Patent Description:
The objective of the present invention is to provide means and methods to chemically synthesize amanin or derivatives thereof. <CIT> discloses the synthesis of amatoxin derivatives and their respective use in the manufacture of antibody-drug conjugates.

This objective is attained by the subject-matter of the independent claim of the present specification.

The invention relates to a method for preparation of a compound of formula (I)
<CHM>.

Amino acid sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, <NUM>rd ed. Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids.

The term AA in the context of the present specification relates to amino acid.

The term "protecting group" in the context of the present specification relates to a moiety covalently attached to a functional group (particularly the carboxylic acid moiety, the amino moiety or the hydroxyl moiety of the molecules discussed herein) that can be selectively attached to the functional group and selectively removed without affecting the integrity or chiral orientation of the carbon backbone of the molecule the protecting group is attached to, nor cleaving particular other protecting groups attached to other protecting groups attached to the molecule.

The term "deprotection agent" in the context of the present specification relates to an agent which is able to cleave a certain protecting group. The skilled person is able to select the deprotection agent according to the protecting group. The conditions under which the protecting group is cleavable constitute the deprotection agent, e.g. if the protecting group is cleavable under acidic conditions, then the deprotection agent is an acid.

The term "preactivated carboxylic group" in the context of the present specification relates to a carboxylic moiety being reacted into an active ester susceptible for the nucleophilic attack of an amine group in order to form a peptide bond.

The term "preactivated amino group" in the context of the present specification relates to an amino group being reacted into a N-trimethylsilyl amine with increased nucleophilicity to attack a carboxylic acid moiety in order to form a peptide bond.

A comprehensive review of modern protecting group chemistry, particularly as it pertains to the compounds disclosed herein, is available in <NPL>.

<CIT> - "Protecting groups useful in the synthesis of polysaccharides, natural products, and combinatorial libraries" and <CIT> - "Deprotection method" are cited.

Standard convention of organic chemistry, by which a non-designated position in a formula is deemed to be a saturated carbon, is followed herein.

A first aspect of the invention relates to a method for preparation of a compound of formula (I)
<CHM>
wherein.

In certain embodiments, the <NUM>-<NUM> AA peptide is composed of proteinogenic amino acids. In certain embodiments, a compound of formula (III)
<CHM>
is reacted with a compound of formula (IV)
<CHM>
wherein.

wherein (III) and (IV) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (e) to yield the compound characterized by (IIa).

In certain embodiments, a compound of formula (V)
<CHM>
is reacted with a compound of formula (VI)
<CHM>
wherein.

In certain embodiments, for a compound of formula (Idesox)
<CHM>
wherein.

In certain embodiments, the oxidation of the sulfur atom is performed using PPO, dibenzyolperoxide, tert-butyl peroxybenzoate, or lauroyl peroxide. Preparation of PPO is described in (<NPL>.

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta-chloroperoxybenzoic acid) in isopropanol/ethanol (<NUM>:<NUM>).

In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (<NPL>).

In certain embodiments, the oxidation of the sulfur atom is performed with non-enantioselective agents or simply with oxygen or hydrogenperoxide.

In certain embodiments, the oxidation of the sulfur atom is performed using iodine and oxygen.

The oxidation of the sulfur atom is performed in a reaction step (h) to yield the compound (lox)
<CHM>.

In certain embodiments, for a compound of formula (Vdesox).

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

A solution of L-cystine-(OtBu)<NUM> (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ) in a <NUM>:<NUM> mixture of H<NUM>O/dioxane (<NUM>) was treated with NaHCO<NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and Boc<NUM>O (<NUM>, <NUM> mmol, <NUM> eq. ) and the reaction mixture was stirred for <NUM> at r. The reaction mixture was concentrated under reduced pressure and the aqueous layer was extracted with EtOAc (<NUM> x <NUM>). The organic layer was washed with brine (<NUM>), dried over Na<NUM>SO<NUM> and the solvent was removed under reduced pressure to afford <NUM> (<NUM>, <NUM> mmol, quant. ) as a pale yellow solid.

HRMS (ESI): m/z calc for C<NUM>H<NUM>N<NUM>O<NUM>S<NUM> (M+H)+ <NUM>, found <NUM>.

To a solution of (N-Boc)<NUM>-L-Cystin-(OtBu)<NUM> (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ) in CHCl<NUM> (<NUM>) was added SO<NUM>Cl<NUM> (<NUM>µL, <NUM> mmol, <NUM> eq. After the reaction mixture was stirred for <NUM> at r. the solvent was removed under reduced pressure. The residue was redissolved in CHCl<NUM> (<NUM>) and cooled to <NUM> and added to an ice cold solution of <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and NaHCO<NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in CHCl<NUM> (<NUM>) dropwise over a periode of <NUM>. Afterwards the reaction mixture was stirred for <NUM> at <NUM> and <NUM> at r. The organic layer was washed with H<NUM>O (<NUM>) and sat. NaHCO<NUM>-solution (<NUM>). After drying of the organic layer with Na<NUM>SO<NUM> and removal of the solvent under reduced pressure the crude product of <NUM> was used in the next step without further purification.

HRMS (ESI): m/z calc. for C<NUM>H<NUM>Cl<NUM>N<NUM>O<NUM>SSi (M+H)+ <NUM>, found <NUM>.

To a solution of (N-Boc)<NUM>-L-Cystin-(OtBu)<NUM> (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ) in CHCl<NUM> (<NUM>) was added SO<NUM>Cl<NUM> (<NUM>, <NUM> mmol, <NUM> eq. After the reaction mixture was stirred for <NUM> at r. the solvent was removed under reduced pressure. The residue was redissolved in CHCl<NUM> (<NUM>), cooled to <NUM> and added dropwise to an ice cold solution of <NUM> (<NUM>, <NUM> mmol <NUM> eq. ) and NaHCO<NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in CHCl<NUM> (<NUM>) over a periode of <NUM>. Afterwards the reaction mixture was stirred for <NUM> at <NUM> and <NUM> at r. The organic layer was washed with H<NUM>O (<NUM> x <NUM>) and sat. NaHCO<NUM>-solution (<NUM> x <NUM>). After drying of the organic layer with Na<NUM>SO<NUM> and removal of the solvent under reduced pressure the crude product of <NUM> was used in the next step without further purification.

A solution of tryptathionine derivative <NUM> (<NUM> mmol, <NUM> eq. ) in DMF (<NUM>) was treated with CH<NUM>COOH (<NUM>) and zinc (<NUM>, <NUM> mmol, <NUM> eq. ) for <NUM> at <NUM>. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (<NUM>) and washed with <NUM>% KHSO<NUM> solution (<NUM> x <NUM>) and brine (<NUM> x <NUM>). After drying over Na<NUM>SO<NUM> and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>% gradient) to give compound <NUM> as a yellow solid (<NUM>, <NUM>% over <NUM> steps).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM>SSi (M+H)+ <NUM>, found <NUM>.

A solution of tryptathionine derivative <NUM> (<NUM> mmol, <NUM> eq. ) in DMF (<NUM>) was treated with CH<NUM>COOH (<NUM>) and zinc (<NUM>, <NUM> mmol, <NUM> eq. ) for <NUM> at <NUM>. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (<NUM>) and washed with <NUM>% KHSO<NUM> solution (<NUM> x <NUM>) and brine (<NUM> x <NUM>). After drying over Na<NUM>SO<NUM> and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>% gradient) to afford compound <NUM> as a yellow oil (<NUM>, <NUM>%. over <NUM> steps).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>Cl<NUM>N<NUM>O<NUM>SSi (M+H)+<NUM>, found <NUM>.

To a solution of Cbz-glycine (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ) in acetone (<NUM>) was added a suspension of L-isoleucine (<NUM>, <NUM> mmol, <NUM> eq. ) and NaHCO<NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in water (<NUM>). The reaction mixture was stirred at r. for <NUM> and concentrated under reduced pressure. The aqueous layer was carefully acidified to pH = <NUM> by dropwise addition of <NUM> HCI and extracted with EtOAc (<NUM> x150 mL). The organic phase was then washed with brine (<NUM> x <NUM>), dried over Na<NUM>SO<NUM> and evaporated under reduced pressure to afford the product <NUM> as a colourless oil (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM> (M+H)+ <NUM>, found <NUM>.

Dipeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and benzyl glycinate (<NUM>, <NUM> mmol, <NUM> eq. ) were dissolved in dry DMF (<NUM>). Then, COMU (<NUM>, <NUM> mmol, <NUM> eq. ) and DIPEA (<NUM>, <NUM> mmol, <NUM> eq. ) were added at <NUM>. The reaction mixture was allowed to warm to r. overnight and diluted with EtOAc (<NUM>) afterwards. After washing with a solution of <NUM>% KHSO<NUM>-solution (2x100 mL) the fully protected tripeptide precipitated in the organic phase. The organic phase was cooled to <NUM> for <NUM> in order for the peptide to precipitate, then the precipitate was filtered and washed with cold EtOAc. The precipitate was redissolved in a <NUM>:<NUM> mixture of water and THF (<NUM>). Pd/C (<NUM>) was added to the solution after degassing with N<NUM> for <NUM>. Then, the reaction mixture was degassed with hydrogen for <NUM>. After vigorous stirring at room temperature under <NUM> atm of hydrogen overnight, the catalyst was filtered through a pad of Celite. Afterwards, the mixture was concentrated under reduced pressure to obtain the product <NUM> as a white solid (<NUM>, <NUM> %).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM> (M+H)+<NUM>, found <NUM>.

A solution of thioether building block <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in AcN (<NUM>) was treated with collidine (<NUM>µL, <NUM> mmol, <NUM> eq) and N,N'-disuccinimidyl carbonate (<NUM>, <NUM> mmol, <NUM> eq. ) and stirred for <NUM> at r. A solution of tripeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq) in a <NUM>:<NUM> mixture of AcN/H<NUM>O (<NUM>) was added and the reaction mixture was stirred for <NUM> at r. Afterwards, the mixture was diluted with EtOAc (<NUM>), <NUM>% KHSO<NUM>-solution (<NUM>) was added and the aqueous layer was extracted with EtOAc (<NUM> x <NUM>). The organic layer was washed with brine (<NUM> x <NUM>), dried over Na<NUM>SO<NUM> and evaporated under reduced pressure which afforded pentapeptide <NUM> as a yellow solid (<NUM>, <NUM>%).

A solution of tryptathionine building block <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in AcN (<NUM>) was treated with collidine (<NUM>µL, <NUM> mmol, <NUM> eq) and N,N'-disuccinimidyl carbonate (<NUM>, <NUM> mmol, <NUM> eq. ) and stirred for <NUM> at r. A solution of tripeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in a <NUM>:<NUM> mixture of AcN/H<NUM>O (<NUM>) was added and the reaction mixture was stirred for <NUM> at r. Afterwards, the mixture was diluted with EtOAc (<NUM>), <NUM>% KHSO<NUM>-solution (<NUM>) was added and the aqueous layer was extracted with EtOAc (2x50 mL). The organic layer was washed with brine (<NUM> x <NUM>), dried over Na<NUM>SO<NUM> and evaporated under reduced pressure which afforded pentapeptide <NUM> as a yellow solid (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for (M+H)+ C<NUM>H<NUM>Cl<NUM>NeO<NUM>S <NUM>, found <NUM>.

Pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) was dissolved in <NUM> of a solution of p-toluenesulfonic acid in THF (<NUM>) and was stirred for <NUM> at r. Then, the reaction mixture was neutralized by the addition of DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq) and diluted with DCM (<NUM>). Afterwards, DIPEA (<NUM>µL, <NUM>µmol, <NUM> eq. ) and T3P (<NUM>% in EtOAc, <NUM>µL, <NUM>µmol, <NUM> eq. ) were added. After the solution was stirred for <NUM> at r. <NUM>/<NUM> of the the solvent was concentrated under reduced pressure. The organic phase was washed with <NUM>% KHSO<NUM>-solution (<NUM>), sat. NaHCO<NUM>-solution (<NUM>), water (<NUM>) and brine (<NUM>). The organic layer was dried over Na<NUM>SO<NUM> and the solvent was removed under reduced pressure.

The crude product was purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>% gradient) to afford cyclic pentapeptide <NUM> as a yellow solid (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>NeO<NUM>SSi (M+H)+ <NUM>, found <NUM>.

Pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) was dissolved in <NUM> of <NUM> HCI in dioxane and stirred for <NUM> at r. Then, the reaction mixture diluted with <NUM> of dioxane and the solvent was evaporated under reduced pressure. The precipitate was dissolved in <NUM> DMF and diluted with <NUM> DCM. Afterwards, DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq. ) and T3P (<NUM>% in EtOAc, <NUM>µL, <NUM> mmol, <NUM> eq. ) were added. After the solution was stirred for <NUM> at r. , <NUM>/<NUM> of the the solvent was concentrated under reduced pressure. The organic phase was washed with <NUM>% KHSO<NUM>-solution (<NUM>), sat. NaHCO<NUM>-solution (<NUM>), water (<NUM>) and brine (<NUM>). The organic layer was dried over Na<NUM>SO<NUM> and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>% gradient) to afford cyclic pentapeptide <NUM> as a yellow solid (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM>SSi (M+H)+<NUM>, found <NUM>.

Monocyclic Pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) was stirred in TFA/DCM/TIPS (<NUM>:<NUM>:<NUM>) for <NUM> at r. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>%) to afford the fully deprotected monocyclic pentapeptide <NUM> as a white powder (<NUM>, quant.

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM>S (M+H)+ <NUM>, found <NUM>.

A solution of fully deprotected monocyclic pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and MSA (<NUM>µL, <NUM> mmol, <NUM> eq. ) in DMA (<NUM>) was stirred for <NUM> at <NUM>. Simultaneously, a solution of Fmoc-DHIIe(TBS)<NUM>-OH (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ), COMU (<NUM>, <NUM> mmol, <NUM> eq. ) and DIPEA (15µL, <NUM> mmol, <NUM> eq. ) in DMA (<NUM>) was stirred for <NUM> at <NUM>. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for <NUM> at <NUM> then at r. Afterwards, the mixture was diluted with EtOAc (<NUM>) and washed with <NUM>% KHSO<NUM> solution (<NUM> x <NUM>). The organic phase was washed with brine (<NUM> x <NUM>), dried over NaSO<NUM> and evaporated under reduced pressure. The crude product of <NUM> was used in the next step without any further purification.

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM>SSi<NUM> (M+H)+ <NUM>, found <NUM>.

A solution of fully deprotected monocyclic pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and MSA (<NUM>µL, <NUM> mmol, <NUM> eq. ) in DMA (<NUM>) was stirred for <NUM> at <NUM>. Simultaneously, a solution of Fmoc-DHIIe(TBS)<NUM>-OH (<NUM>, <NUM>, <NUM> mmol, <NUM> eq. ), COMU (<NUM>, <NUM> mmol, <NUM> eq. ) and DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq. ) in DMA (<NUM>) was stirred for <NUM> at <NUM>. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for <NUM> at <NUM> then at r. Afterwards, the mixture was diluted with EtOAc (<NUM>) and washed with <NUM>% KHSO<NUM> solution (<NUM> x <NUM>). The organic phase was washed with brine (<NUM> x <NUM>), dried over NaSO<NUM> and evaporated under reduced pressure. The crude product of <NUM> was used in the next step without any further purification.

A solution of N-Boc-protected (<NUM>,4R)-<NUM>-hydroxyproline <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) in DMF (<NUM>) was added dropwise to a solution of <NUM>-fluorenemethanol (<NUM>, <NUM> mmol, <NUM> eq. ), EDC*HCI (<NUM>, <NUM> mmol, <NUM> eq. ) and DMAP (<NUM>, <NUM> mmol, <NUM> eq. ) in DCM (<NUM>). The reaction mixture was stirred at r. Then, <NUM>% KHSO<NUM> solution (<NUM>) was added. The organic phase was washed with brine (<NUM>) and dried over NaSO<NUM>. Afterwards, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = <NUM>:<NUM>) and treated with <NUM> HCI in dioxane for <NUM>. Evaporation of the solvent under reduced pressure afforded the product <NUM> as a white solid (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>NO<NUM> (M+H-HCI)+ <NUM>, found <NUM>.

Boc-I-aspartic acid <NUM>-allyl ester (<NUM>, <NUM> mmol, <NUM> eq. ), compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) and HATU (<NUM>, <NUM> mmol, <NUM> eq. ) were dissolved in DMF (<NUM>) at <NUM>. Then, DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq. ) was added and reaction mixture was stirred at r. Subsequently, the reaction mixture was diluted with EtOAc (<NUM>). The organic phase was washed with <NUM>% KHSO<NUM> solution (<NUM> x <NUM>), sat. NaHCO<NUM> (<NUM>) and brine (<NUM>). After drying over NaSO<NUM> and removal of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = <NUM>:<NUM>) and treated with <NUM> HCI in dioxane for <NUM> afterwards. Evaporation of the solvent under reduced pressure afforded the product <NUM> as a yellow oil (<NUM>, <NUM>%).

HRMS (ESI): m/z calc. for C<NUM>H<NUM>N<NUM>O<NUM> (M+H-HCI)+ <NUM> , found <NUM>.

Fully deprotected monocyclic pentapeptide <NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) was dissolved in DMA (<NUM>) and MSA (<NUM>µL, <NUM> mmol, <NUM> eq. ) was added. The solution was stirred for <NUM> at <NUM>. A solution of Fmoc-Dhl(OTBS)<NUM>-OH (<NUM>, <NUM> mmol, <NUM> eq. ), COMU (<NUM>, <NUM> mmol, <NUM> eq. ) and DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq. ) in DMA (<NUM>) was stirred for <NUM> at <NUM>. The silylated monocyclic pentapeptide was added to the preactivated amino acid and stirred for <NUM>. Then, H-Asp(OAII)-Hyp-OFm*HCI (<NUM>, <NUM> mmol, <NUM> eq. ) and HATU (<NUM>, <NUM> mmol, <NUM> eq) were added to the reaction mixture at <NUM>. DIPEA (<NUM>µL, <NUM> mmol, <NUM> eq. ) was added and the reaction mixture was stirred for <NUM>, then diluted with EtOAc (<NUM>) and washed with <NUM>% citric acid (<NUM> x <NUM>) and sat. NaHCO<NUM> (<NUM> x <NUM>). The organic phase was washed with brine (<NUM> x <NUM>), dried over NaSO<NUM> and evaporated under reduced pressure. The crude product <NUM> was submitted to the next step without any further purification.

Monocyclic octapeptide <NUM> which was obtained from above reaction without further chromatographic purification step, was dissolved in DMF/ACN (<NUM>). Then Et<NUM>NH (<NUM>µL, <NUM> mmol, <NUM> eq. ) was added and stirred for <NUM> at r. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (<NUM>). Then, a solution of TBAF in THF (<NUM>, <NUM>, <NUM> eq) was added and the reaction mixture was stirred for <NUM> at r. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography (AcN/H<NUM>O <NUM>% to <NUM>%) to afford the product <NUM> as a white solid (<NUM>, <NUM>% after four steps).

Monocyclic octapeptide <NUM> (<NUM>, <NUM>µmol, <NUM> eq. ) was dissolved in DMF (<NUM>). Then, DIPEA (<NUM>µL, <NUM>µmol, <NUM> eq. ) and HATU (<NUM>, <NUM>µmol, <NUM> eq) were added at <NUM>. The reaction mixture was stirred for <NUM> during which it was allowed to warm to r. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD <NUM>, 50x150 mm column, gradient A) to afford allyl protected Deoxyamanin-precursor <NUM> (<NUM>, <NUM>%) as a white powder.

Deoxyamanin precursor <NUM> (<NUM>, <NUM>µmol, <NUM> eq. ) was dissolved in THF (<NUM>). Then, morpholine (<NUM>µL, <NUM>µmol, <NUM> eq. ) and Pd(PPh<NUM>)<NUM> (<NUM>µg, <NUM>µmol, <NUM> eq) were added. The reaction mixture was stirred vigorously for <NUM>. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD <NUM>, 50x150 mm column, gradient B) to afford (S)-Deoxyamanin (<NUM>) (<NUM>, <NUM>%) as a white powder.

Claim 1:
A method for preparation of a compound of formula (I)
<CHM>
wherein
a) a compound of formula (IIa)
<CHM>
is reacted with a peptide bond forming reagent,
particularly with a coupling reagent selected from a carbodiimide, an imidazolinium reagent, a phosphonium salt, an organo-phosphorous reagent, an uronium salt, a pyridinium reagent, and a phosphonic acid,
more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU,
in a reaction step (a),
and the compound is reacted with a deprotection agent removing RPGP and/or RPGOH and/or RAL and/or RAMIN,
wherein
• X<NUM>, X<NUM>, X<NUM>, and X<NUM>, and Y<NUM>, Y<NUM>, Y<NUM>, and Y<NUM> are H, or
one, two, three or all of Y<NUM>, Y<NUM>, Y<NUM>, and Y<NUM> are OH or NH<NUM> and all other Y are H and the corresponding X is NHRAMIN or ORPGP and all other X are H, wherein RPGP is a protecting group for phenolic OH groups, particularly a phenolic OH-protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, or RAMIN is a protecting group for phenolic amino groups, particularly a phenolic amino protecting group not acid- or alkali-labile, more particularly cleavable under reductive conditions, or
one, two, three or all of Y<NUM>, Y<NUM>, Y<NUM>, and Y<NUM> and the corresponding X are selected from F, CI, Br, I, CN, NO<NUM>, acyl, N<NUM>, or alkin, and all other X and Y are H,
particularly X and Y are H, or one of Y<NUM>, Y<NUM>, Y<NUM>, and Y<NUM> is OH and the corresponding X is ORPGP and all other X and Y are H;
• Z and Ware H, or
Z is OH and W is ORPGOH, wherein RPGOH is a protecting group for hydroxyl-groups, particularly a hydroxyl-protecting group cleavable with fluoride ions,
• RAL is a protected carboxyl-group or a <NUM>-5AA peptide, particularly RAL is O-allyl or O-methylester or a <NUM>-5AA peptide, more particularly RAL is O-allyl or O-methylester, most particularly RAL is O-allyl;
• V is OH or a <NUM>-5AA peptide, particularly V is OH;
• Q is S.