. All organic solvents were eliminated under reduced pressure, the peptide crude was dissolved in H2O-ACN ( 1 : 1), and the resulting solution was lyophilized to provide 42.
  • the crude was partially purified by semi-analytical HPLC-PDA using a XBridgeTM Prep BEH130 C 18 5 ⁇ 10x 100 mm column and a linear gradient (0% to 30% over 5 min and 30% to 32% over 20 min) of ACN (0.036% TFA) into H 2 0 (0.045% TFA) with a flow rate of 3.0 mL/min.
  • H-D-Asp(0-resin)-OAUyl 38 was prepared by the procedure described in Example 4.
  • Octanoic acid (317.8 ⁇ L, 2.01 mmol), H-D-Asp(tBu)-OBzl 34 (466.8 mg, 1.67 mmol), HOAt (273.0 mg, 2.01 mmol) and EDC*HC1 (384.4 mg, 2.01 mmol) were placed in a round-bottom flask.
  • a mixture of dry DCM:DMF ( 1 : 1 , 12 mL) was added and, after checking that the pH of the reaction solution was around 6, it was allowed to react for 12 h at room temperature.
  • the Fmoc group was kept in place until ester bond formation.
  • An aliquot of the resin 83 was treated with a solution of TFA-H 2 0-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the octapeptide Fmoc- diMeGln-D-a/Zo-Thr-D-a/Zo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 90% as checked by HPLC-PDA.
  • Fresh Alloc-pipecolic-OH 1 (86.4 mg, 0.405 mmol, 15 equiv) was dissolved in dry DCM ( 1.3 mL) and DIPCDI (62.7 ⁇ L, 0.405 mmol, 15 equiv) was added. The mixture was poured into the resin 83 followed by addition of
  • the Fmoc group of diMeGln moiety in resin 84 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL). The resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (48.0 mg, 0.068 mmol, 2.5 equiv) quickly added to the resin.
  • This aa was introduced using HATU (25.7 mg, 0.068 mmol, 2.5 equiv), HOAt (9.2 mg, 0.068 mmol, 2.5 equiv) and DIEA (23.5 ⁇ L, 0. 135 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling.
  • the resin was then subjected to the following washings/treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • An aliquot of the resin 85 was treated with a solution of TFA-H2O- TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate.
  • the peptide-resin 87 was treated with Pd(PPh 3 ) 4 (6.24 mg, 0.005 mmol, 0.2 equiv) and PhSiH 3 (66.6 ⁇ L, 0.540 mmol, 20 equiv) dissolved in DCM (0.4 mL) under N 2 . After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times.
  • the macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 ⁇ ⁇ , 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin 88 followed by the addition of solid PyBOP (56.2 mg, 0. 108 mmol, 4 equiv) and it was allowed to react for 2.5 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5, 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS.
  • Resin 48 was obtained following the procedures described in Example 1.
  • the peptide-resin 90 was treated with Pd(PPh 3 ) 4 (6.24 mg, 0.005 mmol, 0.2 equiv) and PhSiH 3 (66.6 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.4 mL) under N 2 . After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times.
  • the macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 ⁇ L, 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin 91 followed by the addition of solid PyBOP (56.2 mg, 0. 108 mmol, 4 equiv) and it was allowed to react for 2.5 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL).
  • the cyclization step was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS.
  • EXAMPLE 10 BIOASSAYS FOR THE DETECTION OF ANTITUMOR ACTIVITY
  • the aim of this assay is to evaluate the in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) activity of the samples being tested.
  • a colorimetric assay, using sulforhodamine B (SRB) reaction has been adapted to provide a quantitative measurement of cell growth and viability (following the technique described by Skehan et al. J. Natl. Cancer Inst. 1990, 82, 1 107-1 1 12).
  • This form of assay employs SBS-standard 96-well cell culture microplates (Faircloth et al. Methods in Cell Science, 1988, 1 1 (4), 201-205; Mosmann et al, Journal of Immunological Methods, 1983, 65(1-2), 55-63). All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and derive from different types of human cancer.
  • ATCC American Type Culture Collection
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • 2mM L-glutamine 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C, 5% C0 2 and 98% humidity.
  • cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.
  • Cells were seeded in 96 well micro titer plates, at 5 x 10 3 cells per well in aliquots of 150 ⁇ ⁇ , and allowed to attach to the plate surface for 18 hours (overnight) in drug free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 ⁇ ⁇ aliquots of 4X stock solutions in complete culture medium plus 4% DMSO) using ten serial dilutions (concentrations ranging from 10 to 0.00262 ⁇ g/mL) and triplicate cultures ( 1% final concentration of DMSO).
  • the antitumor effect was measured by using the SRB methodology: Briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS, and stained in 0.4% SRB solution for 30 min at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm. Effects on cell growth and survival were estimated by applying the NCI algorithm (Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91 - 104).
  • GI50 compound concentration that produces 50% cell growth inhibition, as compared to control cultures
  • TGI total cell growth inhibition (cytostatic effect), as compared to control cultures
  • LC50 compound concentration that produces 50% net cell killing (cytotoxic effect).
  • Table III illustrates data on the biological activity of compounds of the present invention.
  • Table III Cytotoxicity assay -Activity Data (Molar) of Pipecolidepsin A'.

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Abstract

(I) wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, n and Y are as described. The invention provides a process for the synthesis of complex pipecolidepsin and related compounds of formula (I), opening a new field of compounds with useful biological properties. The invention also provides intermediates, useful in the synthesis of compounds of formula (I).

Description

SYNTHETIC PROCESS FOR THE MANUFACTURE OF PIPECOLIDEPSIN
COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to synthetic processes, and in particular to the first synthetic process for the manufacture of Pipecolidepsins and analogues thereof, to intermediates useful for such syntheses, and to analogues of Pipecolidepsins.
BACKGROUND OF THE INVENTION
Pipecolidepsins are a group of marine natural compounds which are disclosed to have cytotoxic properties. Examples of pipecolidepsins are provided by Pipecolidepsin A, B and C. Their isolation and cytotoxic properties against lung carcinoma (NSCLC), colorectal adenocarcinoma and breast adenocarcinoma human tumor cell lines have been described in WO 2010/070078. These natural compounds have been originally isolated from a sponge of the order Lithistida, family Neopeltidae, genus Homophymia, species Homophymia lamellosa Vacelet & Vasseur, 1971. However, the limited availability of natural material has resulted in the search for alternative synthetic methods being sought for the natural compounds and related analogs.
The structural characteristics of Pipecolidepsins A, B and C are represented in Figure I: Exocyclic linear region
Figure imgf000003_0001
Macrocyclic region
Figure imgf000003_0002
Figure I As shown, the structure of pipecolidepsins is complex, comprising eight amino acids as a macrocyclic region (aal-aa8) and an exocyclic linear region of three amino acids (aa9-aal l) with a N-terminal polyketide-derived moiety. The distinguishing structural characteristics of this family of cyclic depsipeptides include a preponderance of unusual amino acid residues, including non- proteinogenic amino acids, and an unique structural arrangement, a "head-to- side-chain" macrocyclic region through an ester bond, terminated in an exocyclic linear region with a N-terminal polyketide-derived moiety.
The synthesis of these cyclic depsipeptides is hampered by the presence of the several unusual residues, usually N-alkyl and β-branched residues, and the construction of the synthetic core comprising an ester bond formed through a hindered secondary hydroxyl group constituting the "head-to-side- chain" arrangement of the macrocyclic region.
In view of their interesting biological properties and the limited availability of biological material there is a need to provide alternative synthetic processes for the preparation of natural pipecolidepsins and related compounds in an efficient way.
OBJECT OF THE INVENTION
There is a need for synthetic methods for pipecolidepsin compounds. Such methods may provide a synthetic route to the natural compounds as well as permitting the preparation of new related active compounds.
SUMMARY OF THE INVENTION The inventors have developed for the first time a process for the synthesis of complex pipecolidepsin and related compounds, opening a new field of compounds with useful biological properties.
In a first aspect, the present invention is directed to a process for the synthesis of pipecolidepsin compounds of general formula I:
Figure imgf000005_0001
wherein Ri is selected from substituted or unsubstituted Ci-Cie alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each R2 and R3 is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl;
R4 is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl;
R5 is selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; each R6 and R7 is independently selected from hydrogen, CORa, COORa, CONRaRb, C(=NRa)NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; each Rg and Rio is independently selected from substituted or unsubstituted C1-C12 alkyl; each Rg and R12 is independently selected from ORc, NRaRb, CORa, NRaCONRaRb, NRaC(=NRa)NRaRb, halogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group;
R11 is selected from hydrogen, CORa, COORa, CONRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; n is 3 or 4;
Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000006_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000006_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy and R6y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each F¾y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group; and
Rc is selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; and each Ra and Rb is independently selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the process comprises a macrolactamization step performed between positions 1 and 2 of an intermediate of formula Ila to give a compound of formula la in accordance with Scheme I:
Figure imgf000008_0001
Ila la
Scheme I
wherein Ri, R2, R3 , R4, Re, Rio and n are as defined for compounds of formula I;
R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3; R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3; Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3;
R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and a suitable insoluble support;
Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000009_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000009_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy, and R6y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each R2y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or R2y with R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted 1 ,3- dioxolane; or each R2y and R3y is independently selected as PG3; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or when each R4y and R5y is independently selected from ORc and NHRb then each Rb and Rc in such groups may also be independently selected as PG3; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
In another aspect, the present invention provides intermediates formula II:
Figure imgf000010_0001
wherein
X is selected from PG 1 and a group of formulae (f), (g) or (h):
Figure imgf000010_0002
Formula (f) Formula (g) Formula (h) Zi is selected from hydrogen and PG 1 ;
Z is selected from hydrogen, PG2 and a suitable insoluble support;
Ri , R , R3, R4, Re, Rio and n are as defined for compounds of formula I;
R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3;
R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3;
Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3; R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and a suitable insoluble support; provided that when Z2 is a suitable insoluble support then R12 is not a suitable insoluble support;
Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000012_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000012_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy and R6y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each R2y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or R2y with R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted 1 ,3- dioxolane; or each R2y and R3y is independently selected as PG3; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or when each R4y and R5y is independently selected from ORc and NHRb then each Rb and Rc in such groups may also be independently selected as PG3; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group; each PG1 is independently selected as a a-amino protecting group; provided that when there are several PG 1 groups they are orthogonal protecting groups;
PG2 is a a-carboxy protecting group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
In one particular aspect, the invention relates to the use of intermediates of formula II in the manufacture of compounds of formula I.
In a further aspect, the invention relates to a process performed in solid phase for the synthesis of intermediates of formula II which comprise the step of forming an ester bond performed at the branching position 5 of a compound of formula III to provide an intermediate of formula lib in accordance with Scheme II:
Figure imgf000014_0001
III lib
Scheme II wherein X is PG 1 ;
Z is selected from PG2 and a suitable insoluble support; R3, R4, Re, Rio and n are as defined for compounds of formula I;
R5, and R11 are as defined for compounds of formula I or may be independently selected as PG3; R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3; Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3;
R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and a suitable insoluble support; provided that when Z2 is a suitable insoluble support then R1 is not a suitable insoluble support; each PG1 is independently selected as a a-amino protecting group; provided that when there are several PG 1 groups they are orthogonal protecting groups;
PG2 is a a-carboxy protecting group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In the compounds defined by Markush formulae in this specification, the groups can be selected in accordance with the following guidance:
Alkyl groups may be branched or unbranched, and preferably have from 1 to about 18 carbon atoms. One more preferred class of alkyl groups has from 1 to about 12 carbon atoms; and even more preferably from 1 to about 6 carbon atoms. Methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl are particularly preferred alkyl groups in the compounds of the present invention. Another preferred class of alkyl groups has from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms. As used herein, the term alkyl, unless otherwise stated, refers to both cyclic and acyclic groups, although cyclic groups will comprise at least three carbon ring members.
Preferred alkenyl and alkynyl groups in the compounds of the present invention may be branched or unbranched, have one or more unsaturated linkages and from 2 to about 18 carbon atoms. One more preferred class of alkenyl and alkynyl groups has from 2 to about 12 carbon atoms; and even more preferably from 2 to about 6 carbon atoms. Alkenyl and alkynyl groups having 2, 3, 4 or 5 carbon atoms are particularly preferred. Another preferred class of alkenyl and alkynyl groups has from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms. The terms alkenyl and alkynyl as used herein refer to both cyclic and acyclic groups, although cyclic groups will comprise at least three carbon ring members.
Suitable aryl groups in the compounds of the present invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups. Typical aryl groups contain from 1 to 3 separated and/ or fused rings and from 6 to about 18 carbon ring atoms. Preferably aryl groups contain from 6 to about 10 carbon ring atoms. Specially preferred aryl groups include substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted phenanthryl and substituted or unsubstituted anthryl. Suitable heterocyclic groups include heteroaromatic and heteroalicyclic groups containing from 1 to 3 separated and/ or fused rings and from 5 to about 18 ring atoms. Preferably heteroaromatic and heteroalicyclic groups contain from 5 to about 10 ring atoms. Suitable heteroaromatic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., coumarinyl including 8- coumarinyl, quinolyl including 8-quinolyl, isoquinolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Suitable heteroalicyclic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., pyrrolidinyl, tetrahydrofuryl, dihydro furyl, tetrahydro thienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1 ,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3. l .OJhexyl, 3- azabicyclo[4. 1.0]heptyl, 3H-indolyl, and quinolizinyl.
The groups above mentioned may be substituted at one or more available positions by one or more suitable groups such as OR', =0, SR', SSR', SOR', S02R', S03R', OSO2R', OSO3R', N02, NHR', N(R 2, =N-R', NfR'JCOR', NiCOR^, NfR'JCONHR', NfR'JCONiR'^, N(R')S02R', N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, COOCOR', COOCOOR', COOCONHR', COOCONiR'Ja, CONHR', CONiR'Ja, CONNOR', CONiR'JCOR', CONiR'JCOOR', CONfR'JCONHR', CONiR'JCONiR'Ja, CON(R')S02R', POiOR^, POiOR^R', PO(OR')(N(R')R'), substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R' groups is independently selected from the group consisting of hydrogen, OH, N02, NH2, SH, CN, halogen, CHO, COalkyl, COOH, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list.
Suitable halogen groups or substituents in the compounds of the present invention include F, CI, Br, and I.
The term "pharmaceutically acceptable salts" refers to any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts can be carried out by methods known in the art. For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and -toluenesulfonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N- dialkylenethanolamine, triethanolamine and basic aminoacids salts. Trifluoroacetate is one of the preferred pharmaceutically acceptable salts in the compounds of the invention.
Any compound referred to herein is intended to represent such specific compound as well as certain variations or forms. In particular, compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention. Thus, any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms, and mixtures thereof. Particularly, the compounds of the present invention represented by the above described formulae I, II, and III may include enantiomers depending on their asymmetry or diastereoisomers. Stereoisomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E) -isomer or (2)-isomer. If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same as, or different to, the stereoisomerism of the other double bonds of the molecule. The single isomers and mixtures of isomers fall within the scope of the present invention.
Furthermore, any compound referred to herein may exist as tautomers. Specifically, the term tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily converted from one isomeric form to another. Common tautomeric pairs are amine-imine, amide- imidic acid, keto-enol, lactam-lactim, etc. Additionally, any compound referred to herein is intended to represent hydrates, solvates, and polymorphs, and mixtures thereof when such forms exist in the medium. In addition, compounds referred to herein may exist in isotopically-labelled forms. All geometric isomers, tautomers, atropisomers, hydrates, solvates, polymorphs, and isotopically labelled forms of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.
In the present description and definitions, when there are several groups Ra, Rb, Rc, PG 1 , PG2 or PG3 present in the compounds of the invention, and unless it is stated explicitly so, it should be understood that they can be each independently different within the given definition, i.e. Ra does not represent necessarily the same group simultaneously in a given compound of the invention. In the context of the invention, the term "residue" refers to a specific monomer within the peptide chain. Therefore, "residue" refers to specific amino acids present in the peptide under construction which are numbered from aal to aal l for clarity purposes. On the other hand, the term "moiety" refers to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. For example, the N-terminal fragment present in the linear region of compounds of general formula I is herein referred to as "N-terminal moiety".
The processes according to the invention may make use of solid phase peptide synthesis (SPPS), i.e. where the peptide is bound to an insoluble support (R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 2149). Following this strategy, any un-reacted reagents left at the end of any synthetic step and soluble by-products can be removed by a simple wash procedure, greatly decreasing the time required for synthesis. Therefore, reagents can be used in excess allowing the reactions to proceed to completion in a minimum time resulting in faster synthesis of peptides with high purity. The couplings typically proceed in a C→N terminal direction to allow the use of racemisation limiting a-amine protection for the activated species. Although less frequent, it is also possible for the peptide synthesis to proceed in the reverse N→C direction, thus needing a-carboxy protection for the activated species. Accordingly, compounds of formula I and intermediates of formula II, III can be obtained from suitable starting materials in a stereo- controlled and fast manner. Moreover, the method is also amenable to automation. See for example, Lloyd -Williams, P., et al. in Chemical Approaches to the Synthesis of Peptides and Proteins; CRC Press, Boca Raton (FL), 1997.
The insoluble support may be any support known in the art which is suitable for use in SPPS. See for example, James, I. W. Tetrahedron 1999, 55, 4855-4946; Guillier, F., Orain, D., Bradley, M. Chem. Rev. 2000, 100, 2091 - 2157 and Kates S.A. and Albericio, F. in Solid-Phase synthesis A practical guide; 1st Ed. Boca Raton: CRC Press 2000. These references are incorporated here by reference in their entirety. Typically, the insoluble support comprises a matrix polymer based resin, optionally with a linker group to which the growing peptide is attached during synthesis and which can be cleaved under desired conditions to release the target peptide from the support. Typically the matrix polymer based resin may be made from one or more polymers, copolymers or combinations of polymers, such as crosslinked polystyrene (PS)- , crosslinked polyamide (PA)-, crosslinked polyethylene glycol (PEG)-, crosslinked polylysine, composite PS-Polyethylene glycol (PEG)- or composite PA-Polyethylene glycol (PEG) -based resins . Suitable linker groups may link the C-terminal function of the first amino acid residue to the insoluble support through an ester or an amide bond depending on the C-terminal functional group of the target peptide (respectively peptide-acid or peptide-amide). Suitable linker groups may comprise trityl, amino or hydroxy moieties. Therefore, suitable insoluble supports for the syntyhesis of peptide-acids may include 4-hydroxymethylphenoxymethyl polystyrene Wang resin (Wang, S.S., J. Am. Chem. Soc. 1973, 95, 1328- 1333; J. Org. Chem. 1975, 40, 1235-1239; Albericio, F., Barany, G. Int. J. Pept. Protein Res. 1985, 26, 92-97), Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin (Atherton, E., et. al. J. Chem. Soc. Chem. Comm. 1978, 537-539), benzhydryl bromide resins, SASRIN resin (Mergler, M., Nyfeler, R., Tanner, R., Gosteli, J., Grogg, P. Tetrahedron Lett. 1988, 29, 4009), 2-chlorotritylchloride (CTC) Barlos resin (Barlos, K., et al. Int. J. Pept. Protein Res. 1991 , 37, 513-520; 38, 555), trityl ChemMatrix and the like. Suitable insoluble supports for the syntyhesis of peptide-amides may include NovaSyn® TGR resin, Rink amide resin, Rink amide MBHA resin, Rink amide AM resin, Rink amide PEGA resin, Rink amide NovaGel® resin, Rink amide ChemMatrix resin, PAL ChemMatrix, Sieber amide resin, NovaSyn® TG Sieber resin, Sieber amide ChemMatrix resin, Ramage amide resin, Ramage amide MBHA resin, Ramage amide PEGA resin, Ramage amide AM resin, Ramage amide ChemMatrix resin and the like. The mention of these insoluble supports should not be interpreted as a limitation of the scope of the invention, since they have been mentioned as a mere illustration of suitable insoluble supports for peptide synthesis, but further insoluble supports having said function may be known by the skilled person in the art, and they are to be understood to be also encompassed by the present invention.
The process according to the invention is preferably carried out in the presence of appropriate protecting groups unless otherwise noted. Protecting groups may refer to side chain protecting groups (including protecting groups for hydroxyl, 1 ,2-diols, carboxyl, amino, amide and thiol groups), a-carboxy, or a-amino protecting groups for the amino acids used in the process according to the invention. Suitable protecting groups are well known by the skilled person in the art. For instance, general reviews of protecting groups in organic chemistry are provided by Wuts, P. G. M. and Greene T. W. in Greene's Protective groups in Organic Synthesis, 4th Ed. John Wiley & Sons Inc. 2006 and Kocienski, P. J. in Protecting Groups, 3rd Ed. Georg Thieme Verlag: New York, 2005. These references provide sections on protecting groups for hydroxyl (including 1 ,2-diols), carboxyl, amino, amide and thiol groups. The nature and use of protecting groups for amino acids in peptide synthesis is well known in the art. See for example, Albericio, F. et al. Chem. Rev. 2009, 109, 2455-2504. All these references are incorporated here by reference in their entirety. Protecting groups are chosen following an orthogonal protecting scheme. An orthogonal protecting scheme is defined as one based on completely different classes of protecting groups such that each class of groups can be removed in any order and in presence of all other classes of protecting groups (Barany, G.; Albericio, F. J. Am. Chem. Soc. 1985, 107, 4936-4942). Therefore the a-amino and the side chain protecting groups are typically not the same. It also applies when a-carboxy protection is needed. In some cases, and depending on the type of reagents used in solid phase synthesis and other peptide processing, an amino acid may not require the presence of a side-chain protecting group.
A a-amino protecting group refers to a chemical moiety coupled to the alpha amino group of an amino acid. Typically, the a-amino protecting group is removed in a deprotection reaction prior to the coupling of the next amino acid to be incorporated to the growing peptide chain. Examples of a-amino protecting groups are provided by 9-fluorenylmethoxycarbonyl (Fmoc), 2-(4- nitrophenylsulfonyl)ethoxycarbonyl (Nsc), ( 1 , l -dioxobenzo[fo]thiphene-2- yl)methyloxycarbonyl (Bsmoc), ( 1 , 1 -dioxonaphtol[ 1 ,2-b]thiophene-2- yl)mehyloxycarbonyl (a-Nsmoc), l -(4,4-dimethyl-2,6-dioxocyclohex- l -ylidine)- 3-methylbutyl (ivDde), 2,7-di-t-butyl-Fmoc (Fmoc*), 2-fluoro-Fmoc (Fmoc(2F)), 2-monoisooctyl-Fmoc (mio-Fmoc), 2,7-diisooctyl-Fmoc (dio-Fmoc), tetrachloronaphtaloyl (TCP), 2-
[phenyl(methyl)sulfonio]ethyloxycarbonyltetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps), t-butyloxycarbonyl (Boc), triphenylmethyl (Trt), 2-(4- biphenyl)isopropoxycarbonyl (Bpoc), 2-nitrophenylsulfenyl (Nps), benzyloxycarbonyl (Z), a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz), allyloxycarbonyl (Alloc), o-nitrobemzenesulfonyl (oNBS), 2,4- dinitrobenzenesulfonyl (dNBS), benzothiazole-2-sulfonyl (Bts), 2,2,2- trichloroethyloxycarbonyl (Troc), dithiasuccinoyl (Dts), p- nitrobenzyloxycarbonyl (pNZ), a-azidoacids, propargyloxycarbonyl (Poc), o- nitrobenzyloxycarbonyl (oNZ), 4-nitroveratryloxycarbonyl (NVOC), 2-(2- nitrophenyl)propyloxycarbonyl (NPPOC), 2-(3,4-methylenedioxy-6- nitrophenyl)propyloxycarbonyl (MNPPOC), 9-(4-bromophenyl)-9-fluorenyl (BrPhF), azidomethoxycarbonyl (Azoc) and hexafluoroacetone (HFA). In one embodiment, preferred a-amino protecting groups are selected from 9- fluorenylmethoxycarbonyl (Fmoc), 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc), ( 1 , l -dioxobenzo[fo]thiphene-2-yl)methyloxycarbonyl (Bsmoc), (1 , 1 - dioxonaphtol[ 1 ,2-b]thiophene-2-yl)mehyloxycarbonyl (a-Nsmoc), 1 -(4,4- dimethyl-2,6-dioxocyclohex- 1 -ylidine)-3-methylbutyl (ivDde), 2,7-di-t-butyl- Fmoc (Fmoc*), 2-fluoro-Fmoc (Fmoc(2F)), 2-monoisooctyl-Fmoc (mio-Fmoc), 2,7-diisooctyl-Fmoc (dio-Fmoc), tetrachloronaphtaloyl (TCP), 2- [phenyl(methyl)sulfonio]ethyloxycarbonyltetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), and 2-(4- sulfophenylsulfonyl)ethoxycarbonyl (Sps). More preferred a-amino protecting groups are selected from from 9-fluorenylmethoxycarbonyl (Fmoc), 2-(4- nitrophenylsulfonyl)ethoxycarbonyl (Nsc) and 2-fluoro-Fmoc (Fmoc(2F)). Most preferred a-amino protecting group is 9-fluorenylmethoxycarbonyl (Fmoc). In another embodiment, preferred a-amino protecting groups for pipecolic acid and pipecolic residue are selected from allyloxycarbonyl (Alloc), azidomethoxycarbonyl (Azoc) and -nitrobenzyloxycarbonyl (pNZ). More preferred a-amino protecting group is allyloxycarbonyl (Alloc).
A α-carboxy protecting group refers to a chemical moiety coupled to the alpha carboxy group of an amino acid. Typically in SPPS the terminal a- carboxy group is usually linked to the insoluble support, and therefore, the linker/ handle acts as protecting group. Nevertheless, in some synthetic strategies such as strategies where the peptide is linked to the support by an amino acid side chain, and/ or where the peptide synthesis proceed in the reverse N->C direction, α-carboxy protection is needed. Examples of a-carboxy protecting groups are provided by t-butyl (£Bu), benzyl (Bn), 2-chlorotrityl (2- Cl-Trt), 2,4-dimethoxybenzyl (Dmb), 2-phenylisopropyl (2-PhiPr), 5-phenyl-3,4- ethylenedioxythenyl derivatives (Phenyl-EDOTn), 9-fluorenylmethyl (Fm), 4-(N- [ l -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino)benzyl
(Dmab), methyl (Me), ethyl (Et), carbamoylmethyl (Cam), Allyl, phenacyl (Pac), -nitrobenzyl (pNB), 2-trimethylsilylethyl (TMSE), (2-phenyl-2- trimethylsilyl) ethyl (PTMSE), 2-(trimethylsilyl)isopropyl (Tmsi), 2,2,2,- trichloroethyl (Tee), -hydroxyphenacyl ( HP), 4,5-dimethoxy-2-nitrobenzyl (Dmnb), 1 , 1 -dimethylallyl (Dma), and pentaamine cobalt (III). Preferred a- carboxy protecting groups are selected from allyl and 1 , 1 -dimethylallyl (Dma) . More preferred a-carboxy protecting group is allyl.
A side chain protecting group refers to a chemical moiety coupled to the side chain of an amino acid that prevents a portion of the side chain from reacting with chemicals used in steps of peptide synthesis, processing etc. In the context of the invention, a side chain protecting group may refer to a protecting group for hydroxyl (including 1 ,2-diols), carboxyl, amino, amide or thiol side chain groups. Examples of hydroxyl side chain protecting groups are provided by benzyl (Bn), 2,6-dichlorobenzyl (Deb), 2-bromobenzyl (BrBn), o- nitrobenzyl (oNB), benzyloxycarbonyl (Z), 2-bromobenzyloxycarbonyl (BrZ), 4- (3,6,9-trioxadecyl)oxybenzyl (TEGBn), i-butyloxycarbonyl (Boc), Boc-N-methyl- N-[2-(methylamino)ethyl]carbamoyl (Boc-Nmec), 3-pentyl (Pen), cyclohexyl (cHx), i-butyl (iBu), allyl, triphenylmethyl (Trt), 2-chlorotriphenylmethyl (2-C1- Trt), t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), 4,5- dimethoxy-2-nitrobenzyloxycarbonyl (Dmnb), propargyloxycarbonyl (Poc), and pseudoprolines (ΨΡΓΟ), such as dimethyloxazolidines (ΨΜε>ΜεΡΓθ) . Preferred hydroxyl side chain protecting groups are selected from i-butyl (iBu), i- butyloxycarbonyl (Boc), triphenylmethyl (Trt), 2-chlorotriphenylmethyl (2-C1- Trt), and dimethyloxazolidines (ΨΜε>ΜεΡΓθ). More preferred are i-butyl (iBu), triphenylmethyl (Trt), and 2-chlorotriphenylmethyl (2-Cl-Trt). Most preferred is i-butyl (iBu). Examples of protected 1 ,2-diol side chain groups include cyclic acetals and ketals, chiral ketones, cyclic ortho esters, silyl derivatives, cyclic carbonates and cyclic boronates. In the case of cyclic acetals and ketals the protecting group for the 1 ,2-diol can be selected from methylene, ethylidene, i- butylmethylidene, 1 -i-Butylethylidene, 1 -Phenylethylidene, 2-
(Methoxycarbonyl)ethylidene, 2-(i-Butylcarbonyl)ethylidene, phenylsulfonylethylidene, 2,2,2-trichloroethylidene, 3-(benzyloxy)propylidene, acrolein, acetonide (isopropylidene), cyclopentylidene, cyclohexylidene, cycloheptylidene, benzylidene, -methoxybenzylidene, l -(4- methoxyphenyl)ethylidene, 2,4-dimethoxybenzylidene, 3,4- dimethoxybenzylidene, -acetoxybenzylidene, 4-(£- butyldimethylsilyloxy)benzylidene, 2-nitrobenzylidene, 4-nitrobenzylidene, mesitylene, 6-bromo-7-hydroxycoumarin-2-ylmethylidene, 1 -naphthaldehyde acetal, 2 -naphthaldehyde acetal, 9-anthracene acetal, benzophenone ketal, di- ( -anisyl)methylidene ketal, xanthen-9-ylidene ketal, and 2,7- dimethylxanthen-9-ylidene ketal. In the case of chiral ketones the protecting group for the 1 ,2-diol can be selected from camphor and menthone. In the case of cyclic ortho esters the protecting group for the 1 ,2-diol can be selected from methoxymethylene, ethoxymethylene, 2-oxacyclopentylidene, dimethoxymethylene, 1 -methoxyethylidene, 1 -ethoxyethylidine, methylidene, phthalide, 1 ,2-dimethoxyethylidene, a-methoxybenzylidene, 1 -(N,N- dimethylamino)ethylidene derivative, a-(N,N-dimethylamino)benzylidene derivative, butane-2,3-bisacetal, cyclohexane- l ,2-diacetal and dispiroketals. In the case of silyl derivatives the protecting group for the 1 ,2-diol can be selected from di-i-butylsilylene group, dialkylsilylene groups, 1 ,3-( 1 , 1 ,3,3- tetraisopropyldisiloxanylidene) derivative, 1 , 1 ,3,3-tetra-i- butoxydisiloxanylidene derivative, methylene-bis-(diisopropylsilanoxanylidene), l , l ,4,4-tetraphenyl- l ,4-disilanylidene, o-xylyl ether, 3,3 - oxybis(dimethoxytrityl) ether and l ,2-ethylene-3,3-bis(4 "4 -dimethoxytrityl) ether. In the case of cyclic boronates the protecting group for the 1 ,2-diol can be selected from methyl boronate, ethyl boronate, phenyl boronate and o- acetamidophenyl boronate. Preferred 1 ,2-diol side chain protecting groups are selected from cyclic acetals and ketals. More preferred are selected from ethylidene, i-butylmethylidene, 1 -i-Butylethylidene, 2,2,2-trichloroethylidene, acetonide, acetonide (isopropylidene), cyclopentylidene, cyclohexylidene and cycloheptylidene. Most preferred is acetonide. Examples of amino side chain protecting groups are provided by formyl, benzyloxycarbonyl (Z) , 2- chlorobenzyloxycarbonyl (Cl-Z) , i-butyloxycarbonyl (Boc), cyclohexyloxycarbonyl (Hoc) , triphenylmethyl (Trt) , 4-methyltriphenylmethyl (Mtt) , monomethoxytriphenymethyl (Mmt) , dimethoxytriphenylmethyl (Dmt) , 9- fluorenylmethoxycarbonyl (Fmoc), 1 -(4,4-dimethyl-2,6-dioxoocyclohex- 1 - ylidene) -3 -methyl-butyl (ivDde), 2-(methylsulfonyl)ethoxycarbonyl (Msc), tetrachlorophthaloyl (TCP), allyloxycarbonyl (Alloc), 2-chlorobenzyloxycarbonyl (Cl-Z), -nitrobenzyloxycarbonyl ( NZ), 2-nitrobenzyloxycarbonyl (oNZ), 6- nitroveratryloxycarbonyl (NVOC), phenyldisulfanylethyloxycarbonyl (Phdec, and 2-pyridyldisulfanylethyloxycarbonyl (Pydec). Preferred amino side chain protecting groups are selected from i-butyloxycarbonyl (Boc) and 4- methyltritryl (Mtt). More preferred is i-butyloxycarbonyl (Boc). Examples of carboxy side chain protecting groups are provided by benzyl (Bn), cyclohexyl (cHx), i-butyl (iBu), -menthyl (Men), /3-3-methylpen-3-yl (Mpe), 2- phenylisopropyl (2-PhiPr), 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBn), 9- fluorenylmethyl (Fm), 4-(N-[ 1 -(4,4-dimethyl-2,6-dioxocyclohexylidene)-3- methylbutyl]amino)benzyl (Dmab), allyl, -nitrobenzyl (pNB), 2- (trimethylsilyl) ethyl (TMSE), (2-phenyl-2-trimethylsilylyl)ethyl (PTMSE), and 4,5-dimethoxy-2nitrobenzyl (Dmnb). Preferred carboxy side chain protecting groups are selected from benzyl (Bn), cyclohexyl (cHx), i-butyl (iBu), -menthyl (Men), /3-3-methylpen-3-yl (Mpe), and 2-phenylisopropyl (2-PhiPr). More preferred is i-butyl (iBu). Examples of amide side chain protecting groups are provided by 9-xanthenyl (Xan), triphenylmethyl (Trt), 4 -methyltriphenylmethyl (Mtt), cyclopropyldimethylcarbinyl (Cpd), 4, 4 '-dimethoxybenzhydryl (Mbh) and 2,4,6-trimethoxybenzyl (Tmob). Preferred amide side chain protecting groups are selected from 9-xanthenyl (Xan), triphenylmethyl (Trt), and 4- methyltriphenylmethyl (Mtt). More preferred are triphenylmethyl (Trt) or 4- methyltriphenylmethyl (Mtt). Most preferred is triphenylmethyl (Trt). Examples of thiol side chain protecting groups are provided by alkyl, acetamidomethyl (Acm), phenylacetamidomethyl (PhAcm), benzyl (Bn), -methylbenzyl (Meb), p- methoxybenzyl (Mob), 2,4,6,-trimethoxybenzyl (Tmob), o-nitrobenzyl (oNB), 9- fluorenylm ethyl (Fm), 9-xanthenyl (Xan), triphenylmethyl (Trityl, Trt), monomethoxytrityl (Mmt), S-alkyl disulfide 2,2,4,6,7-pentamethyl-5- dihydrobenzofuranylmethyl (Pmbf), 2-(2,4-dinitrophenyl)ethyl (Dnpe), 2- quinolyl, 4-picolyl, i-butyl (£Bu), 5-iert-butylmercapto (S'Bu), 1 -adamantyl ( 1 - Ada), t-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), N-allyloxycarbonyl-N- [2,3,5,6-tetrafluoro-4-(phenylthio)phenyl]aminomethyl (Fsam), 9- fluorenylmethoxycarbonyl (Fmoc), 3-nitro-2-pyridinesulfenyl (Npys), 2- piridinesulfenyl (S-Pyr) and ninhydrin (Nin) . Preferred thiol side chain protecting groups are selected from acetamidomethyl (Acm) , phenylacetamidomethyl (PhAcm) , benzyl (Bn) , -methylbenzyl (Meb) , triphenylmethyl (Trityl, Trt) and monomethoxytrityl (Mmt) . More preferred are selected from acetamidomethyl (Acm), phenylacetamidomethyl (PhAcm), triphenylmethyl (Trityl, Trt) and monomethoxytrityl (Mmt) . Most preferred is triphenylmethyl (Trityl, Trt) .
Suitable coupling reagents and additives for peptide synthesis are well known for the skilled person in the art. For instance a review for peptide coupling reagents is provided in Han, S-. Y. and Kim Y-. A. Tetrahedron 2004, 60, 2447-2467. Examples of coupling reagents are Ν,Ν'- dicyclohexylcarbodiimide (DCC) , N-(3-dimethylaminopropyl)-N- ethylcarbodiimide (EDC) and its salts, l -[3-(dimethylamino)propyl] -3- ethylcarbodiimide methiodide (EDC methiodide), N,N'-diisopropylcarbodiimide (DIPCDI) , l -ieri-butyl-3 -ethyl carbodiimide, N-cyclohexyl-N'-(2- morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC), N,N'-di-tert- butylcarbodiimide, 1 ,3-Di-p-tolylcarbodiimide, Ι , Γ-carbonyldiimidazole (CDI) , l , l '-carbonyl-di-( l ,2 ,4-triazole) (CDT), oxalic acid diimidazolide, 2-chloro- l ,3- dimethylimidazolidinium chloride (DMC) , 2-chloro- l ,3- dimethylimidazolidinium tetrafluorob orate (CIB), 2-chloro- l ,3- dimethylimidazolidinium hexafluorophosphate (CIP) , 2-fluoro- l ,3- dimethylimidazolidinium hexafluorophosphate (DFIH) , (benzotriazol- 1 - yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) , (benzotriazol- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), [(7-azabenzotriazol- 1 -yl)oxy]tripyrrolidinophosphonium hexafluorophos-phate (PyAOP), bromotris(dimethylamino)phosphonium hexafluorophos-phate (BroP) , chlorotripyrrolidinophosphonium hexafluorophosphate (PyClOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 3-(diethoxyphosphoryloxy)- l ,2,3-benzotriazin- 4 (3 H) -one (DEPBT), O- (benzotriazol- 1 -yl)-N,N,N',N-tetramethyluronium hexafluorophosphate (HBTU) , 0-(benzotriazol- l -yl)-N,N,N',N- tetramethyluronium tetrafluo-roborate (TBTU) , 0-(7-azabenzotriazol- l -yl)- N,N,N',N'-tetramethyluro-nium hexafluorophosphate (HATU), 0-(benzotriazol- l -yl)-N,N,N',N-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), O- benzotriazol- 1 -yl-N,N,N',N'-bis(pentamethylene)uronium hexafluoro-phosphate (HBPipU), (benzotriazol- 1 -yloxy)dipiperidinocarbenium tetrafluoroborate (TBPipU), 0-(6-chlorobenzotriazol- l -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), 0-(6-chloro-benzotriazol- 1 -yl)-N,N,N',N'- tetramethyluronium (TCTU), 0-(3,4-dihydro-4-oxo- l ,2,3-benzotriazin-3-yl)- N,N,N',N'-tetramethyluronium tetrafluoro-borate (TDBTU), 0-(2-oxo- 1 (2 H)pyridyl)-N,N,N',N -tetramethyluronium tetrafluoroborate (TPTU), O- [(ethoxycarbonyl)cyanomethylenamino]-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HOTU) , 0-[(ethoxycarbonyl)cyanomethylenamino] - Ν,Ν,Ν',Ν -tetramethyluronium tetrafluoroborate (TOTU), N,N,N',N-tetramethyl- 0-(N-succinimidyl)u-ronium hexafluorophosphate (HSTU), Ν,Ν,Ν',Ν'- tetramethyl-0-(N-succinimidyl)uronium tetrafluoroborate (TSTU), dipyrrolidino(N-succinimidyloxy)carbenium (HSPyU), S-( 1 -oxido-2-pyridyl)- N,N,N',N'-tetramethylthiouronium tetrafluoroborate (TOTT) and l -[( l -(cyano-2- ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino)]uranium hexafluorophosphate (COMU). Examples of additives are 1 - hydroxybenzotriazole (HOBt), 1 -hydroxy-7-azabenzotriazole (HO At), 3,4- dihydro-3-hydroxy-4-oxo- l , 2, 3-benzotriazine (HODhbt), N-hydroxytetrazole, ethyl- 1 -hydroxy- l H- 1 , 2, 3-triazole-4-carboxylate (HOCt), ethyl 2-cyano-2- (hydroxyimino)acetate (Oxyma) and benzyltriphenylphosphonium dihydrogen trifluoride (PTF).
The mention of these groups should not be interpreted as a limitation of the scope of the invention, since they have been mentioned as a mere illustration of protecting groups, coupling reagents and additives for peptide synthesis, but further groups having said functions may be known by the skilled person in the art, and they are to be understood to be also encompassed by the present invention. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about". It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/ or measurement conditions for such given value.
In a first aspect, the compounds of formula I can be obtained synthetically from intermediates of formula Ila following a sequence of key reactions that comprise a macrolactamization step performed between positions 1 and 2 of the intermediate Ila to give a compound of formula la.
Moreover, this process can further comprise the step of cleaving a compound of formula la from an insoluble support to which said compound may be attached through terminal R1 side chain group.
Moreover, this process may further comprise the step of removing side chain protecting groups performed over a compound of formula la.
The macrolactamization step performed between positions 1 and 2 of the intermediate Ila to give a compound of formula la is typically effected using suitable coupling reagents. Suitable coupling reagents are as defined above, typically PyBOP/HOAt coupling system is used. The macrolactamization step may be effected using solid or solution phase synthesis, being solid phase synthesis more preferred.
When necessary, the step of cleaving a compound of formula la from a insoluble support to which said compound may be attached through terminal R12 side chain group is typically carried out following standard procedures known in solid phase peptide synthesis. The precise conditions required to cleave said compound from the insoluble support may vary with the nature of the side chain functional group attached to the support and the linker group in the support and are similar to those known in the art. Preferably the insoluble support is selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like; more preferably the insoluble support is selected from 4- hydroxymethylphenoxymethyl polystyrene Wang resin and Wang ChemMatrix resin; being 4 -hydroxymethylphenoxymethyl polystyrene Wang resin most preferred. When the insoluble support is 4 -hydroxymethylphenoxymethyl polystyrene Wang resin, the cleavage step is typically effected by using acidic conditions; preferably by using TFA-H2O-TIS.
The step of removing side chain protecting groups performed over a compound of formula la is typically effected following standard procedures known in the art. The precise conditions may vary with the nature of the side chain protecting groups and are known in the art. The side chain removal step may be effected before, at the same time or after the link between the insoluble support and the compound of formula la has been cleaved. In a preferred embodiment, the step of cleaving said compound from the insoluble support and the step of removing side chain protecting groups are carried out by a single step.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein Ri is preferably selected from substituted or unsubstituted Ci-Cie alkyl and substituted or unsubstituted C2-C 18 alkenyl, which may be branched or unbranched. More preferred alkyl and alkenyl groups, which may be branched or unbranched, are those having from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12 , 13, 14, 15 or 16 carbon atoms. It is particularly preferred that the alkyl and alkenyl groups are substituted by one or more suitable substituents, being the substituents preferably selected from OR', =0, SR', SSR', SOR', SO2R', SO3R', OSO2R', OSO3R', NO2, NHR', N(R 2, =N-R', NfR'JCOR', NfCOR^, N(R')S02R', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, CONHR', CONiR'Ja, CONNOR', CON(R')S02R', POiOR^, POiOR^R', PO(OR')(N(R')R'), substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2- C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R' groups is independently selected from the group consisting of hydrogen, OH, NO2, N¾, SH, CN, halogen, CHO, COalkyl, COOH, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferably, substituents for the above mentioned alkyl and alkenyl groups are selected from OR', OS02R', OSO3R', halogen, OCOR', OCOOR', OCONHR', OCONiR'Ja, CONHR', and CONfROa, wherein each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; and even more preferred the substituent is OH. More preferably Ri is a substituted alkyl group having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms wherein the substituent is OH. Even more preferably Ri is selected from 2 -hydroxy -pentyl, 2-hydroxy-hexyl, 2-hydroxy-heptyl, 2-hydroxy-octyl, 2-hydroxy-nonyl, 2- hydroxy-decyl, 2 -hydroxy -undecyl, 2-hydroxy-dodecyl, 2-hydroxy-tridecyl, 2- hydroxy-tetradecyl, 2-hydroxy-pentadecyl, 2-hydroxy-hexadecyl, 3-methyl-2- hydroxy-butyl, 4-methyl-2-hydroxy-pentyl, 5-methyl-2-hydroxy-hexyl, 6- methyl-2-hydroxy-heptyl, 7-methyl-2-hydroxy-octyl, 8 -methyl-2 -hydroxy - nonyl, 9 -methyl-2 -hydroxy-decyl, 10-methyl-2-hydroxy-undecyl, l l -methyl-2- hydroxy-dodecyl, 12-methy-2-hydroxy-tridecyl, 13 -methyl-2 -hydroxy - tetradecyl, 14 -methyl-2 -hydroxy-pentadecyl, 2-hydroxy-l ,3,5-trimethylhexyl and 2-hydroxy-l , 3, 5, 7-tetramethyloctyl; being 2-hydroxy- l ,3,5-trimethylhexyl and 2 -hydroxy- 1 ,3, 5, 7-tetramethyloctyl the most preferred. Particularly preferred processes according to invention are those that provide compounds of formula I wherein R2 and R3 are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2, 3, 4, 5 or 6 carbon atoms. It is particularly preferred that the alkyl group is substituted at one or more available positions by one or more suitable substituents, being the substituents preferably selected from OR', =0, SR', SSR', NHR', NfR^, NfR'JCOR', NfCOR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, COOCOR', COOCOOR', COOCONHR', COOCONiR'Ja, CONHR', CONiR'Ja, CONiR'JCOR', CONiR COOR', CONfR'JCONHR', CONiR'JCONiR'Ja, substituted or unsubstituted C2-Ci2 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; more preferably, substituents for the above mentioned alkyl group is selected from OR', SR', SSR', NHR', NfR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', COOR', CONHR', CONiR'Ja, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; being most preferred OR', COOR', CONHR' and CON^; wherein preferably each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group; more preferably each of the R' groups is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; even more preferred is hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl; being most preferred hydrogen. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferably R2 is selected from hydrogen and a substituted alkyl having 1 carbon atom wherein substituents for the alkyl group are selected from OR', COOR', CONHR' and CON^ wherein R' is as defined above. Even more preferably R2 is selected from hydrogen, -CH2COOR', -CH R^COOR', CH2CONHR', -CH2CON(R')2, -CHiOR^CONHR' and -CHiOR^CONiR^, wherein R' is as defined above. Most preferred R2 is selected from hydrogen, - CH2COOH, -CH(OH)COOH, -CH2CONH2, and -CH(OH)CONH2. More preferably R3 is a substituted alkyl having 2, 3 or 4 carbon atoms, which may be branched or unbranched, wherein the substituents for the alkyl group are selected from -CONHR' and CONiR^ wherein R' is as defined above. Even more preferred R3 is selected from -CH2CH2CONHR', -CHaCHaCONiR'Ja, - CH(CH3)CH2CONHR', -CHiCHaJCHaCONiR'Ja, -CH2CH(CH3)CONHR', CHaCHiCHaJCONiR'Ja, CH(CH3)CH(CH3)CONHR' and -CHiCHaJCHiCHaJCONiR'Ja wherein R' is as defined above. Most preferred R3 is selected from - CH2CH2CONH2, -CH(CH3)CH2CONH2, -CH2CH(CH3)CONH2 and CH(CH3)CH(CH3)CONH2. Particularly preferred processes according to invention are those that provide compounds of formula I wherein R4 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferably R4 is an alkyl group, which may be branched or unbranched, having 1 , 2, 3, 4, 5 or 6 carbon atoms. Even more preferred R4 is selected from methyl, ethyl, n-propyl, iso-propyl, butyl, including n-butyl, tert- butyl, sec-butyl and iso-butyl, and pentyl, including n-pentyl, 1 -methyl-butyl, 2 -methyl-butyl, 3 -methyl-butyl, 1 , 1 -dimethyl-propyl, 1 ,2-dimethyl-propyl, 1 - ethyl-propyl, neopentyl and cyclohexyl; being methyl and 1 ,2-dimethyl-propyl the most preferred.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein R5 is independently selected from hydrogen, substituted or unsubstituted C 1-C6 alkyl, CORa, and COORa, wherein Ra is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably R5 is hydrogen. Particularly preferred processes according to invention are those that provide compounds of formula I wherein R6, and R7 are each independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, CORa, CONRaRb, and C(=NRa)NRaRb, wherein Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n- propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso- butyl. More preferably R6 and R7 are hydrogen.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein Re and Rio are each independently selected from substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred alkyl groups, which may be branched or unbranched, are those having 1 , 2, 3, or 4 carbon atoms; being methyl and ethyl the most preferred. Preferably Re and Rio have different meaning in the compounds of the invention.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein Rn is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. More preferably Rn is independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl. Even more preferably Rn is independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl; being hydrogen the most preferred group.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein Rg and R12 are each independently selected from NRaRb and ORc, wherein Rc is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, CORa, and COORa, and wherein Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C1-C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl. More preferably Rc is hydrogen. Preferably Rg and R12 are each independently selected from OH and NH2.
Particularly preferred processes according to invention are those that provide compounds of formula I wherein Y is selected from a group of formulae (a), (b), (c), (d) and (e); wherein particularly preferred Riy and R6y are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2, 3, 4, 5 or 6 carbon atoms. It is particularly preferred that the alkyl group is substituted at one or more available positions by one or more suitable substituents, being the substituents preferably selected from OR', =0, SR', SSR', NHR', NfR^, NfR'JCOR', I^COR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, COOCOR', COOCOOR', COOCONHR', COOCONiR'Ja, CONHR', CONfROa, CONiR'JCOR', CONiR'JCOOR', CONfR'JCONHR', CONiR'JCONiR'Ja, substituted or unsubstituted C2-Ci2 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; more preferably, substituents for the above mentioned alkyl group is selected from OR', SR', SSR', NHR', NfR^, NiR^CONHR', Ν^ΟΟΝ^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', COOR', CONHR', CONiR'Ja, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; being most preferred OR', COOR', CONHR', CONiR'Ja, NfR'JCONHR', NfR'JCONiR'Ja, N(R')C(=NR')NHR' and N(R')C(=NR')N(R')R'; wherein preferably each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; more preferably each of the R' groups is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; even more preferred is hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec- butyl and iso-butyl; being most preferred hydrogen. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferably each Riy and R6y is selected from hydrogen and a substituted alkyl having 1 , 2, 3 or 4 carbon atoms wherein substituents for the alkyl group are independently selected from OR', COOR', CONHR', CONiR^, NfR'JCONHR', Ν^ΟΟΝ^, N(R')C(=NR')NHR' and N(R')C(=NR')N(R')2, wherein R' is as defined above. Even more preferably each Riy and R6y is independently selected from hydrogen, -CH2CONHR', - CHaCONiR'Ja -CHaCHaCONHR', -CHaCHaCONiR'Ja, - CH2(OR') and - CHfOROCHa. Most preferred Rly is selected from -CH2CONH2, -CH2CH2CONH2 and -CH(OH)CH3. Most preferred R6y is - CH2OH. particularly preferred R2y and R3y are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, CORa and COORa, wherein Ra is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably F¾y and R3y are hydrogen. particularly preferred each R4y and Rsy is independently selected from hydrogen, substituted or unsubstituted C 1 -C6 alkyl, NRaRb and ORc, wherein Rc is selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, CORa, and COORa, and wherein Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C 1 -C 12 alkyl. Particularly preferred Ra and Rb are each independently selected from hydrogen and substituted or unsubstituted C 1 -C6 alkyl; and even more preferred are each independently selected from hydrogen, methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. Particularly preferred Rc is hydrogen. More preferably each R4y and Rsy is independently selected from hydrogen, NRaRb, ORc, and a substituted alkyl group having 1 or 2 carbon atoms wherein the substituent is OH. Most preferred each R4y and Rsy is independently selected from hydrogen, N¾ , OH and -CH2OH; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted heteroalicyclic group; being a substituted or unsubstituted pirrolidine most preferred.
More preferred processes according to the invention are those that provide compounds of formula I wherein Y is selected from a group of formulae (a), (c) and (e).
In additional preferred embodiments, the preferences described above for the different substituents are combined. The process of the present invention is also directed to such combinations of preferred substitutions in the formula I above.
More preferred processes for the synthesis of compounds of formula I are those that give compounds of formula: 36
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
In another aspect, with this invention we provide novel intermediate compounds of formula II:
Figure imgf000041_0002
wherein X, Zi , Z2, R3, R4, R5, 6, R7, Rs, R9, Rio, R11 , R12 and n are as defined above in the previous description of intermediates of formula II.
In compounds of formula II, particularly preferred X is a group of formula (h): O R2 o
Formula (h)
In compounds of formula II, particularly preferred Ri is selected from substituted or unsubstituted Ci-Cie alkyl and substituted or unsubstituted C2-C 18 alkenyl, which may be branched or unbranched. More preferred alkyl and alkenyl groups, which may be branched or unbranched, are those having from 3 to about 18 carbon atoms; and even more preferably 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms. It is particularly preferred that the alkyl and alkenyl groups are substituted by one or more suitable substituents, being the substituents preferably selected from OR', =0, SR', SSR', SOR', SO2R', SO3R', OSO2R', OSO3R', NO2, NHR', N(R 2, =N-R', NfR'JCOR', NfCOR^, N(R')S02R', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, CONHR', CONiR'Ja, CONNOR', CON(R')S02R', POiOR^, POiOR^R', PO(OR')(N(R')R'), substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2- C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, wherein each of the R' groups is independently selected from the group consisting of hydrogen, OH, NO2, N¾, SH, CN, halogen, CHO, COalkyl, COOH, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferably, substituents for the above mentioned alkyl and alkenyl groups are selected from OR', OSO2R', OSO3R', halogen, OCOR', OCOOR', OCONHR', OCONiR'Ja, CONHR', and CONfROa, wherein each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; and even more preferred the substituent is OH. More preferably Ri is a substituted alkyl group having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms wherein the substituent is OH. Even more preferably Ri is selected from 2 -hydroxy -pentyl, 2-hydroxy-hexyl, 2-hydroxy-heptyl, 2-hydroxy-octyl, 2-hydroxy-nonyl, 2- hydroxy-decyl, 2 -hydroxy -undecyl, 2-hydroxy-dodecyl, 2-hydroxy-tridecyl, 2- hydroxy-tetradecyl, 2-hydroxy-pentadecyl, 2-hydroxy-hexadecyl, 3 -methyl-2 - hydroxy-butyl, 4-methyl-2-hydroxy-pentyl, 5-methyl-2-hydroxy-hexyl, 6- methyl-2-hydroxy-heptyl, 7-methyl-2-hydroxy-octyl, 8 -methyl-2 -hydroxy - nonyl, 9-methyl-2-hydroxy-decyl, 10-methyl-2 -hydroxy -undecyl, l l -methyl-2- hydroxy-dodecyl, 12 -methy-2 -hydroxy-tridecyl, 13 -methyl-2 - hydroxy - tetradecyl, 14 -methyl-2 -hydroxy-pentadecyl, 2-hydroxy- l ,3 ,5-trimethylhexyl and 2-hydroxy- l ,3,5,7-tetramethyloctyl; being 2-hydroxy- l ,3,5-trimethylhexyl and 2-hydroxy- l ,3,5,7-tetramethyloctyl the most preferred.
In compounds of formula II, particularly preferred R2 and R3 are each independently selected from hydrogen, and substituted or unsubstituted Ci- C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2 , 3 , 4, 5 or 6 carbon atoms. It is particularly preferred that the alkyl group is substituted at one or more available positions by one or more suitable substituents, being the substituents preferably selected from OR', =0, SR', SSR', NHR', N(R 2, NfR'JCOR', NfCOR^, NfR'JCONHR', NiR'JCONiR^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', OCOR', OCOOR', OCONHR', OCONiR'Ja, COOCOR', COOCOOR', COOCONHR', COOCONiR^, CONHR', CON^, CONR'COR', CONR'COOR', CONR'CONHR', CONR'CONiR'Ja, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; more preferably, substituents for the above mentioned alkyl group is selected from OR', SR', SSR', NHR', NfR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', COOR', CONHR', CONiR'Ja, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; being most preferred OR', COOR', CONHR' and CON^; wherein preferably each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, or when R' is attached to S, O or N atoms then R' may also be selected from a side chain protecting group; more preferably each of the R' groups is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl or a side chain protecting group; even more preferred is hydrogen, methyl, ethyl, n- propyl, iso-propyl, butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl, or a side chain protecting group; being most preferred hydrogen or a side chain protecting group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. Most preferred R2 is selected from hydrogen, -CH2COOR', -CHfOROCOOR', -CH2CONHR', and - CHiOR'JCONHR', wherein R' is selected from hydrogen and a hydroxyl side chain protecting group when R' is linked to an O atom, a carboxy side chain protecting group when R' is linked to a COO- group or an amide side chain protecting group when R' is linked to a CONH- group. Most preferred R3 is selected from -CH2CH2CONHR', -CH(CH3)CH2CONHR', -CH2CH(CH3)CONHR' and -CH(CH3)CH(CH3)CONHR', wherein R' is selected from hydrogen and an amide side chain protecting group. The different side chain protecting groups are selected following the guidance and preferences given above.
In compounds of formula II, particularly preferred R4 is selected from hydrogen and a substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred R4 is an alkyl group, which may be branched or unbranched, having 1 , 2, 3, 4, 5 or 6 carbon atoms. Even more preferred R4 is selected from methyl, ethyl, n-propyl, iso-propyl, butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl, and pentyl, including n- pentyl, 1 -methyl-butyl, 2 -methyl-butyl, 3 -methyl-butyl, 1 , 1 -dimethyl-propyl, 1 ,2 -dimethyl-propyl, 1 -ethyl-propyl, neopentyl and cyclohexyl; being methyl and 1 ,2 -dimethyl-propyl the most preferred.
In compounds of formula II, particularly preferred R5 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, CORa, and COORa, wherein Ra is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably R5 is PG3. PG3 is a side chain protecting group and is selected following the guidance and preferences given above. In compounds of formula II, particularly preferred R6 and R7 are each independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, CORa, CONRaRb, and C(=NRa)NRaRb wherein each Ra and Rb is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; or when R6 is hydrogen then R7 is selected from PG3, CONHRb, and C(=NH)NHRb wherein Rb is independently selected as PG3. More preferred alkyl groups are substituted or unsubstituted C1-C6 alkyl; and even more preferred alkyl groups are each independently selected from methyl, ethyl, n-propyl, iso- propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably, R6 is hydrogen and R7 is selected from PG3, CONHRb and C(=NH)NHRb wherein Rb is independently selected as PG3. Most preferred, R6 is hydrogen and R7 is PG3. PG3 is a side chain protecting group and is selected following the guidance and preferences given above.
In compounds of formula II, particularly preferred Re and Rio are each independently selected from substituted or unsubstituted C1-C6 alkyl, which may be branched or unbranched. More preferred alkyl groups, which may be branched or unbranched, are those having 1 , 2, 3, or 4 carbon atoms; being methyl and ethyl the most preferred. Preferably Re and Rio have different meaning in the compounds of the invention.
In compounds of formula II, particularly preferred R9 is selected from NRaRb and ORc, wherein Rc is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, CORa, and COORa, and wherein Ra is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl and PG3. More preferred alkyl groups are substituted or unsubstituted C1-C6 alkyl; and even more preferred alkyl groups are each independently selected from methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably each Rb and Rc is independently selected from hydrogen and PG3. More preferably R9 is selected from ORc and NHRb wherein each Rb and Rc is independently selected from hydrogen and PG3. PG3 is a side chain protecting group and is selected following guidance and preferences given above. In compounds of formula II, particularly preferred Rn is selected from hydrogen, PG3 and substituted or unsubstituted C1-C12 alkyl. More preferably Rn is independently selected from hydrogen, PG3 and substituted or unsubstituted C1-C6 alkyl. Even more preferably Rn is independently selected from hydrogen, PG3, methyl, ethyl, n-propyl, iso-propyl and butyl, including n- butyl, ieri-butyl, sec-butyl and iso-butyl; being PG3 the most preferred group. PG3 is a side chain protecting group and is selected following the guidance and preferences given above.
In compounds of formula II, particularly preferred R12 is selected from NRaRb and ORc, wherein Rc is preferably selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, a suitable insoluble support, CORa, and COORa, and wherein Ra is independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, PG3 and a suitable insoluble support. More preferred alkyl groups are substituted or unsubstituted C1-C6 alkyl; and even more preferred alkyl groups are each independently selected from methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferably each Rb and Rc is independently selected from PG3 and a suitable insoluble support. More preferably R12 is selected from ORc and NHRb wherein each Rb and Rc is independently selected from PG3 and a suitable insoluble support. PG3 is a side chain protecting group and is selected following the guidance and preferences given above. The suitable insoluble support is preferably selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like.; more preferably the insoluble support is selected from 4- hydroxymethylphenoxymethyl polystyrene Wang resin and Wang ChemMatrix resin; being 4-hydroxymethylphenoxymethyl polystyrene Wang resin most preferred. In compounds of formula II, particularly preferred Y is selected from formulae (a), (b) , (c), (d) and (e); wherein particularly preferred Riy and R6y are each independently selected from hydrogen, and substituted or unsubstituted C1-C12 alkyl, which may be branched or unbranched. More preferred alkyl groups are those having 1 , 2 , 3 , 4, 5 or 6 carbon atoms. It is particularly preferred that the alkyl group is substituted at one or more available positions by one or more suitable substituents, being the substituents preferably selected from OR', OCOR', OCOOR', OCONHR', OCONiR'Ja, =0, SR', SSR', NHR', N(R 2, NfR'JCOR', NiCOR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', CN, halogen, COR', COOR', COOCOR', COOCOOR', COOCONHR', COOCONiR^, CONHR', CON^, CONR'COR', CONR'COOR', CONR'CONHR', CONR'CONiR^, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; more preferably, substituents for the above mentioned alkyl group is selected from OR', SR', SSR', NHR', NfR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR', N(R')C(=NR')N(R')R', COOR', CONHR', CONiR^, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; being most preferred OR', COOR', CONHR', CONiR^, NfR'JCONHR', NfR'JCONiR'^, N(R')C(=NR')NHR' and N(R')C(=NR')N(R')R'; wherein each of the R' groups is independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group or when R' is attached to S, O or N atoms then R' may also be selected from a side chain protecting group; more preferably each of the R' groups is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl or a side chain protecting group; even more preferred is hydrogen, methyl, ethyl, n-propyl, iso-propyl, butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl, or a side chain protecting group; being most preferred hydrogen or a side chain protecting group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. More preferably each Riy and R6y is selected from hydrogen and a substituted alkyl having 1 , 2, 3 or 4 carbon atoms wherein substituents for the alkyl group are independently selected from OR', COOR', CONHR', NfR'JCONHR' and N(R')C(=NR')NHR', wherein R' is as defined above. Even more preferably each Riy and R6y is independently selected from hydrogen, -CH2CONHR', -CH2CH2CONHR', - CH2(OR') and
Figure imgf000048_0001
wherein R' is independently selected from hydrogen and a hydroxyl side chain protecting group when R' is linked to an O atom or an amide side chain protecting group when R' is linked to a CONH- group; most preferred R' is independently selected from a hydroxyl side chain protecting group and an amide side chain protecting group. Most preferred Riy is selected from - CH2CONHR', -CH2CH2CONHR' and -CHiOR CHa, wherein R' is selected from a hydroxyl side chain protecting group and an amide side chain protecting group. Most preferred R6y is - CH2(OR'), wherein R' is a hydroxyl side chain protecting group. The different side chain protecting groups are selected following the guidance and preferences given above; particularly preferred R2y and R3y are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, PG3, CORa and COORa, wherein Ra is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl. Particularly preferred Ra is substituted or unsubstituted C1-C6 alkyl; and even more preferred is methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, ieri-butyl, sec-butyl and iso-butyl. More preferred R2y and R3y are each independently selected as PG3, even more preferred R2y and R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted dioxolane; being 1 , 1 -dimethyl- 1 ,3 -dioxolane most preferred. PG3 is a side chain protecting group and is selected following guidance and preferences given above. particularly preferred each R4y and Rsy is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, NRJ¾ and ORc, wherein Rc is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, PG3, CORa, and COORa, and wherein Ra is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl and Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl and PG3. More preferably Ra is selected from hydrogen and substituted or unsubstituted Ci-C-6 alkyl; and even more preferred is hydrogen. More preferably, each Rb and Rc is independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl and PG3; and even more preferred are each independently selected from hydrogen and PG3. More preferably each R4y and Rsy is independently selected from hydrogen, NHRb, ORc and a substituted alkyl group having 1 or 2 carbon atoms wherein the substituent is ORc; wherein each Rc and Rb is independently selected from hydrogen and PG3. PG3 is a side chain protecting group and is selected following guidance and preferences given above; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted heteroalicyclic group; being a substituted or unsubstituted pirrolidine most preferred.
In compounds of formula II, particularly preferred Zi is hydrogen and particularly preferred Z2 are hydrogen and a suitable insoluble support. Most preferred Z is hydrogen.
More preferred compounds of formula II are those of formula Ila wherein X is a group of formula (h) and Y is a group selected from the group of formulae (a), (c) and (e).
In additional preferred embodiments, the preferences described above for the different substituents are combined. The process of the present invention is also directed to such combinations of preferred substitutions in the formula II above.
Suitable starting materials for the synthesis of the intermediates of formula II include proteinogenic and non-pro teinogenic amino acids. Such starting materials may be either commercially available or synthetically prepared as previously described or as decribed herein. See for example, J. Org. Chem. 2003, 68, 7841 ; Tetrahedron 2001 , 57, 6353; Org. Lett. 2000, 2, 4157; J. Org. Chem 2006, 71 , 6351 and Amino Acid 2010, 39, 161 , which are incorporated herein by reference in their enterity.
In a further aspect, intermediates of formula II are obtained by a process performed in solid phase following a sequence of key reactions that comprise a step of forming an ester bond performed at the branching position 5 of a compound of formula III to provide an intermediate of formula lib wherein X is PG 1. Moreover, this process can further comprise the step of removing terminal PG 1 group of the exocyclic chain and elongating said exocyclic chain performed over a compound of formula lib wherein X is PG 1 to provide a compound of formula lib wherein X is a group of formula (f) ; or performed over a compound of formula lib wherein X is a group of formula (f) to provide a compound of formula lib wherein X is a group of formula (g); or performed over a compound of formula lib wherein X is a group of formula (g) to provide a compound of formula lib wherein X is a group of formula (h).
Moreover, this process further comprises the step of removing PG 1 group performed over an intermediate of formula lib wherein X is a group of formula (h) and Z2 is selected from PG2 and a suitable insoluble support.
Moreover, this process can further comprise the step of removing PG2 group performed over an intermediate of formula lib wherein X is a group of formula (h) and Z2 is PG2.
Moreover, this process can further comprise the step of cleaving a compound of formula lib wherein X is a group of formula (h) and Z2 is a suitable insoluble support.
Moreover, this process further comprises the step of elongating the peptidic chain performed over conveniently protected aal residue of the macrocyclic region which is attached to a suitable insoluble support to provide a compound of formula III.
Moreover, this process further comprises the step of attaching conveniently protected aa 1 amino acid of the macrocyclic region to a suitable insoluble support to be used as starting point for elongating the peptidic chain to provide a compound of formula III . The step of forming the ester bond is performed between secondary hydroxyl group at at branching position 5 of a compound of formula III and free carboxy group of a-amino protected pipecolic acid to provide an intermediate of formula lib wherein X is PG l . This step is typically effected using suitable coupling reagents and may required to apply temperature depending on the esteric hindrance around the branching position 5. In a preferred embodiment this step is effected using a carbodiimide in the presence of catalytic amounts of a base as coupling reagents; being DIPCDI in the presence of catalytic amounts of DMAP more preferred. In another preferred embodiment this step is effected at a temperature from about 25 to about 65 °C; being a temperature from about 35 to about 55 °C more preferred. Suitable a-amino protecting groups (PG l) to be used in pipecolic acid are selected following guidance and preferences given above. The exocyclic chain elongation step performed over a compound of formula lib wherein X is selected from PG 1 and a group of formulae (f) or (g) is typically effected by cycles of a-amino deprotection and coupling reactions in a C→N terminal direction to finally provide a compound of formula lib wherein X is a group of formula (h). The protecting groups for a-amino and for side chain functionalities that may be present are chosen following the guidance and preferences given above. The coupling reactions are carried out using suitable coupling reagents. Suitable coupling reagents are selected following guidance given above, typically HATU/HOAt or PyBOP/HOAt coupling systems are independently used in each coupling reaction. In a preferred embodiment, the last amino acid and N- terminal moiety of the exocyclic chain are incorporated as one unit. More preferably, the last amino acid and terminal moiety are incorporated as one unit using a phosphonium salt together with HOAt additive as coupling system; being PyBOP/HOAt coupling system most preferred.
The step of removing PG 1 group performed over a compound of formula lib wherein X is a group of formula (h) and Z2 is selected from PG2 and a suitable insoluble support is typically effected by standard procedures known in the art. The precise conditions required may vary depending the nature of PG 1. PG 1 is selected following guidance and preferences given above.
The step of removing PG2 group performed over a compound of formula lib wherein X is a group of formula (h) and Z2 is PG2 is typically effected by standard procedures known in the art. The precise conditions required may vary depending the nature of PG2. PG2 is selected following guidance and preferences given above.
When necessary, the step of cleaving a compound of formula lib wherein X is a group of formula (h) and Z2 is a suitable insoluble support from said insoluble support is typically carried out following standard procedures known in solid phase peptide synthesis. The precise conditions required to cleave said compound from the insoluble support may vary with the nature of the support and the linker group in the support and are similar to those known in the art. Preferably the insoluble support to be used in Z2 is selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin, Wang ChemMatrix resin, HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, 4-hydroxymethylphenoxyacetyl- poly(dimethylacrylamide) Atherton resin, benzhydryl bromide resins, SASRIN resin, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix and the like.; more preferably the insoluble support is selected from HMPB polystyrene resin, HMPB ChemMatrix resin, NovaPEG HMPB resin, benzhydryl bromide resins, 2-chlorotritylchloride (CTC) Barlos resin, trityl ChemMatrix; being benzhydryl bromide resins and 2-chlorotritylchloride (CTC) Barlos resin most preferred. When the insoluble support is selected from benzhydryl bromide resins and 2-chlorotritylchloride (CTC) Barlos resin, the cleavage step is typically effected by using acidic conditions; preferably by using TFA.
In a preferred embodiment, the steps of removing PG 1 and PG2 groups performed over an intermediate of formula lib wherein X is a group of formula (h) and Z is PG2 to provide a compound of formula Ila are carried out by a single step. When PG1 is Alloc and PG2 is Allyl, this step is typically carried out by using neutral reductive conditions; preferably by using Pd(PPh3)4 in the presence of a suitable scavenger such as PhSiH3, ΗβΝ-ΒΗβ or Me2NH-BH3; being PhSiH3 more preferred. The step of elongating the peptidic chain is typically effected by repetitive cycles of a-amino deprotection and coupling reactions in a C→N terminal direction performed over conveniently protected aal residue of the macrocyclic region which is attached to a suitable insoluble support as starting point to provide a compound of formula III. Typically, the coupling reactions are carried out using suitable coupling reagents that are known in the art and are selected following the guidance given above; typically HATU/HOAt or DIPCDI/HOBt coupling systems are independently used in each coupling reaction. The protecting groups for a-amino, a-carboxy and side chain functionalities that may be present are chosen following the guidance and preferences given above.
The step of attaching conveniently protected aal amino acid of the macrocyclic region to a suitable insoluble support to be used as starting point for elongating the peptidic chain to provide a compound of formula III is performed by attaching said aal amino acid through terminal R1 side chain functional group or through its a-carboxy functional group to a suitable insoluble support. In the case of attaching aal amino acid to a suitable insoluble support through terminal R1 side chain functional group, the nature of the linkage formed may vary depending on the desired terminal R1 side chain functional group in the target peptide (acid or amide). This step is typically effected using methods known in the art which are described in standard texts on solid phase peptide synthesis such as Kates, S. A. and Albericio, F. in Solid-Phase synthesis. A practical guide; 1st Ed. Boca Raton: CRC Press 2000. In a preferred embodiment, the aal amino acid of the macrocyclic region is attached to a suitable insoluble support through terminal R1 side chain functional group forming an ester or amide linkage The protecting groups for a-amino and α-carboxy functionalities are chosen following the guidance and preferences given above. Suitable insoluble supports are selected following the guidance and preferences given above.
Particularly preferred processes according to invention are those that provide compounds of formula II according to the preferences previously described for intermediates of formula II.
This invention also relates to the use of intermediates of formula II the manufacture of compounds of formula I, and in particular in 1 manufacture of:
Figure imgf000054_0001
Figure imgf000054_0002
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
EXAMPLES ABBREVIATIONS
Nomenclature used for cyclic peptides and precursors as described by Spengler et al. (see Spengler, J., Jimenez, J. C, Burger, K., Giralt, E., Albericio, F. "Abbreviated nomenclature for cyclic and branched homo- and hetero-detic peptides" J. Peptide Res. 2005, 65, 550-555). The "&" symbol is used in the nomenclature for cyclic peptides and precursors. The appearance of "&" in a given position of the one-line formula represents the point at which one end of a chemical bond is located and the second "&" indicates the point to which this bond is attached. Thus, "&" represents the start or the end of a chemical bond, which is "cut" with the aim of visualazing a complex formula more easily. In this way, two "&" symbols represent one chemical bond.
GENERAL PROCEDURES Protected amino acid derivatives were obtained from Bachem
(Bubendorf, Switzerland) and Iris-Biotech. Coupling reagents, linkers and resins were purchased to MedalChemy, Aldrich (Milwaukee, WI), Luxembourg Industries (Tel Aviv, Israel) and NovaBiochem (Laufelfingen, Switzerland). Solvents and piperidine were obtained from SDS (Peypin, France). Reagents of common use were obtained from Aldrich (Milwaukee, WI) and Panreac (Barcelona, Spain). All commercial reagents and solvents were used as received.
Solution reactions were performed in round-bottomed flasks. Organic layers were dried over anhydrous Na2S04 or MgS04, and the solvent removed under reduced pressure at temperatures below 40 °C. All reactions were monitored by TLC on MERCK TLC plates silica gel 60 F254 under UV light (254 and 365 nm), and/or stained with ethanolic phosphomolybdic acid ( 12 g in 100 mL) or ninhydrin solution ( 1.5 g ninhydrin, 3 mL AcOH, 100 mL EtOH). Flash column chromatography was carried out using SDS Siliga gel 60A (35- 70 μηι). Solid phase syntheses were undertaken in polypropylene syringes fitted with two polyethylene filter discs. All solvents and soluble reagents were removed by suction. Washings between deprotection, coupling and cyclization steps were carried out with DMF (3 x 1 min) and DCM (3 x 1 min) using 4 mL/ 100 mg resin solvent for each wash. When not specified, all transformations and washes were performed at 25 °C.
Analytical HPLC was performed on a Waters instrument comprising a separation module (Waters 2695), automatic injector, photodiode array detector (Waters 996 or Waters 2998), and system controller (Millenium32 login). The columns used were Xbridge™ C 18 reversed-phase analytical column 2.5 μιη x 4.6 mm x 75 mm and Xbridge™ BEH130 C 18 reversed- phase analytical column 3.5 μιη x 4.6 mm x 100 mm. UV detection was at 220 and 254 nm, and linear gradients of ACN (+0.036% TFA) into H20 (+0.045% TFA) were run at a 1 mL/min flow rate over 8 min. Semi-preparative HPLC was carried out on the same Waters instrument but using a Xbridge™ BEH 130 C 18 reversed-phase semipreparative column 5 μιη x 10 mm x 100 mm and linear gradients with a flow rate of 3 mL/min over 20 min. HPLC-MS analysis was performed on a Waters instrument comprising a separation module (Waters 2695), automatic injector, photodiode array detector (Waters 2998), a Waters Micromass ZQ spectrometer and a system controller (Masslynx v4. 1). The column used was a Sunfire™ C 18 reversed- phase analytical column 3.5 mm x 2. 1 mm x 100 mm. UV detection was at 220 and 254 nm, and linear gradients of ACN (+0.07% Formic acid) into H20 (+0.1% Formic acid) were run at a 0.3 mL/min flow rate over 8 min.
Ή and 13C NMR spectra were recorded on a Varian MERCURY 400 (400 MHz for iH NMR, 100 MHz for NMR) spectrometer and a Bruker 600 Avance III Ultrashielded, provided with a cryoprobe TCI (600 MHz for Ή NMR, 150 MHz for 13C NMR) spectrometer. Chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilyl chloride. Coupling constants are expressed in Hertz.
EXAMPLE 1 : SYNTHESIS OF NON-PROTEINOGENIC AMINO ACIDS
Synthesis of Alloc-Pipecolic-OH 1
To a solution of H-pipecolic-OH (500 mg, 3.87 mmol) in 2% aqueous Na2C03- dioxane ( 1 : 1 , 20 mL), Alloc-Cl (823 μΐ^, 7.74 mmol) was dropped and the pH readjusted to 8-9 by addition of 3 M aqueous NaOH solution. The reaction mixture was stirred overnight at room temperature. The pH was re-adjusted again to 8-9, the dioxane was eliminated under reduced pressure, and the aqueous layer was washed with TBME (x 3), acidified to pH 2 by adding 4 M aqueous HC1 and extracted with EtOAc (x 3). The combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-MeOH = 100:0 to 95:5) to give 775.2 mg (94%) of Alloc-pipecolic-OH 1 as a yellowish oil; Ή NMR (400 MHz, CDC13) δ 1.34 ( 1H, m, CH2 Y), 1.42 ( 1H, m, CH2 8), 1.67 ( 1H, m, CH2 8), 1.71 ( 1H, m, CH2 P), 1.73 ( 1H, m, CH2 Y), 2.26 ( 1H, m, CH2 P), 3.02 ( 1H, m, CH2 e), 4.07 ( 1H, dd, J = 26.8, 12.8 Hz, CH2 e), 4.62 (2H, m, CH2, Allyl), 4.94 ( 1H, dd, J = 34.6, 4.1 Hz, CHa), 5.22 ( 1H, dd, J = 18.9, 9.0 Hz, CH2, H^, Allyl), 5.31 ( 1H, d, J = 16.5 Hz, CH2, Hds, Allyl), 5.93 ( 1H, m, CH, Allyl); NMR ( 100 MHz, CDCI3) δ 20.67 (ΟΗ2 γ), 24.63 (CH2 8), 26.53 (CH2 P), 41.83 (CH2 e), 54.07 (CHa), 66.42 (CH2, Allyl), 1 17, 44 (CH2, Allyl), 132.76 (CH, Allyl), 156,53 (OCONH), 176,95 (COO).
Synthesis of [(4i?,5i?)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4- (tritylamino)butyl) -2, 2 -dimethyl- 1 ,3-dioxolane-4-carboxylic acid] (Fmoc- DADHOHAiTrt, Acetonide)-OH) 10 and (4S,5S) diastereomer 10b
ROUTE A
Figure imgf000061_0001
Scheme III. a) i. EDC, HOBt, dry DMF, 25 °C, 30 min; ii. Ν,Ο- dimethylhydroxylamine, DIEA, dry DMF, 25 °C, 3 h, 96%; b) LiAlH4, dry THF, 0 °C, 30 min, 97%; c) (CF3CH20)2P(0)CH2COOMe, KHMDS, 18-crown-6, dry THF, -78 °C, 3 h, 67%; d) cat. Os04, NMO, THF-H20 (9: 1), 25 °C, 10 days, 75% (6) and 77% (diastereomer 6b); e) (CH3)2C(OCH3)2, cat. PPTS, dry toluene, 25→ 60 °C, 36 h, 77% (7) and 96% (diastereomer 7b); f) LiOH, THF-H20 (7:3), 0 °C, 2 h, 98% (8) and 96% (diastereomer 8b); g) H2 ( 1 atm), Pd-C ( 10%), dry MeOH, 25 °C, 5 h, 94% (9) and 93% (diastereomer 9b); h) Fmoc-Cl, NaN3, dioxane-H20 ( 1 : 1), pH 9, 0 °C, 5 d, 46% ( 10) and 45% (diastereomer 10b).
Synthesis of [(S)-NJ-methoxy-NJ-methyl-2-(2-phenylacetamido)-N5- tritylpentanediamide] 3
To a solution of Z-L-Gln(Trt)-OH 2 (5 g, 9.57 mmol) in dry DMF ( 15 mL), HOBt*H20 ( 1.758 g, 1 1.48 mmol) and EDC (2.201 g, 1 1.48 mmol) were added at room temperature. After 30 min of stirring under Ar, a solution of Ν, Ο- dimethylhydroxylamine hydrochloride ( 1.213 g, 12.44 mmol) and DIEA (2. 17 mL, 12.43 mmol) in dry DMF ( 10 mL) was added dropwise and the reaction mixture was reacted for 3 h. Saturated aqueous NaHCC was added and the aqueous layer was extracted with EtOAc (x 3) . The combined organic layer was washed twice with 0. 1 M aqueous HC1, once with brine, dried over Na2S04, filtrated and concentrated under vacuo to give 5.20 g of the Weinreb amide 3 (96%) as a white solid which was used in the next reaction without further purification: Ή NMR (400 MHz, CDC13) δ 1.83 ( 1H, m, CH2 3), 2. 14 ( 1H, m, CH23) , 2.37 (2H, m, CH2 4), 3. 17 (3H, s, N-CH3), 3.65 (3H, s, O-CH3) , 4.75 (1H, m, CH2), 5.08 (2H, m, Z), 5.66 ( 1H, d, J = 8.4 Hz, OCONH), 6.93 ( 1H, s, CONH), 7. 19-7.36 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDC13) δ 29.20 (CH2 3), 32.34 (N-CH3) , 33.63 (CH2 4), 50.86 (CH2), 61.77 (O-CH3) , 67.18, (CH2, Z), 70.81 (Cq, Trt), 127. 15 (CH, Ar, Trt), 128. 12 (CH, Ar, Trt), 128.38 (CH, Ar, Z), 128.71 (CH, Ar, Z), 128.98 (CH, Ar, Trt), 136.50 (Cq, Z), 144.97 (Cq, Ar, Trt), 156.66 (OCONH), 171.03 (CO), 172.29 (CO); HRMS (NanoESI) m/z calculated for C34H36N3O5 [M+H]+ 566.2650, found 566.2651.
Synthesis of (S)-5-oxo-4-(2-phenylacetamido)-N-tritylpentanamide 4 To a solution of Weinreb amide 3 ( 1.54 g, 1.66 mmol) in dry THF ( 10 mL) at 0 °C, a pre-cooled suspension of LiAlH4 ( 164.9 mg, 4.35 mmol) in dry THF (5 mL) was added dropwise. After 30 min of stirring at 0 °C, the reaction was quenched with a 5% aqueous KHSO4 solution keeping the ice bath. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed once with brine, dried over Na2S04, filtrated and concentrated under vacuo to give 1.34 g of crude aldehyde 4 (97%) as a white solid, which was used in the next reaction without further purification: Ή NMR (400 MHz, CDCI3) δ 1.84 ( 1H, m, CH2 3), 2.22 ( 1H, m, CH2 3), 2.37 ( 1H, m, CH2 4), 2.48 (1H, m, CH24) , 4. 17 ( 1H, m, CH2), 5. 10 (2H, d, J = 2.5 Hz, Z), 5.71 ( 1H, d, J = 6. 1 Hz, OCONH), 6.86 ( 1H, s, CONH), 7.16-7.37 (20H, m, Trt, Z), 9.46 ( 1H, s, CHO); i3C NMR ( 100 MHz, CDC13) δ 24.64 (CH2 3), 32.72 (CH24) , 59.68 (CH2), 67. 17 (CH2, Z), 70.72 (Cq, Trt), 127.07 (CH, Ar, Trt), 127.96 (CH, Ar, Trt), 128.21 (CH, Ar, Z), 128.26 (CH, Ar, Z), 128.53 (CH, Ar, Z), 128.61 (CH, Ar, Trt), 136.07 (Cq, Z), 144.46 (Cq, Ar, Trt), 156.51 (OCONH), 170.85 (CONH), 198.84 (CHO); HRMS (NanoESI) m/z calculated for C32H31N2O4 [M+H]+ 507.2278, found 507.2281.
Synthesis of [(S,2)-methyl 7-oxo-4-(2-phenylacetamido)-7-(tritylamino)hept-2- enoate] 5
To a solution of 18-Crown-6 (3.07 g, 1 1.60 mmol) and methyl , -bis(2,2,2- trifluoroethyl)phosphonoacetate ( 1.5 mL, 6.85 mmol) in dry THF (40 mL) cooled down at -78 °C and under Ar, a solution 0.5 M of KHMDS in dry toluene ( 13.7 mL, 6.85 mmol) was added dropwise. After 30 min of stirring at - 78 °C, a solution of aldehyde 4 ( 1.34 g, 2.64 mmol) in dry THF (40 mL) was cannulated into the reaction mixture and allowed to react for 3 h. Saturated aqueous NH4CI was used to quench the reaction that didn't reach completion. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (hexane-TBME = 7:3 to 1 : 1 ; isocratic hexane-EtOAc = 6:4) to give 1.02 g of the Z-alkene 5 (67%) as a yellowish solid: Ή NMR (400 MHz, CDC13) δ 1.93 (2H, m, CH25), 2.42 (2H, m, CH2 6), 3.70 (3H, s, COOCH3), 5.07 (2H, s, Z), 5. 15 (1H, m, CH4), 5.56 ( 1H, d, J = 6.2 Hz, OCONH), 5.82 (1H, d, J = 1 1.5 Hz, CH2), 6.04 ( 1H, dd, J = 10.8, 9.2 Hz, CH3), 6.73 ( 1H, s, CONH), 7. 15-7.38 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDCI3) δ 28.89 (CH2 5), 33.78 (CH2 6), 49.76 (CH4), 51.46 (COOCH3), 66.68 (CH2, Z), 70.66 (Cq, Trt), 1 19.97 (CH2), 127.00 (CH, Ar, Trt), 127.93 (CH, Ar, Trt), 128.04 (CH, Ar, Z), 128.17 (CH, Ar, Z), 128.44 (CH, Ar, Z), 128.64 (CH, Ar, Trt), 136.46 (Cq, Z), 144.56 (Cq, Ar, Trt), 149.75 (CH3), 156.04 (OCONH), 166.21 (COO), 171.47 (CONH); HRMS (NanoESI) m/z calculated for C35H35N2O5 [M+H]+ 563.2541 , found 563.2527.
Synthesis of [(2i?,3i?,4S)-methyl 2,3-dihydroxy-7-oxo-4-(2-phenylacetamido)-7- (tritylamino)heptanoate] 6 and its (2S,3S,4S) diastereomer 6b
4-Methylmorpholine N-oxide (780.0 mg, 5.77 mmol) and Os04 (cat.) were added to a solution of alkene 5 ( 1.05 g, 1.86 mmol) in THF-H2O (9: 1 , 30 mL) at room temperature, and the resulting reaction mixture was stirred for 10 days at room temperature. Dihydroxylation completion was monitored by HPLC- PDA and HPLC-ESMS analysis. The reaction was quenched with a 40% aqueous NaHSC solution, and the resulting mixture was stirred further for 30 min. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-EtOAc = 1 : 1 to 2:8) to give 418.1 mg of the diastereomer (2R,3R,4S) 6 (75%) and 432.7 mg of the diastereomer (2S,3S,4S) 6b (77%). Diastereomer (2R,3R,4S) 6: Ή NMR (400 MHz, CDC13) δ 1.92 (2H, ddd, J = 7.1 , 7.1 , 6.9 Hz, CH2 5), 2.39 (2H, m, CH2 6), 3.66 ( 1H, d, J = 8.2 Hz, CH3), 3.78 (3H, s, COOCH3) , 3.95 ( 1H, m, CH4), 3.96 ( 1H, m, CH2), 5.08 (2H, s, Z), 5.28 ( 1H, d, J = 9.4 Hz, OCONH), 6.78 ( 1H, s, CONH), 7.15-7.35 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDC13) δ 26.95 (CH2 5), 33.63 (CH2 6), 51. 17 (CH4), 52.64 (COOCH3) , 67.29 (CH2, Z), 70.72 (Cq, Trt), 71.1 1 (CH2), 73.22 (CH3), 127.05 (CH, Ar, Trt), 127.97 (CH, Ar, Trt), 128. 15 (CH, Ar, Z), 128.26 (CH, Ar, Z), 128.55 (CH, Ar, Z), 128.63 (CH, Ar, Trt), 136.05 (Cq, Z), 144.48 (Cq, Ar, Trt), 157.63 (OCONH), 171.59 (COO), 173.71 (CONH); HRMS (NanoESl) m/z calculated for C35H37N2O7 [M+H]+ 597.2595, found 597.2601. Diastereomer (2S,3S,4S) 6b: Ή NMR (400 MHz, CDC13) δ 1.71 ( 1H, m, CH2 5), 2.08 ( 1H, m, CH25), 2.37 (2H, m, CH2 6), 3.68 (3H, s, COOCH3) , 3.81 ( 1H, m, CH3), 3.85 ( 1H, m, CH4), 4.20 ( 1H, m, CH2), 5.06 ( 1H, d, J = 12. 1 Hz, Z), 5. 1 1 ( 1H, d, J = 12.2 Hz, Z), 5.24 ( 1H, d, J = 9.0 Hz, OCONH), 6.87 ( 1H, s, CONH), 7.16-7.36 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDC13) δ 25.63 (CH2 5), 33.37 (CH2 6), 51.93 (CH4), 52.58 (COOCH3) , 67.17 (CH2, Z), 70.72 (Cq, Trt), 72.41 (CH2), 74.86 (CH3), 127.01 (CH, Ar, Trt), 127.93 (CH, Ar, Trt), 128.20 (CH, Ar, Z), 128.25 (CH, Ar, Z), 128.49 (CH, Ar, Z), 128.68 (CH, Ar, Trt), 136.20 (Cq, Z), 144.50 (Cq, Ar, Trt), 156.97 (OCONH), 172.00 (CO), 173. 10 (CO); HRMS (NanoESl) m/z calculated for CssHayNaO? [M+H]+ 597.2595, found 597.2593.
Synthesis of [(4i?,5i¾-methyl 2,2-dimethyl-5-((S)-4-oxo-l -(2-phenylacetamido)- 4-(tritylamino) butyl) l ,3-dioxolane-4-carboxylate] 7 A solution of (2R,3R,4S) diol 6 (746.6 mg, 1.25 mmol) and acetone dimethyl acetal ( 15 mL) in dry toluene (30 mL) was placed in a round bottomed flask equipped with a condenser and, after addition of catalytic PPTS, the mixture was heated at 60 °C for 36 h. The toluene was removed under reduced pressure, the residue re-dissolved in EtOAc and the organic layer washed with saturated aqueous NH4C1 and brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-EtOAc = 97.5:2.5 to 9: 1) to give 579.4 mg of acetal 7 (77%) as a white solid: Ή NMR (400 MHz, CDC13) δ 1.34 (3H, s, CH3, acetal), 1.60 (3H, s, CH3, acetal), 1.83 ( 1H, m, CH2 5), 1.91 ( 1H, m, CH2 5), 2.29 (2H, m, CH26) , 3.40 (3H, s, COOCH3) , 4.04 ( 1H, td, J = 10.3, 10. 1 , 2.5 Hz, CH4), 4.34 ( 1H, d, J = 7.8 Hz, CH3), 4.57 ( 1H, d, J = 7.8 Hz, CH2), 4.97 ( 1H, d, J = 9.4 Hz, OCONH), 5.02 ( 1H, d, J = 12.2 Hz, Z), 5.09 ( 1H, d, J = 12.2 Hz, Z), 7.21 -7.39 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDC13) δ 24.50 (CH3, acetal), 26.39 (CH3, acetal), 31.90 (CH2 5), 33.86 (CH2 6), 48.50 (CH4), 51.99 (COOCH3) , 67.00 (CH2, Z), 70.51 (Cq, Trt), 75.08 (CH2), 79. 19 (CH3), 1 10.29 (Cq, acetal), 126.81 (CH, Ar, Trt), 127.78 (CH, Ar, Trt), 128. 19 (CH, Ar, Z), 128.40 (CH, Ar, Z), 128.44 (CH, Ar, Z), 128.79 (CH, Ar, Trt), 136.26 (Cq, Z), 144.82 (Cq, Ar, Trt), 156.58 (OCONH), 169.59 (COO), 171.48 (CONH); HRMS (NanoESI) m/z calculated for C38H4iN207 [M+H]+ 637.2908, found 637.2921.
Synthesis of [(4S,5S)-methyl 2,2-dimethyl-5-((S)-4-oxo-l -(2-phenylacetamido)- 4-(tritylamino) butyl) l ,3-dioxolane-4-carboxylate] 7b A solution of (2S,3S,4S) diol 6b (774.4 mg, 1.30 mmol) and acetone dimethyl acetal ( 15 mL) in dry toluene (30 mL) was placed in a round bottomed flask equipped with a condenser and, after addition of catalytic PPTS, the mixture was heated at 60 °C for 36 h. The toluene was removed under reduced pressure, the residue re-dissolved in EtOAc and the organic layer washed with saturated aqueous NH4C1 and brine, dried over Na2S04, filtrated and concentrated under vacuo to give 795.6 mg of the acetal 7b (96%) as a white solid which was used in the next reaction without further purification: Ή NMR (400 MHz, CDC ) δ 1.33 (3H, s, CH3, acetal), 1.54 (3H, s, CH3, acetal), 1.71 ( 1H, m, CH25) , 2.01 ( 1H, m, CH2 5), 2.31 (2H, m, CH2 6), 3.54 (3H, s, COOCH3) , 3.80 ( 1H, m, CH4), 4.30 ( 1H, dd, J = 6.8, 6.8 Hz, CH3), 4.63 ( 1H, d, J = 6.8 Hz, CH2), 4.97 ( 1H, d, J = 9.4 Hz, OCONH), 5.02 ( 1H, d, J = 12.3 Hz, Z), 5. 10 ( 1H, d, J = 12.3 Hz, Z), 6.89 ( 1H, s, CONH), 7. 16-7.35 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CDC13) δ 25.29 (CH3, acetal), 26.55 (CH3, acetal), 27.68 (CH2 5), 33.76 (CH26), 51.20 (CH4), 52.41 (COOCH3), 66.89 (CH2, Z), 70.56 (Cq, Trt), 75.99 (CH2), 79.46 (CH3), 1 10.93 (Cq, acetal), 126.92 (CH, Ar, Trt), 127.88 (CH, Ar, Trt), 128. 14 (CH, Ar, Z), 128. 17 (CH, Ar, Z), 128.47 (CH, Ar, Z), 128.72 (CH, Ar, Trt), 136.33 (Cq, Z), 144.71 (Cq, Ar, Trt), 156.23 (OCONH), 170.05 (COO), 171.26 (CONH); HRMS (NanoESI) m/z calculated for C38H41N2O7 [M+H]+ 637.2908, found 637.2913.
Synthesis of [(4i?,5i¾-2,2-dimethyl-5-((S)-4-oxo- 1 -(2-phenylacetamido)-4- (tritylamino)butyl)- l ,3-dioxolane-4-carboxylic acid] 8
To a solution of acetal 7 (529.4 mg, 0.83 mmol) in THF-H2O (7:3, 8 mL) at 0 °C, 1.6 eq of LiOH*¾0 (55.8 mg, 1.33 mmol) were added and the resulting mixture was stirred for 2 h at 0 °C. The reaction was allowed to warm to room temperature and acidified to pH 5 by the addition of 1 M aqueous HCl. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo to give 507.4 mg of crude acid 8 (98%) as a white solid, which was used in the next reaction without further purification: Ή NMR (400 MHz, CD3OD) δ 1.34 (3H, s, CH3, acetal), 1.53 (3H, s, CH3, acetal), 1.75 ( 1H, m, CH2 5), 1.90 ( 1H, m, CH25), 2.32 (2H, m, CH2 6), 4.00 ( 1H, ddd, J = 9.6, 3.7, 3.7 Hz, CH4), 4.40 ( 1H, dd, J = 7.6, 3.6 Hz, CH3), 4.62 ( 1H, d, J = 7.7 Hz, CH2), 5.01 ( 1H, d, J = 12.4, Z), 5. 1 1 ( 1H, d, J = 12.4 H, Z), 7. 17-7.39 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CD3OD) δ 24.76 (CH3, acetal), 26.80 (CH3, acetal), 30.67 (CH2 5), 34.73 (CH26), 51.85 (CH4), 67.69 (CH2, Z), 71.59 (Cq, Trt), 77.26 (CH2), 80. 1 1 (CH3), 1 10.46 (Cq, acetal), 127.73 (CH, Ar), 128.69 (CH, Ar), 128.93 (CH, Ar), 129.45 (CH, Ar), 130.09 (CH, Ar), 138.42 (Cq, Z), 146. 1 1 (Cq, Ar, Trt), 158.64 (OCONH), 174.98 (CO); HRMS (NanoESI) m/z calculated for CsyHasNaNaO? [M+Na]+ 645.2571 , found 645.2586. Synthesis of [(4S,5S)-2,2-dimethyl-5-((S)-4-oxo- 1 -(2-phenylacetamido)-4- (tritylamino)butyl)- l ,3-dioxolane-4-carboxylic acid] 8b To a solution of acetal 7b (745.6 mg 1. 17 mmol) in THF-H20 (7:3, 10 mL) at 0 °C, 1.6 eq of LiOH*H20 (78.6 mg, 1.87 mmol) were added and the resulting mixture was stirred for 2 h at 0 °C. The reaction was allowed to warm to room temperature and acidified to pH 5 by the addition of 1 M aqueous HC1. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo to give 698.4 mg of crude acid 8b (96%) as a white solid, which was used in the next reaction without further purification: Ή NMR (400 MHz, CD3OD) δ 1.32 (3H, s, CH3, acetal), 1.48 (3H, s, CH3, acetal), 1.66 ( 1H, m, CH25), 1.94 ( 1H, m, CH25), 2.23 ( 1H, m, CH2 6), 2.36 ( 1H, m, CH2 6), 3.88 ( 1H, m, CH4), 4.36 ( 1H, dd, J = 7.3, 4.9 Hz, CH3), 4.61 ( 1H, d, J = 7.3 Hz, CH2), 5.05 (2H, s, Z), 7. 16-7.38 (20H, m, Trt, Z); i3C NMR ( 100 MHz, CD3OD) δ 25.35 (CH3, acetal), 26.94 (CH3, acetal), 28.88 (CH2 5), 34.93 (CH2 6), 53.65 (CH4), 67.52 (CH2, Z), 71.58 (Cq, Trt), 78.29 (CH2), 80.61 (CH3), 1 10.73 (Cq, acetal), 127.70 (CH, Ar, Trt), 128.69 (CH, Ar, Trt), 128.94 (CH, Ar, Z), 129.45 (CH, Ar, Z), 130. 10 (CH, Ar, Trt), 138.46 (Cq, Ar, Z), 146. 1 1 (Cq, Ar, Trt), 158.62 (OCONH), 175. 19 (CO); HRMS (NanoESI) m/z calculated for C37H38N2Na07 [M+Na]+ 645.2571 , found 645.2586.
Synthesis of [(4JR,5JR)-5-((S)- l -amino-4-oxo-4-(tritylamino)butyl)-2,2-dimethyl- l ,3-dioxolane-4-carboxylic acid] 9
A solution of acid 8 (413.0 mg, 0.66 mmol) in dry MeOH (7 mL) was stirred with a catalytic amount of Pd-C (10%) under an atmosphere of H2 (atmospheric pressure) for 5 h. EtOAc was added and the mixture was filtrated through celite and concentrated under vacuo to give 304.6 mg of crude amine 9 (94%) as a yellow solid which was used in the next reaction without further purification: Ή NMR (400 MHz, CD3OD) δ 1.36 (3H, s, CH3, acetal), 1.54 (3H, s, CH3, acetal), 1.75 ( 1H, m, CH2 5), 1.89 ( 1H, m, CH2 5), 2.50 (2H, m, CH2 6), 3.05 ( 1H, m, CH4), 4.34 ( 1H, dd, J = 7.5, 1.8 Hz, CH3), 4.63 ( 1H, d, J = 7.6 Hz, CH2), 7. 19-7.30 ( 15H, m, Trt); i3C NMR ( 100 MHz, CD3OD) δ 24.69 (CH3, acetal), 26.56 (CH3, acetal), 28.90 (CH2 5), 34. 19 (CH2 6), 53.32 (CH4), 71.69 (Cq, Trt), 77.83 (CH3), 79.63 (CH2), 1 10.86 (Cq, acetal), 127.86 (CH, Ar, Trt), 128.76 (CH, Ar, Trt), 130.05 (CH, Ar, Trt), 145.96 (Cq, Ar, Trt), 174.51 (CO); HRMS (NanoESI) m/z calculated for C29H33N2O5 [M+H]+ 489.2384, found 489.2393.
Synthesis of [ (4 S, 5 S] -5 - ( ( S] - 1 -amino-4 -oxo-4 - (tritylamino)butyl) -2,2 -dimethyl- l ,3-dioxolane-4-carboxylic acid] 9b
A solution of acid 8b (633.6 mg, 1.02 mmol) in dry MeOH ( 15 mL) was stirred with a catalytic amount of Pd-C (10%) under an atmosphere of H2 (atmospheric pressure) for 24 h. EtOAc was added and the mixture was filtrated through celite and concentrated under vacuo to give 462.8 mg of crude amine 9b (93%) as a yellow solid which was used in the next reaction without further purification: Ή NMR (400 MHz, CD3OD) δ 1.34 (3H, s, CH3, acetal), 1.50 (3H, s, CH3, acetal), 1.65 ( 1H, m, CH2 5), 2.04 ( 1H, m, CH2 5), 2.51 (2H, m, CH26), 3.02 ( 1H, m, CH4), 4.19 ( 1H, dd, J = 7.0, 7.0 Hz, CH3), 4.57 ( 1H, d, J = 7.2 Hz, CH2), 7.18-7.30 ( 15H, m, Trt); 13C NMR (100 MHz, CD3OD) δ 25.41 (CH3, acetal), 27. 12 (CH3, acetal), 28.66 (CH2 5), 34.51 (CH2 6), 53.47 (CH4), 71.65 (Cq, Trt), 79.15 (CH3), 80.44 (CH2), 1 10.68 (Cq, acetal), 127.82 (CH, Ar, Trt), 128.74 (CH, Ar, Trt), 130.06 (CH, Ar, Trt), 146.00 (Cq, Ar, Trt), 175.22 (CO); HRMS (NanoESI) m/z calculated for C29H33N205 [M+H]+ 489.2384, found 489.2394.
Synthesis of (4i?,5i?)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4- (tritylamino)butyl) -2, 2 -dimethyl- 1 ,3-dioxolane-4-carboxylic acid 10
To a pre-cooled solution of NaN3 (93.0 mg, 1.43 mmol) in H20 (2 mL) Fmoc-Cl (246.6 mg, 0.95 mmol) dissolved in dioxane (2.5 mL) was added dropwise at 0 °C and the resulting mixture was stirred for 2 h at 0 °C. This solution was then dropped at 0 °C into a solution of amine 9 (232.9 mg, 0.48 mmol) in H20- 2% Na C03 ( 1 : 1 , 4 mL). More dioxane (3 mL) was added to complete solubilisation of the reagents, and the pH was re-adjusted to 9 by addition of 0.1 M aqueous HCl. The reaction mixture was allowed to react for 6 days at room temperature. The solution was acidified to pH 5 by the addition of 1 M aqueous HCl, the aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-hexane = 1 : 1 to DCM-MeOH 9: 1) to give 156.0 mg of the Fmoc-protected diol moiety 10 (46%) as a white solid: Ή NMR (400 MHz, CD3OD) δ 1.35 (3H, s, CH3, acetal), 1.57 (3H, s, CH3, acetal), 1.76 ( 1H, m, CH25), 1.88 ( 1H, m, CH25), 2.29 (2H, m, CH2 6), 4.01 ( 1H, m, CH4), 4.24 ( 1H, dd, J = 6.9, 6.9 Hz, CH, Fmoc), 4.33 (2H, m, CH2, Fmoc), 4.38 ( 1H, m, CH3), 4.62 ( 1H, d, J = 7.6 Hz, CH2), 7. 16-7.31 ( 17H, m, Fmoc, Trt), 7.37 (2H, dt, J = 7.4, 7.4, 3.3 Hz, CH, Ar, Fmoc), 7.64 (1H, d, J = 7.3 Hz, CH, Ar, Fmoc), 7.69 ( 1H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.78 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); 13C NMR ( 100 MHz, CD3OD) δ 24.86 (CH3, acetal), 26.82 (CH3, acetal), 30.99 (CH2 5), 34.67 (CH26), 49.33 (CH, Fmoc), 51.53 (CH4), 67.95 (CH2, Fmoc), 71.61 (Cq, Trt), 77.00 (CH2), 80.43 (CH3), 1 10.73 (Cq, acetal), 120.92 (CH, Ar, Fmoc), 126.37 (CH, Ar, Fmoc), 126.59 (CH, Ar, Fmoc), 127.74 (CH, Ar, Trt), 128. 19 (CH, Ar, Fmoc), 128.70 (CH, Ar, Trt), 128.75 (CH, Ar, Fmoc), 128.82 (CH, Ar, Fmoc), 130.08 (CH, Ar, Trt), 142.55 (Cq, Fmoc), 142.70 (Cq, Fmoc), 145. 1 1 (Cq, Fmoc), 145.83 (Cq, Fmoc), 146.09 (Cq, Ar, Trt), 158.71 (OCONH), 174.92 (CO); HRMS (NanoESI) m/z calculated for C44H43N207 [M+H]+ 71 1.3065, found 71 1.3077.
Synthesis of (4S,5S)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4- (tritylamino)butyl) -2, 2 -dimethyl- 1 ,3-dioxolane-4-carboxylic acid 10b
To a pre-cooled solution of NaN3 (99.8 mg, 1.54 mmol) in H20 (2.5 mL) Fmoc- CI (264.7 mg, 1.02 mmol) dissolved in dioxane (3 mL) was added dropwise at 0 °C and the resulting mixture was stirred for 2 h at 0 °C. This solution was then dropped at 0 °C into a solution of amine 9b (250.0 mg, 0.51 mmol) in Η2θ-2% Na2C03 (1 : 1 , 4 mL). More dioxane (3 mL) was added to complete solubilisation of the reagents, and the pH was re-adjusted to 9 by addition of 0.1 M aqueous HCl. The reaction mixture was allowed to react for 6 days at room temperature. The solution was acidified to pH 5 by the addition of 1 M aqueous HCl, the aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-hexane = 1 : 1 to DCM-MeOH 9: 1) to give 165.4 mg of the Fmoc-protected diol moiety 10b (45%) as a white solid: Ή NMR (400 MHz, CD3OD) δ 1.35 (3H, s, CH3, acetal), 1.53 (3H, s, CH3, acetal), 1.59 ( 1H, m, CH25), 2.02 ( 1H, m, CH25), 2.26 (2H, m, CH2 6), 3.84 ( 1H, ddd, J = 10.7, 8.4, 2.7 Hz, CH4), 4.21 ( 1H, dd, J = 6.8, 6.8 Hz, CH, Fmoc), 4.31 ( 1H, m, CH3), 4.37 (2H, dd, J = 6.8, 2.2 Hz, CH2, Fmoc), 4.63 ( 1H, d, J = 6.8 Hz, CH2), 7.16-7.32 ( 17H, m, Fmoc, Trt), 7.37 (2H, dt, J = 7.5, 7.4, 2.1 Hz, CH, Ar, Fmoc), 7.63 ( 1H, d, J = 7.5 Hz, CH, Ar, Fmoc), 7.67 ( 1H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.78 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc); i3C NMR ( 100 MHz, CD3OD) δ 25.65 (CH3, acetal), 27. 14 (CH3, acetal), 30.33 (CH2 5), 34.33 (CH2 6), 48.69 (CH, Fmoc), 52.26 (CH4), 67.61 (CH2, Fmoc), 71.62 (Cq, Trt), 77.67 (CH2), 80.80 (CH3), 1 1 1.92 (Cq, acetal), 120.95 (CH, Ar, Fmoc), 126.30 (CH, Ar, Fmoc), 126.40 (CH, Ar, Fmoc), 127.73 (CH, Ar, Trt), 128. 15 (CH, Ar, Fmoc), 128. 17 (CH, Ar, Fmoc), 128.72 (CH, Ar, Trt), 128.74 (CH, Ar, Fmoc), 128.82 (CH, Ar, Fmoc), 130.08 (CH, Ar, Trt), 142.62 (Cq, Fmoc), 142.73 (Cq, Fmoc), 145. 15 (Cq, Fmoc), 145.75 (Cq, Fmoc), 146.07 (Cq, Ar, Trt), 158.60 (OCONH), 173.07 (CO), 174.95 (CO); HRMS (NanoESI) m/z calculated for C44H43N207 [M+H]+ 71 1.3065, found 71 1.3082. ROUTE B
Figure imgf000071_0001
10
Scheme IV. a) EDC, HOBt, dry DMF, 25 °C, 30 min; Ν,Ο- dimethylhydroxylamine, DIEA, dry DMF, 25 °C, 3 h, 98%; b) LiAlH4 ( 1M in dry THF), dry THF, 0 °C, 30 min, 96%; c) (CF3CH20)2P(0)CH2COOMe, KHMDS, 18- crown-6, dry THF, -78 °C, 3.5 h, 72%; d) cat. Os04, NMO, THF-H20 (9: 1), 25 °C, 7 days, 76% (15) and 70% (diastereomer 15b); e) (CH3)2C(OCH3)2, cat. PPTS, dry toluene, 60 °C, 24 h, 77% (16) and quantitative (diastereomer 16b); f) LiOH, THF-H20 (7:3), 0 °C, 2 h; g) Fmoc-OSu, pH = 8, THF-H2O (7:3), 25 °C, overnight, 57% (10) and 60% (diastereomer 10b).
Synthesis of [(S)-(9H-fluoren-9-yl)methyl l -(methoxy(methyl)amino)- l ,5-dioxo- 5-(tritylamino)pentan-2-ylcarbamate] 12
To a solution of Fmoc-L-Gln(Trt)-OH (5 g, 8. 19 mmol) in dry DMF (20 mL), HOBt*¾0 ( 1.505 g, 9.82 mmol) and EDC ( 1.883 g, 9.82 mmol) were added at room temperature. After 30 min of stirring under Ar, a solution of Ν,Ο- dimethylhydroxylamine hydrochloride ( 1.038 g, 10.64 mmol) and DIEA ( 1.82 mL, 10.64 mmol) in dry DMF ( 10 mL) was added dropwise and the reaction mixture was reacted for 3 h. Saturated aqueous NaHCC was added and the aqueous layer was extracted with EtOAc (x 3). The combined organic layer was washed twice with 0. 1 M aqueous HC1, once with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (hexane-EtOAc = 10:0 to 2:8) to give 5.249 g of the Weinreb amide 12 (98%) as a white solid: Ή NMR (400 MHz, CDC13) δ 1.83 ( 1H, m, CH23), 2. 16 ( 1H, m, CH2 3), 2.36 (2H, m, CH2 4), 3. 18 (3H, s, N-CH3), 3.64 (3H, s, O-CH3), 4.20 ( 1H, dd, J = 6.9, 6.9 Hz, CH, Fmoc), 4.38 (2H, d, J = 7.0 Hz, CH2, Fmoc), 4.77 ( 1H, m, CH2), 5.69 ( 1H, d, J = 8.3 Hz, OCONH), 6.95 ( 1H, s, CONH), 7.19-7.34 ( 17H, m, Trt, Fmoc), 7.38 (2H, dd, J = 7.3, 7.3 Hz, CH, Ar, Fmoc), 7.58 (2H, J = 7.5, 7.5 Hz, CH, Ar, Fmoc), 7.76 (2H, d, J = 7.6 Hz, CH, Ar, Fmoc); i3C NMR ( 100 MHz, CDC13) δ 29.16 (CH2 3), 32. 13 (N-CH3), 33.39 (CH24), 47.22 (CH, Fmoc), 50.53 (CH2), 61.59 (O-CH3), 66.91 (CH2, Fmoc), 70.58 (Cq, Trt), 1 19.95 (CH, Ar, Fmoc), 1 19.98 (CH, Ar, Fmoc), 125.12 (CH, Ar, Fmoc), 126.92 (CH, Ar, Trt), 127.02 (CH, Ar, Fmoc), 127.05 (CH, Ar, Fmoc), 127.69 (CH, Ar, Fmoc), 127.87 (CH, Ar, Trt), 128.75 (CH, Ar, Trt), 141.26 (Cq, Fmoc), 141.33 (Cq, Fmoc), 143.68 (Cq, Fmoc), 143.92 (Cq, Fmoc), 144.73 (Cq, Ar, Trt), 156.45 (OCONH), 170.80 (CO), 171. 13 (CO); HRMS (NanoESI) m/z calculated for C41H40N3O5 [M+H]+ 654.2968, found 654.2974.
Synthesis of [(S)-(9H-fluoren-9-yl)methyl l ,5-dioxo-5-(tritylamino)pentan-2- ylcarbamate] 13
To a solution of Weinreb amide 12 ( 1.896 g, 2.90 mmol) in dry THF ( 10 mL) at 0 °C, a pre-cooled 1M solution of LiAlH4 in dry THF (3.77 mL, 3.77 mmol) was added dropwise. After 30 min of stirring at 0 °C, the reaction was quenched with a 5% aqueous KHSO4 solution keeping the ice bath. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed once with brine, dried over Na2S04, filtrated and concentrated under vacuo to give 1.654 g of crude aldehyde 13 (96%) as a white solid, which was used in the next reaction without further purification: Ή NMR (400 MHz, CDC13) δ 1.83 ( 1H, m, CH23), 2.24 ( 1H, m, CH2 3), 2.37 (2H, m, CH2 4), 4. 13 ( 1H, m, CH2), 4.21 ( 1H, t, J = 6.5, 6.5 Hz, CH, Fmoc), 4.46 (2H, m, CH2, Fmoc), 5.57 ( 1H, d, J = 6.2 Hz, OCONH), 6.80 ( 1H, s, CONH), 7. 16-7.42 ( 19H, m, Trt, Fmoc), 7.58 (2H, d, J = 7.3 Hz, CH, Ar, Fmoc), 7.71 (2H, t, J = 7.0, 7.0 Hz, CH, Ar, Fmoc), 9.44 ( 1H, s, CHO); i3C NMR (100 MHz, CDC13) δ 24.59 (CH2 3), 32.46 (CH^), 47.30 (CH, Fmoc), 59.44 (CH2), 66.73 (CH2, Fmoc), 70.71 (Cq, Trt), 1 19.98 (CH, Ar, Fmoc), 125.03 (CH, Ar, Fmoc), 127.08 (CH, Ar, Trt), 127.73 (CH, Ar, Fmoc), 127.96 (CH, Ar, Trt), 128.62 (CH, Ar, Trt), 141.33 (Cq, Fmoc), 141.36 (Cq, Fmoc), 143.64 (Cq, Fmoc), 143.76 (Cq, Fmoc), 144.47 (Cq, Ar, Trt), 156.44 (OCONH), 170.88 (CONH), 198.56 (CHO); HRMS (NanoESl) m/z calculated for C39H35N2O4 [M+H]+ 595.2591 , found 595.2600.
Synthesis of [(S,2)-methyl 4-(((9H-fluoren-9-yl)methoxy)carbonylamino)-7-oxo- 7-(tritylamino)hept-2-enoate] 14
To a solution of 18-Crown-6 (4.432 g, 16.77 mmol) and methyl P, P-bis (2,2,2 - trifluoroethyl)phosphonoacetate (2. 1 mL, 9.91 mmol) in dry THF (65 mL) cooled down at -78 °C and under Ar, a solution 0.5 M of KHMDS in toluene ( 19.8 mL, 9.91 mmol) was added dropwise. After 30 min of stirring at -78 °C, a solution of aldehyde 13 (2.266 g, 3.81 mmol) in dry THF (65 mL) was cannulated into the reaction mixture and allowed to react for 3.5 h. Saturated aqueous NH4CI was used to quench the reaction that didn't reach completion. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (isocratic DCM-EtOAc-hexane = 7:2: 1) to give 1.817 g of the Z-alkene 14 (72%) as a yellowish solid: Ή NMR (400 MHz, CDC13) δ 1.65 (2H, m, CH2 5), 2.28 (2H, m, CH26), 3.64 (3H, s, CH3), 4.21 ( 1H, t, J = 6.8, 6.8 Hz, CH, Fmoc), 4.30 (2H, dd, J = 6.7, 3.5 Hz, CH2, Fmoc), 5.08 ( 1H, m, CH4), 5.81 ( 1H, d, J = 1 1.7 Hz, CH2), 6.08 ( 1H, dd, J = 1 1.5, 9.1 Hz, CH3), 7. 14-7.47 ( 19H, m, Trt, Fmoc), 7.68 (2H, d, J = 7.3 Hz, CH, Ar, Fmoc), 7.89 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc), 8.57 ( 1H, s, CONH); NMR ( 100 MHz, CDC13) δ 29.96 (CH2 5), 32.47 (CH2 6), 46.64 (CH, Fmoc), 48.51 (CH4), 51.05 (CH3), 65. 15 (CH2, Fmoc), 69.06 (Cq, Trt), 1 18.60 (CH2), 120.03 (CH, Ar, Fmoc), 124.99 (CH, Ar, Fmoc), 125.03 (CH, Ar, Fmoc), 126. 16 (CH, Ar, Trt), 126.94 (CH, Ar, Fmoc), 127.31 (CH, Ar, Trt), 127.50 (CH, Ar, Fmoc), 128.41 (CH, Ar, Trt), 140.62 (Cq, Fmoc), 143.74 (Cq, Fmoc), 144.82 (Cq, Ar, Trt), 155.38 (OCONH), 165.36 (COO), 171.30 (CONH); HRMS (NanoESl) m/z calculated for C42H39N205 [M+H]+ 651.2854, found 651.2853. Synthesis of [(2i?,3i?,4S)-methyl 4-(((9H-fluoren-9-yl)methoxy)carbonylamino)- 2,3-dihydroxy-7-oxo-7-(tritylamino)heptanoate] 15 and its (2S,3S,4S) diastereomer 15b 4-Methylmorpholine N-oxide (225.2 mg, 1.67 mmol) and Os04 (cat.) were added to a solution of alkene 14 (349.8 mg, 0.54 mmol) in THF-H20 (9: 1 , 10 mL) at room temperature, and the resulting reaction mixture was stirred for 7 days at room temperature. Dihydroxylation completion was monitored by HPLC-PDA and HPLC-ESMS analysis. The reaction was quenched with a 40% aqueous NaHSC solution, and the resulting mixture was stirred further for 30 min. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (isocratic DCM-EtOAc = 8:2) to give 282.3 mg of the diastereomer 15 (2R,3R,4S) (76%) and 259.0 mg of the diastereomer 15b (2S,3S,4S) (70%). Diastereomer 15 (2R,3R,4S): Ή NMR (400 MHz, CDC13) δ 1.91 (2H, m, CH25), 2.34 (2H, m, CH2 6), 3.55 ( 1H, br s, OH), 3.64 ( 1H, d, J = 8.5 Hz, CH3), 3.79 (3H, s, OCH3), 3.85 ( 1H, d, J = 8.7 Hz, CH2), 3.92 ( 1H, m, CH4), 3.92 ( 1H, br s, OH), 4. 17 ( 1H, t, J = 6.5, 6.5 Hz, CH, Fmoc), 4.43 ( 1H, dd, J = 10.7, 6.5 Hz, CH2, Fmoc), 4.50 ( 1H, dd, J = 10.7, 6.6 Hz, CH2, Fmoc), 5.23 ( 1H, d, J = 9.3 Hz, OCONH), 6.78 ( 1H, s, CONH), 7. 15-7.34 ( 17H, m, Trt, Fmoc), 7.38 (2H, t, J = 7.4, 7.4 Hz, CH, Ar, Fmoc), 7.57 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.75 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); 13C NMR ( 100 MHz, CDC13) δ 27. 12 (CH2 5), 33.62 (CH2 6), 47.34 (CH, Fmoc), 51.03 (CH4), 52.66 (OCH3), 66.71 (CH2, Fmoc), 70.71 (Cq, Trt), 71.00 (CH2), 73.22 (CH3), 120.00 (CH, Ar, Fmoc), 120.01 (CH, Ar, Fmoc), 124.94 (CH, Ar, Fmoc), 124.99 (CH, Ar, Fmoc), 127.04 (CH, Ar, Trt), 127.71 (CH, Ar, Fmoc), 127.72 (CH, Ar, Fmoc), 127.96 (CH, Ar, Trt), 128.64 (CH, Ar, Trt), 141.34 (Cq, Fmoc), 141.36 (Cq, Fmoc), 143.61 (Cq, Fmoc), 143.67 (Cq, Fmoc), 144.47 (Cq, Ar, Trt), 157.58 (OCONH), 171.62 (COO), 173.81 (CONH); HRMS (NanoESI) m/z calculated for C42H4iN207 [M+H]+ 685.2908, found 685.2929. Diastereomer 15b (2S,3S,4S): iH NMR (400 MHz, CDCI3) δ 1.67 ( 1H, m, CH2s), 2.07 ( 1H, m, CH2 5), 2.31 (2H, m, CH26) , 3.66 (3H, s, OCH3), 3.78 ( 1H, m, CH3), 3.82 ( 1H, m, CH4), 4. 17 ( 1H, m, CH2), 4. 19 ( 1H, m, CH, Fmoc), 4.45 (2H, d, J = 6.5 Hz, CH2, Fmoc), 5. 19 ( 1H, d, J = 8.9 Hz, OCONH), 6.89 ( 1H, s, CONH), 7. 15-7.32 ( 17H, m, Trt, Fmoc), 7.38 (2H, dt, J = 7.2, 7.2, 7.3 Hz, CH, Ar, Fmoc), 7.57 (2H, dd, J = 7.3, 3.0 Hz, CH, Ar, Fmoc), 7.74 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); HRMS (NanoESI) m/z calculated for C42H41N2O7 [M+H]+ 685.2908, found 685.291 1.
Synthesis of [(4JR,5JR)-methyl-5-((S)- l -(((9H-fluoren-9- yl)methoxy)carbonylamino)-4-oxo-4-(tritylamino)butyl)-2,2-dimethyl- l ,3- dioxolane-4-carboxylate] 16
A solution of (2R,3R,4S) diol 15 (916.4 mg, 1.34 mmol) and acetone dimethyl acetal ( 18 mL) in dry toluene (36 mL) was placed in a round bottomed flask equipped with a condenser and, after addition of catalytic PPTS, the mixture was heated at 60 °C for 24 h. The toluene was removed under reduced pressure, the residue re-dissolved in EtOAc and the organic layer washed with saturated aqueous NH4CI and brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (DCM-EtOAc = 97.5:2.5 to 9: 1) to give 746.6 mg of acetal 16 (77%) as a white solid: Ή NMR (400 MHz, CDC13) δ 1.37 (3H, s, CH3, acetal), 1.66 (3H, s, CH3, acetal), 1.84 ( 1H, m, CH2 5), 1.93 ( 1H, m, CH2 5), 2.26 (2H, m, CH26), 3.50 (3H, s, OCH3), 4.06 ( 1H, td, J = 10. 1 , 10. 1 , 2.4 Hz, CH4), 4.23 ( 1H, t, J = 7.0,CH, Fmoc), 4.37 (2H, m, CH2, Fmoc), 4.37 ( 1H, m, CH3), 4.61 ( 1H, d, J = 7.8 Hz, CH2), 4.99 ( 1H, d, J = 9.4 Hz, OCONH), 7. 18-7.43 ( 17H, m, Trt, Fmoc), 7.61 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.63 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc), 7.76 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); 13C NMR (100 MHz, CDC13) δ 24.53 (CH3, acetal), 26.44 (CH3, acetal), 31.87 (CH2 5), 33.80 (CH2 6), 47.27 (CH, Fmoc), 48.53 (CH4), 52. 18 (OCH3), 66.90 (CH2, Fmoc), 70.53 (Cq, Trt), 75.16 (CH2), 79.25 (CH3), 1 10.37 (Cq, acetal), 1 19.95 (CH, Ar, Fmoc), 1 19.99 (CH, Ar, Fmoc), 125. 19 (CH, Ar, Fmoc), 126.82 (CH, Ar, Trt), 127.06 (CH, Ar, Fmoc), 127.07 (CH, Ar, Fmoc), 127.70 (CH, Ar, Fmoc), 127.75 (CH, Ar, Fmoc), 127.78 (CH, Ar, Trt), 128.77 (CH, Ar, Trt), 141.25 (Cq, Fmoc), 141.35 (Cq, Fmoc), 143.54 (Cq, Fmoc), 144.03 (Cq, Fmoc), 144.79 (Cq, Ar, Trt), 156.60 (OCONH), 169.58 (COO), 171.43 (CONH); HRMS (NanoESI) m/z calculated for C45H45N2O7 [M+H]+ 725.3221 , found 725.3213.
Synthesis of [(4S,5S)-methyl-5-((S)- l -(((9H-fluoren-9- yl)methoxy)carbonylamino)-4-oxo-4-(tritylamino)butyl)-2,2-dimethyl- l ,3- dioxolane-4-carboxylate] 16b
A solution of (2S,3S,4S) diol 15b (847.0 mg, 1.24 mmol) and acetone dimethyl acetal ( 17 mL) in dry toluene (34 mL) was placed in a round bottomed flask and, after addition of catalytic PPTS, the mixture was stirred for 36 h. The toluene was removed under reduced pressure, the residue re-dissolved in EtOAc and the organic layer washed with saturated aqueous NH4CI and brine, dried over Na2S04, filtrated and concentrated under vacuo to give 893.9 mg of the acetal 16b (quantitative) as a white solid which was used in the next reaction without further purification: Ή NMR (400 MHz, CDC13) δ 1.34 (3H, s, CH3, acetal), 1.56 (3H, s, CH3, acetal), 1.70 ( 1H, m, CH2 5), 2.02 ( 1 H, m, CH2 5), 2.24 (2H, m, CH2 6), 3.57 (3H, s, OCH3), 3.77 ( 1H, m, CH4), 4. 19 ( 1H, t, J = 6.5, 6.5 Hz, CH, Fmoc), 4.31 ( 1H, t, J = 6.8, 6.8 Hz, CH3), 4.63 ( 1H, d, J = 6.6 Hz, CH2), 4.93 ( 1H, d, J = 9.2 Hz, OCONH), 6.86 ( 1H, s, CONH), 7.13-7.33 ( 17H, m, Trt, Fmoc), 7.37 (2H, t, J = 7.2, 7.2 Hz, CH, Ar, Fmoc), 7.58 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.73 (2H, dd, J = 7.5, 4.0 Hz, CH, Ar, Fmoc); 13C NMR ( 100 MHz, CDC ) δ 25.31 (CH3, acetal), 26.56 (CH3, acetal), 27.61 (CH2 5), 33.62 (CH26) , 47.33 (CH, Fmoc), 51.06 (CH4), 52.45 (OCH3), 66.48 (CH2, Fmoc), 70.57 (Cq, Trt), 76.05 (CH2), 79.40 (CH3), 1 10.96 (Cq, acetal), 1 19.97 (CH, Ar, Fmoc), 124.99 (CH, Ar, Fmoc), 125.01 (CH, Ar, Fmoc), 126.93 (CH, Ar, Trt), 127.03 (CH, Ar, Fmoc), 127.05 (CH, Ar, Fmoc), 127.70 (CH, Ar, Fmoc), 127.72 (CH, Ar, Fmoc), 127.88 (CH, Ar, Trt), 128.72 (CH, Ar, Trt), 141.34 (Cq, Fmoc), 143.66 (Cq, Fmoc), 143.86 (Cq, Fmoc), 144.70 (Cq, Ar, Trt), 156.20 (OCONH), 170. 12 (COO), 171.26 (CONH); HRMS (NanoESI) m/z calculated for C45H45N207 [M+H]+ 725.3221 , found 725.3214. Synthesis of [(4i?,5i?)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4- (tritylamino)butyl) -2, 2 -dimethyl- 1 ,3-dioxolane-4-carboxylic acid] 10
To a solution of acetal 16 (717.6 mg, 0.99 mmol) in THF-H20 (7:3, 10 mL) at 0 °C, 1.1 eq of LiOH*H20 (45.7 mg, 1.09 mmol) were added and the resulting mixture was stirred for 1 h at 0 °C. After analyzing the reaction crude by TLC, 0.5 more eq (20.8 mg, 0.49 mmol) of LiOH*H20 were added and the mixture was allowed to react for another hour at 0 °C. Then, after checking complete conversion of the starting material, the reaction mixture was acidified to pH 8 by the addition of 0.1 M aqueous HC1 and 2 eq of Fmoc-OSu (667.9 mg, 1.98 mmol) were added. The solution was allowed to react at 25 °C and pH = 8 overnight. After analyzing the crude by HPLC-PDA and HPLC-ESMS to find that there wasn't any unprotected product, the solution was acidified to pH = 5 with 1 M aqueous HC1 and extracted with EtOAc (x 3). The combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was washed with cold hexane, purified by reverse phase using a C 18 Porapak Rxn RP 60 cc (Waters) column and then lyophilized to give 401.4 mg (57%) of the Fmoc-protected diol moiety 10 as a white solid: Ή NMR (400 MHz, CD3OD) δ 1.35 (3H, s, CH3, acetal), 1.57 (3H, s, CH3, acetal), 1.76 ( 1H, m, CH2 5), 1.88 ( 1H, m, CH2 5), 2.29 (2H, m, CH2 6), 4.01 ( 1H, m, CH4), 4.24 ( 1H, dd, J = 6.9, 6.9 Hz, CH, Fmoc), 4.32 (2H, m, CH2, Fmoc), 4.38 ( 1H, m, CH3), 4.62 ( 1H, d, J = 7.6 Hz, CH2), 7. 16-7.31 ( 17H, m, Fmoc, Trt), 7.37 (2H, dt, J = 7.4, 7.4, 3.3 Hz, CH, Ar, Fmoc), 7.64 ( 1H, d, J = 7.3 Hz, CH, Ar, Fmoc), 7.69 ( 1H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.78 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); 13C NMR ( 100 MHz, CD3OD) δ 24.86 (CH3, acetal), 26.82 (CH3, acetal), 30.99 (CH2 5), 34.67 (CH2e), 49.33 (CH, Fmoc), 51.53 (CH4), 67.95 (CH2, Fmoc), 71.61 (Cq, Trt), 77.00 (CH2), 80.43 (CH3), 1 10.73 (Cq, acetal), 120.92 (CH, Ar, Fmoc), 126.37 (CH, Ar, Fmoc), 126.59 (CH, Ar, Fmoc), 127.74 (CH, Ar, Trt), 128.19 (CH, Ar, Fmoc), 128.70 (CH, Ar, Trt), 128.75 (CH, Ar, Fmoc), 128.82 (CH, Ar, Fmoc), 130.08 (CH, Ar, Trt), 142.55 (Cq, Fmoc),
142.70 (Cq, Fmoc), 145.1 1 (Cq, Fmoc), 145.83 (Cq, Fmoc), 146.09 (Cq, Ar, Trt),
158.71 (OCONH), 174.92 (CO); HRMS (NanoESI) m/z calculated for C44H43N207 [M+H]+ 71 1.3065, found 71 1.3077. Synthesis of [(4S,5S)-5-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4- (tritylamino)butyl) -2, 2 -dimethyl- 1 ,3-dioxolane-4-carboxylic acid] 10b To a solution of acetal 16b (721.4 mg, 1.00 mmol) in THF-H20 (7:3, 10 mL) at 0 °C, 1. 1 eq of LiOH*H20 (45.9 mg, 1.09 mmol) were added and the resulting mixture was stirred for 1 h at 0 °C. After analyzing the reaction crude by TLC, 0.5 more eq (20.9 mg, 0.50 mmol) of LiOH*H20 were added and the mixture was allowed to react for another hour at 0 °C. Then, after checking complete conversion of the starting material, the reaction mixture was acidified to pH 8 by the addition of 0.1 M aqueous HC1 and 2 eq of Fmoc-OSu (671.4 mg, 1.99 mmol) were added. The solution was allowed to react at r.t. and pH = 8 overnight. After analyzing the crude by HPLC-PDA and HPLC-ESMS to find that there wasn't any unprotected product, the solution was acidified to pH = 5 with 1 M aqueous HC1 and extracted with EtOAc (x 3). The combined organic layer was washed with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was washed with cold hexane, purified by reverse phase using a C 18 Porapak Rxn RP 60 cc (Waters) column and then lyophilized to give 477.6 mg (60%) of the Fmoc-protected diol moiety 10b as a white solid: Ή NMR (400 MHz, CD3OD) δ 1.35 (3H, s, CH3, acetal), 1.53 (3H, s, CH3, acetal), 1.59 ( 1H, m, CH2 5), 2.02 ( 1H, m, CH2 5), 2.26 (2H, m, CH2 6), 3.84 ( 1H, ddd, J = 10.7, 8.4, 2.7 Hz, CH4), 4.21 ( 1H, dd, J = 6.8, 6.8 Hz, CH, Fmoc), 4.31 ( 1H, m, CH3), 4.37 (2H, dd, J = 6.8, 2.2 Hz, CH2, Fmoc), 4.63 ( 1H, d, J = 6.8 Hz, CH2), 7. 16-7.32 ( 17H, m, Fmoc, Trt), 7.37 (2H, dt, J = 7.5, 7.4, 2. 1 Hz, CH, Ar, Fmoc), 7.63 ( 1H, d, J = 7.5 Hz, CH, Ar, Fmoc), 7.67 ( 1H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.78 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc); 13C NMR ( 100 MHz, CD3OD) δ 25.65 (CH3, acetal), 27.14 (CH3, acetal), 30.33 (CH2 5), 34.33 (CH2e), 48.69 (CH, Fmoc), 52.26 (CH4), 67.61 (CH2, Fmoc), 71.62 (Cq, Trt), 77.67 (CH2), 80.80 (CH3), 1 1 1.92 (Cq, acetal), 120.95 (CH, Ar, Fmoc), 126.30 (CH, Ar, Fmoc), 126.40 (CH, Ar, Fmoc), 127.73 (CH, Ar, Trt), 128. 15 (CH, Ar, Fmoc), 128. 17 (CH, Ar, Fmoc), 128.72 (CH, Ar, Trt), 128.74 (CH, Ar, Fmoc), 128.82 (CH, Ar, Fmoc), 130.08 (CH, Ar, Trt), 142.62 (Cq, Fmoc), 142.73 (Cq, Fmoc), 145. 15 (Cq, Fmoc), 145.75 (Cq, Fmoc), 146.07 (Cq, Ar, Trt), 158.60 (OCONH), 174.95 (CO); HRMS (NanoESI) m/z calculated for C44H43N207 [M+H]+ 71 1.3065, found 71 1.3082.
Synthesis of i2-R,3-R,4-R)-2-{[i9H-fluoren-9-yl)methoxy1carbonylamino|-3- hydroxy-4,5-dimethylhexanoic acid (Fmoc-D-aito-AHDMHA-OH) 23
Figure imgf000079_0001
Scheme V. a) i. 2-bromo-3-methyl-2-butene, iBuLi, Et20, -78 °C, 3.3 h; ii. Garner's aldehyde, Et20, -78→ 25 °C, 1.2 h, 34%; b) H2 (4 atm), Pd(C), MeOH, - 78 °C, 2 d, 78%; c) 2,6-di-ieri-butyl pyridine, Tf20, DCM, 0 °C, 10 min, 90%; d) i. Jones reagent, acetone, 0 °C, 15 h; ii. CH2N2, Et20, 25 °C, 67%; e) HClCOnc, reflux, 72 h, 100%; f) Fmoc-OSu, 0.5% Na2C03-dioxane ( 1 : 1), 25 °C, 15 h, 91%.
Synthesis of (4S)-[( 1 S)- 1 -Hydroxy-2,3-dimethyl-but-2-enyl]-2,2-dimethyl- oxazolidine-3-carboxylic acid ieri-butyl ester 18 and its ( 1 R) analogue 18b
To a precooled solution of 2-bromo-3-methyl-but-2-ene (6.0 mL, 52.4 mmol) in dry Et20 (60 mL) at -78 °C, was added dropwise a 1.7 M solution of ieri-butyl lithium in hexane (31 mL, 52.4 mmol) over 20 min. The resultant yellowish solution was stirred for an additional 3 h at -78 °C, and then a solution of Garner's aldehyde 17 (3.0 g, 13. 1 mmol) in dry Et20 ( 15 mL) was added over a period of 1 h at the same temperature. The reaction flask was allowed to warm up to r.t. and left stirring (approx. 15 h). The reaction mixture was quenched with saturated aqueous NH4C1. The organic layer was washed with H20 (x 2), dried over MgSC , filtrated and concentrated under vacuo. The residue was purified by column chromatography allowing the separation of the diastereomers to yield 18 ( 1.33 g, 34 % yield) as first eluating product followed by 18b ( 1.44 g, 36 % yield). Pure 18 is a dense oil that transforms slowly into a waxy solid. Ή NMR (400 MHz, CDC13) δ 1.45- 1.49 ( 12H, m), 1.58 (3H, br s); 1.61 (3H, br s), 1.65 (3H, br s), 1.72 (3H, br m), 3.57 ( 1H, d, J = 9.0 Hz), 3.78 ( 1H, m), 4.07 ( 1H, br m), 4.65 ( 1H, d, J = 9.0 Hz), 4.74 ( 1H, br s); 13C NMR ( 100 MHz, CDC ) δ 155.7, 129.9, 127.7, 94.3, 81.6, 74.9, 65.0, 61.3, 28.4, 27.4, 24.2, 21. 1 , 20.2, 12.2; IR (CHCI3) 3404, 2982, 2936, 1656, 1402, 1369, 1 175, 1 106 cm 1; HRMS (NanoESI) m/z calculated for C16H30NO4 [M+H]+ 300.2175, found 300.2177. 18b. White solid. Ή NMR (400 Mhz, DMSO-d6) δ 4.89 (br s, 1H), 4.36 (m, 1H), 4.05 (br m, 1H), 3.79 (m, 2H), 1.64- 1.49 (m, 12H), 1.38 (br s, 3H), 1.36 (br s, 9H); NMR ( 100 MHz, DMSO-d6) δ 151.8, 129.2 and 128.9 (rotamer), 125.1 and 124.9 (rotamer), 93.7 and 93.0 (rotamer), 78.6, 68.4, 64.2 and 63.8 (rotamer), 58.9 and 58.7 (rotamer), 28.0 and 27.9 (rotamer), 27.2 and 26.6 (rotamer), 24.3 and 22.9 (rotamer), 21. 1 and 21.0 (rotamer), 20.3, 13.2 and 12.9 (rotamer). HRMS (NanoESI) m/z calculated for C16H30NO4 [M+H]+ 300.2175, found 300.2176.
Synthesis of (4S)-[( 1 S,2R - 1 -Hydroxy-2, 3 -dimethyl-butyl] -2,2 -dimethyl- oxazolidine-3-carboxylic acid ieri-butyl ester 19 A solution of 18 ( 1.54 g, 5. 14 mmol) in MeOH ( 100 mL) was stirred with Pd-C ( 10 %, 0.2 g) in a ¾ atmosphere (4 atm). The reaction was complete after 2 days (monitored with Ή NMR). It was filtered through Celite and purified by column chromatography on silica. The product 19 ( 1.21 g, 78 %) was obtained as a colorless dense oil; Ή NMR (400 MHz, acetone-d6) δ 0.85-0.92 (9H, m), 1.26 ( 1H, br m), 1.49 ( 12H, s), 1.56 (3H, s), 1.65 ( 1H, m), 3.76 (2H, m), 4.00 (2H, br m); NMR (100 MHz, acetone-d6) δ 1 1.00, 21.55, 22.08, 25.65, 28.70, 33.08, 43.31 , 62.94, 66.63, 76.41 , 82.41 , 95.54, 150.7; HRMS (NanoESI) m/z calculated for C16H32NO4 [M+H]+ 302.2331 , found 302.2329.
Synthesis of (lJR,7aS)-5,5-dimethyl- l -((i?)-3-methylbutan-2-yl)dihydro- lH- oxazolo[3,4-c]oxazol-3(5H)-one 20 To a solution of 19 (0.78 g, 2.59 mmol) and 2,6-di-ieri-butyl pyridine ( 1. 17 g, 5.69 mmol) in dry DCM ( 15 mL) was added Tf20 (0.48 mL, 2.85 mmol) at 0 °C. The resulting pulp was stirred for 10 min, and then the solvent was removed under vacuo. The residue was treated with Et20 (50 mL) and the solution was fitered off. After concentration, the reddish residue was purified by column chromatography to yield 20 as white crystalline solid (0.52 g, 90 %); JH NMR (400 MHz, CDC13) δ 0.73 (3H, d, J = 6.9 Hz), 0.83 (3H, d, J = 6.9 Hz), 0.93 (3H, d, J = 7.0 Hz), 1.44 (3H, s), 1.71 ( 1H, m), 1.74 (3H, s), 2. 13 ( 1H, m), 3.66 ( 1H, dd, J = 9.6, 8.3 Hz), 3.98 ( 1H, dd, J = 8.2, 6.0 Hz), 4.24 ( 1H, ddd, J= 9.7, 7.6, 6.0 Hz), 4.41 ( 1H, dd, J = 1 1.2, 7.6 Hz); NMR (100 MHz, CDC13) δ 8.97, 14.88, 20.36, 23.47, 27. 13, 28.78, 38.71 , 61. 10, 63.64, 75.94, 94.84, 157.26; HRMS (NanoESI) m/z calculated for C12H22NO3 [M+H]+ 228.1594, found 228.1598.
Synthesis of (4i?,5i?)-methyl-5-[(i?)-3-methylbutan-2-yl]-2-oxo-oxazolidine-4- carboxylate 21
To a solution of 20 (0.46 g, 2.02 mmol) in acetone (50 mL) was added dropwise Jones reagent ( 10 mL) drop by drop at 0 °C. The orange solution becomes turbid, and was then left to stir and reach r.t. for 15 h. Then, 2-PrOH ( 10 mL) was added. When it became clear it was decanted from the precipitate. The solution was concentrated under vacuo and re-dissolved in EtOAc (30 mL). The organic layer was washed with an aqueous solution of NaHCOa (5%, 2 x 10 mL) and then, diazomethane in Et20 (approx. 5-fold excess, 30 mL) was added until a yellow color persists. The solvents were evaporated and the residue was purified by column chromatography to give 21 as a white solid (0.29 g, 67 %); Ή NMR (400 MHz, CDC13) δ 0.83 (3H, d, J = 6.8 Hz), 0.84 (3H, d, J = 6.8 Hz), 0.91 (3H, d, J = 7.0 Hz), 1.62 ( 1H, m), 2. 16 ( 1H, m), 3.81 (3H, s), 4.31 ( 1H, d, J = 7. 1 Hz), 4.47 ( 1H, dd, J = 1 1.0, 7. 1 Hz), 5.58 ( 1H, br s); i3C NMR ( 100 MHz, CDCI3) δ 8.7, 15.03, 20.29, 27.22, 38.46, 52.49, 58.54, 81.72, 159.84, 170.26; HRMS (NanoESI) m/z calculated for Ci0Hi8NO4 [M+H]+ 216. 1230, found 216.1236. Synthesis of (2i?,3i?,4i?)-2-amino-3-hydroxy-4,5-dimethylhexanoic acid 22
The methyl ester 21 (0.24 g, 1. 1 1 mmol) was refluxed with HC1 cone ( 15 mL) for 72 h. Then, the solution was concentrated to give the hydrochloride of 21 as a white solid (0.23 g, 100 %) which was used without further purification for the next step. A sample of the hydrochloride was dissolved in EtOH (6 mL) and propylen oxide (3 mL) and heated for 1 h to 90 °C. The volatiles were removed under vacuo, and the residue was recrystallized from dioxane / water to give 22 as colorless needles; Ή NMR (400 MHz, acetone-d6) δ 0.80 (3H, d, J = 7.0 Hz), 0.85 (3H, d, J = 6.9 Hz), 0.94 (3H, d, J = 7.0 Hz), 1.84 ( 1H, m), 2.22 ( 1H, m), 3.65 ( 1H, dd, J = 10.0, 2.7 Hz), 3.70 ( 1H, d, J= 2.6 Hz); NMR ( 100 MHz, acetone-d6) δ 9.68, 14.93, 21.78, 27.30, 41.63, 57.17, 74.20, 169.46;
Synthesis of (2i?,3i?,4i?)-2-{[(9H-fluoren-9-yl)methoxy]carbonylamino}-3- hydroxy-4,5-dimethylhexanoic acid 23
Crude hydrochloride 22 (240 mg, 1. 1 1 mmol) was dissolved together with Na C03 ( 135 mg, 1.27 mmol) in water (30 mL) and dioxane (30 mL) to give a clear solution. Solid Fmoc-OSu (376 mg, 1. 1 1 mmol) was added at 0 °C and the resulting suspension was stirred at r.t. until it was completely dissolved (approx. 15 h). It was acidified with IN HC1 and most of the dioxane was removed with the rotavaporator. Then, it was extracted with EtOAc (3 x 20 mL). The organic phase was dried with MgS04, filtered and the solvents were evaporated to give an oil. After purification by column chromatography the fractions with product were pooled, the solvent was removed and the residue was lyophilized to give 23 as a white solid (400 mg, 91 %); Ή NMR (400 MHz, acetone-d6) δ 0.79 (3H, d, J = 6.9 Hz), 0.89 (3H, d, J = 6.9 Hz), 0.92 (3H, d, J = 7.0 Hz), 1.85 ( 1H, m), 2.25 ( 1H, m), 3.71 ( 1H, dd, J = 9.7, 2.7 Hz), 4.25 ( 1H, m), 4.36 (2H, m), 4.49 ( 1H, m), 6.54 ( 1H, d, J = 8.9 Hz), 7.33 (2H, m), 7.42 (2H, m), 7.73 (2H, m), 7.86 (2H, d, J = 7.5 Hz); NMR (100 MHz, acetone-d6) δ 1 1.04, 16.57, 22.87, 27.99, 42.27, 49.00, 58.95, 68.33, 77.28, 121.80, 127.1 1 , 127.15, 128.92, 128.94, 129.52, 143.09, 145.97, 146.07, 157.83, 172.8; HRMS (NanoESI) m/z calculated for C23H28NO5 [M+H]+ 398. 1962, found 398. 1974. EXAMPLE 2: SYNTHESIS OF (2i?,3i?,4i?)-3-HYDROXY-2,4,6- TRIMETHYLHEPTANOIC ACID (HTMHA) 31 HTMHA was synthesized following a modified procedure reported in the literature. See J. Org. Chem. 2003, 68, 7841 -7844.
The described synthetic strategy is shown in Scheme VI:
Figure imgf000083_0001
30a: R = Bn 29a: R = Bn
30b: R : TBS 29b: R : TBS
Scheme VI; Lipton's synthesis of (2i?,3JR,4JR)-3-hydroxy-2,4,6- trimethylheptanoic acid, a) LDA, LiCl, dry THF, -78 °C; l -iodo-2- methylpropane, -78 °C, 98%; b) LiAlH(OEt)3, dry hexanes-THF, 0 °C; TFA, 1 M HCl, 86%; c) LAB, dry THF; d) TPAP, NMO, 4 A MS, dry DCM, 77% (two steps); e) i-BuOK, trans-2 -butene, n-BuLi, -78 to -57 °C; (-)-B- methoxydiisopinocampheylborane; BF3-Et20, 27, -78 °C, 75%; f) benzyl 2,2,2- trichloroacetimidate, cat. TfOH, dry cyclohexane-DCM, 96%; g) TBSC1, imidazole, dry DCM, 98%; h) cat. Os04, NaI04, NMO, dioxane-H20; NaC102, H2NSO3H, 95%; i) cat. NaI04/RuCl3-H20, CC14-ACN-H20, 98%. The final synthetic approach used to synthesize HTMHA converts the tertiary amide to the primary alcohol, which is subsequently re-oxidized to the corresponding aldehyde by means of the Dess-Martin reagent. Protection of the hydroxyl group of intermediate 28 was considered dispensable and therefore suppressed. The synthesis was finally performed as detailed in Scheme VII.
Figure imgf000083_0002
31 28 Scheme VII: Modified Lipton's synthesis of (2i?,3i?,4i?)-3-hydroxy-2,4,6- trimethylheptanoic acid, a) DIPA, n-BuLi, LiCl, dry THF, -78 °C, 1 h; l -iodo-2- methylpropane, -78 °C, 3.5 h; b) DIPA, n-BuLi, H3B-NH3, dry THF, -78→ 25 °C, 2 h; c) Dess-Martin, dry DCM, 25 °C, 30 min; d) i-BuOK, trans-2 -butene, n-BuLi, -78→ -57 °C; (-)-B-methoxydiisopinocampheylborane; BF3-Et20, 27, - 78 °C; e) cat. Os04, NaI04, NMO, dioxane-H20, 25 °C, 3 h; NaC102, H2NSO3H, 0 ^ 25 °C, 2 h.
See Org. Lett. 2009, 2(21), 4767-4769 for detailed intermediate 26 characterization.
Synthesis of i2-R,3-R,4-R)-3-hydroxy-2,4,6-trimethylheptanoic acid (HTM HA) 31
To a solution of the alkene 28 ( 139.6 mg, 0.82 mmol, 1 equiv) in H20-dioxane ( 1 : 1 , 8 mL), 4-methyl-morpholine N-oxide ( 143.9 mg, 1.23 mmol, 1.5 equiv) and Os04 (4% in H20, 104 \iL, 0.033 mmol, 0.04 equiv) were added. After 1 h, NaI04 (263. 1 mg, 1.23 mmol, 1.5 equiv) was added and the resulting milky suspension was stirred for 2 h more. Then, the reaction mixture was cooled down to 0 °C and quenched by adding NaC102 (368.9 mg, 3.28 mmol, 4 equiv) and H2NS03H (318.1 mg, 3.28 mmol, 4 equiv) to obtain a bright yellow suspension that was stirred for 2 h at room temperature. Then, 5% aqueous HC1 was added and the aqueous layer was extracted with DCM (x 3). The combined organic extracts were washed with 5% aqueous HC1 (x 1) and brine, dried over MgS04, filtrated and concentrated under vacuo to obtain crude HTMHA 31 as a yellowish oil that was used without further purification. Ή NMR (400 MHz, CDCI3) δ 0.83 (3H, d, J = 6.5 Hz, CH36) , 0.91 (3H, d, J = 6.6 Hz, CH37) , 0.94 (3H, d, J = 6.7 Hz, CH34) , 1. 13 ( 1H, m, CH2s) , 1.23 (3H, d, J = 7.2 Hz, CH32) , 1.24 ( 1H, m, CH2 5), 1.65 ( 1H, m, CH6) , 1.69 ( 1H, m, CH4), 2.69 ( 1H, m, CH2) , 3.44 ( 1H, dd, J = 6.5, 5. 1 Hz, CH3); i3C NMR (100 MHz, CDC13) δ 14.71 (CH32) , 16.56 (CH34) , 21.24 (CH37) , 24.24 (CH36) , 25.16 (CH6) , 33.55 (CH4), 39.51 (CH25) , 41.96 (CH2), 78. 15 (CH3).
EXAMPLE 3: SYNTHESIS OF DIPEPTIDE BUILDING BLOCK: HTMHA-D- Asp(tBU)-OH 36
Figure imgf000085_0001
Scheme VIII. a) i) Cs2C03, dry MeOH, 25 °C, 20 min, ii) BzlBr, dry DMF, 25 °C, 2 h; b) Piperidine-DCM ( 1 :4), 25 °C, 40 min; c) HTMHA, EDC*HC1, HOBt*H20, dry DCM-DMF ( 1 : 1), 25 °C, 8 h; d) H2 ( 1 atm), Pd(C), dry MeOH, 25 °C, overnight.
Synthesis of Fmoc-D-Asp(tBu)-OBzl 33
To a solution of Fmoc-D-Asp(tBu)-OH 32 ( 1.5 g, 3.65 mmol) in dry MeOH (20 mL) CS2CO3 (593.8 mg, 1.82 mmol) was added, and the resulting solution was stirred under Ar at room temperature for 20 min. Then the solvent was removed under reduced pressure to fulfil the cesium carboxilate as a white solid. It was re-dissolved in dry DMF ( 17 mL) and BzlBr ( 1.3 mL, 10.94 mmol) was dropped under Ar. The resulting mixture was stirred for 2 h. Saturated aqueous NH4C1 was used to quench the reaction. The aqueous layer was extracted with EtOAc (x 3) and the combined organic layer was washed once with brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (hexane-EtOAc = 10:0 to 8:2) to give 1.75 g of Fmoc-D-Asp(tBu)-OBzl 33 (95%) as a white solid: Ή NMR (400 MHz, CDCI3) δ 1.42 (9H, s, CH3), 2.79 ( 1H, dd, J = 17.0, 4.5 Hz, CH2 P), 2.97 ( 1H, dd, J = 17.0, 4.6 Hz, CH2 P), 4.23 ( 1H, t, J = 7.2, 7.2 Hz, CH, Fmoc), 4.33 ( 1H, dd, J = 10.4, 7.5 Hz, CH2, Fmoc), 4.42 ( 1H, dd, J = 10.5, 7.3 Hz, CH2, Fmoc), 4.65 ( 1H, td, J = 8.9, 4.5, 4.5 Hz, CHa), 5. 17 ( 1H, d, J = 12.3 Hz, CH2, Bzl), 5.24 ( 1H, d, J = 12.2 Hz, CH2, Bzl), 5.83 ( 1H, d, J = 8.6 Hz, CONH), 7.30 (2H, td, J = 7.5, 7.5, 0.9 Hz, CH, Ar, Fmoc), 7.32-7.36 (5H, m, CH, Ar, Bzl), 7.40 (2H, t, J = 7.5, 7.5 Hz, CH, Ar, Fmoc), 7.59 (2H, d, J = 7.4 Hz, CH, Ar, Fmoc), 7.76 (2H, d, J = 7.5 Hz, CH, Ar, Fmoc); 13C NMR (100 MHz, CDC13) δ 27.99 (CH3), 37.73 (CH2 P), 47.09 (CH, Fmoc), 50.64 (CHa), 67.28 (CH2, Fmoc), 67.46 (CH2, Bzl), 81.88 (Cq, tBu), 1 19.96 (CH, Ar, Fmoc), 125. 1 1 (CH, Ar, Fmoc), 125. 16 (CH, Ar, Fmoc), 127.06 (CH, Ar, Fmoc), 127.70 (CH, Ar, Fmoc), 128.23 (CH, Ar, Bzl), 128.41 (CH, Ar, Bzl), 128.57 (CH, Ar, Bzl), 135.24 (Cq, Bzl), 141.25 (Cq, Fmoc), 141.27 (Cq, Fmoc), 143.70 (Cq, Fmoc), 143.90 (Cq, Fmoc), 155.97 (OCONH), 169.94 (COO), 170.78 (COO).
Synthesis of H-D-Asp(tBu)-OBzl 34
A solution of piperidine-DCM ( 1 :4, 15 mL) was added to the solid Fmoc-D- Asp(tBu)-OBzl 33 ( 1.72 g, 3.42 mmol) and the resulting mixture was stirred for 40 min at room temperature. The solvents were removed under reduced pressure and the residue obtained purified by flash column chromatography (hexane-EtOAc = 95:5 to 0: 100) to give 765.0 mg (80%) of H-D-Asp(tBu)-OBzl 34 as a white solid: Ή NMR (400 MHz, CDC13) δ 1.42 (9H, s, CH3), 2.69 (1H, dd, J = 16.6, 6.7 Hz, CH2 P), 2.77 ( 1H, dd, J = 16.6, 4.7 Hz, CH2 P), 3.82 ( 1H, dd, J = 6.6, 4.8 Hz, CHa), 5. 15 ( 1H, d, J = 12.2 Hz, CH2, Bzl), 5.20 ( 1H, d, J = 12.3 Hz, CH2, Bzl), 7.32-7.37 (5H, m, Bzl); NMR ( 100 MHz, CDC13) δ 28.03 (CH3), 39.62 (CH2 P), 51.33 (CHa), 67.07 (CH2, Bzl), 81.41 (Cq, tBu), 128.30 (CH, Ar, Bzl), 128.40 (CH, Ar, Bzl), 128.59 (CH, Ar, Bzl), 135.49 (Cq, Bzl), 170.28 (COO), 173.90 (COO);
Synthesis of HTMHA-D-Asp(tBu)-OBzl 35
HTMHA 31 ( 120.0 mg, 0.64 mmol), H-D-Asp(tBu)-OBzl 34 (359.0 mg, 1.29 mmol), HOBt*H20 ( 196.8 mg, 1.29 mmol) and EDC*HC1 (246.3 mg, 1.29 mmol) were placed in a round-bottom flask. A mixture of dry DCM:DMF (1 : 1 , 10 mL) was added and, after checking that the pH of the reaction solution was around 6, it was allowed to react for 8 h at room temperature. DCM was removed under reduced pressure, EtOAc added and the organic layer washed with saturated aqueous NH4C1 solution (x 3), saturated aqueous NaHC03 solution (x 3) and brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (hexane- EtOAc = 95:5 to 80:20) to give 68.9 mg (24%) of HTMHA-D-Asp(tBu)-OBzl 35 as a yellowish oil: Ή NMR (400 MHz, CDC13) δ 0.83 (3H, d, J = 6.5 Hz, CH3 ¾, HTMHA), 0.91 (3H, d, J = 6.7 Hz, CH3 ei, HTMHA), 0.91 (3H, d, J = 6.6 Hz, CH3 Y, HTMHA), 1. 12 (2H, m, CH2 8, HTMHA), 1. 19 (3H, d, J = 7.0 Hz, CH3 a, HTMHA), 1.39 (9H, s, CH3, tBu, Asp), 1.62 ( 1H, m, CHe, HTMHA), 1.65 ( 1H, m, CHY, HTMHA), 2.47 ( 1H, m, CHa, HTMHA), 2.73 ( 1H, dd, J = 17.0, 4.4 Hz, CH2 P, Asp), 2.95 ( 1H, dd, J = 17.0, 4.8 Hz, CH2 P, Asp), 3.36 ( 1H, m, CHD, HTMHA), 4.88 ( 1H, dt, J = 8.8, 4.6, 4.6 Hz, CHa, Asp), 5. 14 ( 1H, d, J = 12.2 Hz, CH2, Bzl), 5.22 ( 1H, d, J = 12.2 Hz, CH2, Bzl), 6.77 (1H, d, J = 8.4 Hz, CONH), 7.31 -7.36 (5H, m, Bzl); NMR ( 100 MHz, CDC13) δ 15.14 (CH3 a, HTMHA), 16.70 (CH3 Y, HTMHA), 21.33 (CH3 ¾, HTMHA), 24.32 (CH 1, HTMHA), 25.20 (CHe, HTMHA), 27.96 (CH3, tBu), 33.78 (CHY, HTMHA), 37.06 (CH2 P, Asp), 43. 17 (CHa, HTMHA), 48.56 (CHa, Asp), 67.60 (CH2, Bzl), 78.71 (CHP, HTMHA), 81.95 (Cq, tBu), 128.35 (CH, Ar, Bzl), 128.49 (CH, Ar, Bzl), 128.60 (CH, Ar, Bzl), 135. 12 (Cq, Bzl), 170.02 (COO), 170.95 (COO), 176.33 (CONH);
Synthesis of HTMHA-D-Asp(tBu)-OH 36
A solution of HTMHA-D-Asp(tBu)-OBzl 35 (68.9 mg, 0. 15 mmol) in dry MeOH (5 mL) was stirred with a catalytic amount of Pd-C (10%) under an atmosphere of H2 (atmospheric pressure) for 12 h. EtOAc was added and the mixture was filtrated through celite and concentrated under vacuo to give 53.7 mg of crude HTMHA-D-Asp(tBu)-OH 36 (97%) as a yellow oil. By HPLC-PDA [conditions: linear gradient (5% to 100%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 6.61 min), at 220 nm] it had a purity of 60% but was used in the next reaction without further purification: iH NMR (400 MHz, CDC13) δ 0.82 (3H, d, J = 6.6 Hz, CH3 ¾, HTMHA), 0.90 (3H, d, J = 6.6 Hz, CH3 £i, HTMHA), 0.94 (3H, d, J = 6.8 Hz, CH3 Y, HTMHA), 1. 12 (3H, d, J = 7.0 Hz, CH3 a, HTMHA), 1. 15 (2H, m, CH2 8, HTMHA), 1.43 (9H, s, CH3, tBu), 1.61 ( 1H, m, CHe, HTMHA), 1.68 ( 1H, m, CHY, HTMHA), 2.54 ( 1H, dt, J = 15. 1 , 7.4, 7.4 Hz, CH°, HTMHA), 2.75 ( 1H, dd, J = 16.7, 4.9 Hz, CH2 P, Asp), 2.87 ( 1 H, dd, J = 16.9, 5.5 Hz, CH2 P, Asp), 3.53 ( 1H, dd, J = 7.3, 4. 1 Hz, CHP, HTMHA), 4.84 ( 1H, m, CHa, Asp), 7. 13 ( 1H, d, J = 8.3 Hz, CONH); NMR ( 100 MHz, CDC13) δ 14.60 (CH3 a, HTMHA), 16.79 (CH3 Y, HTMHA), 21. 19 (CH3 ¾, HTMHA), 24.35 (CHs'1, HTMHA), 25.09 (CHe, HTMHA), 27.99 (CH3, tBu), 32.76 (CHY, HTMHA), 37.02 (CH2 P, Asp), 38.82 (CH2 8, HTMHA), 43.67 (CHa, HTMHA), 48.74 (CHa, Asp), 78.66 (CHP, HTMHA), 82.01 (Cq, tBu), 170.30 (COO), 173.69 (COO), 176.97 (CONH). EXAMPLE 4: SOLID-PHASE SYNTHESIS OF PIPECOLIDEPSIN A 51
Non-proteinogenic amino acids appropriately protected Alloc-Pipecolic- OH 1, Fmoc-DADHOHA(Trt, Acetonide)-OH 10, and (Fmoc-D-ctZZo-AHDMHA- OH 23, were synthesized as described in Example 1.
The dipeptide building block (HTMHA-D-Asp(tBu)-OH) 36 was synthesized as described in Examples 2 and 3.
Fmoc-DiMe-Gln-OH 45 was synthesized by procedures known in the literature. See for example Tetrahedron 2001 , 57, 6353 and Org. Lett. 2000, 2, 4157. In addition, a-amino protection was carried out under standard procedures known in the literature.
Fmoc-NMe-Gln-OH 40 was synthesized by procedures known in the literature. See for example J. Org. Chem 2006, 71 , 6351.
Fmoc-ihreo-p-EtO-Asn(Trt)-OH 39 was synthesized as previously described by the inventors. See Amino Acid 2010, 39, 161. Experimental Protocol:
Synthesis of [3- (4 -hydroxymethylphenoxy)propionyl1 aminopolvstyrene Wang like resin
Figure imgf000089_0001
Aminomethyl resin (75. 1 mg, 0.36 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). 3-(4- Hydroxymethylphenoxy)propionic acid ( 15.9 mg, 0.081 mmol, 3 equiv) and HOBt*H20 ( 12.4 mg, 0.081 mmol, 3 equiv) were dissolved in DMF (0.3 mL) and DIEA (41.6 uL, 0.243 mmol, 9 equiv) first and HBTU (30.7 mg, 0.081 mmol, 3 equiv) second were added. The resulting mixture was shaken for a minute (pre-activation) and added to the resin. The mixture was reacted for 1.5 h (3 min of manual stirring and 87 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). A Kaiser test indicated the completion of the coupling reaction. Synthesis of H-D-Asp(0-resin)-OAllyl 38
Figure imgf000089_0002
Fmoc-D-Asp(OH)-OAllyl 37 (53.4 mg, 0. 135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0. 135 mmol, 5 equiv) were dissolved in DCM (0.2 mL) and DIPCDI (20.9 uL, 0. 135 mmol, 5 equiv) and DMAP ( 1.6 mg, 0.014 mmol, 0.5 equiv) in DMF (0. 15 mL) were added. The resulting mixture was added to the resin and stirred for 3 h (3 min of manual stirring and 177 min on a shaker) at 25 °C. After filtration and washes with DCM ( l x l min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL), the aa was re-coupled under the same conditions. The excess sites on the AB linker were quenched with a solution of DIEA (23 1 μL, 1.350 mmol, 50 equiv) and Ac20 ( 128 μL, 1.350 mmol, 50 equiv) in DMF (0. 1 mL) for 16 min. The solvents were removed and the resin subjected to the following washings/treatments: DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The loading, as calculated by UV absorbance at 290 nm, was 0.36 mmol/g.
Synthesis of H-D - αίίο-AHDMHA-D- αΖΖο-Thr ( ¾ul -D -Lys (Boc) -Leu-NMe-Gln- threo- β -EtO - Asn(Trt) -D -Asp (O -resin) -O Allyl 44
Figure imgf000090_0001
Sequential incorporation of Fmoc-ihreo-p-EtO-Asn(Trt)-OH 39 (43.2 mg, 0.068 mmol, 2.5 equiv), Fmoc-NMe-Gln-OH 40 (36.1 mg, 0.095 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (42.9 mg, 0.122 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (44.3 mg, 0.095 mmol, 3.5 equiv), Fmoc-D-ctZZo-Thr(tBu)-OH 43 (32.2 mg, 0.081 mmol, 3 equiv) and Fmoc-D-ctZZo-AHDMHA-OH 23 (26.8 mg, 0.068 mmol, 2.5 equiv) to the previously obtained H-D-Asp(0-resin)-OAllyl 38 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.3 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- AHDMHA-D - aZZo-Thr-D -Lys-Leu-NMe-Gln- i reo-β -EtO -Asn-D -Asp-O Allyl was obtained with a purity of 91% as checked by HPLC-PDA. Conditions: linear gradient (20% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.08 min). HPLC-ESMS(+) analysis showed: m/z calculated for C43H76N10O15 972.55; [M+H]+ found, 974.31.
Synthesis of Fmoc-(3S, 4i?)-3, 4-άίΜ6-01η-Ρ-αΖΖο-ΑΗΡΜΗΑ-Ρ-αΖΖο-Τ1ΐΓ(^ι)-Ρ- LysiBoc)-Leu-i\/Me-Gln-t reo-B-EtO-AsniTrt)-D-AspiO-resin)-OAllyl 46
Figure imgf000091_0001
Fmoc-diMeGln-OH 45 (48.2 mg, 0. 122 mmol, 4.5 equiv) was coupled using DIPCDI ( 19.0 μL, 0. 122 mmol, 4.5 equiv) and HOBt*H20 ( 18.6 mg, 0. 122 mmol, 4.5 equiv) in DMF (0.6 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin was treated with a solution of TFA-H2O-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc-diMeGln-D-ctZZo- AHDMHA-D - aZZo-Thr-D -Lys-Leu-NMe-Gln- i reo-β -EtO -Asn-D -Asp-O Allyl was obtained with a purity of 87% as checked by HPLC-PDA. Conditions: linear gradient (40% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 2.96 min). HPLC-ESMS(+) analysis showed: m/z calculated for
Figure imgf000091_0002
1350.71 ; [M+H]+ found, 1352.93.
Synthesis of {[ Fmoc-(3S,4^)-3,4-diMe-Gln-D- ZZo-AHDMHA(&)-D- ZZo-Thr(tBu) -D -Lys (Boc) -Leu-NMe-Gln- threo- β -EtO -Asn(Trt) -D -Asp (O -resin) -O Allyl] [Alloc- pipecolic&ll 47
Figure imgf000092_0001
After washings with DCM (3 x 1 min; 4 mL) the resin was dried under vacuum for 20 min and transferred to a PYREXR culture tube provided with a magnetic stirrer. Fresh Alloc-pipecolic-OH 1 (86.4 mg, 0.405 mmol, 15 equiv) was dissolved in dry DCM ( 1.5 mL) and DIPCDI (63.4 mL, 0.405 mmol, 15 equiv) was added. The mixture was cooled down to 0 °C, filtered through a 0.45 μπι filter and added to the resin. DMAP ( 1.6 mg, 0.014 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL) was finally added to the tube. The mixture was reacted for 2.3 h at 45-47 °C with smooth stirring. The reaction mixture was transferred to the syringe, the solvents removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL), followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The nonapeptide Fmoc- diMeGln-D-a//o-AHDMHA(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln- i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 67% as checked by HPLC-PDA. Conditions: linear gradient (40% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.85 min). HPLC-ESMS(+) analysis showed: m/z calculated for C75H111N13O22 1545.80; [M+H]+ found, 1548.20. Synthesis of rH-(2i?,3i?,4S)-DADHOHA(Trt, Acetonide)-(3S,4i?)-3,4-diMe-Gln-D- allo- AHDMHA(&) -D - ZZo-Thr ( ¾u) -D -Lys (Boc) -Leu-NMe-Gln- threo- β -EtO - Asn(Trt)-D-Asp(0-resin)-OAllyl1 [Alloc-pipecolic&l} 48
Figure imgf000093_0001
The Fmoc group of diMeGln was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL). The resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (48.0 mg, 0.068 mmol, 2.5 equiv) quickly added to the resin. This aa was introduced using HATU (25.7 mg, 0.068 mmol, 2.5 equiv), HOAt (9. 19 mg, 0.068 mmol, 2.5 equiv) and DIEA (23.5 μL, 0.135 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings /treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-DADHOHA-diMeGln-D - alio- AHDMHA(Alloc-pipecolic) -D - allo- Thr-D-Lys-Leu-NMe-Gln-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 60% (the decapeptide protected with an acetal group was also considered to calculate purity) as checked by HPLC-PDA. Conditions: linear gradient (30% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.57 min). HPLC-ESMS(+) analysis showed: m/z calculated for C67H113N15O24 151 1.81 ; [(M+2H+)/2] found, 757.80.
Synthesis of [{(2^3^4^)-ΗΤΜΗΑ-Ρ-Α8Ρ(Μ-(2^3^45)-ΡΑΡΗΟΗΑ(ΤΓΪ, Acetonide) - (3 SAR) -3 , 4 -diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ( ¾u) -D - LysiBoc)-Leu-i\/Me-Gln-t reo-B-EtO-AsniTrt)-D-AspiO-resin)-OAllyl1 [Alloc- pipecolic&ll 49
Figure imgf000094_0001
The last two residues were incorporated to the growing peptide chain as one unit. HTMHA-D-Asp(£Bu)-OH 36 (60% purity, 80.9 mg, 0.135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0.135 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (47.0 μΐ^, 0.270 mmol, 10 equiv) was added. The reaction mixture was added to the resin followed by the addition of solid PyBOP (70.3 mg, 0.135 mmol, 5 equiv) and it was allowed to react for 3.5 h (5 min of manual stirring and 205 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) (0.35 mL, 1 x 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The dodecapeptide HTMHA-D- Asp-DADHOHA-diMeGln-D - alio- AHDMHA(Alloc-pipecolic) -D - ctZZo-Thr-D -Lys-
Leu-NMe-Gln-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 37% as checked by HPLC-PDA. Conditions: linear gradient (30% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 5.47 min). HPLC-ESMS(+) analysis showed: m/z calculated for C8iHi36Ni6029 1796.97; [M+H]+ found, 1800.73.
Synthesis of r{i2Jg,3Jg,4i¾-HTMHA-D-Aspi¾u)-i2Jg,3Jg,4.S|-DADHOHAiTrt,
Acetonide) - (3 SAR\ -3 , 4 -diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ( ¾u) -D - Lys(Boc)-Leu-i\ e-Gln-t¾reo-B-EtO^
50
Figure imgf000095_0001
Next, to remove the Allyl and Alloc groups, the peptide-resin 49 was treated with Pd(PPh3)4 (6.2 mg, 0.005 mmol, 0.2 equiv) and PhSiH3 (66.6 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.3 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor 50 was obtained with a purity of 32% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.21 min). HPLC-ESMS(+) analysis showed: m/z calculated for C74H128N16O27 1672.91 ; [(M+2H+)/2] found, 838.80.
Figure imgf000095_0002
The macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 μΐ^, 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin followed by the addition of solid PyBOP (56.2 mg, 0.108 mmol, 4 equiv) and it was allowed to react for 3 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC- ESMS.
The cleavage of the peptide and the elimination of the side-chain protecting groups were accomplished simultaneously by treating the resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1.5 h, 3 x 2 min; 4 mL). All the filtrates were collected in a round-bottom flask, evaporated to dryness under reduced pressure and lyophilized in H2O-ACN ( 1 : 1 , 25 mL) to give 37.6 mg of crude with a purity of 15% as checked by HPLC-PDA. Conditions: linear gradient (20% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 6.71 min). HPLC-ESMS(+) analysis showed: m/ z calculated for C74H126N16O26 1654.90; [M+H]+ found, 1657.84.
The crude was purified by semi-analytical HPLC-PDA using a XBridge™ Prep BEH 130 C 18 5 μιη 10x 100 mm column and a linear gradient (5% to 35% over 5 min and 35% to 37% over 20 min) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 3.0 mL/min (iR = 14.13 min) to give 1.46 mg of HTMHA-D-Asp-DADHOHA-diMe-Gln-D-a//o-AHDMHA(S6)-D-a//o-Thr-D-Lys- Leu-NMe-Gln-i reo-p-EtO-Asn-D-Asp-pipecolic(fi6) (Pipecolidepsin A) 51 (3.3%). HRMS (NanoESI) analysis showed: m/z calculated for C74H126O26N16 1654.9029, found 1654.9025 [M]. The product co-eluted by HPLC-PDA with a natural sample of Pipecolidepsin A. Conditions: linear gradient (36% to 38%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 40 min, with a flow rate of 1.0 mL/min (iR = 19.53 min) in a Phenomenex Column C 18 250 x 4.60 mm 5 micron. The Ή and 13C NMR (600 MHz, CD3OH) data are shown in Table I. Table I. Ή and 13C NMR data of Pipecolidepsin A 51 (CD3OH)
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000098_0002
Pipecolidepsin A 51
EXAMPLE 4: SOLID-PHASE SYNTHESIS OF PIPECOLIDEPSIN A' 60
Non-proteinogenic amino acids appropriately protected Alloc-Pipecolic- OH 1, Fmoc-DADHOHA(Trt, Acetonide)-OH) 10, and (Fmoc-D-aZZo-AHDMHA- OH 23, were synthesized as described in Example 1.
HTMHA 31 was synthesized as described in Example 2. Fmoc-DiMe-Gln-OH 45 was synthesized by procedures known in the literature. See for example Tetrahedron 2001 , 57, 6353 and Org. Lett. 2000, 2, 4157. In addition, a-amino protection was carried out under standard procedures known in the literature. Fmoc-ihreo-P"EtO-Asn(Trt)-OH 39 was synthesized as previously described by the inventors. See Amino Acid 2010, 39, 161.
Synthesis of [3- (4 -hydroxymethylphenoxylpropionyll aminopolvstyrene Wang like resin
Figure imgf000099_0001
Aminomethyl resin ( 150 mg, 0.36 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). 3-(4- Hydroxymethylphenoxy)propionic acid (31.8 mg, 0. 162 mmol, 3 equiv) and HOBt*H20 (24.8 mg, 0. 162 mmol, 3 equiv) were dissolved in DMF (0.5 mL) and DIEA (84.7 μL, 0.486 mmol, 9 equiv) first and HBTU (61.4 mg, 0. 162 mmol, 3 equiv) second were added. The resulting mixture was shaken for a minute (pre-activation) and added to the resin. The mixture was reacted for 1.5 h (3 min of manual stirring and 87 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). A Kaiser test indicated the completion of the coupling reaction. Synthesis of H-D-Asp(0-resin)-OAllyl 38
Figure imgf000100_0001
Fmoc-D-Asp(OH)-OAllyl 37 ( 106.8 mg, 0.270 mmol, 5 equiv) and HOAt (38.8 mg, 0.270 mmol, 5 equiv) were dissolved in DCM (0.3 mL) and DIPCDI (41.8 μΐ., 0.270 mmol, 5 equiv) and DMAP (3.3 mg, 0.027 mmol, 0.5 equiv) in DMF (0.2 mL) were added. The resulting mixture was added to the resin and stirred for 3 h (3 min of manual stirring and 177 min on a shaker) at 25 °C. After filtration and washes with DCM ( l x l min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL), the aa was re-coupled under the same conditions. The excess sites on the AB linker were quenched with a solution of DIEA (470 μL, 2.700 mmol, 50 equiv) and Ac20 (255 μL, 2.700 mmol, 50 equiv) in DMF (0.2 mL) for 16 min. The solvents were removed and the resin subjected to the following washings/treatments: DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The loading, as calculated by UV absorbance at 290 nm, was 0.36 mmol/g.
Synthesis of H-D - αίίο-AHDMHA-D- a o-Thr ( ¾ul -D -Lys (Boc) -Leu-NMe-Glu( ¾u) - threo- β -EtO -Asn(Trt) -D -Asp (O -resin) -O Allyl 53
Figure imgf000101_0001
38 53
Sequential incorporation of Fmoc-ihreo-p-EtO-Asn(Trt)-OH 39 (86.5 mg, 0. 135 mmol, 2.5 equiv), Fmoc-iVMe-Glu(<Bu)-OH 52 (83. 1 mg, 0. 189 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (85.9 mg, 0.243 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (88.6 mg, 0. 189 mmol, 3.5 equiv), Fmoc-D-ctZZo-Thr(tBu)-OH 43 (64.4 mg, 0. 162 mmol, 3 equiv) and Fmoc-D-ctZZo-AHDMHA-OH 23 (53.7 mg, 0. 135 mmol, 2.5 equiv) to the previously obtained H-D-Asp(0-resin)-OAllyl 38 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.5 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- AHDMHA-D-aZZo-Thr-D-Lys-Leu-NMe-Glu-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 100% as checked by HPLC-PDA. Conditions: linear gradient ( 10% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.78 min). HPLC-ESMS(+) analysis showed: m/z calculated for C43H75N9O16 973.53; [M+H]+ found, 975.39.
Synthesis of Fmoc- (3 S, 4-R) -3 , 4 -diMe-Gln-D - ZZo-AHDMHA-D - ZZo-Thr ( ¾ul -D - Lvs(Boc)-Leu-NMe-Glu(tBu)-t reo-B-EtO-Asn(Trt)-D-Asp(0-resin)-OAllyl 54
Figure imgf000102_0001
Fmoc-diMeGln-OH 45 (96.3 mg, 0. 243 mmol, 4.5 equiv) was coupled using DIPCDI (37.6 μΐ., 0.243 mmol, 4.5 equiv) and HOBt*¾0 (37.2 mg, 0.243 mmol, 4.5 equiv) in DMF ( 1.0 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc-diMeGln-D-aZZo- AHDMHA-D-a//o-Thr-D-Lys-Leu-NMe-Glu-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 90% as checked by HPLC-PDA. Conditions: linear gradient (30% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.61 min). HPLC-ESMS(+) analysis showed: m/z calculated for C65H97N11O20 1351.69; [M+H]+ found, 1352.53.
Synthesis of {[Fmoc- (3 SAR\ -3 , 4 - diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ( ¾u) - D-Lys(Boc)-Leu-i\/Me-Glu(tBu)-t reo-B-EtO-Asn(Trt)-D-Asp(0-resin)- OAUylUAUoc-pipecolic&l) 55
Figure imgf000103_0001
After washings with DCM (3 x 1 min; 4 mL) the resin was dried under vacuum for 20 min and transferred to a PYREXR culture tube provided with a magnetic stirrer. Fresh Alloc-pipecolic-OH 1 ( 172.7 mg, 0.810 mmol, 15 equiv) was dissolved in dry DCM (2 mL) and DIPCDI ( 125.4 mL, 0.810 mmol, 15 equiv) was added. The mixture was cooled down to 0 °C, filtered through a 0.45 μπι filter and added to the resin. DMAP (3.3 mg, 0.027 mmol, 0.5 equiv) dissolved in dry DMF (0.3 mL) was finally added to the tube. The mixture was reacted for 2.3 h at 45-47 °C with smooth stirring. The reaction mixture was transferred to the syringe, the solvents removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL), followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The nonapeptide Fmoc-diMeGln-D- a//o-AHDMHA(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Glu-i reo-p-EtO- Asn-D-Asp-OAllyl was obtained with a purity of 80% as checked by HPLC- PDA. Conditions: linear gradient (30% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 6.89 min). HPLC-ESMS(+) analysis showed: m/z calculated for C75H110N12O23 1546.78; [M+H]+ found, 1548.07.
Synthesis of {[H-(2^3^4S)-DADHOHA(Trt, Acetonide)-(3S,4i?)-3,4-diMe-Gln- D - alio- AHDMHA(&) -D - a o-Thr (tBu) -D -Lys (Boc) -Leu-NMe-Glu( ¾u) - threo- β -EtO - Asn(Trt)-D-Asp(0-resin)-OAllyl1 [Alloc-pipecolic&l} 56
Figure imgf000104_0001
The Fmoc group of diMeGln was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL). The resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (96.0 mg, 0.135 mmol, 2.5 equiv) quickly added to the resin. This aa was introduced using HATU (51.3 mg, 0.135 mmol, 2.5 equiv), HOAt ( 18.4 mg, 0.135 mmol, 2.5 equiv) and DIEA (47.0 μL, 0.270 mmol, 5 equiv) dissolved in DMF (0.5 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings /treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-DADHOHA-diMeGln-D - alio- AHDMHA(Alloc-pipecolic) -D - allo- Thr-D-Lys-Leu-NMe-Glu-i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 68% (the decapeptide protected with an acetal group was also considered to calculate purity) as checked by HPLC-PDA. Conditions: linear gradient (20% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.66 min). HPLC-ESMS(+) analysis showed: m/z calculated for C67H112N14O25 1512.79; [(M+2H+)/2] found, 757.85.
Synthesis of {(2^3^4^)-HTMHA-D-Asn(Trt)-(2^3^4S)-DADHOHA(Trt,
Acetonide) - (3 SAR\ -3 , 4 -diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ( ¾u) -D - LvsiBocl-Leu-NMe-GluitBul-t reo-B-EtO-AsniTrtl-D-AspiO-resinl-OAllyllfAlloc- pipecolic&ll 58
Figure imgf000105_0001
The last two residues were incorporated stepwise to the growing peptide chain. Fmoc-D-Asn(Trt)-OH 57 (112.8 mg, 0.189 mmol, 3.5 equiv) and HOAt (25.7 mg, 0.189 mmol, 3.5 equiv) were dissolved in DMF (0.5 mL) and HATU (71.9 mg, 0.189 mmol, 3.5 equiv) and DIEA (65.8 μL, 0.378 mmol, 7 equiv) were added. After 1 min of pre-activation, the reaction mixture was added to the resin. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings/treatments: piperidine-DMF (1:4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Once the amino group was unprotected, the terminating acid was incorporated. HTMHA 31 (30.5 mg, 0.162 mmol, 3 equiv), DIPCDI (25.1 μL, 0.162 mmol, 3 equiv) and HOBt (8.3 mg, 0.162 mmol, 3 equiv) were dissolved in DMF (0.5 mL) and added to the resin without pre- activation. After 3.25 h of coupling (5 min of manual stirring and 190 min on a shaker) more reagents were added (HTMHA-DIPCDI-HOBt, 1:1:1) and the resulting mixture was stirred for 1 h more. Then, the solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.35 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The undecapeptide HTMHA-D-Asn-DADHOHA- diMeGln-D-a//o-AHDMHA(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Glu- i reo-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 28% as checked by HPLC-PDA. Conditions: linear gradient (30% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 6.27 min). HPLC-ESMS(+) analysis showed: m/ z calculated for C81H136N16O29 1796.97; [M+H]+ found, 1797.93.
Synthesis of {(2^3^4^)-HTMHA-D-Asn(Trt)-(2^3^4S)-DADHOHA(Trt, Acetonide) - (3 SAR) -3 , 4 -diMe-Gln-D - alio- AHDMHA(&) -D - a o-Thr ftBu) - D - Lys(Boc)-Leu-i\/Me-Glu(tBu)-t reo-B-EtO-Asn(Trt)-D-Asp(0-resin)-OH1[H- pipecolic&ll 59
Figure imgf000106_0001
Next, to remove the Allyl and Alloc groups, the peptide-resin 58 was treated with Pd(PPh3)4 ( 12.5 mg, 0.01 1 mmol, 0.2 equiv) and PhSiH3 ( 133.3 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.5 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor 59 was obtained with a purity of 26% as checked by HPLC-PDA. Conditions: linear gradient (20% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.65 min). HPLC-ESMS(+) analysis showed: m/z calculated for C74H128N16O27 1672.91 ; [(M+2H+)/2] found, 837.49.
Synthesis of PIPECOLIDEPSIN A' 60
Figure imgf000107_0001
The macrolactamization step was carried out on solid phase. HO At (29.4 mg, 0.2 16 mmol, 4 equiv) was dissolved in DMF (0.5 mL) and DIEA (75.2 μΐ^, 0.432 mmol, 8 equiv) was added. The reaction mixture was then added to the resin followed by the addition of solid PyBOP ( 1 12.4 mg, 0.2 16 mmol, 4 equiv) and it was allowed to react for 3 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5, 1 h, 0.35 mL) followed by analysis of the crude by HPLC-PDA and HPLC- ESMS.
The cleavage of the peptide and the elimination of the side-chain protecting groups were accomplished simultaneously by treating the resin with TFA-H20-Tis (95:2.5:2.5, 1 x 1.5 h, 3 x 2 min; 4 mL). All the filtrates were collected in a round-bottom flask, evaporated to dryness under reduced pressure and lyophilized in H2O-ACN ( 1 : 1 , 25 mL) to give 82.6 mg of crude with a purity of 15% as checked by HPLC-PDA. Conditions: linear gradient (20% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 6.71 min). HPLC-ESMS(+) analysis showed: m/ z calculated for C74H126N16O26 1654.90; [M+H]+ found, 1655.76.
The crude was purified by semi-analytical HPLC-PDA using a XBridge™ Prep BEH 130 C 18 5 μιη 10x 100 mm column and a linear gradient (5% to 36% over 5 min and 36% to 42% over 20 min) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 3.0 mL/min (iR = 1 1.69 min) to give 4. 19 mg of HTMHA-D-Asn-DADHOHA-diMeGln-D-a//o-AHDMHA(S6)-D-a//o-Thr-D-Lys-Leu- NMe-Glu-i reo-p-EtO-Asn-D-Asp-D-Asp-pipecolic(fi6) (Pipecolidepsin A 60 (4.7%). HRMS (NanoESl) analysis showed: m/ z calculated for C74H1 6O26N16 1654.9029, found 1654.9046 [M]. The Ή and 13C NMR (600 MHz, CD3OH) data are shown in Table II.
Table II. Ή and 13C NMR data of Pipecolidepsin A' 60 (CD3OH)
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000110_0002
EXAMPLE 5: SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 71
Figure imgf000111_0001
71
Experimental protocol Synthesis of Fmoc-D-As tBul-O-resin 61
Figure imgf000111_0002
61
2-Chlorotrityl chloride resin (75 mg, 1.6 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). Fmoc-D- Asp(£Bu)-OH (15.4 mg, 0.038 mmol) and DIEA (43.5 iL, 0.25 mmol) were dissolved in DCM (0.3 mL). The resulting mixture was added to the resin and manually stirred for 5 min. Then, more DIEA (21.8 μΐ^, 0.125 mmol) was added and the mixture was reacted for 40 min at 25 °C on a shaker. The excess sites on the 2-CTC resin were quenched by adding 800 μΐ^/g resin of MeOH (60 μΐ^) and stirring the resulting mixture 10 min at 25 °C. The solvents were removed and the resin subjected to the following washings/ treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF (1:4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The loading, as calculated by UV absorbance at 290 nm, was 0.40 mmol/g.
Synthesis of H-D - aZZo-Thr(OH) -D - aZZo-Thr ( *Bu) -D -Lvs (Boc) -Leu-NMe-Gln- β -EtO - AsniTrtl-D-AspitBul-O-resin 63
Figure imgf000112_0001
63
Sequential incorporation of Fmoc-p-EtO-Asn(Trt)-OH 39 (48. 1 mg, 0.075 mmol, 2.5 equiv), Fmoc-NMe-Gln-OH 40 (40.2 mg, 0.105 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (47.7 mg, 0.135 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (49.2 mg, 0.105 mmol, 3.5 equiv), Fmoc-D-ctZZo-Thr(tBu)-OH 43 (35.8 mg, 0.090 mmol, 3 equiv) and Fmoc-D-aZZo-Thr(OH)-OH 62 (30.7 mg, 0.090 mmol, 3 equiv) to the previously obtained H-D-Asp(fBu)-0-resin 61 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.3 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin 63 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- Thr-D-aZZo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 86% as checked by HPLC-PDA. Conditions: linear gradient (5% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.60 min). HPLC-ESMS(+) analysis showed: m/z calcd for C36H64NioO i5 876.46; [M+H]+ found, 877.56. Synthesis of Fmoc-QS^jei-S^-diMe-Gln-D-aZZo-ThriOH -D-aZZo-ThritBu -D-
LysiBocl-Leu-NMe-Gln-B-EtO-AsniTrtl-D-AspitBul-O-resin 64
Figure imgf000113_0001
63 64
Fmoc-diMeGln-OH 45 (53.5 mg, 0. 135 mmol, 4.5 equiv) was coupled with resin 63 using DIPCDI (20.9 iL, 0. 135 mmol, 4.5 equiv) and HOBt*H20 (20.7 mg, 0. 135 mmol, 4.5 equiv) in DMF (0.5 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C without pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin 64 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc- diMeGln-D-a//o-Thr-D-a//o-Thr-D-Lys-Leu-i\/Me-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 78% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.90 min). HPLC-ESMS(+) analysis showed: m/z calcd for
Figure imgf000113_0002
1254.61 ; [M+H]+ found, 1255.81.
Synthesis of {[Fmoc- (3 S, 4R) -3 , 4 -diMe-Gln-D - a o-Thr (&) -D - a o-Thr ( ¾u) -D - Lys (Boc) -Leu-NMe-Gln- β -EtO -Asn(Trt) -D -Asp ( ¾u) -O -resin] [ Alloc-pipecolic&l) 65
Figure imgf000114_0001
Fresh Alloc-pipecolic-OH 1 (51.2 mg, 0.240 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (37.2 μΐ., 0.240 mmol, 8 equiv) was added. The mixture was poured into the resin 64 followed by addition of DMAP ( 1.8 mg, 0.015 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL), followed by analysis of the crude by HPLC-PDA and HPLC-MS. The nonapeptide Fmoc-diMeGln-D-aZZo-Thr(Alloc-pipecolic)-D-aZZo-Thr-D-Lys-Leu- NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 77% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 5.04 min). HPLC-ESMS(+) analysis showed: m/z calcd for C68H99N13O22 1449.70; [M+H]+ found, 1450.92.
Synthesis of {[H-Gln(Trt)-Ser(tBu)-(3S,4^)-3,4-diMe-Gln-D- ZZo-Thr(&)-D- ZZo- Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-Asn(Trt)-D-Asp(tBu)-0-resin[Alloc- pipecolic&ll 68
Figure imgf000114_0002
The Fmoc group of diMeGln moiety in 65 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL) . The resin was shortly washed with DMF (x 2 ; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-Ser(£Bu)-OH 66 (40.3 mg, 0. 105 mmol, 3.5 equiv) quickly added to the resin. This aa was introduced using HATU (39.9 mg, 0. 105 mmol, 3.5 equiv), HOAt ( 14.3 mg, 0. 105 mmol, 3.5 equiv) and DIEA (36.6 iL, 0.210 mmol, 7 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings/ treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . An aliquot of the resin was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-Ser-diMeGln-D-a//o-Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p- EtO-Asn-D-Asp-OH was obtained with a purity of 54% as checked by HPLC- PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 2.74 min). HPLC-ESMS(+) analysis showed: m/z calcd for C56H94N14O22 13 14.67; [M+H]+ found, 13 13.93. Next, Fmoc-Gln(Trt)-OH 67 (64. 1 mg, 0. 105 mmol, 3.5 equiv) was coupled using HATU (39.9 mg, 0. 105 mmol, 3.5 equiv), HOAt ( 14.3 mg, 0. 105 mmol, 3.5 equiv) and DIEA (36.6 μL, 0.2 10 mmol, 7 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings /treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL) , DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . An aliquot of the resin 68 was treated with a solution of TFA-H2O-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The undecapeptide H-Gln-Ser-diMeGln-D - ctZZo-Thr (Alloc-pipecolic) -D - aZZo-Thr-D - Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 67% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 2.69 min). HPLC-ESMS(+) analysis showed: m/z calcd for C61H102N16O24 1442.73 [M+H]+ found, 1442.52.
Synthesis of {i2Jg,3Jg,4J¾-HTMHA-D-Aspi¾u)-GlniTrt)-Seri¾u)-i3S,4J?|-3,4- diMe-Gln-D- ZZo-Thri&l-D-gZZo-ThritBul-D-LvsiBocl-Leu-NMe-Gln-B-EtO- AsniTrtl-D-AspitBul-O-resinl [Alloc-pipecolic&l) 69
Figure imgf000116_0001
The last two residues were incorporated as a single unit to the growing peptide chain to avoid aspartimides formation. HTMHA-D-Asp(£Bu)-OH 36 (53.9 mg, 0. 150 mmol, 5 equiv) and HOAt (20.4 mg, 0. 150 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (52.3 μΐ., 0.300 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 68, and solid PyBOP (78.0 mg, 0.150 mmol, 5 equiv) was added. After 3.5 h of coupling (3 min of manual stirring and 227 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.3 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The dodecapeptide HTMHA-D-Asp-Gln-Ser- diMeGln-D-a//o-Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn- D-Asp-OH was obtained with a purity of 46% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.29 min). HPLC-ESMS(+) analysis showed: m/z calcd for C75H125N17O29 1727.88 [M+H]+ found, 1728.67.
Synthesis of {i2Jg,3Jg,4J¾-HTMHA-D-Aspi¾u)-GlniTrt)-Seri¾u)-i3S,4J?|-3,4- diMe-Gln-D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-
AsniTrt)-D-Aspi¾u)-0-resini rH-pipecolic&1l 70
Figure imgf000117_0001
Next, to remove the Alloc group, the peptide-resin 69 was treated with
Pd(PPh3)4 (3.47 mg, 0.003 mmol, 0. 1 equiv) and PhSiH3 (37.0 \iL, 0.300 mmol, 10 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor was obtained with a purity of 60% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 2.96 min). HPLC-ESMS(+) analysis showed: m/z calcd for C71H121N17O27 1643.86 [M+H]+ found, 1643.47.
Synthesis of Gln-Ser analog 71
Before performing the macrolactamization step in solution, the suitably protected linear precursor was cleaved from the resin 70 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL). All the filtrates were collected in a round-bottom flask provided with 20 mL of H2O in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 52.9 mg of fully protected linear precursor.
Figure imgf000118_0001
The macrolactamization step was then carried out in solution. The peptide crude was dissolved in dry DCM (25 mL) and then a solution of HO At (5.5 mg, 0.040 mmol, 2 equiv) in DMF (0.05 mL) and DIEA ( 14 μL, 0.080 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP (20.8 mg, 0.040 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C. The reaction mixture was washed with 5% aqueous NaHCOa solution (x 3), saturated aqueous NH4C1 solution (x 3) and brine (x 1), dried over Na2S04, filtrated and concentrated under vacuo. To fully remove the side-chain protecting groups, the protected cyclopeptide was treated with TFA- H20-Tis (95:2.5:2.5) (1 x 1.5 h, 4 mL) at 25 °C. The final deprotection step was monitored by analysis of the crude by HPLC-PDA and HPLC-ESMS. All organic solvents were eliminated under reduced pressure, the peptide crude was dissolved in H2O-ACN ( 1 : 1), and the resulting solution was lyophilized to provide 53.9 mg of Gln-Ser-analog 71 with a purity of 36% as checked by HPLC-PDA. Conditions: linear gradient (20% to 70%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.51 min). HPLC-ESMS(+) analysis showed: m/z calcd for C71H119N17O26 1625.85 [M+H]+ found, 1625.84.
The crude was partially purified by semi-analytical HPLC-PDA using a XBridge™ Prep BEH130 C 18 5 μιη 10x 100 mm column and a linear gradient (0% to 30% over 5 min and 30% to 33% over 20 min) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 3.0 mL/min. Final HTMHA-D-Asp-Gln- Ser-diMeGln-D-a//o-Thr(fi6)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp- pipecolic(&) was obtained with a purity above 95% as checked by HPLC-PDA analysis. Conditions: linear gradient (25% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.76 min). HPLC-ESMS(+) analysis showed: m/z calcd for C71H119N17O26 1625.85 [M+H]+ found, 1625.84. EXAMPLE 6: SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 77
Figure imgf000119_0001
77 Experimental protocol:
Synthesis of Fmoc-D-AspftBul-O-resin 61
Figure imgf000119_0002
61
2-Chlorotrityl chloride resin (75 mg, 1.6 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL) . Fmoc-D- Asp('Bu)-OH ( 15.4 mg, 0.038 mmol) and DIEA (43.5 iL, 0.25 mmol) were dissolved in DCM (0.3 mL) . The resulting mixture was added to the resin and manually stirred for 5 min. Then, more DIEA (2 1 .8 μL, 0. 125 mmol) was added and the mixture was reacted for 40 min at 25 °C on a shaker. The excess sites on the 2-CTC resin were quenched by adding 800 μL/ g resin of MeOH (60 μL) and stirring the resulting mixture 10 min at 25 °C. The solvents were removed and the resin subjected to the following washings/ treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The loading, as calculated by UV absorbance at 290 nm, was 0.42 mmol/g.
Synthesis of H-D - aZZo-Thr(OH) -D - aZZo-Thr ( ¾ul -D -Lvs (Boc) -Leu-NMe-Gln- β -EtO -
AsniTr^-D-AspiM-O-resin 63
Figure imgf000120_0001
63
Sequential incorporation of Fmoc-p-EtO-Asn(Trt)-OH 39 (50.5 mg, 0.079 mmol, 2.5 equiv), Fmoc-NMe-Gln-OH 40 (42.2 mg, 0. 1 10 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (50. 1 mg, 0. 141 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (51.7 mg, 0. 1 10 mmol, 3.5 equiv), Fmoc-D-ctZZo-Thr(tBu)-OH 43 (37.6 mg, 0.095 mmol, 3 equiv) and Fmoc-D-aZZo-Thr(OH)-OH 62 (32.2 mg, 0.095 mmol, 3 equiv) to the previously obtained H-D-Asp(fBu)-0-resin 61 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.3 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin 63 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- Thr-D-aZZo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 89% as checked by HPLC-PDA. Conditions: linear gradient (5% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 3.57 min). HPLC-ESMS(+) analysis showed: m/z calcd for C36H64NioO i5 876.46; [M+H]+ found, 877.59.
Synthesis of Fmoc-GlniTrtl-D- ZZo-ThriOHl-D- ZZo-ThritBul-D-LysiBocl-Leu- NMe-Gln- β -EtO -Asn(Trt) -D -Asp ( tBu) -O -resin 72
Figure imgf000121_0001
63 72
Fmoc-Gln(Trt)-OH 67 (76.9 mg, 0. 126 mmol, 4 equiv) was coupled with resin 63 using HATU (47.9 mg, 0. 126 mmol, 4 equiv), HOAt ( 17.2 mg, 0. 126 mmol, 4 equiv) and DIEA (43.9 μΐ., 0.252 mmol, 8 equiv) in DMF (0.4 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C with 1 min of pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin 72 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc-Gln-D - ctZZo-Thr-D - ctZZo-Thr-D -Lys-Leu-NMe-Gln-β -EtO - Asn-D-Asp-OH was obtained with a purity of 85% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.71 min). HPLC-ESMS(+) analysis showed: m/z calcd for C56H82N12O 19 1226.58 [M+H]+ found, 1227.22.
Synthesis of {[Fmoc-Gln(Trt) -D - aZZo-Thr (&) -D - ZZo-Thr ( ¾u) -D -Lys (Boc) -Leu- NMe-Gln- β -EtO -Asn(Trt) -D -Asp (tBu) -O -resin] [ Alloc-pipecolic&l) 73
Figure imgf000122_0001
Fresh Alloc-pipecolic-OH 1 (53.8 mg, 0.252 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (39. 1 μΐ., 0.252 mmol, 8 equiv) was added. The mixture was poured into the resin 72 followed by addition of DMAP ( 1.9 mg, 0.016 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL), followed by analysis of the crude by HPLC-PDA and HPLC-MS. The nonapeptide Fmoc-Gln-D-aZZo-Thr(Alloc-pipecolic)-D-aZZo-Thr-D-Lys-Leu-NMe- Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 81% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.86 min). HPLC-ESMS(+) analysis showed: m/z calcd for C66H95N13O22 142 1.67 [M+H]+ found, 1422.25.
Synthesis of {[H-(2^3^4S)-DADHOHA(Trt, Acetonide)-Gln(Trt)-D- ZZo-Thr(&)- D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-Asn(Trt)-D-Asp(tBu)-0- resin [Alloc-pipecolic&l} 74
Figure imgf000122_0002
The Fmoc group of Gin in resin 73 was then removed following a common deprotection treatment with piperidine-DMF ( 1 :4) (2 x 1 min, 2 >< 5 min, 1 x 10 min, 4 mL). The resin was washed with DMF (3 x 1 min; 4 mL) and DCM ( 3 x 1 min; 4 mL) and the reaction mixture of the next residue Fmoc- DADHOHA(Trt, Acetonide)-OH 10 (55.9 mg, 0.079 mmol, 2.5 equiv) added to the resin. This aa was introduced using HATU (29.9 mg, 0.079 mmol, 2.5 equiv), HOAt ( 10.7 mg, 0.079 mmol, 2.5 equiv) and DIEA (27.4 \iL, 0. 158 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings/ treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). An aliquot of the resin 74 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-DADHOHA-Gln-D-aZZo- Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 79% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 4.378 min and 4.569 min, the second peak corresponding to our desired intermediate with the diol function protected as an acetal). HPLC-ESMS(+) analysis showed: m/z calcd for C58H97N15O24 1387.68 [M+H]+ found, 1388.07. For the intermediate with the diol function protected as an acetal: m/z calcd for C61H101N15O24 1427.71 [M+H]+ found, 1427.96.
Synthesis of {(2^3^4^-ΗΤΜΗΑ-Ρ-Α8Ρ(¾^-ΡΑΡΗΟΗΑ(ΤΓΪ, Acetonide)-
Gln(Trt)-D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-Asn(Trt)-
D -Asp (tBu)-O -resin] [Alloc-pipecolic&j]) 75
Figure imgf000124_0001
The last two residues were incorporated as a single unit to the growing peptide chain to avoid aspartimides formation. HTMHA-D-Asp(£Bu)-OH 36 (56.6 mg, 0. 158 mmol, 5 equiv) and HOAt (21.4 mg, 0. 158 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (54.9 μΐ., 0.315 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 74, and solid PyBOP (81.9 mg, 0.158 mmol, 5 equiv) was added. After 3.5 h of coupling (3 min of manual stirring and 227 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.3 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The undecapeptide HTMHA-D-Asp-DADHOHA-Gln- D - ctZZo-Thr (Alloc-pipecolic) -D - ctZZo-Thr-D -Lys-Leu-NMe-Gln-β -EtO -Asn-D -Asp- OH was obtained with a purity of 62% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 5.86 min). HPLC-ESMS(+) analysis showed: m/z calcd for C72H120N16O29 1672.84; [M+H]+ found, 1673.29. Synthesis of {(2^3^4^-ΗΤΜΗΑ-Ρ-Α8ρ(ίΒ^-ΡΑΡΗΟΗΑ(ΤΓΪ, Acetonide)- Gln(Trt)-D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO- AsniTr^-D-AspitB^-O-resinl fH-pipecolic&l} 76
Figure imgf000124_0002
Next, to remove the Alloc group, the peptide-resin 75 was treated with Pd(PPh3)4 (3.64 mg, 0.003 mmol, 0.1 equiv) and PhSiH3 (38.8 \iL, 0.315 mmol, 10 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL) , DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) (1 x 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor was obtained with a purity of 71% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/ min (ip> = 2.71 min) . HPLC-ESMS(+) analysis showed: m/z calcd for C68Hi i6Ni6027 1588.82 [M+H]+ found, 1589.23.
Synthesis of Gin analog 77
Before performing the macrolactamization step in solution, the suitably protected linear precursor was cleaved from the resin 76 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL) . All the filtrates were collected in a round-bottom flask provided with 20 mL of ¾0 in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 68. 1 mg of fully protected linear
Figure imgf000125_0001
The macrolactamization step was then carried out in solution. The peptide crude was dissolved in dry DCM (52 mL) and then a solution of HOAt (7.1 mg, 0.052 mmol, 2 equiv) in DMF (0.05 mL) and DIEA ( 18 μL, 0. 103 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP (27.0 mg, 0.052 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C. The reaction mixture was washed with 5% aqueous NaHCC solution (x 3), saturated aqueous NH4C1 solution (x 3) and brine (x 1), dried over Na2S04, filtrated and concentrated under vacuo. To fully remove the side-chain protecting groups, the protected cyclopeptide was treated with TFA- H20-Tis (95:2.5:2.5) (1 x 1.5 h, 4 mL) at 25 °C. The final deprotection step was monitored by analysis of the crude by HPLC-PDA and HPLC-ESMS. All organic solvents were eliminated under reduced pressure, the peptide crude was dissolved in H2O-ACN ( 1 : 1), and the resulting solution was lyophilized to provide 66. 1 mg of Gin-analog with a purity of 55% as checked by HPLC-PDA. Conditions: linear gradient (20% to 40%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 6.25 min). HPLC-ESMS(+) analysis showed: m/z calcd for CesHi
Figure imgf000126_0001
1570.81 [M+H]+ found, 1570.97. The crude was partially purified by semi-analytical HPLC-PDA using a
XBridge™ Prep BEH 130 C 18 5 μιη 10x 100 mm column and a linear gradient (0% to 30% over 5 min and 30% to 33% over 20 min) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 3.0 mL/min. Final HTMHA-D-Asp- DADHOHA-Gln-D-a//o-Thr(fi6)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D- Asp-pipecolic(&) was obtained with a purity above 95% as checked by HPLC- PDA analysis. Conditions: linear gradient (20% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 5.28 min). HPLC-ESMS(+) analysis showed: m/z calcd for CesHi
Figure imgf000126_0002
1570.81 [M+H]+ found, 1570.97.
EXAMPLE 7: SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 81
NH2 \
81
Experimental Protocol: Synthesis of Fmoc-D-AspftBul-O-resin 61
Figure imgf000127_0001
61
2-Chlorotrityl chloride resin (50 mg, 1.6 mmol/g) was placed in a 5 mL- polypropylene syringe fitted with two polyethylene filter discs. The resin was then washed with DMF (5 x 1 min; 4 mL) and DCM (5 x 1 min; 4 mL). Fmoc-D- Asp(£Bu)-OH ( 10.3 mg, 0.025 mmol) and DIEA (29 μΐ., 0. 17 mmol) were dissolved in DCM (0.3 mL). The resulting mixture was added to the resin and manually stirred for 5 min. Then, more DIEA ( 14.5 μΐ^, 0.083 mmol) was added and the mixture was reacted for 40 min at 25 °C on a shaker. The excess sites on the 2-CTC resin were quenched by adding 800 μΐ^/g resin of MeOH (40 μΐ^) and stirring the resulting mixture 10 min at 25 °C. The solvents were removed and the resin subjected to the following washings/treatments: DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL), DCM (3 x 1 min; 4 mL), piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The loading, as calculated by UV absorbance at 290 nm, was 0.43 mmol/g. Synthesis of H-D - aZZo-Thr(OH) -D - aZZo-Thr ( ¾ul -D -Lvs (Boc) -Leu-NMe-Gln- β -EtO -
AsniTr^-D-AspiM-O-resin 63
Figure imgf000128_0001
63
Sequential incorporation of Fmoc-p-EtO-Asn(Trt)-OH 39 (34.4 mg, 0.054 mmol, 2.5 equiv), Fmoc-NMe-Gln-OH 40 (28.8 mg, 0.075 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (34.2 mg, 0.097 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (35.3 mg, 0.075 mmol, 3.5 equiv), Fmoc-D-aZZo-Thr(tBu)-OH 43 (25.6 mg, 0.065 mmol, 3 equiv) and Fmoc-D-aZZo-Thr(OH)-OH 62 (22.0 mg, 0.065 mmol, 3 equiv) to the previously obtained H-D-Asp(fBu)-0-resin 61 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.3 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin 63 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- Thr-D-aZZo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 89% as checked by HPLC-PDA. Conditions: linear gradient (5% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.47 min). HPLC-ESMS(+) analysis showed: m/z calcd for C36H64NioO i5 876.95; [M+H]+ found, 876.66. Synthesis of Fmoc-OS^jei-S^-diMe-Gln-D-aZZo-ThriOH -D-aZZo-ThritBu -D- LysiBocl-Leu-NMe-Gln-B-EtO-AsniTrtl-D-AspitBul-O-resin 64
Figure imgf000129_0001
Fmoc-diMeGln-OH 45 (38.4 mg, 0. 097 mmol, 4.5 equiv) was coupled with resin 63 using DIPCDI ( 15.0 \iL, 0.097 mmol, 4.5 equiv) and HOBt*¾0 ( 14.8 mg, 0.097 mmol, 4.5 equiv) in DMF (0.4 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C with 1 min of pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin 64 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc-diMeGln-D - ctZZo-Thr-D - ctZZo-Thr-D -Lys-Leu-NMe-Gln-β -EtO - Asn-D-Asp-OH was obtained with a purity of 85% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.88 min). HPLC-ESMS(+) analysis showed: m/z calcd for CssHgeNiaOig 1255.37; [M+H]+ found, 1255.81.
Synthesis of {[Fmoc- (3 S, 4R) -3 , 4 -diMe-Gln-D - aZZo-Thr (&) -D - aZZo-Thr ( ¾u) -D -
Lys (Boc) -Leu-NMe-Gln- β -EtO -Asn(Trt) -D -Asp (tBu) -O -resin] [Alloc-pipecolic&l) 65
Figure imgf000130_0001
Fresh Alloc-pipecolic-OH 1 (36.7 mg, 0. 172 mmol, 8 equiv) was dissolved in dry DCM (0.8 mL) and DIPCDI (26.6 μΐ., 0. 172 mmol, 8 equiv) was added. The mixture was poured into the resin 64 followed by addition of DMAP ( 1.3 mg, 0.01 1 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL), followed by analysis of the crude by HPLC-PDA and HPLC-MS. The nonapeptide Fmoc-diMeGln-D-aZZo-Thr(Alloc-pipecolic)-D-aZZo-Thr-D-Lys-Leu- NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 77% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 5.00 min). HPLC-ESMS(+) analysis showed: m/z calcd for C68H99N13O22 1450.59; [M+H]+ found, 1449.66.
Synthesis of {[H-(2^3^4S)-DADHOHA(Trt, Acetonide)-(3S,4i?)-3,4-diMe-Gln- D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-Asn(Trt)-D- Asp ( tBu) -O -resin [Alloc-pipecolic&l} 78
Figure imgf000131_0001
The Fmoc group of diMeGln moiety in resin 65 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL) . The resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (38.2 mg, 0.054 mmol, 2.5 equiv) quickly added to the resin. This aa was introduced using HATU (20.4 mg, 0.054 mmol, 2.5 equiv) , HOAt (7.3 mg, 0.054 mmol, 2.5 equiv) and DIEA ( 18.7 μΐ., 0. 108 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings/treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL) . An aliquot of the resin 78 was treated with a solution of TFA-H2O- TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-DADHOHA-diMeGln-D-a//o-Thr(Alloc- pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OH was obtained with a purity of 62% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 2.589 min and 2.856 min, the second peak corresponding to our desired intermediate with the diol function protected as an acetal) . HPLC-ESMS(+) analysis showed: m/ z calcd for C60H101N15O24 1415.71 [M+H]+ found, 1416.87. For the intermediate with the diol function protected as an acetal: m/ z calcd for C63H105N15O24 1455.75 [M+H]+ found, 1456.92. Synthesis of {(2 ,3 ^-ΗΤΜΗΑ-Ρ-Α8ρ(ίΒ^-ΡΑΡΗΟΗΑ(ΤΓΪ, Acetonide)- i3S^i¾-3^-diMe-Gln-D- ZZo-Thri&)-D- ZZo-ThritBu)-D-LvsiBoc)-Leu-i\/Me-Gln-B-
EtO-AsniTrtl-D-AspitBul-O-resinl [Alloc-pipecolic&l} 79
Figure imgf000132_0001
The last two residues were incorporated as a single unit to the growing peptide chain to avoid aspartimides formation. HTMHA-D-Asp(£Bu)-OH 36 (38.6 mg, 0. 108 mmol, 5 equiv) and HOAt ( 14.6 mg, 0. 108 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (37.4 μΐ., 0.2 15 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 78, and solid PyBOP (55.9 mg, 0. 108 mmol, 5 equiv) was added. After 3.5 h of coupling (3 min of manual stirring and 227 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.3 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The undecapeptide HTMHA-D-Asp-DADHOHA- diMeGln-D-a//o-Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn- D-Asp-OH was obtained with a purity of 44% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.42 min). HPLC-ESMS(+) analysis showed: m/z calcd for C74H124N16O29 1700.87 [M+H]+ found, 1700.98. Synthesis of {(2^3^4^-ΗΤΜΗΑ-Ρ-Α8ρ(ίΒ^-ΡΑΡΗΟΗΑ(ΤΓΪ, Acetonide)-
(3S,4i?)-3,4-diMe-Gln -D-a;;o-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln- β -EtO -Asn(Trt) -D -Asp ( 'Bui -O -resin] [H-pipecolic&ll 80
Figure imgf000133_0001
Next, to remove the Alloc group, the peptide-resin 79 was treated with Pd(PPh3)4 (2.48 mg, 0.002 mmol, 0. 1 equiv) and PhSiH3 (26.5 \iL, 0.215 mmol, 10 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor was obtained with a purity of 56% as checked by HPLC-PDA. Conditions: linear gradient (5% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.41 min). HPLC-ESMS(+) analysis showed: m/z calcd for C70H120N16O27 1616.85 [M+H]+ found, 1616.78. Synthesis of Ό-allo-Thr analog 81
Before performing the macrolacatamization step in solution, the suitably protected linear precursor was cleaved from the resin 80 under very mild conditions (TFA: DCM (2:98), 5 2 min, 4 mL). All the filtrates were collected in a round-bottom flask provided with 20 mL of H2O in order the keep the percentage of TFA to an innocuous 2%. After evaporation of the organic phase under reduced pressure, ACN was added to dissolve all the peptide, and the final solution was lyophilized to provide 31.6 mg of fully protected linear precursor.
Figure imgf000134_0001
The macrolactamization step was then carried out in solution. The peptide crude was dissolved in dry DCM (26 mL) and then a solution of HO At (3.6 mg, 0.026 mmol, 2 equiv) in DMF (0.03 mL) and DIEA (9. 1 μΐ., 0.052 mmol, 4 equiv) were added. Once a clear solution was obtained, solid PyBOP ( 13.6 mg, 0.026 mmol, 2 equiv) was added. The reaction mixture was allowed to react for 3 h at 25 °C. The reaction mixture was washed with 5% aqueous NaHCOa solution (x 3), saturated aqueous NH4C1 solution (x 3) and brine (x 1), dried over Na2S04, filtrated and concentrated under vacuo. To fully remove the side-chain protecting groups, the protected cyclopeptide was treated with TFA- H20-Tis (95:2.5:2.5) (1 x 1.5 h, 4 mL) at 25 °C. The final deprotection step was monitored by analysis of the crude by HPLC-PDA and HPLC-ESMS. All organic solvents were eliminated under reduced pressure, the peptide crude was dissolved in H2O-ACN ( 1 : 1), and the resulting solution was lyophilized to provide 42. 1 mg of D-ctZZo-Thr-analog 81 with a purity of 45% as checked by HPLC-PDA. Conditions: linear gradient (30% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (fe = 2.99 min). HPLC-ESMS(+) analysis showed: m/z calcd for C70H118N16O26 1598.84 [M+H]+ found, 1598.30.
The crude was partially purified by semi-analytical HPLC-PDA using a XBridge™ Prep BEH130 C 18 5 μιη 10x 100 mm column and a linear gradient (0% to 30% over 5 min and 30% to 32% over 20 min) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 3.0 mL/min. Final HTMHA-D-Asp- DADHOHA-diMeGln-D-aZZo-Thr(fi6)-D-aZZo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn- D-Asp-pipecolic(&) was obtained with a purity above 95% as checked by HPLC-PDA analysis. Conditions: linear gradient (30% to 50%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.15 min). HPLC-ESMS(+) analysis showed: m/z calcd for C70H118N16O26 1598.84 [M+H]+ found, 1598.30. EXAMPLE 8: SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 89
Figure imgf000135_0001
89
H-D-Asp(0-resin)-OAUyl 38 was prepared by the procedure described in Example 4.
Experimental Protocol:
Synthesis of Octanoic-D-Asp(tBu)-OH 86
Octanoic acid (317.8 μL, 2.01 mmol), H-D-Asp(tBu)-OBzl 34 (466.8 mg, 1.67 mmol), HOAt (273.0 mg, 2.01 mmol) and EDC*HC1 (384.4 mg, 2.01 mmol) were placed in a round-bottom flask. A mixture of dry DCM:DMF ( 1 : 1 , 12 mL) was added and, after checking that the pH of the reaction solution was around 6, it was allowed to react for 12 h at room temperature. DCM was removed under reduced pressure, EtOAc added and the organic layer washed with saturated aqueous NH4C1 solution (x 3), saturated aqueous NaHCOa solution (x 3) and brine, dried over Na2S04, filtrated and concentrated under vacuo. The residue was purified by flash column chromatography (hexane-EtOAc = 90: 10 to 80:20) to give 552.0 mg (81%) of Octanoic-D-Asp(tBu)-OBzl as a yellowish oil: Ή NMR (400 MHz, CDC13) δ 0.87 (3H, t, CH3, octanoic), 1.28 (8H, m, CH2, octanoic), 1.39 (9H, s, CH3, tBu, Asp), 1.62 (2H, m, CH2, octanoic), 2.22 (2H, m, CH2, octanoic), 2.75 ( 1H, dd, J = 17. 1 , 4.5 Hz, CH2 P, Asp), 2.95 ( 1H, dd, J = 17. 1 , 4.4 Hz, CH2 P, Asp), 4.86 ( 1H, dt, J = 8.4, 4.0, 4.0 Hz, CHa, Asp), 5. 14 ( 1H, d, J = 12.3 Hz, CH2, Bzl), 5.22 ( 1H, d, J = 12.3 Hz, CH2, Bzl), 6.46 ( 1H, d, J = 8.1 Hz, CONH), 7.31 -7.36 (5H, m, Bzl); NMR ( 100 MHz, CDC13) δ 14.01 (CH3, octanoic), 22.40 (CH2, octanoic), 25.42 (CH2, octanoic), 27.83 (CH3, tBu), 29.00 (CH2, octanoic), 31.47 (CH2, octanoic), 36.51 (CH2, octanoic), 37.26 (CH2 P, Asp), 48.48 (CHa, Asp), 67.25 (CH2, Bzl), 128.24 (CH, Ar, Bzl).
A solution of Octanoic-D-Asp(tBu)-OBzl (552.0 mg, 1.36 mmol) in dry MeOH ( 10 mL) was stirred with a catalytic amount of Pd-C ( 10%) under an atmosphere of H2 (atmospheric pressure) for 12 h. EtOAc was added and the mixture was filtrated through celite and concentrated under vacuo to give quantitatively 429. 1 mg of crude Octanoic-D-Asp(tBu)-OH 86 as a yellow oil, which was used in the next reaction without further purification: Ή NMR (400 MHz, CDC ) δ 0.87 (3H, t, J = 6.9 Hz, CH3, octanoic), 1.28 (8H, m, CH2, octanoic), 1.44 (9H, s, CH3, tBu), 1.62 (2H, m, CH2, octanoic), 2.24 (2H, t, J = 7.6 Hz, CH2, octanoic), 2.75 ( 1H, dd, J = 17.0, 4.9 Hz, CH2 P, Asp), 2.94 ( 1H, dd, J = 17.0, 4.4 Hz, CH2 P, Asp), 4.81 ( 1H, m, CHa, Asp), 6.67 ( 1H, d, J = 7.7 Hz, CONH); NMR ( 100 MHz, CDCb) δ 13.97 (CH3, octanoic), 22.40 (CH2, octanoic), 25.38 (CH2, octanoic), 27.85 (CH3, tBu), 28.89 (CH2, octanoic), 31.54 (CH2, octanoic), 36.35 (CH2, octanoic), 36.94 (CH2 P, Asp), 48.82 (CHa, Asp).
Synthesis of H-D - allo-Thr (OH) -D - allo-Thr ( ¾ul -D -Lys (Boc) -Leu-NMe-Gln- β -EtO - Asn(Trt)-D-Asp(0-resin)-OAllyl 82
Figure imgf000136_0001
82 Sequential incorporation of Fmoc-p-EtO-Asn(Trt)-OH 39 (43.2 mg, 0.068 mmol, 2.5 equiv), Fmoc-NMe-Gln-OH 40 (36. 1 mg, 0.095 mmol, 3.5 equiv), Fmoc-Leu-OH 41 (42.9 mg, 0. 122 mmol, 4.5 equiv), Fmoc-D-Lys(Boc)- OH 42 (44.3 mg, 0.095 mmol, 3.5 equiv), Fmoc-D-ctZZo-Thr(tBu)-OH 43 (32.2 mg, 0.081 mmol, 3 equiv) and Fmoc-D-aZZo-Thr(OH)-OH 62 (27.7 mg, 0.081 mmol, 3 equiv) to the previously obtained H-D-Asp(fBu)-(0-resin)-0-allyl 38 was accomplished using HATU/HOAt and DIEA (aa-HATU-HOAt-DIEA 1 : 1 : 1 :2) in DMF (0.3 mL) with 1 min of pre-activation. In all cases, Kaiser's test showed a quantitative coupling after 60 min of treatment. The Fmoc-Leu-OH was re- coupled by default and chloranil's test was used to check the completion of the reaction. Fmoc removal was carried out treating the resin with piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL). An aliquot of the resin 82 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The heptapeptide H-D-aZZo- Thr-D-aZZo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 92% as checked by HPLC-PDA. Conditions: linear gradient (5% to 100%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 3.50 min). HPLC-ESMS(+) analysis showed m/ z calcd for C39H68N10O15 916.49; [M+H]+ found, 917.50.
Synthesis of Fmoc-QS^^-S^-diMe-Gln-D- ZZo-ThriO^-D- ZZo-ThritB^-D- Lvs(Boc)-Leu-NMe-Gln-B-EtO-Asn(Trt)-D-Asp(0-resin)-OAllyl 83
Figure imgf000137_0001
82 83 Fmoc-diMeGln-OH 45 (48.2 mg, 0. 122 mmol, 4.5 equiv) was coupled to resin 82 using DIPCDI ( 18.8 \iL, 0. 122 mmol, 4.5 equiv) and HOBt*¾0 ( 18.6 mg, 0. 122 mmol, 4.5 equiv) in DMF (0.4 mL) for 1 h (3 min of manual stirring and 57 min on a shaker) at 25 °C with 1 min of pre-activation. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The Fmoc group was kept in place until ester bond formation. An aliquot of the resin 83 was treated with a solution of TFA-H20-TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The octapeptide Fmoc- diMeGln-D-a/Zo-Thr-D-a/Zo-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 90% as checked by HPLC-PDA. Conditions: linear gradient (30% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3. 16 min). HPLC-ESMS(+) analysis showed m/z calcd for CeiHgoNiaOig 1294.64; [M+H]+ found, 1295.74.
Synthesis of {[Fmoc- (3 SAR-3, 4 -diMe-Gln-D - a o-Thr (&) -D - ZZo-Thr ( ¾u) -D - Lvs(Boc)-Leu-NMe-Gln-B-EtO-Asn(Trt)-D-Asp(0-resin)-OAllyl1 [Alloc-pipecolic&1} 84
Figure imgf000138_0001
83 84
Fresh Alloc-pipecolic-OH 1 (86.4 mg, 0.405 mmol, 15 equiv) was dissolved in dry DCM ( 1.3 mL) and DIPCDI (62.7 μL, 0.405 mmol, 15 equiv) was added. The mixture was poured into the resin 83 followed by addition of
DMAP ( 1.7 mg, 0.014 mmol, 0.5 equiv) dissolved in dry DMF (0.2 mL). The mixture was reacted for 2 h 15 min at 25 °C on a shaker. The solvents were removed by filtration and the resin washed with DCM (3 x 1 min; 4 mL), DMF
(3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Ester bond formation was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL), followed by analysis of the crude by HPLC-PDA and HPLC-MS. The nonapeptide Fmoc-diMeGln-D-aZZo-Thr(Alloc-pipecolic)-D-aZZo-Thr-D-Lys- Leu-NMe-Gln-p-EtO-Asn-D-Asp-OAllyl was obtained with a purity of 80% as checked by HPLC-PDA. Conditions: linear gradient (30% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.48 min). HPLC-ESMS(+) analysis showed m/z calcd for C71H103N13O22 1489.73; [M+H]+ found, 1490.94.
Synthesis of {[H-(2^3^4S)-DADHOHA(Trt, Acetonide)-(3S,4^)-3,4-diMe-Gln-
D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO-Asn(Trt)-D-
Asp ( O -resin) -O Allyl [Alloc-pipecolic&j]) 85
Figure imgf000139_0001
The Fmoc group of diMeGln moiety in resin 84 was then removed following a short deprotection treatment with piperidine-DMF ( 1 :4) (2 x 2 min, 4 mL). The resin was shortly washed with DMF (x 2; 4 mL) and DCM (x 1 ; 4 mL) and the reaction mixture of the next residue Fmoc-DADHOHA(Trt, Acetonide)-OH 10 (48.0 mg, 0.068 mmol, 2.5 equiv) quickly added to the resin. This aa was introduced using HATU (25.7 mg, 0.068 mmol, 2.5 equiv), HOAt (9.2 mg, 0.068 mmol, 2.5 equiv) and DIEA (23.5 μL, 0. 135 mmol, 5 equiv) dissolved in DMF (0.3 mL) with 1 min of pre-activation. After 1 h of coupling (3 min of manual stirring and 57 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The Kaiser's test proved quantitative coupling. The resin was then subjected to the following washings/treatments: piperidine-DMF ( 1 :4) (2 x 1 min, 2 x 5 min, 1 x 10 min; 4 mL), DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). An aliquot of the resin 85 was treated with a solution of TFA-H2O- TIS (95:2.5:2.5, 0.3 mL) for 1 h to provide the cleaved and unprotected peptide intermediate. The decapeptide H-DADHOHA-diMeGln-D-a//o-Thr(Alloc- pipecolic) -D - aZZo-Thr-D -Lys-Leu-NMe-Gln-β -EtO -Asn-D -Asp-O Allyl was obtained with a purity of 65% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 3.244 min and 3.442 min, the second peak corresponding to our desired intermediate with the diol function protected as an acetal). HPLC-ESMS(+) analysis showed m/ z calcd for C63H105N15O24 1455.75; [(M+2H)/2]+ found, 729.14. For the intermediate with the diol function protected as an acetal: m/z calcd for C66H109N15O24 1495.78 [(M+2H)/2]+ found, 749. 18.
Synthesis of {Octanoic-D-AspftBu -DADHOHAiTrt, Acetonide)-(3S,4-R)-3,4- diMe-Gln-D- ZZo-Thr(&)-D- ZZo-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO- Asn(Trt)-D-Asp(0-resin)-OAllyl1 [Alloc-pipecolic&l} 87
Figure imgf000140_0001
The last two residues were incorporated as a single unit to the growing peptide chain to avoid aspartimides formation. Octanoic-D-Asp(£Bu)-OH 86 (42.6 mg, 0. 135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0.135 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (47.0 μL, 0.270 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 85, and solid PyBOP (70.3 mg, 0.135 mmol, 5 equiv) was added. After 3.5 h of coupling (3 min of manual stirring and 227 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.3 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The undecapeptide Octanoic-D-Asp-DADHOHA- diMeGln-D-a//o-Thr(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn- D-Asp-OAllyl was obtained with a purity of 60% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.79 min). HPLC-ESMS(+) analysis showed m/z calcd for C75H124N16O28 1696.88; [M+H]+ found, 1698.15.
Synthesis of {Octanoic-D-AspitBu^DADHOHAiTrt, Acetonide)-(3S,4-R)-3,4- diMe-Gln -D - allo-Thr (&) -D - allo-Thr (tBu) -D -Lys (Boc) -Leu-NMe-Gln- β -EtO -
Asn(Trt)-D-Asp(0-resin)-OAllyl1 [H-pipecolic&1} 88
Figure imgf000141_0001
Next, to remove the Allyl and Alloc groups, the peptide-resin 87 was treated with Pd(PPh3)4 (6.24 mg, 0.005 mmol, 0.2 equiv) and PhSiH3 (66.6 \L, 0.540 mmol, 20 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor was obtained with a purity of 38% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 3.00 min). HPLC-ESMS(+) analysis showed m/z calcd for CeeHneNieOae 1572.82; [M+H]+ found, 1573.96.
Synthesis of D- allo-Thr analog 89
Figure imgf000142_0001
The macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 μΐ^, 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin 88 followed by the addition of solid PyBOP (56.2 mg, 0. 108 mmol, 4 equiv) and it was allowed to react for 2.5 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The cyclization step was monitored by cleavage of an aliquot of resin with TFA-F O-Tis (95:2.5:2.5, 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS.
The cleavage of the peptide and the elimination of the side-chain protecting groups were accomplished simultaneously by treating the resin with TFA-H20-Tis (95:2.5:2.5, 1 x 1.5 h, 3 x 2 min; 4 mL). All the filtrates were collected in a round-bottom flask, evaporated to dryness under reduced pressure and lyophilized in H2O-ACN ( 1 : 1 , 25 mL) to give 59.7 mg of crude 89 with a purity of 36% as checked by HPLC-PDA. Conditions: linear gradient (30% to 60%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 3.00 min).
The crude was partially purified by analytical HPLC-PDA using a SunFire™ C 18 3.5 μιη 4.6x 100 mm reversed-phase column and a linear gradient (5% to 29% over 5 min and 29% to 32% over 15 min at 60 °C) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 1.0 mL/min (iR = 7. 153 min and 8.552 min) . Two peaks with the mass of our final product Octanoic-D-Asp-DADHOHA-diMeGln-D-a//o-Thr(fi6)-D-a//o-Thr-D-Lys-Leu-NMe- Gln-p-EtO-Asn-D-Asp-D-Asp-pipecolic(&) were isolated and biologically assayed. HPLC-ESMS(+) analysis showed m/z calcd for CesHi
Figure imgf000142_0002
1554.81 ; [M+H]+ found, 1556.90. EXAMPLE 9: SOLID PHASE SYNTHESIS OF PIPECOLIDEPSIN ANALOGUE 92
Figure imgf000143_0001
92
Resin 48 was obtained following the procedures described in Example
4.
Experimental Protocol:
Synthesis of {Octanoic-D-Asp(mri-DADHOHA(Trt, Acetonide)-(3S,4-R)-3,4- diMe-Gln-D- //o-AHDMHAi&)-D- //o-ThritBu)-D-LvsiBoc)-Leu-i\/Me-Gln-B-EtO- Asn(Trt)-D-Asp(0-resin)-OAUyl1 [Alloc-pipecolic&l) 90
Figure imgf000143_0002
The last two residues were incorporated as a single unit to the growing peptide chain to avoid aspartimides formation. Octanoic-D-Asp(£Bu)-OH 86 (42.6 mg, 0. 135 mmol, 5 equiv) and HOAt ( 18.4 mg, 0.135 mmol, 5 equiv) were dissolved in DMF (0.3 mL) and DIEA (47.0 μΐ., 0.270 mmol, 10 equiv) was added. The remaining mixture was poured into the resin 48, and solid PyBOP (70.3 mg, 0.135 mmol, 5 equiv) was added. After 3.5 h of coupling (3 min of manual stirring and 227 min on a shaker) the solvents were removed and the resin was washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). Coupling completion was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 0.3 mL, 1 h) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS, as the Kaiser's test is not reliable at this point of the growing peptide chain. The undecapeptide Octanoic-D-Asp-DADHOHA- diMeGln-D-a//o-AHDMHA(Alloc-pipecolic)-D-a//o-Thr-D-Lys-Leu-NMe-Gln-p- EtO-Asn-D-Asp-OAllyl was obtained with a purity of 18% as checked by HPLC- PDA. Conditions: linear gradient (30% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (iR = 4.22 min).
Synthesis of {Octanoic-D-AspitBu^DADHOHAiTrt, Acetonide)-(3S,4-R)-3,4- diMe-Gln -D- ;;o-AHDMHA(&)-D-^o-Thr(tBu)-D-Lys(Boc)-Leu-i\/Me-Gln-B-EtO- Asn(Trt)-D-Asp(0-resin)-OAllyl1 [H-pipecolic&1} 91
Figure imgf000144_0001
Next, to remove the Allyl and Alloc groups, the peptide-resin 90 was treated with Pd(PPh3)4 (6.24 mg, 0.005 mmol, 0.2 equiv) and PhSiH3 (66.6 iL, 0.540 mmol, 20 equiv) dissolved in DCM (0.4 mL) under N2. After 15 min, the solvents were removed and the resin washed with DCM ( l x l min; 4 mL), DMF ( l x l min; 4 mL) and DCM ( l x l min; 4 mL). The treatment was repeated three times. Deprotection completion was monitored by cleavage of an aliquot of resin with TFA- H20-Tis (95:2.5:2.5) ( 1 x 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS. The linear precursor was obtained with a purity of 19% as checked by HPLC-PDA. Conditions: linear gradient (20% to 80%) of ACN (0.036% TFA) into H20 (0.045% TFA) over 8 min, with a flow rate of 1.0 mL/min (ip> = 3.60 min).
Synthesis of Octanoic analog 92
Figure imgf000145_0001
The macrolactamization step was carried out on solid phase. HO At ( 14.7 mg, 0. 108 mmol, 4 equiv) was dissolved in DMF (0.3 mL) and DIEA (37.6 μL, 0.216 mmol, 8 equiv) was added. The reaction mixture was then added to the resin 91 followed by the addition of solid PyBOP (56.2 mg, 0. 108 mmol, 4 equiv) and it was allowed to react for 2.5 h (5 min of manual stirring and 175 min on a shaker) at 25 °C. The solvents were removed and the resin washed with DMF (3 x 1 min; 4 mL) and DCM (3 x 1 min; 4 mL). The cyclization step was monitored by cleavage of an aliquot of resin with TFA-H20-Tis (95:2.5:2.5, 1 h, 0.3 mL) followed by analysis of the crude by HPLC-PDA and HPLC-ESMS.
The cleavage of the peptide and the elimination of the side-chain protecting groups were accomplished simultaneously by treating the resin with TFA-H20-Tis (95:2.5:2.5, 1 x 1.5 h, 3 x 2 min; 4 mL). All the filtrates were collected in a round-bottom flask, evaporated to dryness under reduced pressure and lyophilized in H2O-ACN ( 1 : 1 , 25 mL) to give 77.5 mg of crude 92.
The crude was purified by analytical HPLC-PDA using a SunFire™ C 18 3.5 μπι 4.6x 100 mm reversed-phase column and a linear gradient (20% to 60% over 15 min at 60 °C) of ACN (0.036% TFA) into H20 (0.045% TFA) with a flow rate of 1.0 mL/min (ip> = 7.24 min and 7.93 min). Two peaks with the mass of our final product Octanoic-D-Asp-DADHOHA-diMeGln-D-a//o-AHDMHA(S&)-D- a//o-Thr-D-Lys-Leu-NMe-Gln-p-EtO-Asn-D-Asp-D-Asp-pipecolic(S6) were isolated.
EXAMPLE 10: BIOASSAYS FOR THE DETECTION OF ANTITUMOR ACTIVITY
The aim of this assay is to evaluate the in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) activity of the samples being tested.
CELL LINES
Figure imgf000146_0001
EVALUATION OF CYTOTOXIC ACTIVITY USING THE SBR COLORIMETRIC ASSAY
A colorimetric assay, using sulforhodamine B (SRB) reaction has been adapted to provide a quantitative measurement of cell growth and viability (following the technique described by Skehan et al. J. Natl. Cancer Inst. 1990, 82, 1 107-1 1 12).
This form of assay employs SBS-standard 96-well cell culture microplates (Faircloth et al. Methods in Cell Science, 1988, 1 1 (4), 201-205; Mosmann et al, Journal of Immunological Methods, 1983, 65(1-2), 55-63). All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and derive from different types of human cancer.
Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C, 5% C02 and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.
Cells were seeded in 96 well micro titer plates, at 5 x 103 cells per well in aliquots of 150 μΐ^, and allowed to attach to the plate surface for 18 hours (overnight) in drug free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 μΐ^ aliquots of 4X stock solutions in complete culture medium plus 4% DMSO) using ten serial dilutions (concentrations ranging from 10 to 0.00262 μg/mL) and triplicate cultures ( 1% final concentration of DMSO). After 72 hours treatment, the antitumor effect was measured by using the SRB methodology: Briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS, and stained in 0.4% SRB solution for 30 min at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm. Effects on cell growth and survival were estimated by applying the NCI algorithm (Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91 - 104).
Using the mean + SD of triplicate cultures, a dose-response curve was automatically generated using nonlinear regression analysis. Three reference parameters were calculated (NCI algorithm) by automatic interpolation: GI50 = compound concentration that produces 50% cell growth inhibition, as compared to control cultures; TGI = total cell growth inhibition (cytostatic effect), as compared to control cultures, and LC50 = compound concentration that produces 50% net cell killing (cytotoxic effect).
Table III illustrates data on the biological activity of compounds of the present invention. Table III. Cytotoxicity assay -Activity Data (Molar) of Pipecolidepsin A'.
Figure imgf000148_0001

Claims

1. A process for the synthesis of pipecolidepsin compounds of general formula I:
Figure imgf000149_0001
wherein Ri is selected from substituted or unsubstituted Ci-Cie alkyl, substituted or unsubstituted C2-C18 alkenyl, substituted or unsubstituted C2-C18 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each R2 and R3 is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl;
R4 is selected from hydrogen and substituted or unsubstituted C1-C12 alkyl;
R5 is selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; each R6 and R7 is independently selected from hydrogen, CORa, COORa, CONRaRb, C(=NRa)NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; each Rg and Rio is independently selected from substituted or unsubstituted C1-C12 alkyl; each Rg and R12 is independently selected from ORc, NRaRb, CORa, NRaCONRaRb, NRaC(=NRa)NRaRb, halogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group;
R11 is selected from hydrogen, CORa, COORa, CONRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; n is 3 or 4;
Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000150_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000150_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy and R6y is independently selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl and substituted or unsubstituted C2-C 12 alkynyl; each I¾y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group; and
Rc is selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, and substituted or unsubstituted C2-C 12 alkynyl; and each Ra and Rb is independently selected from hydrogen, substituted or unsubstituted C 1 -C 12 alkyl, substituted or unsubstituted C2-C 12 alkenyl, substituted or unsubstituted C2-C 12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the process comprises a macrolactamization step performed between positions 1 and 2 of an intermediate of formula Ila to give a compound of formula la in accordance with Scheme I:
Figure imgf000152_0001
Ila la
Scheme I
wherein Ri , R2, R3, R4, Re, Rio and n are as defined for compounds of formula I;
R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3; R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3; Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3;
R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and an insoluble support;
Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000153_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000153_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy, and R6y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each R2y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or R2y with R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted 1 ,3- dioxolane; or each R2y and R3y is independently selected as PG3; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or when each R4y and R5y is independently selected from ORc and NHRb then each Rb and Rc in such groups may also be independently selected as PG3; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
2. A process as defined in claim 1 , wherein the macrolactamization step performed between positions 1 and 2 of the intermediate Ila to give a compound of formula la is effected using coupling reagents, preferably a PyBOP/HOAt coupling system.
3. A process as defined in claim 1 or 2, wherein the macrolactamization step is performed in solid phase.
4. A process according to claim 3, wherein the process further comprises the step of cleaving the compound of formula la from the insoluble support to which said compound is attached.
5. A process according to claims 3 or 4, wherein the insoluble support is attached through terminal R1 side chain group.
6. A process according to claim 5, wherein the insoluble support is selected from 4-hydroxymethylphenoxymethyl polystyrene Wang resin and Wang ChemMatrix resin.
7. A process according to claim 6, wherein the insoluble support is 4- hydroxymethylphenoxymethyl polystyrene Wang.
8. A process according to any of claims 4 to 7, wherein the step of cleaving the compound of formula la from the insoluble support to which said compound is attached is effected by using acidic conditions, preferably by using TFA-H2O- TIS.
9. A process according to any preceding claim, wherein the process further comprises the step of removing side chain protecting groups performed over a compound of formula la.
10. A process according to claim 9, wherein the step of removing side chain protecting groups performed over the compound of formula la and the step of cleaving said compound from the insoluble support to which it is attached, are carried out by a single step.
1 1. A process according to any of the preceding claims, wherein Ri is a substituted alkyl group having 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 carbon atoms wherein the substituent is OH.
12. A process according to claim 1 1 , wherein Ri is a group selected from 2- hydroxy-l ,3,5-trimethylhexyl and 2-hydroxy- l ,3,5,7-tetramethyloctyl.
13. A process according to any of claims 1 to 1 1 , wherein R4 is selected from methyl and 1 ,2-dimethylpropyl.
14. A compound of formula II:
Figure imgf000155_0001
wherein X is selected from PG 1 and a group of formulae (f), (g) or (h):
Figure imgf000156_0001
Formula (f) Formula (g) Formula (h)
Zi is selected from hydrogen and PG 1 ;
Z is selected from hydrogen, PG2 and an insoluble support; Ri , P2, 3, R4, e, Rio and n are as defined for compounds of formula I;
R5 and R11 are as defined for compounds of formula I or may be independently selected as PG3;
R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3; Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3;
R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and an insoluble support; provided that when Z2 is an insoluble support then R12 is not an suitable insoluble support; Y is linked to the rest of the molecule through amide bonds and is selected from a group of formulae (a), (b), (c), (d) and (e):
Figure imgf000157_0001
Formula (a) Formula (b) Formula (c)
Figure imgf000157_0002
Formula (d) Formula (e) wherein the dotted line represents an additional bond; each Riy and R6y is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl and substituted or unsubstituted C2-C12 alkynyl; each R2y and R3y is independently selected from hydrogen, CORa, COORa, CONRaRb, S02Ra, S03Ra, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or R2y with R3y together with the oxygen atoms to which they are attached and carbons 3 and 4 form a substituted or unsubstituted 1 ,3- dioxolane; or each R2y and R3y is independently selected as PG3; each R4y and Rsy is independently selected from hydrogen, ORc, NRaRb, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or when each R4y and R5y is independently selected from ORc and NHRb then each Rb and Rc in such groups may also be independently selected as PG3; provided that in formula (b) when R4y is ORc then Rsy is not ORc; and when the carbons to which R4y and Rsy are attached form a double bond then R4y and Rsy are not selected from ORc and NRaRb; or
Riy and Rsy of formula (b) together with the corresponding carbon atoms to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, or a substituted or unsubstituted heteroalicyclic group; each PG1 is independently selected as a a-amino protecting group; provided that when there are several PG 1 groups they are orthogonal protecting groups;
PG2 is a a-carboxy protecting group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
15. The use of a compound of formula II in the manufacture of a compound of formula I.
16. A process performed in solid phase for the synthesis of intermediates of formula II which comprise the step of forming an ester bond performed at the branching position 5 of a compound of formula III to provide an intermediate of formula lib in accordance with Scheme II:
Figure imgf000158_0001
III lib
Scheme II wherein X is PG 1 ;
Z is selected from PG2 and an insoluble support;
R3, R4, Re, Rio and n are as defined for compounds of formula I;
R5, and R11 are as defined for compounds of formula I or may be independently selected as PG3;
R6 and R7 are as defined for compounds of formula I; or when R6 is hydrogen then R7 may also be selected from a PG3 group; or when R6 is hydrogen and R7 is selected from COORa, CONHRb and C(=NH)NHRb then each Ra and Rb in such groups may also be independently selected as PG3;
Rg is as defined for compounds of formula I; or when Rg is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected as PG3; R12 is as defined for compounds of formula I or when R1 is selected from ORc, NHRb, NHCONHRb and NHC(=NH)NHRb then each Rb and Rc in such groups may also be independently selected from PG3 and an insoluble support; provided that when Z is an insoluble support then R12 is not an insoluble support; each PG1 is independently selected as a a-amino protecting group; provided that when there are several PG 1 groups they are orthogonal protecting groups;
PG2 is a a-carboxy protecting group;
PG3 is a side chain protecting group; wherein PG3 groups may be the same or different side chain protecting groups; or a tautomer or stereoisomer thereof.
17. A process as defined in claim 16, wherein the step of forming an ester bond between secondary hydroxyl group at branching position 5 of a compound of formula III and free a-carboxy group of a-amino protected pipecolic acid to provide a compound of formula lib wherein X is PG 1 is effected using coupling reagents, preferably a carbodiimide in the presence of catalytic amounts of a base as coupling system.
18. A process according to claims 16 or 17, wherein said step is effected at a temperature from about 25 °C to about 65 °C.
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