The present invention relates to the chemical synthesis of amanin and its derivatives. The present invention also relates to intermediate products of the amanin synthesis.

Background of the Invention The objective of the present invention is to provide means and methods to chemically synthesize amanin or derivatives thereof. This objective is attained by the subject-matter of the independent claims of the present specification.

Summary of the Invention

A first aspect of the invention relates to a method for preparation of a compound of formula (I)

Other aspects relate to intermediate products of the amanin synthesis. Brief Description of the Figures

Fig. 1 Synthesis of the dipeptide building block H-Asp(OAII)-Hyp-OFm°HCI (3). Fig. 2 Synthesis of Deoxyamanin. Detailed Description of the Invention

Terms and definitions

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, 3 ^rd ed. p. 21). 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 Peter G. M. Wuts, Greene's Protective Groups in Organic Synthesis, 5th Edition, Wiley 2014.

US 6693178 B2 - "Protecting groups useful in the synthesis of polysaccharides, natural products, and combinatorial libraries" and US 20160024143 A1 - "Deprotection method" are incorporated herein by reference.

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) wherein a) a compound of formula (I la)

(I la) 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 uranium 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 where applicable, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH and/or R ^AL and/or R ^AMIN , or wherein b) a compound of formula (lib) 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 uranium salt, a pyridinium reagent, and a phosphonic acid, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (b), and where applicable, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH and/or R ^AL and/or R ^AMIN , or wherein c) a compound of formula (lie) 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 uranium salt, a pyridinium reagent, and a phosphonic acid, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (c), and where applicable, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH and/or R ^AL and/or R ^AMIN , or wherein d) a compound of formula (lid) 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 uranium salt, a pyridinium reagent, and a phosphonic acid, more particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (d), and where applicable, the compound is reacted with a deprotection agent removing R ^PGP and/or R ^PGOH and/or R ^AL and/or R ^AMIN , wherein

• X ¹ , X ² , X ³ , and X ⁴ , and Y ¹ , Y ² , Y ³ , and Y ⁴ are H, or one, two, three or all of Y ¹ , Y ² , Y ³ , and Y ⁴ are OH or NH2 and all other Y are H and the corresponding X is NHR ^AMIN or OR ^PGP and all other X are H, wherein R ^PGP 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, most particularly Cbz, or R ^AMIN 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, most particularly Cbz, or one, two, three or all of Y ¹ , Y ² , Y ³ , and Y ⁴ and the corresponding X are selected from F, Cl, Br, I, CN, NO2, acyl, N ₃ , or alkin, and all other X and Y are H, particularly X and Y are H, or one of Y ¹ , Y ² , Y ³ , and Y ⁴ is OH and the corresponding X is OR ^PGP and all other X and Y are H;

• Z and W are H, or

Z is OH and W is OR ^PGOH , wherein R ^PGOH is a protecting group for hydroxyl- groups, particularly a hydroxyl-protecting group cleavable with fluoride ions,

• R ^AL is a protected carboxyl-group or a 1-5AA peptide, particularly R ^AL is O- allyl or O-methylester or a 1-5AA peptide, more particularly R ^AL is O-allyl or O-methylester, most particularly R ^AL is O-allyl;

• V is OH or a 1-5AA peptide, particularly V is OH;

• Q is S or SO.

In certain embodiments, the method is performed via step a) or step b).

In certain embodiments, the 1-5 AA peptide is composed of proteinogenic amino acids.

In certain embodiments, a compound of formula (III)

is reacted with a compound of formula (IV) wherein

• X ¹ , X ² , X ³ , X ⁴ , W, Q, and R ^AL have the same meaning as defined above;

• R ^COOY js a carboxyl-protecting group, particularly fluorenylmethyl or benzyl, more particularly fluorenylmethyl; 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 (I la).

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

• X ¹ , X ² , X ³ , X ⁴ , Q, and W have the same meaning as defined above;

• R ^NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, wherein the amino-group of (V) is preactivated, particularly with MSA, and preactivated (V) and (VI) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, more particularly with COMU, in a reaction step (f) to yield the compound (III).

In certain embodiments, a compound of formula (V) is reacted with a compound of formula (VII) wherein

X ¹ , X ² , X ³ , X ⁴ , W, Q, R ^NHB , and R ^AL have the same meaning as defined above; wherein the amino-group of (V) is preactivated, particularly with MSA, and preactivated (V) and (VII) are reacted with a peptide bond forming reagent, particularly with HATU, or the amino-group of (V) is preactivated, particularly with MSA, and the carboxyl-group of compound (VII) is preactivated, particularly with an O-PFP- ester, O-PCP-ester, or OSu-ester, and preactivated (V) and preactivated (VII) are reacted, in a reaction step (g) to yield the compound (lib).

For coupling compounds (V) and (VII), the acid-COOH group of compound (V) does not need to be protected. No significant side reactions were observed without protecting group. In certain embodiments, a compound of formula (V) is reacted with a compound of formula (VII) wherein

• X ¹ , X ² , X ³ , X ⁴ , W, Q, R ^NHB , and R ^AL have the same meaning as defined above; wherein (V) and (VII) are reacted with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, to yield the compound (lie).

In certain embodiments, a compound of formula (XVI) is reacted with a compound of formula (XVII) wherein

• X ¹ , X ² , X ³ , X ⁴ , W, Q, and R ^AL have the same meaning as defined above; • R ^NHB2 js an amino-protecting group, particularly an amino-protecting group cleavable under acidic conditions, more particularly Boc; wherein

• the amino-group of (XVI) is preactivated, particularly with MSA, and preactivated (XVI) and (XVII) are reacted with a peptide bond forming reagent, particularly with HATU, or

• the amino-group of (XVI) is preactivated, particularly with MSA, and the carboxyl-group of compound (XVII) is preactivated, particularly with an O- PFP-ester, O-PCP-ester, or OSu-ester, and preactivated (XVI) and preactivated (XVII) are reacted, to yield the compound (lid).

In certain embodiments, a compound of formula (V) is reacted with a compound of formula (XVIII) (XVIII) wherein

• X ¹ , X ² , X ³ , X ⁴ , W, Q, R ^NHB , and R ^AL have the same meaning as defined above; wherein • the amino-group of (V) is preactivated, particularly with MSA, and preactivated (V) and (XVIII) are reacted with a peptide bond forming reagent, particularly with HATU, or

• the amino-group of (V) is preactivated, particularly with MSA, and the carboxyl-group of compound (XVIII) is preactivated, particularly with an O-

PFP-ester, O-PCP-ester, or OSu-ester, and preactivated (V) and preactivated (XVIII) are reacted, to yield the compound (XVI).

In certain embodiments, for a compound of formula (Idesox) wherein

• Y ¹ , Y ² , Y ³ , Y ⁴ , Z, Q, and V have the same meaning as defined above; the sulfur atom is oxidized.

In certain embodiments, the oxidation of the sulfur atom is performed using manganese ions. In certain embodiments, the compound is reacted with a compound of formula (XV) and with Mn(OTf)2 and H2O2,

In certain embodiments, the oxidation of the sulfur atom is performed using PPO, dibenzyolperoxide, tert-butyl peroxy benzoate, or lauroyl peroxide. Preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta- chloroperoxybenzoic acid) in isopropanol/ethanol (8:3).

In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (Rio et al, Org. Lett. 2007, 9,12, 2265-2268). In certain embodiments, the oxidation of the sulfur atom is performed with non-enantio- selective 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 (I ox)

(I ox).

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

(Vdesox) wherein

X ¹ , X ² , X ³ , and X ⁴ have the same meaning as defined above; the sulfur atom is oxidized. In certain embodiments, the oxidation of the sulfur atom is performed using manganese ions. In certain embodiments, the compound is reacted with a compound of formula (XV) and with Mn(OTf)2 and H2O2,

In certain embodiments, the oxidation of the sulfur atom is performed using PPO, dibenzyolperoxide, tert-butyl peroxy benzoate, or lauroyl peroxide. Preparation of PPO is described in (S. Gan, J. Yin, Y. Yao, Y. Liu, D. Chang, D. Zhu, L. Shi, Org. Biomol. Chem. 2017, 15, 2647-2654.).

In certain embodiments, the oxidation of the sulfur atom is performed with mCPBA (meta- chloroperoxybenzoic acid) in isopropanol/ethanol (8:3). In certain embodiments, the oxidation of the sulfur atom is performed with an oxaziridinium salt as described in (Rio et al, Org. Lett. 2007, 9,12, 2265-2268).

In certain embodiments, the oxidation of the sulfur atom is performed with non-enantio- selective 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 (i) to yield the compound (Vox)

(Vox).

In certain embodiments, a compound of formula (VIII) wherein - R ^NHF is an amino protecting group, particularly an amino protecting group cleavable with fluoride ions or strong acids, more particularly Teoc, or an amino protecting group cleavable with alkaline conditions, more particularly Fmoc, RCOOA j _{S a} carboxyl-protecting group, particularly a carboxyl-protecting group cleavable under strongly acidic conditions, more particularly tert-butyl, - X ¹ , X ² , X ³ , and X ⁴ have the same meaning as outlined above, 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 T3P, HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, in a reaction step (j), and the compound is reacted with a deprotection agent removing R ^NHF and R ^COOA in a reaction step (k), particularly with TFA, to yield the compound characterized by (Vdesox) or (V).

In certain embodiments, a compound of formula (IX)

is reacted with a compound of formula (X) wherein - R ^NHA is an amino protecting group, particularly an amino protecting group cleavable under acidic conditions, more particularly Boc,

RCOOA, R ^NHF and X ¹ , X ² , X ³ , and X ⁴ have the same meaning as outlined above, wherein compound (VII) is

• preactivated with a peptide bond forming reagent, particularly with HATU, COMU, HBTU, TBTU, TOMBU, COMBU, or HCTU, followed by a reaction with the silylated compound (VIII), or

• is preactivated as in OSu-ester, followed by a reaction with the compound (VII) in a reaction step (I), and the compound is reacted with a deprotection agent removing R ^NHA in a reaction step (m), particularly with acidic conditions, more particularly at a pH of -3 to 0, even more particularly with HCI or p-toluenesulfonic acid, most particularly with 2 M HOI in Dioxan, to yield the compound characterized by (VIII).

In certain embodiments, a compound of formula (XI) is reacted with a compound of formula (XII) wherein

Re ⁰⁰² is a carboxyl- protecting group, particularly a carboxyl-protecting group cleavable with Zn, more particularly Tee, or R ^cooz is H,

R ^COOA , RNHF RNHA _anc| cΐ c2 c3 _anc| c 4 |- _|ave ^g _same meaning as outlined above, are reacted in a reaction step (n), and if R ^cooz is a carboxyl-protecting group, the compound is reacted with a deprotection agent removing R ^cooz in a reaction step (o), particularly with Zn, to yield the compound characterized by (IX).

A protection group strategy was applied that relies on acid stability. Decreasing pH values were used for deprotection. First, the Tee group of tryptophan (R ^cooz of compound VIII) was removed under reductive conditions using Zn with mildly acidic pH. Afterwards, the Boc group of cysteine (R ^NHA of compound IX) was removed with p-toluenesulfonic acid. Last, Teoc (R ^NHF ) and tert-butyl (R ^COOA ) of compound (V) were removed concomitantly with 95%TFA.

In certain embodiments, a compound of formula (VI) is reacted with a compound of formula (XIII) wherein

R ^AL has the same meaning as described above,

R ^Pep is an active ester, particularly O-pentafluorophenol or OSu-ester, R ^NHB is an amino protecting group, particularly an amino protecting group cleavable under alkaline conditions, more particularly Fmoc, are reacted with solid phase peptide synthesis in a reaction step (p), wherein the carboxyl-group of compound (XIV) may be protected, to yield the compound characterized by (VII).

Another aspect of the invention relates to a compound of the general formula (I) wherein · one, two, or three of Y ¹ , Y ² , and Y ⁴ are OH and all other Y are H, or one, two, three or all of Y ¹ , Y ² , Y ³ , and Y ⁴ are selected from F, Cl, Br, and I and all other Y are H, particularly one of Y ¹ , Y ² , and Y ⁴ is OH and all other Y are H; Z is H, or Z is OH,

• V is OH or a 1-5AA peptide, particularly V is OH,

• Q is S or SO. Another aspect of the invention relates to a compound of the general formula (I la)

(I la) wherein

• X ¹ , X ² , X ³ , and X ⁴ , are H, or one, two, three or all of X ¹ , X ² , X ³ , and X ⁴ are OH and all other X are H, or one, two, three or all of X ¹ , X ² , X ³ , and X ⁴ are selected from F, Cl, Br, and I and all other X are H, particularly X ¹ , X ² , X ³ , and X ⁴ are H, or one of X ¹ , X ² , X ³ , and X ⁴ is OH and all other X are H;

• W is H, or W is OH,

• V is OH or a 1-5AA peptide, particularly V is OH,

• Q is S or SO.

Another aspect of the invention relates to a compound of the general formula (lib) wherein

• X ¹ , X ² , X ³ , and X ⁴ , are H, or one, two, three or all of X ¹ , X ² , X ³ , and X ⁴ are OH and all other X are H, or one, two, three or all of X ¹ , X ² , X ³ , and X ⁴ are selected from F, Cl, Br, and I and all other X are H, particularly X ¹ , X ² , X ³ , and X ⁴ are H, or one of X ¹ , X ² , X ³ , and X ⁴ is OH and all other X are H;

• W is H, or W is OH,

• V is OH or a 1-5AA peptide, particularly V is OH,

• Q is S or SO.

Another aspect of the invention relates to a compound of the general formula (III) • one, two, or all of X ¹ , X ² , and X ⁴ are OH and all other X are H, or one, two, or all of X ¹ , X ² , and X ⁴ are selected from F, Cl, Br, and I and all other X are H, particularly one of X ¹ , X ² , and X ⁴ is OH and all other X are H; · W is H, or

W is OH,

• Q is S or SO.

Another aspect of the invention relates to a compound of the general formula (V) wherein

• one, two, or all of X ¹ , X ² , and X ⁴ are OH and all other X are H, or one, two, or all of X ¹ , X ² , and X ⁴ are selected from F, Cl, Br, and I and all other X are H, particularly one of X ¹ , X ² , and X ⁴ is OH and all other X are H, · Q is S or SO.

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.

Description of the Figures Fig. 1 Synthesis of the dipeptide building block H-Asp(OAII)-Hyp-OFm°HCI (3) .

Fig. 2 Synthesis of Deoxyamanin. Examples

Synthesis of (N-Boc)2-cystine-(OtBu) 2 (35)

35

A solution of L-cystine-(OtBu)2 (34, 10 g, 24 mmol, 1.0 eq.) in a 1:1 mixture of hhO/dioxane (240 mL) was treated with NaHCC>3 (8.06 g, 96.0 mmol, 4.00 eq.) and B0C2O (10.1 mL, 47.0 mmol, 2.00 eq.) and the reaction mixture was stirred for 16 h at r.t. The reaction mixture was concentrated under reduced pressure and the aqueous layer was extracted with EtOAc (3 x 120 mL). The organic layer was washed with brine (100 mL), dried over Na2SC>4 and the solvent was removed under reduced pressure to afford 35 (13.2 g, 24.0 mmol, quant.) as a pale yellow solid.

HRMS (ESI): m/z calc for C24H44N2O8S2 (M+H) ⁺ 553.2612, found 553.2615. tert-butyl S-(6-(benzyloxy)-3-((S)-3-oxo-3-(2,2,2-trichloroethoxy)-2-(( (2-(trimethylsilyl)ethoxy) carbonyl)amino)propyl)-1H-indol-2-yl)-N-(tert-butoxycarbonyl )-L-cysteinate (40)

40

To a solution of (A/-Boc) ₂ -L-Cystin-(OtBu) ₂ (35, 900 mg, 1.63 mmol, 1.00 eq.) in CHCI ₃ (16.3 mL) was added SO ₂ CI ₂ (263 pL, 3.26 mmol, 2.00 eq.). After the reaction mixture was stirred for 1 h at r.t. the solvent was removed under reduced pressure. The residue was redissolved in CHCI3 (16.3 mL) and cooled to 0°C and added to an ice cold solution of 39 (800 mg, 1.67 mmol, 1.00 eq.) and NaHCC>3 (420 mg, 5.00 mmol, 3.00 eq.) in CHCI3 (16.7 mL) dropwise over a periode of 10 min. Afterwards the reaction mixture was stirred for 15 min at 0°C and 1 h at r.t.. The organic layer was washed with H2O (10 mL) and sat. NaHCC>3-solution (10 mL). After drying of the organic layer with Na2SC>4 and removal of the solvent under reduced pressure the crude product of 40 was used in the next step without further purification.

HRMS (ESI): m/z calc for CssHsaChNsOgSSi (M+H) ⁺ 860.2332, found 860.2323. tert-butyl N-(tert-butoxycarbonyl)-S-(3-((S)-3-oxo-3-(2,2,2-trichloroet hoxy)-2-(((2- (trimethylsilyl)ethoxy)carbonyl)amino)propyl)-1H-indol-2-yl) -L-cysteinate (41 )

To a solution of (A/-Boc)2-L-Cystin-(OtBu)2 (39, 5.06 g, 9.15 mmol, 1.00 eq.) in CHCb (92 ml_) was added SO2CI2 (1.48 ml_, 18.3 mmol, 2.00 eq.). After the reaction mixture was stirred for 1 h at r.t. the solvent was removed under reduced pressure. The residue was redissolved in

CHCI3 (92 ml_), cooled to 0°C and added dropwise to an ice cold solution of 38 (4.4 g, 9.17 mmol 1.00 eq.) and NaHCC>3 (2.31 g, 27.5 mmol, 3.00 eq.) in CHCI3 (92 mL) over a periode of 10 min. Afterwards the reaction mixture was stirred for 15 min at 0°C and 1 h at r.t. The organic layer was washed with H2O (2 x 100 mL) and sat. NaHCC>3-solution (2 x 80 mL). After drying of the organic layer with Na2SC>4 and removal of the solvent under reduced pressure the crude product of 41 was used in the next step without further purification.

HRMS (ESI): m/z calc for CsiH^CIsNsOeSSi (M+H) ⁺ 754.1913, found 754.1917. (S)-3-(6-(benzyloxy)-2-(((R)-3-(tert-butoxy)-2-((tert-butoxy carbonyl)amino)-3-oxopropyl)thio)- 1H-indol-3-yl)-2-(((2-(trimethylsilyl)ethoxy)carbonyl)amino) propanoic acid (49):

Boc

A solution of tryptathionine derivative 39 (1.63 mmol, 1.00 eq.) in DMF (8.4 ml_) was treated with CH3COOH (0.8 ml_) and zinc (3.51 g, 53.6 mmol, 33.0 eq.) for 2 h at 45°C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (50 ml_) and washed with 10% KHSO ₄ solution (2 x 25 ml_) and brine (2 x 25 ml_). After drying over NaaSCU and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (ACN/H2O 50% to 100% gradient) to give compound 49 as a yellow solid (840 mg, 83% over 2 steps).

HRMS (ESI): m/z calc for CseHsiNsOgSSi (M+H) ⁺ 730.3183, found 730.3188.

(S)-3-(2-(((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)am ino)-3-oxopropyl)thio)-1H-indol-3-yl)- 2-( ((2-(trimethylsilyl) ethoxy)carbonyl) amino) propanoic acid ( 50)

A solution of tryptathionine derivative 38 (9.15 mmol, 1.00 eq.) in DMF (40 ml_) was treated with CH3COOH (4 ml_) and zinc (20.0 g, 302 mmol, 33.0 eq.) for 2 h at 45°C. The reaction mixture was filtered over Celite and the solvent was removed under reduced pressure. The crude product was dissolved in EtOAc (200 mL) and washed with 10% KHSO ₄ solution (2 x 50 ml_) and brine (2 x 50 ml_). After drying over Na2SC>4 and removing of the solvent under reduced pressure, the crude product was purified by C18 reverse phase chromatography (ACN/H2O 50% to 100% gradient) to afford compound 50 as a yellow oil (5.0 g, 88%. over 2 steps).

HRMS (ESI): m/z calc for Csi^eChNsOsSSi (M+H) ⁺ 624.2769, found 624.2775.

((benzyloxy)carbonyl)glycyl-L-isoleucine (44)

To a solution of Cbz-glycine (42, 10.0 g, 32.7 mmol, 1.00 eq.) in acetone (100 ml_) was added a suspension of L-isoleucine (4.71 g, 35.9 mmol, 1.10 eq.) and NaHCC>3 (8.23 g, 87.9 mmol, 3.00 eq.) in water (100 ml_). The reaction mixture was stirred at r.t. for 3 h and concentrated under reduced pressure .The aqueous layer was carefully acidified to pH = 4 by dropwise addition of 1 M HOI and extracted with EtOAc (3 x150 ml_). The organic phase was then washed with brine (2 x 100 ml_), dried over NaaSCU and evaporated under reduced pressure to afford the product 44 as a colourless oil (10.1 g, 96%).

HRMS (ESI): m/z calc for C^HaaNaOs (M+H) ⁺ 323.1601 , found 323.1606.

Glycyl-L-isoleucylglycine (45) :

Dipeptide 44 (10.1 g, 31.3 mmol, 1.00 eq.) and benzyl glycinate (8.21 g, 40.7 mmol, 1.30 eq.) were dissolved in dry DMF (125 ml_). Then, COMU (17.4 g, 40.7 mmol, 1.30 eq.) and DIPEA (12.6 ml_, 72.1 mmol, 3.00 eq.) were added at 0°C. The reaction mixture was allowed to warm to r.t. overnight and diluted with EtOAc (300 ml_) afterwards. After washing with a solution of 10% KHS0 ₄ -solution (2x100 ml_) the fully protected tripeptide precipitated in the organic phase. The organic phase was cooled to 4°C for 4 h in order for the peptide to precipitate, then the precipitate was filtered and washed with cold EtOAc. The precipitate was redissolved in a 1:1 mixture of water and THF (260 ml_). Pd/C (1 g) was added to the solution after degassing with N2 for 30 min. Then, the reaction mixture was degassed with hydrogen for 1 h. After vigorous stirring at room temperature under 1.0 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 45 as a white solid (5.71 g, 74 %)

HRMS (ESI): m/z calc for C^I-hgNsCU (M+H) ⁺ 246.1448, found 246.1440.

Synthesis of pentapeptide 51:

A solution of thioether building block 49 (111 mg, 0.14 mmol, 1.00 eq.) in AcN (0.7 ml_) was treated with collidine (37 pl_, 0.27 mmol, 2.0 eq) and L/,L/’-disuccinimidyl carbonate (39 mg, 0.15 mmol, 1.1 eq.) and stirred for 1 h at r.t.. A solution of tripeptide 45 (44 mg, 0.18 mmol, 1.3 eq) in a 1:4 mixture of AcN/hhO (1 ml_) was added and the reaction mixture was stirred for

2 h at r.t.. Afterwards, the mixture was diluted with EtOAc (20 ml_), 10% KHSCU-solution (20 ml_) was added and the aqueous layer was extracted with EtOAc (2 x 20 ml_). The organic layer was washed with brine (2 x 20 ml_), dried over Na2S04 and evaporated under reduced pressure which afforded pentapeptide 51 as a yellow solid (115 mg, 90%). HRMS (ESI): m/z calc for C ₄₆ H ₆₈ N ₆ 0 ₁₂ SSi (M+H) ⁺ 957.4458, found 957.4457. Synthesis of pentapeptide 52:

A solution of tryptathionine building block 50 (2.0 g, 2.5 mmol, 1.0 eq.) in AcN (10 ml_) was treated with collidine (659 pl_, 4.95 mmol, 2.00 eq) and L/,L/’-disuccinimidyl carbonate (697 mg, 2.72 mmol, 1.10 eq.) and stirred for 1 h at r.t.. A solution of tripeptide 45 (790 mg, 3.22 mmol, 1.30 eq.) in a 1 :4 mixture of AcN/hhO (18 ml_) was added and the reaction mixture was stirred for 2 h at r.t.. Afterwards, the mixture was diluted with EtOAc (100 mL), 10% KHSCU-solution (20 mL) was added and the aqueous layer was extracted with EtOAc (2x50 mL). The organic layer was washed with brine (2 x 50 mL), dried over Na ₂ S0 ₄ and evaporated under reduced pressure which afforded pentapeptide 52 as a yellow solid (2.15 g, 93%).

HRMS (ESI): m/z calc for (M+H) ⁺ CseHsiCIsNeOnS 851.4039, found 851.4058.

Fully protected cyclic pentapeptide 53 :

Pentapeptide 51 (151 mg, 0.180 mmol, 1.00 eq.) was dissolved in 1 mL of a solution of p-toluenesulfonic acid in THF (1.8 M) and was stirred for 4 h at r.t. Then, the reaction mixture was neutralized by the addition of DIPEA (320 pL, 1.84 mmol, 10 eq) and diluted with DCM (180 mL). Afterwards, DIPEA (60.2 pL, 354 pmol, 2.00 eq.) and T3P (50% in EtOAc, 210 pL, 354 pmol, 0.34 eq.) were added. After the solution was stirred for 16 h at r.t. 2/3 of the the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHS0 ₄ -solution (20 mL), sat. NaHCOs-solution (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over Na2S04 and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/hhO 50% to 100% gradient) to afford cyclic pentapeptide 53 as a yellow solid (82 g, 70%)

HRMS (ESI): m/z calc for C^HseNeOgSSi (M+H) ⁺ 839.3828, found 839.3839.

Fully protected cyclic pentapeptide (54): Pentapeptide 52 (700 mg, 0.822 mmol, 1.00 eq.) was dissolved in 10 ml_ of 2 M HCI in dioxane and stirred for 40 min at r.t. Then, the reaction mixture diluted with 40 ml_ of dioxane and the solvent was evaporated under reduced pressure. The precipitate was dissolved in 8 ml_ DMF and diluted with 82 ml_ DCM. Afterwards, DIPEA (279 pl_, 1.64 mmol, 2.00 eq.) and T3P (50% in EtOAc, 977 mI_, 1.64 mmol, 2.00 eq.) were added. After the solution was stirred for 5 h at r.t., 1/3 of the the solvent was concentrated under reduced pressure. The organic phase was washed with 10% KHSCU-solution (20 ml_), sat. NaHCC>3-solution (20 ml_), water (20 ml_) and brine (20 ml_). The organic layer was dried over NaaSCU and the solvent was removed under reduced pressure. The crude product was purified by C18 reverse phase chromatography (AcN/hhO 50% to 100% gradient) to afford cyclic pentapeptide 54 as a yellow solid (420 mg, 72%).

HRMS (ESI): m/z calc for Ca^NeOeSSi (M+H) ⁺ 733.3409, found 733.3409. Fully deprotected monocyclic pentapeptide 55 :

Monocyclic Pentapeptide 53 (125 mg, 0.17 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:15:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/FhO 20% to 100%) to afford the fully deprotected monocyclic pentapeptide 55 as a white powder (100 mg, quant.). HRMS (ESI): m/z calc for CsiHssNeOyS (M+H) ⁺ 639.2595, found 639.2590.

Fully deprotected monocyclic pentapeptide 56:

Monocyclic Pentapeptide 54 (250 mg, 0.34 mmol, 1.00 eq.) was stirred in TFA/DCM/TIPS (8:15:0.5) for 2 h at r.t. The solvent was removed under reduced pressure and the crude product was purified by C18 reverse phase chromatography (AcN/hhO 10% to 30%) to afford the fully deprotected monocyclic pentapeptide 56 as a white powder (200 mg, quant.).

HRMS (ESI): m/z calc for C^H _t NeOeS (M+H) ⁺ 533.2177, found 533.2188. Monocyclic hexapeptide 67:

A solution of fully deprotected monocyclic pentapeptide 55 (42 g, 0.66 mmol, 1.00 eq.) and MSA (11.6 pL, 0.723 mmol, 1.10 eq.) in DMA (2 mL) was stirred for 2 h at 50°C. Simultaneously, a solution of Fmoc-DHIIe(TBS)2-OH (13, 52 mg, 0.85 mmol, 1.30 eq.), COMU (36 mg, 0.85 mmol, 1.30 eq.) and DIPEA (15 pl_, 0.85 mmol, 1.30 eq.) in DMA (0.4 ml_) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0°C then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (50 ml_) and washed with 10% KHSCU solution (3 x 5 ml_). The organic phase was washed with brine (2 x 20 ml_), dried over NaSCU and evaporated under reduced pressure. The crude product of 67 was used in the next step without any further purification.

HRMS (ESI): m/z calc for CwHeyNyO^SSfe (M+H) ⁺ 1234.5745, found 1234.5745.

Monocyclic hexapeptide 68:

A solution of fully deprotected monocyclic pentapeptide 56 (100 mg, 0.188 mmol, 1.00 eq.) and MSA (33.2 pL, 0.207 mmol, 1.10 eq.) in DMA (4 ml_) was stirred for 2 h at 50°C. Simultaneously, a solution of Fmoc-DHIIe(TBS)2-OH (13, 149 mg, 0.244 mmol, 1.30 eq.), COMU (104 mg, 0.244 mmol, 1.30 eq.) and DIPEA (42.5 pL, 0.244 mmol, 1.30 eq.) in DMA (1.25 ml_) was stirred for 30 min at 0°C. The silylated monocyclic peptide was then added to the activated dihydroxyisoleucine derivative and stirred for 1 h at 0°C then at r.t. overnight. Afterwards, the mixture was diluted with EtOAc (100 mL) and washed with 10% KHSCU solution (3 x 10 mL). The organic phase was washed with brine (2 x 25 mL), dried over NaSCU and evaporated under reduced pressure. The crude product of 68 was used in the next step without any further purification.

HRMS (ESI): m/z calc for CsyHsiNyOnSSh (M+H) ⁺ 1128.5326, found 1128.5316.

Synthesis of (9H-fluoren-9-yl) methyl (2S,4R)-4-hydroxypyrroiidine-2-carboxyiate hydrochloride (2):

A solution of /V-Boc-protected (2S,4R)-4-hydroxyproline 1 (5.0 g, 22 mmol, 1.0 eq.) in DMF (20 ml_) was added dropwise to a solution of 9-fluorenemethanol (8.5 mg, 43 mmol, 2.0 eq.), EDC*HCI (8.3 g, 43 mmol, 2.0 eq.) and DMAP (396 mg, 3.24 mmol, 0.150 eq.) in DCM (220 ml_). The reaction mixture was stirred at r.t. for 2 h. Then, 10% KHSCL solution (50 ml_) was added. The organic phase was washed with brine (50 ml_) and dried over NaSCU. Afterwards, the solvent was removed under reduced pressure and the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 1:1) and treated with 4 m HCI in dioxane for 30 min. Evaporation of the solvent under reduced pressure afforded the product 2 as a white solid (3.7 g, 56%).

HRMS (ESI): m/z calc for C ₁₉ H ₁₉ N0 ₃ (M+H-HCI) ⁺ 310.1438, found 310.1426.

Synthesis of (9H-fluoren-9-yl) methyl (2S,4R)-1-((S)-4-(allyloxy)-2-amino-4-oxobutanoyl)-4- hydroxypyrrolidine-2-carboxylate (3)

Boc-l-aspartic acid 4-allyl ester (287 mg, 1.05 mmol, 1.30 eq.), compound 2 (250 mg,

0.808 mmol, 1.0 eq.) and HATU (614 mg, 1.62 mmol, 2.0 eq.) were dissolved in DMF (2 mL) at 0°C. Then, DIPEA (563 pL, 3.23 mmol, 4 eq.) was added and reaction mixture was stirred at r.t. for 2 h. Subsequently, the reaction mixture was diluted with EtOAc (50 ml_). The organic phase was washed with 10% KHSCU solution (2 x 10 ml_), sat. NaHCC>3 (10 ml_) and brine (10 ml_). After drying over NaSCU and removal of the solvent under reduced pressure the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 2:1) and treated with 4 m HCI in dioxane for 30 min afterwards. Evaporation of the solvent under reduced pressure afforded the product 3 as a yellow oil (344 mg, 85%).

HRMS (ESI): m/z calc for C ₂₆ H ₂₈ N ₂ 0 ₆ (M+H-HCI) ⁺ 465.2020 , found 465.2017.

Synthesis of monocyclic octapeptide 5.

Fully deprotected monocyclic pentapeptide 4 (50 mg, 0.09 mmol, 1.00 eq.) was dissolved in DMA (1 ml_) and MSA (17 pl_, 0.1 mmol, 1.1 eq.) was added. The solution was stirred for 2 h at 50°C. A solution of Fmoc-Dhl(OTBS) ₂ -OH (75 mg, 0.12 mmol, 1.3 eq.), COMU (52 mg, 0.12 mmol, 1.3 eq.) and DIPEA (43 mI_, 0.24 mmol, 2.6 eq.) in DMA (0.5 ml_) was stirred for 30 min at 0°C.The silylated monocyclic pentapeptide was added to the preactivated amino acid and stirred for 1 h. Then, H-Asp(OAII)-Hyp-OFm*HCI (70 mg, 0.14 mmol, 1.5 eq.) and HATU (53 mg, 0.14 mmol, 1.5 eq) were added to the reaction mixture at 0°C. DIPEA (49 mI_, 0.28 mmol, 3.0 eq.) was added and the reaction mixture was stirred for 2 h, then diluted with EtOAc (50 ml_) and washed with 10% citric acid (2 x 10ml_) and sat. NaHCOs (2 x 10 ml_). The organic phase was washed with brine (2 x 20 ml_), dried over NaS0 ₄ and evaporated under reduced pressure. The crude product 5 was submitted to the next step without any further purification.

HRMS (ESI): m/z calc for CssH^NgO^SSh (M+H) ⁺ 1574.7167, found 1574.7134.

Synthesis of monocyclic C- and N-terminally deprotected octapeptide 6.

Monocyclic octapeptide 5 which was obtained from above reaction without further chromatographic purification step, was dissolved in DMF/ACN (2 ml_). Then Et2lMH (191 mI_, 1.85 mmol, 20.0 eq.) was added and stirred for 30 min at r.t. The solvent was removed under reduced pressure and the precipitate was redissolved in THF (1 ml_). Then, a solution of TBAF in THF (1 m, 0.85 ml_, 10 eq) was added and the reaction mixture was stirred for 4 h at r.t. The solvent was evaporated in vacuo and the crude product purified by C18 reverse phase chromatography (ACN/H2O 5% to 70%) to afford the product 6 as a white solid (51 mg, 64% after four steps).

HRMS (ESI): m/z calc for C42H59N9O _M S (M+H) ⁺ 946.3975, found 946.3972.

Synthesis of allylester-protected Deoxyamanin-precursor (7)\

Monocyclic octapeptide 6 (10.0 mg, 10.6 pmol, 1.00 eq.) was dissolved in DMF (5 ml_). Then, DIPEA (3.68 pl_, 21.4 p ol, 2.00 eq.) and HATU (8.04 mg, 21.1 pmol, 2.00 eq) were added at 0°C. The reaction mixture was stirred for 5 h during which it was allowed to warm to r.t.

The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient A) to afford allyl protected Deoxyamanin-precursor 7 (6 mg, 68%) as a white powder.

HRMS (ESI): m/z calc for C ₄₂ H ₅₇ N ₉ 0 ₁₃ S (M+H) ⁺ 928.3869, found 928.3885.

Deoxyamanin precursor 7 (3.0 mg, 3.2 pmol, 1.0 eq.) was dissolved in THF (0.3 ml_). Then, morpholine (5.58 pl_, 64.6 pmol, 20.0 eq.) and Pd(PPh3)4 (747 pg, 0.64 pmol, 0.2 eq) were added. The reaction mixture was stirred vigorously for 3 h. The crude product was purified using preparative HPLC (Sunfire Prep C18 OBD 10pm, 50x150 mm column, gradient B) to afford (S)- Deoxyamanin (8) (1.5 mg, 50%) as a white powder.

HRMS (ESI): m/z calc for CsgHssNgO S (M+H) ⁺ 888.3556, found 888.3529.

Preparative HPLC purification gradients:

Gradient A: 0-30 min 10%-30% B, 30-35 min 30-100% B; 40-45 min 100% B;45-50 min, 100-10% B

0.1% formic acid in water (Solvent A) and 0.1% formic acid in ACN (Solvent B).

Gradient B:

0-30 min 15%-40% B, 30-35 min 40-100% B; 35-40min 100% B, 40-15 min 15% B 0.1% formic acid in water (Solvent A) and 0.1% formic acid in ACN (Solvent B).