RU2817013C1 - Method for synthesis of cyclic depsipeptides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method for synthesis of cyclic depsipeptides, particularly emodepside. Disclosed is a method for the synthesis of cyclic depsipeptides of general formula (I) on an industrial scale from depsipeptides of general formula (II), where B is a protective group of an amine group and A is a protective group of a carboxylic acid group, wherein the method comprises the steps of: removing the protective group of an amine group which is protected by group B, thereby obtaining an unprotected amine group; removing protective group of carboxylic acid, which is protected by group A, thus obtaining unprotected group of carboxylic acid; condensation of an unprotected amine group and an unprotected carboxylic acid group with a binding agent, wherein the binding agent is added slowly, preferably drop by drop, to the reaction solution, thus obtaining cyclic depsipeptide (I), wherein R1-R12 are defined in the claims, where the binding agent is T3P® (propylphosphonic acid anhydride, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6- trioxide, PPACA).

EFFECT: method for synthesis of cyclic depsipeptides is disclosed.

31 cl, 9 dwg, 6 ex

Description

The present invention relates to a method for the synthesis of cyclic depsipeptides, in particular emodepside, including specific carboxylic acid TAG protecting groups.

Emodepside (cyclo[( R )-lactoyl -N -methyl-L-leucyl-( R )-3-( p-morphol inophenyl)lactoyl -N -methyl-L-leucyl-( R )-lactoyl -N -methyl- L-leucyl-( R )-3-( p-morphol inophenyl)lactoyl- N -methyl-L-leucyl) is an anthelmintic drug that is effective against a number of gastrointestinal nematodes. Its molecular structure, which is depicted below, can be described as cyclic octadepsipeptides, a depsipeptide being a peptide in which one or more of its amide groups have been replaced by corresponding ester groups. On an industrial scale, emodepside can be produced by derivatization of the naturally occurring substance PF1022A, in which two hydrogen atoms are replaced by morpholine rings.

WO 93/19053 A1 (EP 0 634 408 A1) discloses a compound of general formula:

where A is a benzyl group which has suitable substituent(s) or a phenyl group which may have suitable substituent(s), A ^a is a benzyl group which has suitable substituent(s) or a phenyl group which may have suitable substituent(s), B and D are each lower alkyl, C is hydrogen or lower alkyl, and a pharmaceutically acceptable salt thereof.

WO 2005/055973 A2 discloses transdermally applied agents containing cyclic depsipeptides and/or praziquantel, in addition to its preparation and its use for the control of endoparasites. Combinations of emodepside and praziquantel or epsiprantel as well as 1,2-isopropylidene glycol as endoparasiticides are disclosed in WO 2006/094664 A1.

WO 2006/053641 A1 relates to the use of endoparasiticidal depsipeptides for the preparation of pharmaceutical preparations for the prevention of vertical infection by endoparasites.

General synthesis, among other things, PF1022A and its derivatives are discussed in the viewing article “CyclodepsipTides: A Rich Source of Biological Compounds for Drug Research” by CherkenBeck, Molecules 2014 , 19 , 12368-12420; DOI: 10.3390/Molecules190812368. For several cyclodepsipeptides, common syntheses have been established in both solution and solid phase, allowing the construction of combinatorial libraries. In addition, the biosynthesis of specific cyclodepsipeptides has been illuminated and used for the chemoenzymatic preparation of non-naturally occurring analogues. The review article also summarizes recent literature data about cyclic tetra-decadepsipeptides, consisting exclusively of α-amino- and α-hydroxy acids.

In the synthesis of polypeptides, the solid-phase strategy has the advantage of simple separation of reaction products, while the liquid-phase strategy has the advantage of homogeneous reaction conditions. The hybrid approach is a TAG-based strategy in which compounds having a TAG group are easily separated from molecules without a TAG group.

In this regard, JP 2000/044493 A1 discloses a protecting group for the synthesis of a library of compounds which consists of compounds capable of binding to the compound to be protected in a 1:1 ratio, having the same molecular structure, and also having a molecular weight >=500, which consists of 3,4,5-tris-(n-octadecyloxy)benzyl alcohol, 3,4,5-tris-(n-octadecyloxy)benzyl chloride or methyl 3,4,5-tris-(n-octadecyloxy)benzoate .

EP 2 003 104 A2 relates to an organic synthesis reagent with which a chemical reaction can be carried out in the liquid phase and the unwanted compound(s) can be easily separated at low cost from the liquid phase after completion of the reaction. The organic synthesis reagent depicted below is said to change reversibly from a liquid phase state to a solid phase state with changes in solution composition and/or solution temperature and is intended for use in organic synthesis reactions.

R ₁ - R ₅ may be the same or different and represent hydrogen, halogen, an alkyl group of 1 to 30 carbon atoms, which may have a substituent group, an alkoxy group of 1 to 30 carbon atoms, which may have a substituent group , an aryl group of 1 to 30 carbon atoms which may have a substituent group, an acyl group of 1 to 30 carbon atoms which may have a substituent group, a thioalkyl group of 1 to 30 carbon atoms which may have a substituent group a substituent, a dialkylamino group having from 1 to 30 carbon atoms, which may have a substituent group, a nitro group or an amino group; and at least two of R ₁ 0 R5 are groups having from 18 to 30 carbon atoms, and X is an active site reactant having one or more atoms selected from the group consisting of a carbon atom, an oxygen atom, a sulfur atom and a nitrogen atom.

In "TAG-Assisted Liquid-Phase Peptide Synthesis Using Hydrophobic Benzyl Alcohols as Supports" by Yohei Okada, Hideaki Suzuki, Takashi Nakae, Shuji Fujita, Hitoshi Abe, Kazuo Nagano, Toshihide Yamada, Nobuyoshi Ebata, Shokaku Kim, and Kazuhiro Chiba, The Journal of Organic Chemistry 2013 , 78 , 320-327; doi: 10.1021/jo302127d; reported that liquid-phase synthesis of soluble peptides by TAG group was successfully developed based on simple hydrophobic benzyl alcohols, which can be easily obtained from natural substances. Each step is reported to obtain excellent precipitation yields by combining the best properties of solid-phase and liquid-phase techniques. It is reported that this approach can be effectively applied to fragment linkages, allowing the chemical synthesis of multiple bioactive peptides.

In "A Novel Protecting Group for Constructing Combinatorial Peptide Libraries" by Hitoshi Tamiaki, Tomoyuki Obata, Yasuo Azefu and Kazunori Toma, Bulletin of the Chemical Society of Japan 2001 , 74 , 733-738; doi http://dx.doi.org/10.1246/bcsj.74.733; discloses 3,4,5-tris(octadecyloxy)benzyl alcohol, HO-Bzl(OC ₁₈ ) _3, which was prepared from gallic acid and stearyl bromide. Using conventional stepwise extension, N,C-protected peptides, Fmoc-AA _n -…-AA ₁ -OBzl(OC ₁₈ ) ₃ , were synthesized. Substituted benzyl esters were selectively cleaved by treatment with 4 M hydrogen chloride in ethyl acetate to give Fmoc-AA _n -...-AA ₁ -OH and HO-Bzl(OC ₁₈ ) ₃ . Thus, the substituted benzyl group is reported to be effective in protecting C-terminal carboxyl groups in liquid-phase peptide synthesis. Since the substituted benzyl group has a moderately high molecular weight, Fmoc-AA _n -...-AA ₁ -OBzl(OC ₁₈ ) ₃ can reportedly be easily purified by size exclusion chromatography; All protected peptides were reported to be eluted into the void fraction of a Sephadex LH-20 gel filtration column. The combination of the carboxyl-protecting group B of Bzl(OC ₁₈ ) ₃ with simple gel filtration purification is reported to provide a new route for the generation of combinatorial peptide libraries in solution phase.

WO 2017/116702 A1 relates to a cyclic depsipeptide compound or pharmaceutically or veterinarily acceptable salt of the following formula:

As an example, the compound designated 7-34A, as depicted below, shows an EC ₅₀ value of between 0.1 µM and 1.0 µM in an in vitro test against the microfilaria Dirofilaria immitis :

An object of the present invention is to provide an improved route for the synthesis of emodepside and related cyclic depsipeptides. Another object of the present invention is to provide new depsipeptides that can be synthesized using this route.

This problem is solved by the method according to paragraph 1 and depsipeptides according to paragraph 22. Preferred embodiments of the present invention are the objects of dependent claims. They may be freely combined unless the context clearly indicates otherwise.

Accordingly, the present invention provides a method for synthesizing cyclic depsipeptides of general formula (I) from depsipeptides of general formula (II):

where B represents an amine protecting group and A represents a carboxylic acid protecting group,

wherein the method includes the stages:

- removing the protecting group of the amine group, which is protected by group B, thus obtaining an unprotected amine group;

- removing the protecting group of the carboxylic acid which is protected by group A, thus obtaining an unprotected carboxylic acid group;

- condensation of an unprotected amine group and an unprotected carboxylic acid group, thus obtaining a cyclic depsipeptide (I)

where x and y are, independently of each other, 0, 1 or 2, provided that x+y ≥ 1 (preferably x and y are 1), and R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen, straight or branched C1-C8 alkyl, in particular methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl , pentyl, isopentyl, sec-pentyl, hexyl, isohexyl, sec-hexyl, heptyl, isoheptyl, sec-heptyl, tert-heptyl, octyl, isooctyl, sec-octyl, straight or branched halogenated C1-C8 alkyl, in particular fluorinated sec -butyl, hydroxy-C1-C6-alkyl, in particular hydroxymethyl, 1-hydroxyethyl, C1-C4-alkanoyloxy-C1-C6-alkyl, in particular acetoxymethyl, 1-acetoxyethyl, C1-C4-alkoxy-C1-C6-alkyl , in particular methoxymethyl, 1-methoxyethyl, aryl-C1-C4-alkyloxy-C1-C6-alkyl, in particular benzyloxymethyl, 1-benzyloxyethyl, mercapto-C1-C6-alkyl, in particular mercaptomethyl, C1-C4-alkylthio-C1 -C6-alkyl, in particular methylthioethyl, C1-C4-alkylsulfinyl-C1-C6-alkyl, in particular methylsulfinylethyl, C1-C4-alkylsulfonyl-C1-C6-alkyl, in particular methylsulfonylethyl, carboxy-C1-C6-alkyl, in in particular carboxymethyl, carboxyethyl, C1-C4-alkoxycarbonyl-C1-C6-alkyl, in particular methoxycarbonylmethyl, ethoxycarbonylethyl, C1-C4-arylalkoxycarbonyl-C1-C6-alkyl, in particular benzyloxycarbonylmethyl, carbamoyl-C1-C6-alkyl, in particular carbamoylmethyl , carbamoylethyl, amino-C1-C6-alkyl, in particular aminopropyl, aminobutyl, C1-C4-alkylamino-C1-C6-alkyl, in particular methylaminopropyl, methylaminobutyl, C1-C4-dialkylamino-C1-C6-alkyl, in particular dimethylaminopropyl , dimethylaminobutyl, guanidino-C1-C6-alkyl, in particular guanidinopropyl, C1-C4-alkoxycarbonylamino-C1-C6-alkyl, in particular tert-butoxycarbonylaminopropyl, tert-butoxycarbonylaminobutyl, 9-fluorenylmethoxycarbonyl(Fmoc)amino-C1-C6-alkyl , in particular 9-fluorenylmethoxycarbonyl(Fmoc)aminopropyl, 9-fluorenylmethoxycarbonyl(Fmoc)aminobutyl, C2-C8-alkenyl, in particular vinyl, allyl, butenyl, C3-C7-cycloalkyl, in particular cyclopentyl, cyclohexyl, cycloheptyl, C3-C7 -cycloalkyl-C1-C4-alkyl, in particular cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, benzyl, substituted benzyl, phenyl, phenyl-C1-C4-alkyl, in particular phenylmethyl, which may be optionally substituted by radicals from the group consisting of halogen, in in particular fluorine, chlorine, bromine or iodine, hydroxyl, C1-C4 alkoxy, in particular methoxy or ethoxy, C1-C4 alkyl, in particular methyl.

Protecting groups A and B may be independent such that when protecting group B is removed, group A is stable; however, it is also an embodiment of the present invention where the protecting groups A and B are removed simultaneously.

In the method according to the present invention, it has surprisingly been found that cyclic depsipeptides (I), in particular emodepside and closely related structures, can be synthesized in high overall yields.

After the protecting group is removed, the amine group and the carboxylic acid group react to form a peptide bond. Suitable coupling agents together with a base such as N,N-diisopropylethylamine include PyBOP (benzotriazol-1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate), BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate), BOP-Cl (bis (2-oxo-3-oxazolidinyl)phosphonium chloride), EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)- he), HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide), HBTU (2-(1H-benzotriazol-1-yl )-1,1,3,3-tetramethyluronium hexafluorophosphate) and, most preferably, T3P® (propylphosphonic anhydride, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4 ,6-trioxide, PPACA).

According to one embodiment of the method according to the present invention, x is 1, y is 1, R1, R4, R7 and R10 are methyl, R6 and R12 are methyl, R5 and R11 are, independently of each other, straight or branched C1-C4 alkyl or straight or branched halogenated C1-C4 alkyl, and R3 and R9 are, independently of each other, benzyl or substituted benzyl. According to one preferred embodiment of the present invention, R3 and/or R9 are p-morpholino-substituted benzyl.

According to an embodiment of the present invention, group A is acid labile while group B is hydrogenolysis labile. Accordingly, according to an embodiment of the present invention, there is provided a method for synthesizing cyclic depsipeptides of general formula (I) from depsipeptides of general formula (IIa):

where Y represents an amine protecting group and X represents a carboxylic acid protecting group,

wherein the method includes the stages:

- removing the protecting group of the amine group, which is protected by the Y group, in the presence of an acid, thereby obtaining an unprotected amine group;

- removing the protecting group of the carboxylic acid, which is protected by group X, by hydrogenolysis, thereby obtaining an unprotected carboxylic acid group;

- condensation of an unprotected amine group and an unprotected carboxylic acid group, thus obtaining a cyclic depsipeptide (I)

In the method according to this embodiment of the present invention, it has surprisingly been found that cyclic depsipeptides (I), in particular emodepside and closely related structures, can be synthesized in high overall yields.

The term "hydrogenolysis" should be understood in its broadest sense, and is clearly not limited to reaction with gaseous and/or molecular hydrogen, although this is one embodiment of the present invention. Suitable catalysis are Pd, Pd/C, Pt, Pt/C. The term "hydrogenolysis" also includes reactions in which hydrogen is produced in situ or only formally, and where hydrogenolysis reagents such as hydrazine or diimide, etc. are used.

According to one embodiment of the present invention, X represents a substituted or unsubstituted -CH ₂ -aryl group. According to one embodiment of the present invention, X is selected from the group of benzoyl (Bn), 4-methoxybenzoyl (PMB), 3,4-dimethoxybenzoyl (DPMB), 4-phenylbenzoyl (PPB), 2-naphthylmethyl (Nap), benzyloxymethyl acetal (BOM).

According to one embodiment of the present invention, Y is tert-butyloxycarbonyl (Boc), trityl (Trt), p-methoxybenzylcarbamate (Moz) or p-nitrobenzylcarbamate (PNZ). Most preferably, Y is Boc.

According to another embodiment of the method according to the present invention, the depsipeptide of general formula (IIa) is obtained from precursors of general formulas (IV) and (III):

through:

- in the precursor (IV), removing the protecting group of the amine group, which is protected by the PG2 group, in the presence of a base, thus obtaining an unprotected amine group;

- in the precursor (III), removing the carboxylic acid protecting group which is protected by the PG3 group in the presence of the acid, thus obtaining an unprotected carboxylic acid group;

- condensation of an unprotected amine group and an unprotected carboxylic acid group, thereby obtaining depsipeptide (IIa);

where R1 - R12, X, Y, x and y are as defined above (in particular, x and y may be 1), PG2 is an amine group protecting group, and PG3 is a carboxylic acid group protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (IIa) to (I) above.

Preferably, the precursor of the general formula (IV) is obtained from the precursors of the general formulas (VI) and (V):

through:

- in the precursor (VI), removing the protecting group of the amine group, which is protected by the PG4 group, in the presence of a base, thus obtaining an unprotected amine group;

- condensation of the unprotected amine group of the precursor (VI) and the carboxylic acid group of the precursor (V), thus obtaining the precursor (IV);

where R7 - R12, X and x are as defined above (in particular, x may be 1), PG2 is as defined above, and PG4 is an amine protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (II) to (I) above.

Also preferably, the precursor of the general formula (III) is obtained from the precursors of the general formulas (VIII) and (VII):

through:

- in the precursor (VII), removing the protecting group of the amine group, which is protected by the PG5 group, in the presence of a base, thus obtaining an unprotected amine group;

- condensation of the unprotected amine group of the precursor (VII) and the carboxylic acid group of the precursor (VIII), thus obtaining the precursor (III);

wherein R1 - R6, Y and y are as defined above (in particular, y may be 1), PG3 is as defined above, and PG5 is an amine protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (II) to (I) above.

Also preferably, the precursor of the general formula (VI) is obtained by esterifying the precursor of the general formula (IX) with X-LG:

where R10 - R12 and X are as defined above, and PG4 is also as defined above, and LG is a leaving group. The introduction of a protecting group or TAG group on the carboxylic acid group in (IX) can be accomplished using standard techniques; LG is often a halide, especially a chloride. Alternatively, LG can be -OH, then standard condensation protocols are often used.

Also preferably, the precursor of the general formula (VII) is obtained by esterifying the precursor of the general formula (X) with PG3-OH:

where R4 - R6 and y have the meaning as defined above, PG3 also has the meaning as defined above, and PG5 also has the meaning as defined above.

Also preferably, the precursors (III) and (IV) are identical.

Also preferably, R3 and R9 are identical, R1 and R7 are identical, R2 and R8 are identical, R4 and R10 are identical, R5 and R11 are identical, and R6 and R12 are identical.

According to an alternative embodiment of the present invention, group A is base labile while group B is acid labile. Therefore, according to an alternative embodiment of the present invention, the present invention provides a method for synthesizing cyclic depsipeptides of general formula (I) from depsipeptides of general formula (IIb):

where PG1 represents an amine group protecting group, and TAG represents a carboxylic acid group protecting group,

wherein the method includes the stages:

- removing the protecting group of the amine group, which is protected by the PG1 group, in the presence of a base, thereby obtaining an unprotected amine group;

- removing the protecting group of the carboxylic acid, which is protected by the TAG group, in the presence of the acid, thereby obtaining an unprotected carboxylic acid group;

- condensation of an unprotected amine group and an unprotected carboxylic acid group, thus obtaining a cyclic depsipeptide (I)

The TAG group contains an aryl-O-(CH ₂ ) _n - moiety, wherein aryl is an aromatic moiety and n is ≥ 13, x and y are independently 0, 1 or 2, provided that x +y ≥ 1 (preferably x and y are 1) and R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen, straight or branched C1-C8 alkyl, in particular methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, hexyl, isohexyl, sec-hexyl, heptyl, isoheptyl, sec- heptyl, tert-heptyl, octyl, isooctyl, sec-octyl, straight or branched halogenated C1-C8 alkyl, in particular fluorinated sec-butyl, hydroxy-C1-C6 alkyl, in particular hydroxymethyl, 1-hydroxyethyl, C1-C4- alkanoyloxy-C1-C6-alkyl, in particular acetoxymethyl, 1-acetoxyethyl, C1-C4-alkoxy-C1-C6-alkyl, in particular methoxymethyl, 1-methoxyethyl, aryl-C1-C4-alkyloxy-C1-C6-alkyl, in particular benzyloxymethyl, 1-benzyloxyethyl, mercapto-C1-C6-alkyl, in particular mercaptomethyl, C1-C4-alkylthio-C1-C6-alkyl, in particular methylthioethyl, C1-C4-alkylsulfinyl-C1-C6-alkyl, in particular methylsulfinylethyl, C1-C4-alkylsulfonyl-C1-C6-alkyl, in particular methylsulfonylethyl, carboxy-C1-C6-alkyl, in particular carboxymethyl, carboxyethyl, C1-C4-alkoxycarbonyl-C1-C6-alkyl, in particular methoxycarbonylmethyl, ethoxycarbonylethyl, C1-C4-arylalkoxycarbonyl-C1-C6-alkyl, in particular benzyloxycarbonylmethyl, carbamoyl-C1-C6-alkyl, in particular carbamoylmethyl, carbamoylethyl, amino-C1-C6-alkyl, in particular aminopropyl, aminobutyl, C1-C4-alkylamino- C1-C6-alkyl, in particular methylaminopropyl, methylaminobutyl, C1-C4-dialkylamino-C1-C6-alkyl, in particular dimethylaminopropyl, dimethylaminobutyl, guanidino-C1-C6-alkyl, in particular guanidinopropyl, C1-C4-alkoxycarbonylamino-C1- C6-alkyl, in particular tert-butoxycarbonylaminopropyl, tert-butoxycarbonylaminobutyl, 9-fluorenylmethoxycarbonyl(Fmoc)amino-C1-C6-alkyl, in particular 9-fluorenylmethoxycarbonyl(Fmoc)aminopropyl, 9-fluorenylmethoxycarbonyl(Fmoc)aminobutyl, C2-C8- alkenyl, in particular vinyl, allyl, butenyl, C3-C7-cycloalkyl, in particular cyclopentyl, cyclohexyl, cycloheptyl, C3-C7-cycloalkyl-C1-C4-alkyl, in particular cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, benzyl, substituted benzyl, phenyl , phenyl-C1-C4-alkyl, in particular phenylmethyl, which may be optionally substituted by radicals from the group consisting of halogen, in particular fluorine, chlorine, bromine or iodine, hydroxyl, C1-C4-alkoxy, in particular methoxy or ethoxy, C1-C4 alkyl, in particular methyl.

In the method of the present invention, it has surprisingly been discovered that cyclic depsipeptides (I), in particular emodepside and closely related structures, can be synthesized in high overall yields using hydrophobic TAG protecting groups on carboxylic acids. These groups can make the molecules they are associated with ("TAG group-bearing" molecules) insoluble in polar solvents such as methanol. Therefore, TAG group-bearing molecules can be precipitated from the reaction mixture, and the TAG group itself, after removal of the protecting group, can also be separated using this technique. In addition, the hydrophobic TAG group allows TAG-bearing molecules to dissolve in non-polar solvents such as dichloromethane.

Deprotection of the amine group by deprotection of PG1 can be accomplished using standard base techniques such as treatment with a solution of piperidine in dichloromethane. Likewise, deprotection of carboxylic acids by removal of the TAG group can be accomplished using benzyl group removal protocols such as treatment with a solution of trifluoroacetic acid (TFA) in dichloromethane.

In another embodiment of the method of the present invention, PG1 is 9-fluorenylmethoxycarbonyl (Fmoc), tert-butylcarbamate (Boc), benzylcarbamate (Z), acetamide, trifluoroacetamide, phthalimide, benzyl (Bn), triphenylmethyl (Tr), benzylidene or p- toluenesulfonamide (Ts)

and TAG represents:

or

where m is from ≥ 15 to ≤ 25, p is from ≥ 8 to ≤ 18, and q is from ≥ 15 to ≤ 25. Preferably, m is 18, 19, 20, 21 or 22, p is 11, 12 or 13, and q represents 21, 22 or 23.

According to another embodiment of the method according to the present invention, the depsipeptide of general formula (IIb) is obtained from precursors of general formulas (IVb) and (IIIb):

through:

- in the precursor (IVb), removing the protecting group of the amine group, which is protected by the PG2 group, in the presence of a base, thus obtaining an unprotected amine group;

- in the precursor (IIIb), removing the carboxylic acid protecting group, which is protected by the PG3 group, in the presence of an acid, thus obtaining an unprotected carboxylic acid group;

- condensation of an unprotected amine group and an unprotected carboxylic acid group, thereby obtaining depsipeptide (IIb);

wherein R1 - R12, TAG, PG1, x and y are as defined above (in particular, x and y may be 1), PG2 is an amine protecting group, and PG3 is a carboxylic acid protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (II) to (I) above.

Preferably, the precursor of the general formula (IVb) is obtained from the precursors of the general formulas (Vb) and (Vb):

through:

- in the precursor (VIb), removing the protecting group of the amine group, which is protected by the PG4 group, in the presence of a base, thus obtaining an unprotected amine group;

- condensation of the unprotected amine group of the precursor (VIb) and the carboxylic acid group of the precursor (Vb), thereby obtaining the precursor (IVb);

where R7 - R12, TAG and x are as defined above (in particular, x may be 1), PG2 is as defined above, and PG4 is an amine protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (IIb) to (I) above.

Also preferably, the precursor of the general formula (IIIb) is obtained from the precursors of the general formulas (VIIIb) and (VIIb):

through:

- in the precursor (VIIb), removing the protecting group of the amine group, which is protected by the PG5 group, in the presence of a base, thus obtaining an unprotected amine group;

- condensation of the unprotected amine group of the precursor (VIIb) and the carboxylic acid group of the precursor (VIIIb), thereby obtaining the precursor (IIIb);

wherein R1 - R6, PG1 and y are as defined above (in particular, y may be 1), PG3 is as defined above, and PG5 is an amine protecting group. Methods for deprotection and condensation may be the same as those mentioned for the reaction of compounds (IIb) to (I) above.

Also preferably, the precursor of the general formula (VIb) is obtained by esterifying the precursor of the general formula (IXb) with TAG-OH:

where R10 - R12 and TAG have the meanings as defined above, and PG4 also has the meaning as defined above. The introduction of a protecting group or the introduction of a TAG group for the carboxylic acid group in (IXb) can be accomplished using a standard condensation system such as DCC/DMAP.

Also preferably, the precursor of the general formula (VIIb) is obtained by esterifying the precursor of the general formula (X) with PG3-OH:

where R4 - R6 and y have the meaning as defined above, PG3 also has the meaning as defined above, and PG5 also has the meaning as defined above.

Also preferably, PG3 is TAG as defined above.

According to another embodiment of the method of the present invention, at least one of the reaction steps leading to the production of a TAG-bearing molecule is accompanied by precipitation of the crude reaction product in methanol, thereby purifying the crude reaction product.

According to another embodiment of the method according to the present invention, at least one of the reaction steps in which the TAG-protected carboxylic acid groups are subjected to deprotection is accompanied by precipitation of the separated TAG-OH in methanol and removal of the precipitate by filtration, thereby purifying the crude reaction product .

Also preferably, the precursors (IIIb) and (IVb) are identical.

Also preferably, R3 and R9 are different from each other, R1 and R7 are identical, R2 and R8 are identical, R4 and R10 are identical, R5 and R11 are identical, and R6 and R12 are identical.

According to another embodiment of the method according to the present invention, the depsipeptide is selected from the depsipeptide of the general formulas (II-1) - (II-14b):

where in formulas (II-11) - (II-14) -LINK- is selected from:

where X1 may be C, N, S, O, X2 and X3 may be C or N;

where X1 may be C, N, S, O, X2, X3 and X4 may be C or N;

where X1, X2, X3 and X4 may be C or N;

and where R13 is selected from SO ₂ NH(CH ₃ ), SO ₂ NH ₂ , OC(O)CH ₃ , CF ₃ or one of the following lactone structures:

In addition, the present invention provides a method for synthesizing cyclic depsipeptides of general formula (1) from depsipeptides of general formula (IIb):

including stages

providing a mixture of compound (IIc) and base in a solvent having an E _T (30) value of ≥30 to ≤43

slowly, preferably dropwise, adding a solution of the binding agent to the solvent to form cyclic depsipeptide (I),

where x and y are, independently of each other, 0, 1 or 2, provided that x+y ≥ 1 (preferably x and y are 1), and R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are each independently hydrogen, straight or branched C1-C8 alkyl, in particular methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl , pentyl, isopentyl, sec-pentyl, hexyl, isohexyl, sec-hexyl, heptyl, isoheptyl, sec-heptyl, tert-heptyl, octyl, isooctyl, sec-octyl, straight or branched halogenated C1-C8 alkyl, in particular fluorinated sec -butyl, hydroxy-C1-C6-alkyl, in particular hydroxymethyl, 1-hydroxyethyl, C1-C4-alkanoyloxy-C1-C6-alkyl, in particular acetoxymethyl, 1-acetoxyethyl, C1-C4-alkoxy-C1-C6-alkyl , in particular methoxymethyl, 1-methoxyethyl, aryl-C1-C4-alkyloxy-C1-C6-alkyl, in particular benzyloxymethyl, 1-benzyloxyethyl, mercapto-C1-C6-alkyl, in particular mercaptomethyl, C1-C4-alkylthio-C1 -C6-alkyl, in particular methylthioethyl, C1-C4-alkylsulfinyl-C1-C6-alkyl, in particular methylsulfinylethyl, C1-C4-alkylsulfonyl-C1-C6-alkyl, in particular methylsulfonylethyl, carboxy-C1-C6-alkyl, in in particular carboxymethyl, carboxyethyl, C1-C4-alkoxycarbonyl-C1-C6-alkyl, in particular methoxycarbonylmethyl, ethoxycarbonylethyl, C1-C4-arylalkoxycarbonyl-C1-C6-alkyl, in particular benzyloxycarbonylmethyl, carbamoyl-C1-C6-alkyl, in particular carbamoylmethyl , carbamoylethyl, amino-C1-C6-alkyl, in particular aminopropyl, aminobutyl, C1-C4-alkylamino-C1-C6-alkyl, in particular methylaminopropyl, methylaminobutyl, C1-C4-dialkylamino-C1-C6-alkyl, in particular dimethylaminopropyl , dimethylaminobutyl, guanidino-C1-C6-alkyl, in particular guanidinopropyl, C1-C4-alkoxycarbonylamino-C1-C6-alkyl, in particular tert-butoxycarbonylaminopropyl, tert-butoxycarbonylaminobutyl, 9-fluorenylmethoxycarbonyl(Fmoc)amino-C1-C6-alkyl , in particular 9-fluorenylmethoxycarbonyl(Fmoc)aminopropyl, 9-fluorenylmethoxycarbonyl(Fmoc)aminobutyl, C2-C8-alkenyl, in particular vinyl, allyl, butenyl, C3-C7-cycloalkyl, in particular cyclopentyl, cyclohexyl, cycloheptyl, C3-C7 -cycloalkyl-C1-C4-alkyl, in particular cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, benzyl, substituted benzyl, phenyl, phenyl-C1-C4-alkyl, in particular phenylmethyl, which may be optionally substituted by radicals from the group consisting of halogen, in particularly fluorine, chlorine, bromine or iodine.

The term "slowly" specifically means that and/or includes that the coupling agent solution is added at a rate of ≤ 2 mol% per minute, preferably ≤ 1 mol% per minute, more preferably ≤ 0.5 mol% per minute. in a minute.

In the method according to the present invention, it has surprisingly been found that cyclic depsipeptides (I), in particular emodepside and closely related structures, can be synthesized in high overall yields. Without being bound by any theory, the present inventors believe that this is due to the poor solubility of the cyclic depsipeptide (I) in solvent in contrast to the open form (IIb).

The term “ _ET (30) value” refers to E _T (30) according to Reichardt, Angew. Chem. 1979,119-131, which provides both a method for determining such values and measured values for many standard solvents.

According to one embodiment of the present invention, the E _T (30) value of the solvent is from ≥ 34 to ≤ 39.

The term “solvent” in the context of the present invention should also include a mixture of solvents.

According to one embodiment of the present invention, the solvent contains ethyl acetate, according to one embodiment of the present invention, the solvent is ethyl acetate.

Suitable binding agents that are embodiments of the present invention are listed above. In accordance with one embodiment of the present invention, the binding agent comprises T3P, in accordance with one embodiment of the present invention, the binding agent is T3P.

According to one embodiment of the present invention, the ratio of the coupling agent to the compound (IIc) before reaction (in mol:mol) is from ≥ 1:1 to ≤ 5:1, preferably from ≥ 2:1 to ≤ 3:1.

According to one embodiment of the present invention, the ratio of base to compound (IIb) before reaction (in mol:mol) is from ≥ 2:1 to ≤ 10:1, preferably from ≥ 4:1 to ≤ 6:1.

Suitable bases that are embodiments of the present invention include N,N -diisopropylethylamine (DIEA), triethylamine, dimethylaminopyridine (DMAP), N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU ), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) or mixtures thereof. Most preferred is N,N -diisopropylethylamine.

According to one embodiment of the present invention, the temperature during addition of the binding agent is ≤25C.

According to one embodiment of the method according to the present invention, x is 1, y is 1, R1, R4, R7 and R10 are methyl, R6 and R12 are methyl, R5 and R11 are, independently of each other, straight or branched C1-C4 alkyl or straight or branched halogenated C1-C4 alkyl, and R3 and R9 are, independently of each other, benzyl or substituted benzyl. According to one preferred embodiment of the present invention, R3 and/or R9 are p-morpholino-substituted benzyl.

According to one embodiment of the method according to the present invention, R3 and R9 are identical, R1 and R7 are identical, R2 and R8 are identical, R4 and R10 are identical, R5 and R11 are identical, and R6 and R12 are identical.

According to a preferred embodiment of the present invention, compound IIb is synthesized from compound II or IIa by removing the protecting group A and B or X and Y, respectively. Compound IIb can be isolated or used in situ for the synthesis of depsipeptide (I).

The present invention also relates to linear or cyclic depsipeptides selected from one of the general formulas (II-1) - (II-14b) or (I-1) - (I-9), as described below, or pharmaceutically or veterinarily acceptable salt:

where in formulas (II-11) - (II-14) and (I-1) - (I-9), respectively:

A, B, X and Y have the meaning as defined above,

-LINK- selected from:

and R13 is selected from SO ₂ NH(CH ₃ ), SO ₂ NH ₂ , OC(O)CH ₃ , CF ₃ or one of the following lactone structures:

The terms "veterinarily acceptable salt" and "pharmaceutically acceptable salt" are used throughout the specification to describe any salts of compounds that are acceptable for administration for pharmaceutical or veterinary applications and that provide the active compound when administered.

Veterinary acceptable salts include salts derived from veterinary acceptable inorganic or organic bases and acids. Suitable salts include alkali metal salts such as lithium, sodium or potassium, alkaline earth metals such as calcium, magnesium and barium. Salts containing transition metals, including, but not limited to, manganese, copper, zinc and iron, are also suitable. In addition, the present invention covers salts containing ammonium cations, as well as substituted ammonium cations in which one or more hydrogen atoms are replaced by alkyl or aryl groups. Particularly useful are salts derived from inorganic acids, including, but not limited to, hydrohalic acids (HCl, HBr, HF, HI), sulfuric acid, nitric acid, phosphoric acid and the like. Suitable inorganic salts also include, but are not limited to, bicarbonate and carbonate salts. In some embodiments, examples of veterinary and agriculturally acceptable salts are organic acid addition salts formed with organic acids, including, but not limited to, maleate, dimaleate, fumarate, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate , α-ketoglutarate and α-glycerophosphate. Of course, other suitable organic acids can be used.

Salts of alkali metal (eg sodium, potassium or lithium) or alkaline earth metal (eg calcium) compounds can also be prepared by reacting a sufficiently acidic residue in the compounds with an alkali metal or alkaline earth metal hydroxide.

Veterinary acceptable salts can be prepared using standard techniques well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acidic functional group present in the compound, or by reacting a suitable acid with a suitable basic functional group on connections according to the present invention.

Examples

The present invention is further described with reference to, but not limited to, the following examples.

1. General method

The initial structure-forming blocks were prepared from commercially available reagents. Unless otherwise stated, reagents and solvents were purchased at the highest commercial grade and used without further purification. Methanol and dry toluene, CH ₂ Cl ₂ were purchased from Kanto Chemical Co., Inc. All reactions were monitored by thin layer chromatography (TLC) using Merck silica gel 60 F254 precoated plates (0.25 mm). Flash chromatography was performed on Kanto Chemical silica gel (Kanto Chemical, silica gel 60N, spherical neutral, 0.040-0.050 mm, cat. no. 37563-84). ¹ H and ¹³ C NMR spectra were recorded on a JEOL JNM-ECA-500 (500 MHz for ¹ H-NMR and 125 MHz for ¹³ C-NMR). Chemical shifts are expressed in ppm down from the inner solvent peaks for CDCl ₃ ( ¹ H; δ = 7.26 ppm, ¹³ C; δ = 77.0 ppm), CD ₃ OD ( ¹ H; δ = 3.31 ppm, ¹³ C; δ = 49.0 ppm ), and J values are given in Hertz. The following abbreviations have been used to denote multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet doublet, ddd = doublet doublet, dt = doublet triplet, dq = doublet quartet, m = multiplet br = wide. All infrared spectra were measured on a Horiba FT-210 spectrometer. High and low resolution mass spectra were measured on a JEOL JMS-AX505 HA, JEOL JMS-700 MStation, and JEOL JMS-T100LP. Optical rotations were measured using a JASCO P-1010 polarimeter. Melting points were measured using YANACO MP-500P or OptiMelt devices (Stanford Research Systems).

General Method for Removing the Fmoc Protecting Group

The Fmoc protected substrate was dissolved in 5% piperidine/CH ₂ Cl ₂ (generally 0.05 M for substrate) at room temperature and the solution was stirred for 3 hours. The reaction mixture was then cooled to -5°C and MeOH was added ( fivefold excess of the reaction solution). The resulting heterogeneous solution was stirred for an additional 30 minutes at -5°C, and the colorless precipitate was filtered and washed with additional MeOH to obtain the corresponding amine compound as a colorless powder.

General method for removing the TAGa functional group

TAG-bearing substrates with the TAGa group were dissolved in 50% TFA/CH2Cl2 (0.05 M for substrate) at room temperature, and the solution was stirred for a total of 1 h. The reaction mixture was then concentrated with toluene (x 3) to remove TFA. CH2Cl2 was then added to the flask at -5°C, then MeOH was added (fivefold excess of CH2Cl2). The resulting heterogeneous solution was stirred for another 30 minutes at -5°C, and the colorless precipitate was filtered and washed with additional MeOH. The combined filtrates were concentrated in vacuo. 4 M HCl/dioxane (0.05 M for product) was added to the resulting product and concentrated with toluene (x 3) to give the carboxylic acid compound as a generally brown oil. The crude material was used in the next step without further purification.

2. Synthesis of emodepside

The synthesis of emodepside is described below using the compound benzyl (R)-2-hydroxy-3-(4-morpholinophenyl)propanoate (termed EMD-8), which is synthesized from p-fluorobenzaldehyde according to the scheme in FIG. 1. Using this compound, emodepside is synthesized according to the scheme in FIG. 2.

4-Morpholinobenzaldehyde (EMD-22) : A solution of 4-fluorobenzaldehyde (EMD-21, 3.6 kg, 29.0 mol, 1.0 eq) in 1-methyl-2-pyrrolidine (36 L, 10.0 vol), morpholine was placed in a 100 L reactor (7.6 kg, 87 mol, 3.0 equiv), K ₂ CO ₃ (10.0 kg, 72.5 mol, 2.5 equiv). The resulting mixture was stirred for at least 6 hours at 125~130°C. The reaction was monitored by TLC until the absence of EMD-21. The reaction mixture was diluted with ethyl acetate (18 L, 5 vol.), H ₂ O (72 L, 20 vol.), and separated. The aqueous phase was extracted with ethyl acetate (18 L x 2), and the organic phases were combined and washed with H ₂ O (36.0 L x 3). The organic extracts were concentrated in vacuo at below 45°C until no more drops of distillate could fall out. The residue was eluted with heptane/ethyl acetate (5:1, v/v, 7.2 L) and concentrated in vacuo below 45°C. Then, heptane/ethyl acetate (5:1, v/v, 21.6 L) was added to the above residue at 20~25°C. The solution was stirred at 20~25°C for 16 hours. The mixture was filtered, and the filter cake was washed with heptane (7.3 L). The solids were dried under vacuum at 40~45°C. 4.67 kg (84.8%) of 4-morpholinobenzaldehyde (EMD-22) was obtained as a yellow solid. MS (ES, m/z ): 192 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 9.74 (s, 1H), 7.74 (d, J = 8.1 Hz, 2H), 7.07(d, J = 8.4 Hz, 2H), 3.75-3.74 (m, 4H ), 3.35-3.33 (m,4H).

( Z )-2-methyl-4-(4-morpholinobenzylidene)oxazol-5(4H)-one (EMD-22B) : A solution of N -acetylglycine (1.22 kg, 10.46 mol, 1.0 eq) was placed in a 100 L reactor tetrahydrofuran (20 l, 10 vol.), acetic anhydride (3.2 kg, 31.38 mol, 3.0 eq), zinc (II) chloride (1.48 kg, 10.46 mol, 1.0 eq). The resulting mixture was stirred at 70°C for 1 h and EMD-22 (2.0 kg, 10.46 mol, 1.0 eq) was added. The mixture was then stirred at 70°C for another 16 hours and monitored by LCMS. After cooling to 20~25°C, H ₂ O (40 L) was added. Then, the mixture was stirred at 0~5°C for 3 hours and filtered. The filter cake was washed with H ₂ O (10 L), and dried under vacuum at 40~45°C. 2.29 kg (80.4%) of ( Z )-2-methyl-4-(4-morpholinobenzylidene)oxazol-5(4H)-one (EMD-22B) was obtained as a brown solid. MS (ES, m/z ): 273 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.84 (s, 1H), 7.47(d, J = 9.0 Hz, 2H), 7.02(d, J = 9.1 Hz, 2H), 3.74-3.71 (m, 4H ), 3.32-3.27 (m, 4H).

( E )-2-hydroxy-3-(4-morpholinophenyl)acrylic acid (EMD-23) : A solution of EMD-22B (2.2 kg, 8.08 mol, 1.0 eq) in 1,4-dioxane was placed in a 50 L reactor ( 8.8 L, 4.0 vol.), HCl (8.8 L, 4.0 vol.) at 20~25°C. The resulting mixture was stirred at 80°C for 3 hours and monitored by LCMS. After cooling to 0~10°C, the mixture was stirred at 0~10°C for 16 hours and filtered. The filter cake was dried under vacuum at 40~45°C. The crude product was eluted with H ₂ O (4.4 L) and stirred at 0~10°C for 2 hours. The mixture was filtered and the filter cake was dried under vacuum at 40~45°C. 1.17 kg (56.0%) of ( E )-2-hydroxy-3-(4-morpholinophenyl)acrylic acid (EMD-23) was obtained as a bluish-gray solid. MS (ES, m/z ): 250 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.66 (d, J = 8.5 Hz, 2H), 6.97(d, J = 8.6 Hz, 2H), 6.35 (s, 1H), 4.05 (s, 1H, - OH), 3.77-3.74 (m, 4H), 3.19-3.16 (m, 4H).

(R)-2-Hydroxy-3-(4-morpholinophenyl)propanoic acid (EMD-24) : A solution of EMD-23 (66.0 g, 0.26 mol) was placed in a 2 L round bottom flask, purged with nitrogen and maintained under an inert nitrogen atmosphere. , 1.0 equiv) in N,N -dimethylformamide (660 ml, 10.0 vol.), Et ₃ N (107.2 g, 1.06 mol, 4.0 equiv), RuCl(R,R)-TsDPEN (1.69 g, 0.0026 mol, 0.01 equiv ) at 20~25°C. Formic acid (36.59 g, 0.79 mol, 3.0 equiv) was added dropwise to the above mixture for 2 hours at 20~25°C under N ₂ atmosphere. Then, the mixture was stirred at 20~25°C and monitored by LCMS. A reaction was noted for M1, which was used in the next step without further purification.

Benzyl (R)-2-hydroxy-3-(4-morpholinophenyl)propanoate (EMD-8) : To the mixture (M1) was added K ₂ CO ₃ (109.0 g, 0.78 mol, 3.0 eq) and benzyl bromide (54.0 g) dropwise , 0.32 mol, 1.2 equiv) at 20~25°C for 1 hour. Then, the mixture was stirred at 55~60°C for another 16 hours. The reaction mixture was diluted with ethyl acetate (330 ml, 5 vol.), H ₂ O (1.2 l, 20 vol.), and separated. The aqueous phase was extracted with ethyl acetate (330 ml x 2), and the organic phases were combined and washed with H ₂ O (660 ml x 3). The organic extracts were concentrated in vacuo at below 45°C until no more drops of distillate could fall out. The residue was eluted with heptane/ethyl acetate (3:1, v/v, 132 ml) and concentrated in vacuo below 45°C. Then, heptane/ethyl acetate (3:1, v/v, 264 ml) was added to the above residue at 20~25°C. The mixture was filtered and the filter cake was washed with heptane (132 ml). The solids were dried under vacuum at 40~45°C. 52 g (58%) of benzyl(R)-2-hydroxy-3-(4-morpholinophenyl)propanoate (EMD-8) was obtained as a yellow solid. MS (ES, m/z ): 342 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.40-7.27 (m, 5H), 7.05 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 8.1 Hz, 2H), 5.17 (s, 2H ), 4.24 (t, J = 7.5 Hz, 1H), 3.74-3.71 (m, 4H), 3.06-3.03 (m, 4H), 2.90-2.73 (m, 2H).

(R)-1-(benzyloxy)-3-(4-morpholinophenyl)-1-oxopropan-2-yl- N- (tert-butoxycarbonyl)-N - methyl-L-leucinate (EMD-9B) : In a reactor of volume A solution of EMD-8 (1.96 kg, 5.75 mol, 1.0 eq) in dichloromethane (14.1 L, 10. vol.), N -(tert-butoxycarbonyl)-N- was placed in a 50 L tank, purged with nitrogen and maintained under an inert atmosphere of nitrogen. methyl-L-leucine (1.41 kg, 5.75 mol, 1.0 eq), DMAP (0.77 kg, 6.32 mol, 1.1 eq), EDCI (1.21 kg, 6.32 mol, 1.1 eq) at 20~25°C. The mixture was stirred at this temperature for at least 3 hours and monitored by LCMS. The mixture was concentrated at below 40°C until no more drops of distillate fell. The residue was dissolved in MTBE (14.1 L) and HCl (aq, 1H, 14.1 L) and filtered. The organic layer was washed with HCl (aq, 1H, 14.1 L), NaHCO ₃ (sat, 14.1 L x 2), and concentrated at below 40°C until no more drops of distillate precipitated. The residue was then dissolved in heptane/MTBE (28.2 L, 8:1, v/v), and concentrated at below 40°C until no more drops of distillate precipitated. To the above residue, heptane/MTBE (15.5 L, 8:1, v/v) was added at 20~25°C and seed crystal (0.2%, w/w), and stirring was maintained overnight at 20~25°C . The mixture was filtered; The filter cake was washed with heptane (7.0 L). The solids were dried under vacuum at 40~45°C. 2.56 kg (78.3%) of (R)-1-(benzyloxy)-3-(4-morpholinophenyl)-1-oxopropan-2-yl- N- (tert-butoxycarbonyl)-N-methyl-L-leucinate (EMD) were obtained -9B) in the form of a light yellow solid. MS (ES, m/z ): 569 (M+H); ¹ H NMR (DMSO-d ₆ , 300 MHz) 7.38-7.26 (m, 5H), 7.10 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.1 Hz, 2H), 5.25-5.22 (m , 1H), 5.12-5.10 (m, 2H), 4.07-3.99 (m, 1H), 3.79-3.76 (m, 4H), 3.19-2.97 (m, 6H), 2.57 (s, 3H), 1.42- 1.34 (m, 11H), 1.24-1.15 (m, 1H), 0.88-0.82 (m, 6H).

(R)-2-(( N -(tert-butoxycarbonyl)- N -methyl-L-leucyl)oxy)-3-(4-morpholinophenyl)propanoic acid (EMD-10B) : Into a 50 L reactor purged with nitrogen and maintained under an inert nitrogen atmosphere, a solution of EMD-9B (400g, 0.7 mol, 1.0 eq) in EtOH (4.0 L, 10.0 vol) was placed at 20~25°C. The reactor was evacuated and purged with nitrogen three times, and Pd/C (28.0 g, 7% w/w) was added. The reactor was then evacuated and purged with nitrogen three times again, and hydrogen was maintained bubbling under the surface of the reaction mixture. The mixture was stirred for at least 4 hours at 20~25°C and monitored by HPLC. After completion of the reaction, hydrogen bubbling was stopped. The mixture was filtered through Celite (2.0 kg) and the filter cake was washed with EA (0.8 L) and the filtrate was concentrated at below 40°C until no more drops of distillate fell. 317.7 g (94.4%) of (R)-2-(( N -(tert-butoxycarbonyl)- N -methyl-L-leucyl)oxy)-3-(4-morpholinophenyl)propanoic acid (EMD-10B) were obtained in dark yellow oil form; MS (ES, m/z ): 479 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.10 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.03-5.02 (m, 1H), 4.81-4.53 (m , 1H), 3.73 (t, J = 7.2 Hz, 4H), 3.05 (t, J = 7.2 Hz, 4H), 2.96-2.90 (m, 1H), 2.63 (s, 3H), 1.42-1.16 (m, 12 H), 0.89-0.84 (m, 6H).

(R)-1-(benzyloxy)-1-oxopropan-2-ylmethyl-L-leucinate (EMD-12B) : A solution of N- (tert- butoxycarbonyl)-N-methyl-L-leucine (240.3 g, 0.98 mol, 1.0 eq) in THF (3.9 L, 16 vol), benzyl (S)-2-hydroxypropanoate (176.5 g, 0.98 mol, 1.0 eq), triphenylphosphine (385.1 g, 1.47 mol, 1.5 equiv) at 20~25°C. After cooling to below 10°C, diisopropyl azodiformate (297 g, 1.47 mol, 1.5 eq) was added dropwise with stirring at below 10°C. Then heated to 20~25°C and stirred for at least 2 hours. The reaction was monitored by HPLC to benzyl (S)-2-hydroxypropanoate less than or equal to 0.5%. The resulting mixture was diluted with ethyl acetate (3.9 L) and washed with NaHCO ₃ (sat, 3.9 L x 2), saline (3.9 L x 2). The organic phase was concentrated at below 40°C until no more drops of distillate dropped out. The residue was exchanged with tert-butyl methyl ether (240 ml x 2), concentrated at below 40°C until no more drops of distillate precipitated, and mixed with tert-butyl methyl ether (960 ml) for at least 3 h. The mixture was filtered and the filter cake was washed with tert-butyl methyl ether (240 ml). The filtrate was concentrated at below 40°C until no more drops of distillate dropped out. The crude product of (R)-1-(benzyloxy)-1-oxopropan-2-yl N-(tert-butoxycarbonyl)-N-methyl-L-leucinate (EMD-1B) was obtained as a yellow oil, which was used as follows: stage without further purification.

A solution of EMD-1B in HCl/EA (1.2 L, 5.0 eq) at below 25°C was placed in a 10 L reactor, purged with nitrogen and maintained under an inert nitrogen atmosphere. The mixture was stirred for at least 1 hour at 20~25°C and monitored by HPLC to an EMD-1B of less than or equal to 0.5%. The solution was concentrated at below 40°C until drops of distillate stopped falling. The residue was exchanged with tert-butyl methyl ether (240 ml x 2), concentrated at below 40°C until no more drops of distillate precipitated, and dissolved in tert-butyl methyl ether (1.92 L). Then a seed crystal (0.1%, w/w) was added and stirred for at least 5 hours at 20~25°C. The mixture was filtered and the filter cake was washed with tert-butyl methyl ether (0.24 L). The solid was dried under vacuum at 40±5°C and 270 g of crude product was obtained as a white solid. The solid was dissolved in ethyl acetate (810 ml) while heating to 40±5°C, and tert-butyl methyl ether (4.05 L) was added. Then cooled to 20~25°C, and a seed crystal (0.1% w/w) was added. The mixture was stirred for at least 5 hours at 20~25°C, filtered, and the filter cake was washed with tert-butyl methyl ether (0.24 L). The solid was dried under vacuum at 40±5°C. 226.7 g (67.3%, two steps) of ( R)-1-(benzyloxy)-1-oxopropan-2-ylmethyl-L-leucinate (EMD-12B) was obtained as a white solid. MS (ES, m/z ): 308 (M+H); ¹ H NMR (DMSO-d ₆ , 300 MHz) 7.41-7.35 (m, 5 H), 5.28 (q, J = 7.1 Hz, 1H), 5.20 (s, 2H), 2.51 (s, 3H), 1.77- 1.71 (m, 3H), 1.50-1.48 (m, 3H), 0.90-0.87 (m, 6H).

(R)-1-(benzyloxy)-1-oxopropan-2-yl N-((R)-2-((N-(tert-butoxycarbonyl)-N-methyl-L-leucyl)oxy)-3-( 4-morpholinophenyl)propanoyl)-N-methyl-L-leucinate (EMD-13B): A solution of EMD-10B (275.0 g, 0.57 mol, 1.0 eq) was placed in a 10 L reactor, purged with nitrogen and maintained under an inert nitrogen atmosphere ) in ethyl acetate (2.2 L, 8.0 vol.), EMD-12B (197.7 g, 0.57 mol, 1.0 eq), N,N -diisopropylethylamine (372.2 g, 2.88 mol, 5.0 eq) at 20~25°C. After cooling to 10 ~20°C, propylphosphonic anhydride (916.4 g, 1.44 mol, 2.5 eq) was added dropwise with stirring at below 25°C. Then, the mixture was stirred at 20~25°C for at least 2.5 hours and monitored by HPLC to EMD-12B equal to or less than 1.0%. The solution was diluted with heptane (2.75 L) at 0~10°C and slowly quenched with HCl (aq, 1.0N, 2.75 L). The organic phase was washed with HCl (aq, 1.0N, 2.75 L), NaHCO ₃ (sat, 2.75 L x 2), and concentrated at below 40°C until no more drops of distillate fell. 429.8 g (97.4%) of (R)-1-(benzyloxy)-1-oxopropan-2-yl N-((R)-2-((N-(tert-butoxycarbonyl)-N-methyl-L-leucyl) were obtained )oxy)-3-(4-morpholinophenyl)propanoyl)-N-methyl-L-leucinate (EMD-13B) as a thick yellow oil. MS (ES, m/z ): 768 (M+H);

(R)-1-(benzyloxy)-1-oxopropan-2-yl-N-methyl-N-((R)-2-((methyl-L-leucyl)oxy)-3-(4-morpholinophenyl)propanoyl )-L-leucinate (EMD-14B) : A solution of EMD-13B (245 g, 0.32 mol, 1.0 eq) in HCl/EA (735, 3.0) was placed in a 5 L reactor, purged with nitrogen and maintained under an inert nitrogen atmosphere. vol.) at below 25°C. The mixture was stirred for at least 1 hour at 20~25°C and monitored by HPLC to EMD-13B less than or equal to 0.5%. The resulting solution was concentrated at below 40°C until no more drops of distillate precipitated and extracted with ethyl acetate (245 ml x 2) at below 40°C. The residue was dissolved in ethyl acetate (735 ml) and N,N -diisopropylethylamine (245 g) was added at 20~25°C. The mixture was washed with NaHCO3 (sat., 735 ml x 2). The organic layer was concentrated at below 40°C until no more drops of distillate dropped out. 186.9 g (89.9%) of (R)-1-(benzyloxy)-1-oxopropan-2-yl-N-methyl-N-((R)-2-((methyl-L-leucyl)oxy)-3 were obtained -(4-morpholinophenyl)propanoyl)-L-leucinate (EMD-14B) as a yellow oil. MS (ES, m/z ): 668 (M+H); ¹ H NMR (DMSO-d ₆ , 300 MHz) 7.41-7.32 (m, 5 H), 7.17 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 5.50-5.47 ( m, 1H), 5.20-5.04 (m, 4H), 4.23-3.99 (m, 1H), 3.74-3.72 (m, 4H), 3.09-2.76 (m, 10H), 2.22-1.99 (m, 3H), 1.63-1.35 (m, 5H), 1.29-1.16 (m, 4H), 0.97-0.70 (m, 12H).

(6S,9R,12S,15R)-6,12-diisobutyl-2,2,5,11,15-pentamethyl-9-(4-morpholinobenzyl)-4,7,10,13-tetraoxo-3,8, 14-Trioxa-5,11-diazahexadecane-16-oic acid (EMD-15B) : A solution of EMD-13B (274.2 g, 0.36 mol, 1.0 equiv) was placed in a 10 L reactor, purged with nitrogen and maintained under an inert nitrogen atmosphere ) in EtOH (2.8 L, 10.0 vol.) at 20~25°C. The reactor was evacuated and purged with nitrogen three times, and Pd/C (19.2 g, 7% w/w) was added. The reactor was then evacuated and purged with nitrogen three times again, and hydrogen was maintained bubbling under the surface of the reaction mixture. The mixture was stirred for at least 4 hours at 20~25°C and monitored by HPLC. After completion of the reaction, hydrogen bubbling was stopped. The mixture was filtered through celite (2.0 kg), and the filter cake was washed with ethyl acetate (0.56 L), and the filtrate was concentrated at below 40°C until no more drops of distillate fell. 237.4 g (98.0%) of (6S,9R,12S,15R)-6,12-diisobutyl-2,2,5,11,15-pentamethyl-9-(4-morpholinobenzyl)-4,7,10,13 were obtained -tetraoxo-3,8,14-trioxa-5,11-diazahexadecane-16-oic acid (EMD-15B) as a yellow oil; MS (ES, m/z ): 678 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.15 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.3 Hz, 2H), 5.52-5.40 (m, 1H), 5.09-5.02 (m , 1H), 4.91-4.53 (m, 2H), 3.74-3.72 (m, 4H), 3.15-3.04 (m, 5H), 2.94-2.86 (m, 4H), 2.65-2.64 (m, 3H), 1.43-1.28 (m, 16H), 0.93-0.77 (m, 12H).

(R)-1-(benzyloxy)-1-oxopropan-2-yl N-methyl-N-((6S,9R,12S,15R,18S,21R)-6,12,18-triisobutyl-2,2, 5,11,15,17-hexamethyl-9,21-bis(4-morpholinobenzyl)-4,7,10,13,16,19-hexaoxo-3,8,14,20-tetraoxa-5,11,17 -triazadocosan-22-oil)-L-leucinate (EMD-16B):A solution of EMD-15B (147.6 g, 0.22 mol, 1.0 equiv) in ethyl acetate (2.2 L, 8.0 vol.), EMD-14B (145.5 g, 0.22 mol, 1.0 equiv),N,N-diisopropylethylamine (139.6 g, 1.08 mol, 5.0 equiv) at 20~25°C. After cooling to 10 ~20°C, propylphosphonic anhydride (346.4 g, 0.54 mol, 2.5 eq) was added dropwise with stirring at below 25°C. Then, the mixture was stirred at 20~25°C for at least 2.5 hours and monitored by HPLC to EMD-12B less than or equal to 0.5%. The solution was diluted with heptane (2.2 L) at 0~10°C and slowly quenched with HCl (aq, 1.0 N, 2.2 L). The organic phase was washed with HCl (aq, 1.0H, 2.2 L), NaHCO₃ (sat., 2.2 L × 2), concentrated at below 40°C until no more drops of distillate precipitated, and extracted with heptane/MTBE (0.44 L, 1.5:1, v/v) at below 40°C. The residue was dissolved in heptane/MTBE (1.65 L, 1.5:1, v/v), and the seed crystal (0.1%, w/w) was added at 20~25°C. The mixture was stirred for at least 16 hours at 20~25°C and filtered. The filter cake was washed with heptane (0.66 L) and dried in vacuum at 40~45°C. 242.4 g (83.4%) of (R)-1-(benzyloxy)-1-oxopropan-2-yl N-methyl-N-((6S,9R,12S,15R,18S,21R)-6.12 were obtained. 18-triiso-butyl-2,2,5,11,15,17-hexamethyl-9,21-bis(4-morpholinobenzyl)-4,7,10,13,16,19-hexaoxo-3,8,14 ,20-tetraoxa-5,11,17-triazadocosan-22-oil)-L-leucinate (EMD-16B) as a white solid: MS (ES,m/z): 1328 (M+H);

(R)-1-(benzyloxy)-1-oxopropan-2-yl N-((2R,5S,8R,11S,14R,17S)-5,11-diisobutyl-6,8,12,19-tetramethyl- 17-(methylamino)-2,14-bis(4-morpholinobenzyl)-4,7,10,13,16-pentaoxo-3,9,15-trioxa-6,12-diazaicosanoyl)-N-methyl-L- leucinate (EMD-20B) : A solution of EMD-16B (376.4 g, 0.28 mol, 1.0 eq) in HCl/EA (1128 ml, 3.0 vol) was placed in a 5 L reactor, purged with nitrogen and maintained under an inert atmosphere of nitrogen. at below 25°C. The mixture was stirred for at least 1 hour at 20~25°C and monitored by HPLC to EMD-16B less than or equal to 0.5%. The resulting solution was concentrated at below 40°C until no more drops of distillate precipitated and extracted with ethyl acetate (376.4 ml x 2) at below 40°C. The residue was dissolved in ethyl acetate (1128 ml), and N,N -diisopropylethylamine (376.4 g) was added at 20~25°C. The mixture was washed with NaHCO3 (sat., 1128 ml x 2). The organic layer was concentrated at below 40°C until no more drops of distillate dropped out. 313.56 g (90.1%) of (R)-1-(benzyloxy)-1-oxopropan-2-yl N-((2R,5S,8R,11S,14R,17S)-5,11-diisobutyl-6 were obtained. 8,12,19-tetramethyl-17-(methylamino)-2,14-bis(4-morpholinobenzyl)-4,7,10,13,16-pentaoxo-3,9,15-trioxa-6,12-diazaicosanoyl )-N-methyl-L-leucinate (EMD-20B) as a yellow oil; MS (ES, m/z ): 1228 (M+H);

(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-8,12,14,20,24-pentamethyl-6,18-bis(4-morpholinobenzyl) -4,7,10,13,16,19,22-heptaoxo-5,11,17,23-tetraoxa-2,8,14,20-tetraazapentacosan-25-oic acid (EMD-18B): In a reactor volume A solution of EMD-20B (313.56 g, 0.26 mol, 1.0 eq) in EtOH (3.2 L, 10.0 vol) was placed in a 10 L tank, purged with nitrogen and maintained under an inert nitrogen atmosphere, at 20~25°C. The reactor was evacuated and purged with nitrogen three times, and Pd/C (21.95 g, 7% w/w) was added. The reactor was then evacuated and purged with nitrogen three times again, and hydrogen was maintained bubbling under the surface of the reaction mixture. The mixture was stirred for at least 4 hours at 20~25°C and monitored by HPLC to EMD-20B less than or equal to 1.0%. After completion of the reaction, hydrogen bubbling was stopped. The mixture was filtered through celite (2.0 kg), and the filter cake was washed with ethyl acetate (0.64 L x 3), and the filtrate was concentrated at below 40°C until no more drops of distillate fell. The residue was mixed with ethyl acetate (0.7 L) for at least 3 hours and filtered; the filter cake was washed with ethyl acetate (0.32 L × 2). The solid was dried under vacuum at 40~45°C. 234.2 g (80.6%) of (3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-8,12,14,20,24-pentamethyl-6,18 were obtained -bis(4-morpholinobenzyl)-4,7,10,13,16,19,22-heptaoxo-5,11,17,23-tetraoxa-2,8,14,20-tetraazapentacosane-25-oic acid (EMD -18B) in the form of an off-white solid; MS (ES, m/z ): 1138 (M+H); ¹ H NMR (DMSO-d ₆ , 300 MHz) 7.34-7.16 (m, 8H), 5.68-5.28 (m, 3H), 5.11-5.04 (m, 3H), 4.93-4.86 (m, 1H), 4.03- 3.99 (m, 1H), 3.85-3.82 (m, 8H), 3.23-3.22 (m, 8H), 3.23-2.76 (m, 13H), 2.49 (s, 2H), 2.07 (s, 2H), 1.66- 1.45 (m, 8H), 1.43-1.16 (m, 10H), 0.99-0.64 (m, 24H).

(3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,18,22-hexamethyl-12,24-bis(4- morpholinobenzyl)-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosane-2,5,8,11,14,17,20,23-octaone (Emodepsid): In a 10 L reactor, purged with nitrogen and maintained in an inert nitrogen atmosphere, a solution of EMD-18B (234.2 g, 0.21 mol, 1.0 eq) in ethyl acetate (3.8 L, 16.0 vol), N,N -diisopropylethylamine (131.84 g, 1.02 mol, 5.0 eq) was placed ) at 20~25°C. After cooling to 10 ~20°C, propylphosphonic anhydride (324.5 g, 0.51 mol, 2.5 eq) was added dropwise with stirring at below 25°C. Then, the mixture was stirred at 20~25°C for at least 2.5 hours and monitored by HPLC to EMD-18B less than or equal to 0.5%. The mixture was diluted with heptane (2.38 L) at 0~10°C and slowly quenched with HCl (aq, 1.0 N, 2.38 L). The organic phase was washed with HCl (aq, 1.0N, 2.38 L), NaHCO ₃ (sat, 2.38 L x 2), concentrated at below 40°C until no more drops of distillate precipitated, and extracted with EtOH (0.48 L × 2) at below 40°C. The residue was dissolved in EtOH (0.72 L) at 45~55°C, and the seed crystal (0.1%, w/w) was added at 20~25°C. The mixture was stirred for at least 16 hours at 20~25°C and filtered. The filter cake was washed with EtOH (0.24L). The solid was dried under vacuum at 40±5°C. The crude product was recrystallized with ethyl acetate. 118.24 g (51.3%) of (3S,6R,9S,12R,15S,18R,21S,24R)-3,9,15,21-tetraisobutyl-4,6,10,16,18,22-hexamethyl-12 were obtained ,24-bis(4-morpholinobenzyl)-1,7,13,19-tetraoxa-4,10,16,22-tetraazacyclotetracosane 2,5,8,11,14,17,20,23-octaone (emodepside) in in the form of a white solid; MS (ES, m/z ): 1120 (M+H); ^1H NMR (DMSO- _d6 , 300 MHz) 7.16 (d, J = 8.7 Hz, 4H), 6.87 (d, J = 8.2 Hz, 4H), 5.68 (q, J = 8.0 Hz, 1H), 5.51- 4.04 (m, 7H), 3.74-3.71 (m, 8H), 3.08-3.04 (m, 8H), 2.99-2.96 (m, 4H), 2.90-2.88 (m, 4H), 2.83-2.82 (m, 4H ), 2.78 (s, 2H), 2.70 (s, 2H), 1.78-1.38 (m, 8H), 1.31-1.14 (m, 6H), 0.97-0.69 (m, 28H).

3. Preparation of PF1022A (method 1; see reaction scheme in Fig. 3 and NMR spectrum in Fig. 4)

3-1. Synthesis technique

N -Fmoc -N -MeLeu-D-Lac-O-TAGa

To a stirred solution of compound HO-TAGa (1.69 g, 1.85 mmol) in CH ₂ Cl ₂ (37 ml) was added a 0.2 M solution in toluene of substance 1 (12.0 ml, 2.40 mmol), 4-dimethylaminopyridine (12 mg, 93.0 µmol) and N,N' -dicyclohexylcarbodiimide (1.30 g, 2.78 mmol) at room temperature under N ₂ atmosphere. After stirring for 2 hours, the reaction mixture was then cooled to -5°C and MeOH (185 ml) was added. The resulting heterogeneous solution was stirred for an additional 15 min at -5°C, and the colorless precipitate was filtered and washed with additional MeOH (500 ml) to obtain the compound N -Fmoc -N -MeLeu-D-Lac-O-TAGa (2.46 g , 100%) in the form of a colorless powder.

melting point: 44 - 45°C

[α] _D ²⁴ :-10.2 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.78-7.74 (complex m, 2H), 7.60-7.56 (complex m, 2H), 7.39 (m, 2H), 7.29 (m, 2H), 6.50 (s , 4/3H), 6.48 (s, 2/3H), 5.12-5.00 (complex m, 4H), 4.67 (dd, J = 6.3, 9.7 Hz, 3/10H), 4.58 (dd, J = 6.3 Hz, 10.9 Hz, 4/10H), 4.50 (dd, J = 6.9 Hz, 10.3 Hz, 6/10H), 4.38-4.34 (complex m, 9/10H), 4.30 (m, 5/10H), 4.23 (t, J = 6.3 Hz, 3/10H), 3.93 (m, 6H), 2.86 (s, 2H), 2.83 (s 1H), 1.76 (m, 6H), 1.64-1.42 (complex m, 9H), 1.31-1.14 (complex m, 87H), 0.96-0.87 (complex m, 14H), 0.78 (d, J = 6.9 Hz, 1H).

HRMS (FAB, NBA matrix)m/z : 1334.0748 (M⁺, calculated for C₈₆H₁₄₃NO₉ : 1334.0763)

N -MeLeu-D-Lac-O-TAGa (1)

Following the procedure described for the general Fmoc deprotection procedure, the compound N -Fmoc -N -MeLeu-D-Lac-O-TAGa (1.00 g, 0.749 mmol) was converted toconnection 1(816 mg, 98%) in the form of a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 6.50 (s, 2H), 5.16 (q, J = 6.9 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 5.04 (d, J = 12.0 Hz, 1H), 3.93 (m, 6H), 3.30 (t, J = 7.5 Hz, 1H), 2.37 (s, 3H), 1.75 (m, 6H), 1.53-1.42 (complex m, 10H), 1.32 -1.25 (complex m, 86H), 0.93-0.86 (complex m, 15H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 170.6, 153.3, 138.4, 130.2, 106.4, 73.5, 69.2, 68.9, 67.6, 61.4, 42.2, 34.5, 32.0, 30.4, 29.8 (x2 ), 29.5 (x2) , 26.2, 25.0, 22.8, 22.6, 22.5, 17.1, 14.2.

HRMS (FAB, NBA matrix)m/z : 1113.0151 [(M+H)⁺, calculated for C₇₁H₁₃₄NO₇ : 1113.0160]

N- Fmoc -N -MeLeu-D-PhLac- N -MeLeu-D-LacO-TAGa (2)

To a stirred solution of compound 1 (800 mg, 0.719 mmol) in CH ₂ Cl ₂ (25 ml) was added compound 2 (426 mg, 0.827 mmol), N,N- diisopropylethylamine (0.429 ml, 2.52 mmol), and PyBroP (496 mg, 1.222 mmol) at room temperature. After stirring for 88 h, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 2 (1.11 g, 96%) as a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.76-7.72 (complex m, 2H), 7.62-7.40 (complex m, 2H), 7.39-7.17 (complex m, 9H), 6.47 (m, 2H), 5.47-5.27 (complex m, 2H), 5.15-4.97 (complex m, 4H), 4.69-4.13 (complex m, 3H), 3.92 (complex m, 6H), 3.07 (m, 2H), 2.85 (complex, 6H ), 1.80-1.25 (complex m, 105H), 1.02-0.72 (complex m, 21H).

HRMS (FAB, NBA matrix) m/z : 1632.2163 [(M+Na) ⁺ , calculated for C ₁₀₂ H ₁₆₄ N ₂ O ₁₂ Na: 1632.2182]

N -MeLeu-D-PhLac- N -MeLeu-D-Lac-OH (3)

Following the procedure described for the general TAGa cleavage procedure, the compound2 (614 mg, 0.381 mmol) was converted to compound3(261 mg, 95%) as a yellow oil, which was used in the next step without further purification.

N -MeLeu-D-PhLac- N -MeLeu-D-Lac-O-TAGa (4)

Following the procedure described for the general Fmoc deprotection procedure, the compound2 (472 mg, 0.293 mmol) was converted to compound4(404 mg, 100%) in the form of a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.27 (m, 5H), 6.49 (s, 2H), 5.50 (dd, J = 5.7, 8.6 Hz, 1H), 5.32 (dd, J = 4.6, 10.9 Hz, 1H), 5.09-5.00 (complex m, 3H), 3.93 (m, 6H), 3.28 (t, J = 6.9 Hz, 1H), 3.09 (m, 2H), 2.92 (s, 3H), 2.27 ( s, 3H), 1.82-1.25 (complex m, 105H), 0.89-0.77 (complex m, 21H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 175.6, 175.1, 175.0, 174.8, 170.9, 170.5 (x2), 170.4, 169.8, 169.7, 166.7, 165.1, 153.3, 138.5, 13 8.3, 135.8 (x2), 130.2 , 129.5, 129.3, 128.7, 128.6, 128.5 (x2), 127.2, 107.0, 106.9, 105.4, 78.1, 73.5, 71.9, 71.6, 69.4, 69.2, 68.9, 67.7, 67.5, 6 6.9, 65.6, 61.3, 61.2, 59.7, 57.7, 54.7, 42.3, 42.2, 42.1, 40.0, 38.8, 37.5, 36.9, 34.4, 34.3, 32.8, 32.0, 31.3, 30.4, 29.8 (x2), 29.6, 29.5 (x2), 26.2, 25. 0, 24.8, 24.7, 24.6, 23.4, 22.9, 22.8, 22.5 (x2), 22.4 (x2), 21.9, 21.4, 20.5, 17.0, 16.9, 16.8, 14.2.

HRMS (FAB, NBA matrix)m/z : 1388.1664 [(M+H)⁺, calculated for C₈₇H₁₅₅N₂O₁₀ : 1388.1682]

N- Fmoc -N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac-O-TAGa (5)

To a stirred solution of compound 2 (330 mg, 0.238 mmol) in CH ₂ Cl ₂ (4.8 ml) was added compound 3 (221 mg, 0.309 mmol), N,N- diisopropylethylamine (0.150 ml, 0.881 mmol), and PyBroP (197 mg , 0.428 mmol) at room temperature. After stirring for 42 hours, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 5 (477 mg, 96%) as a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.74 (m, 2H), 7.53 (m, 2H), 7.38 (m, 2H), 7.32-7.15 (complex m, 12H), 6.48 (m, 2H) , 5.51-4.96 (complex m, 10H), 4.70-4.12 (complex m, 3H), 3.93 (m, 6H), 3.23 (dd, J = 6.9, 13.7 Hz, 1H), 3.08-2.70 (complex m, 15H ), 1.80-1.25 (complex m, 114H), 0.95-0.78 (complex m, 33H).

HRMS (FAB, NBA matrix)m/z : 2106.4949 [(M+Na)⁺, calculated for C₁₂₈H₂₀₂N₄O₁₈ Na₁: 2106.4912]

N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac-O-TAGa

Following the procedure described for the general Fmoc deprotection procedure, the compound5(450 mg, 0.206 mmol) was converted to the corresponding amine (398 mg, 98%) in the form of a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.25 (m, 10H), 6.48 (s, 2H), 5.57-4.99 (complex m, 9H), 3.93 (m, 6H), 3.26-2.78 (complex m , 14H), 2.24 (s, 3H), 1.80-1.25 (complex m, 114H), 0.90-0.78 (complex m, 33H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 175.6, 175.2 (x2), 171.5, 171.1, 170.8 (x2), 170.6, 170.5 (x2), 170.3, 153.3, 138.5 (x2), 138.3, 136.1, 135.9 , 135.5, 130.2, 130.1, 130.0, 129.8, 129.7, 129.6, 129.4 (x2), 128.8, 128.7, 128.6, 128.5, 127.4 (x2), 127.2, 127.1, 126.9, 126 .8, 107.0 (x2), 106.9, 73.5, 72.5, 72.2, 71.7, 71.6, 69.8, 69.4, 69.2, 68.1, 68.0, 67.8, 67.5 (x2), 66.9, 61.4, 57.4, 55.2, 54.8, 54.6 (x2), 38.8, 37.7, 37. 6, 37.4 (x2) , 37.1 (x2), 37.0, 36.9, 34.7, 34.6, 32.2, 32.0, 31.9, 31.7, 31.6, 31.4, 31.3, 31.2, 30.4, 29.8 (x2), 29.6, 29.5 (x2), 26.2, 24.9 , 24.8 ( x2), 24.7, 24.6, 24.5, 23.5, 23.4, 23.3, 23.1 (x2), 23.0, 22.8, 22.7, 22.5, 22.4, 22.3, 22.0, 21.5, 21.4, 21.3 (x2), 20.5, 16.9, 16.8, 16.6 , 14.2.

HRMS (FAB, NBA matrix)m/z : 1862.4425 [(M+H)⁺, calculated for C₁₁₃H₁₉₃N₄O₁₆ : 1862.4412]

N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac-OH

Following the procedure described for the general TAGa cleavage procedure, the compound N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-LacO-TAGa (380 mg, 0.204 mmol) was converted to the corresponding carboxylic acid (198 mg, 98%) as a yellow oil, which was used in the next step without further purification.

PF1022A

Reaction for 0.005M substrate concentration

The crude mixture from the previous reaction (90 mg, 0.0895 mmol) was dissolved in CH ₂ Cl ₂ (18 ml, 0.005 M). N,N- diisopropylethylamine (76 μL , 4.48 mmol) and PyBOP (93 mg, 0.179 mmol) were added to the reaction mixture at room temperature. After stirring for 48 hours, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (18 ml) at 0°C, and the mixture was extracted with CHCl ₃ (20 ml x 2). The combined organic layers were washed with 10% aqueous NaHSO ₄ (60 ml) and brine (60 ml), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl ₃ : MeOH = 400: 1 - 40: 1) to give PF1022A (55 mg, 65%) as a colorless solid.

Reaction for 0.05M substrate concentration

According to the cyclization procedure mentioned above, the crude mixture from the previous reaction (90 mg, 0.0895 mmol) was cyclized in CH ₂ Cl ₂ (1.8 ml, 0.05 M) with N,N- diisopropylethylamine (76 μl, 4.48 mmol) and PyBOP (93 mg, 0.179 mmol) at room temperature for 48 h to obtain PF1022A (40 mg, 49%) as a colorless solid.

melting point: 100-103°C

[α] ²² _D : -99.4°C ( c 0.06, MeOH)

IR (KBr) : 1743, 1666, 1466, 1412, 1265, 1188, 1126, 1080, 1026, 748, 701

¹ H-NMR (500 MHz, CD ₃ OD) δ: 7.29 (m, 10H), 5.82 (t, J = 7.5 Hz, 1H), 5.72 (m, 2H, rotamer), 5.54 (q, J = 6.9 Hz , 1H), 5.44 (dd, J = 4.6, 11.7 Hz, 1H), 5.40 (dd, J = 4.6, 11.7 Hz, 1H, rotamer), 5.23 (dd, J = 4.6, 11.7 Hz, 1H), 5.18 ( q, J = 6.9 Hz, 1H, rotamer), 4.77 (dd, J = 3.4, 11.2 Hz, 1H), 3.14 (m, 4H), 3.00 (s, 5/2H, rotamer), 2.91 (m, 7H, rotamer), 2.82 (s, 5/2H, rotamer), 1.84 (m, 1H), 1.77-1.47 (complex m, 11H), 1.39 (d, J = 6.3 Hz, 3H), 1.05 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.80 (d, J = 6.3 Hz, 3H), 0.95-0.82 (complex m, 18H).

¹³ C-NMR (125 MHz, CD ₃ OD) δ: 174.4, 173.5, 173.1, 173.1, 172.3, 172.0, 171.0, 170.7, 136.4, 136.2, 130.8, 130.7, 130.7, 129.8 , 129.7, 129.7, 128.4, 128.3, 72.5, 72.3, 69.9, 68.5, 58.6, 55.7, 55.5, 55.4, 39.0, 38.9, 38.6, 38.6, 37.8, 37.3, 32.0, 31.3, 31.1, 30.0, 26.2, 26.1, 25. 5, 25.2, 23.9, 23.7, 23.7, 23.6, 21.7, 21.6, 21.4, 21.1, 17.5, 17.2.

HRMS (FAB, NBA matrix)m/z : 971.5353 [(M+Na)⁺, calculated for C₅₂H₇₆N₄O₁₂Na₁ : 971.5357]

*Literature ( J. Antibiot. , 1992 , 45 , 692-697)

melting point: 104-106°C

[α] ²² _D : -102°C ( c 0.1, MeOH)

¹ H-NMR (400 MHz, CD ₃ OD) δ: 7.30 - 7.20 (PhH, 10H), 5.80 and 5.75 (C _α H-PhLac, 1Hx2), 5.54 and 5.16 (C _α H-Lac, 1Hx2), 5.43 , 5.42, 5.22 and 4.78 (C _α H-Leu, 1Hx4), 3.22-3.15 (C _β H ₂ -PhLac, 2Hx2), 3.00, 2.90, 2.88 and 2.80 (N-Me-Leu, 3Hx4), 1.87-1.50 (C _β H ₂ -Leu, 2Hx4), 1.40 (C _γ H-Leu, 1Hx4), 1.38 (C _β H ₃ -Lac, 3H), 1.02-0.75 (C _δ H ₃ -Leu, 6Hx4), 0.88 ( C _β H ₃ -Lac, 3H).

¹³ C-NMR (100 MHz, CD ₃ OD) δ: 174.4, 173.4, 172.4, 172.4, 172.1, 172.1, 171.0, 170.8, 136.5, 136.2, 130.7, 130.7, 130.7, 130.7 , 129.7, 129.7, 129.7, 129.7, 128.3, 128.2, 72.5, 72.3, 69.9, 68.4, 58.6, 55.7, 55.5, 55.4, 39.0, 38.9, 38.6, 38.6, 37.9, 37.4, 32.0, 31.3, 31.1, 29.9, 2 6.2, 26.1, 25.6, 25.2, 23.6, 23.6, 23.5, 23.5, 21.7, 21.6, 21.4, 21.0, 17.5, 17.2.

4. Preparation of PF1022A (method 2; see reaction scheme in Fig. 5)

4-1. Synthesis technique

N -Fmoc -N -MeLeu-D-PhLac-O-TAGa

To a stirred solution of compound HO-TAGa (170 mg, 0.186 mmol) in CH ₂ Cl ₂ (3.7 ml) was added a 0.1 M solution in toluene of substance 2 (2.42 ml, 0.242 mmol), 4-dimethylaminopyridine (1.1 mg, 9.30 μmol ), and N,N' -dicyclohexylcarbodiimide (58 mg, 0.279 mmol) at room temperature under N ₂ atmosphere. After stirring for 1 hour, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain the compound N -Fmoc -N -MeLeu-D-PhLac-O-TAGa (266 mg, 100%) as a colorless powder.

melting point:44 - 45°C

[α] _D ²⁷ :-6.1 ( c 1.74, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.76 (complex m, 2H), 7.54 (m, 2H), 7.39 (m, 2H), 7.30 (m, 1H), 7.26-7.14 (complex m, 4H ), 7.08 (m, 2H) 6.44 (m, 2H), 5.20 (m, 1H), 5.04-4.94 (complex m, 3H), 4.61-4.15 (complex m, 3H), 3.92 (m, 6H), 3.10 (m, 2H), 2.72 (s, 3H), 1.76 (m, 6H), 1.60-1.25 (complex m, 93H), 0.90-0.71 (complex m, 15H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 177.2, 175.2, 169.5, 153.3, 138.4, 135.9, 130.0, 129.6, 129.3, 128.6, 128.4, 127.1, 107.5, 107.2 , 73.5, 73.1, 69.2, 67.8, 61.6 , 42.5, 37.4, 34.5, 32.0, 30.4, 29.8 (x2), 29.6, 29.5, 26.2, 24.8, 22.8, 22.6, 22.5, 14.2.

HRMS (FAB, NBA matrix) m/z : 1410.1063 [(M+H) ⁺ , calculated for C ₉₂ H ₁₄₇ NO ₉ : 1410.1076]

N -MeLeu-D-PhLac-O-TAGa (6)

Following the procedure described for the general Fmoc deprotection procedure, the compound N -Fmoc- N -MeLeu-D-PhLac-O-TAGa (457 mg, 0.324 mmol) was converted to the compound6 (378 mg, 99%) in the form of a colorless powder.

melting point:48 - 49°C

[α] _D ²⁶ :+4.2 ( c 1.10, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.22 (m, 5H), 6.48 (s, 2H), 5.28 (dd, J = 4.0, 10.3 Hz, 1H), 5.08 (d, J = 12.0 Hz, 1H), 5.04 (d, J = 12.0 Hz, 1H), 3.93 (m, 6H), 3.24 (dd, J = 4.0 Hz, 14.3 Hz, 1H), 3.17 (t, J = 6.9 Hz, 1H), 3.07 (dd, J = 9.7, 14.3 Hz, 1H), 2.18 (s, 3H), 1.76 (m, 6H), 1.48-1.25 (complex m, 93H), 0.90-0.76 (complex m, 15H).

HRMS (FAB, NBA matrix) m/z : 1189.0458 [(M+H) ⁺ , calculated for C ₇₇ H ₁₃₈ NO ₇ : 1189.0473]

N -Fmoc -N -MeLeu-D-Lac- N -MeLeu-D-PhLacO-TAGa (7)

To a stirred solution of compound 6 (358 mg, 0.301 mmol) in CH ₂ Cl ₂ (6.0 ml) was added a 0.1 M solution of compound 1 (3.31 ml, 0.331 mmol), NN- diisopropylethylamine (0.15 ml, 0.903 mmol) and PyBroP ( 211 mg, 0.452 mmol) at room temperature. After stirring for 13 hours, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 7 (468 mg, 97%) as a colorless powder.

melting point:47 - 48°C

[α] _D ²⁶ : -13.9 ( from 1.10, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (m, 2H), 7.59 (m, 2H), 7.39 (m, 2H), 7.30-7.09 (complex m, 7H), 6.44 (m, 2H) , 5.36-4.96 (complex m, 6H), 4.71-4.24 (complex m, 3H), 3.93 (t, J = 6.5 Hz, 6H), 3.14 (m, 2H), 2.90-2.81 (complex s, 6H), 1.80-1.25 (complex m, 105H), 0.96-0.76 (complex m, 21H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 171.6, 171.2, 171.1, 170.9, 169.3, 157.0, 153.3, 144.3, 143.9, 141.4 (x2), 138.4, 135.9, 130.0, 12 9.9, 129.8, 129.7, 129.6, 129.5, 129.3, 128.7, 128.5, 128.4, 127.7, 127.6, 127.0, 125.3, 125.1, 125.0, 124.9, 120.0, 107.5, 107.2, 74.1, 74.0, 73.9, 73.8, 73.5, 71.3, 69.2, 67.9, 67.7, 57.4, 57.2, 56.7, 56.6 (x2), 54.5 (x2), 47.4 (x2), 47.3, 37.6, 37.3, 37.2, 37.1, 37.0, 32.0, 31.1, 30.7, 30.6 (x2), 30.5, 30.4, 30.3, 30. 2, 29.8 (x2), 29.6, 29.5 (x2), 26.2, 24.9, 24.8, 24.7, 24.6, 24.4, 23.4, 23.2, 22.9, 22.8, 22.1, 21.4, 21.2, 16.8, 14.2.

HRMS (FAB, NBA matrix) m/z : 1609.2274 (M ⁺ _, calculated for _{C102H168N2O12 : 1609.2284} )

N -MeLeu-D-Lac- N -MeLeu-D-PhLacOH (8)

Following the procedure described for the general TAGa cleavage procedure, the compound7(244 mg, 0.152 mmol) was converted to the compound8(106 mg, 97%) as a yellow oil, which was used in the next step without further purification.

N -MeLeu-D-Lac- N -MeLeu-D-PhLacO-TAGa (9)

Following the procedure described for the general Fmoc deprotection procedure, the compound7 (204 mg, 0.127 mmol) was converted to compound9(176 mg, 100%) in the form of a colorless powder.

melting point:47 - 48°C

[α] _D ²⁶ : -2.1 ( c 1.1, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.19 (m, 5H), 6.45 (s, 2H), 5.45 (q, J = 6.9 Hz, 1H), 5.31 (ddd, J = 4.6, 11.2, 19.6 Hz, 1H), 5.22 (dd, J = 5.2, 6.9 Hz, 1H), 5.03 (m, 2H), 3.94 (m, 6H), 3.33-2.95 (complex m, 3H), 2.80 (s, 3H), 2.38 (s, 3H), 1.82-1.25 (complex m, 105H), 0.96-0.75 (complex m, 21H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 175.0, 171.1, 171.0, 169.2, 153.3, 130.0, 129.8, 129.7, 129.3, 128.7, 128.6, 128.5, 127.4, 127.1 , 126.9, 107.5, 107.2, 73.8, 73.5 , 71.3, 69.2, 67.9, 67.7, 67.4, 67.3, 61.2, 57.5, 54.6, 42.3, 42.2, 37.3, 37.2, 37.0, 34.6, 32.0, 31.1, 30.4, 29.8 (x2), 29.6 , 29.5 (x2), 26.2 , 25.0, 24.8, 23.4, 23.0, 22.8, 22.7, 22.4, 22.1, 21.3, 16.9, 16.8, 14.2.

HRMS (FAB, NBA matrix) m/z : 1388.1676 [(M+H) ⁺ , calculated for C ₈₇ H ₁₅₅ N ₂ O ₁₀ : 1388.1682]

N- Fmoc -N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac-O-TAGa (10)

Compound 8 was added to a stirred solution of compound 9 (155 mg, 96.2 µmol ) in CH ₂ Cl ₂ (1.9 ml) (103 mg, 0.144 mmol), N,N- diisopropylethylamine (73 μl , 0.434 mmol) and PyBroP (112 mg, 0.241 mmol) at room temperature. After stirring for 66 hours, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 10 (201 mg, 100%) as a colorless powder.

melting point:47 - 48°C

[α] _D ²⁷ :-27.2 ( c 1.1, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (m, 2H), 7.59 (m, 2H), 7.38 (m, 2H), 7.30-7.12 (complex m, 12H), 6.44 (m, 2H) , 5.43-4.96 (complex m, 10H), 4.69-4.22 (complex m, 3H), 3.93 (m, 6H), 3.24-2.69 (complex m, 16H), 1.80-1.25 (complex m, 114H), 0.95- 0.75 (complex m, 33H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 174.1, 171.5, 171.4, 171.3, 171.2, 171.1 (x2), 170.9, 170.8, 170.7, 170.6, 170.5, 170.4, 170.0, 16 9.2, 157.0, 156.5, 153.3, 144.3, 144.2, 144.0 (x2), 141.4 (x2), 138.6, 138.4, 136.1, 136.0, 135.9 (x2), 135.7, 135.6, 135.3, 130.0, 129.8, 129.7, 129.6, 12 9.5, 129.3, 128.8, 128.7, 128.6 , 128.4, 127.7, 127.4, 127.1 (x2), 127.0. 125.3, 125.2, 125.1, 125.0, 120.0, 107.5, 107.2, 107.1, 73.9, 73.5, 72.5, 71.3, 69.2, 68.0, 67.7, 57.5, 56.6 (x2), 55.1, 55.0 , 54.9, 54.7, 54.4, 47.4, 47.3 , 40.9, 40.6, 38.7, 37.6, 37.3, 37.2, 37.1, 32.0, 31.9, 31.7, 31.5, 31.3, 31.2, 31.0, 30.7, 30.6, 30.4, 30.3, 29.8 (x2), 29.6 , 29.5 (x2), 25.0 , 24.9, 24.8, 24.7, 24.5, 24.4 (x2), 23.4, 23.3, 23.1, 23.0, 22.9, 22.8, 22.3, 22.1, 22.0, 21.3, 21.2, 16.8, 16.6, 16.5, 14.2 .

HRMS (FAB, NBA matrix) m/z : 2106.4910 [(M+Na) ⁺ , calculated for C ₁₂₈ H ₂₀₂ N ₄ O ₁₈ Na: 2106.4912]

N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac-O-TAGa

Following the procedure described for the general Fmoc deprotection procedure, the compound10 (181 mg, 86.8 µmol) was converted to the corresponding amine (163 mg, 100%) in the form of a colorless powder.

melting point:47 - 48°C

[α] _D ²⁷ : -18.7 ( c 1.3, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.20 (m, 10H), 6.44 (s, 2H), 5.50-4.95 (complex m, 9H), 3.93 (m, 6H), 3.31-2.72 (complex m , 14H), 2.36 (m, 3H), 1.80-1.25 (complex m, 114H), 0.99-0.81 (complex m, 33H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 171.4, 171.2, 171.0, 170.9, 170.6, 170.4, 153.3, 138.3, 136.1, 136.0, 135.9, 135.8, 130.0, 129.8 (x2), 129.7, 129.6 (x3) , 129.5, 129.4, 129.2, 129.2 (x2), 128.8, 128.7, 128.6, 128.4, 127.4, 127.3, 127.2 (x2), 127.1, 127.0, 107.5, 107.2, 107.1, 73. 9, 73.5, 72.3, 72.2, 71.3, 69.2 , 68.0 (x2), 67.7, 67.4, 61.3, 55.2, 55.0, 54.8, 54.5, 54.4, 42.4, 40.6, 38.7, 37.7, 37.2, 37.1, 34.7 (x2), 32.0, 31.7, 31.5, 3 1.0, 30.4, 29.8 (x2), 29.6, 29.5 (x2), 26.2, 25.0, 24.9 (x2), 24.7, 23.5, 23.4 (x2), 23.3, 23.2, 23.0, 22.8, 22.4, 22.1, 21.9, 21.3 (x2), 16.9, 16.8 (x3), 16.7, 16.6, 16.5, 14.2.

HRMS (FAB, NBA matrix) m/z : 1862.4462 [(M+H) ⁺ , calculated for C ₁₁₃ H ₁₉₃ N ₄ O ₁₆ : 1862.4412]

N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac-OH

Following the procedure described for the general TAGa cleavage procedure, the compound N -MeLeu-D-Lac- N -MeLeu-D-PhLac- N -MeLeu-D-Lac- N -MeLeu-D-PhLac-O-TAGa (143 mg, 0.768 mmol) was converted to the corresponding carboxylic acid (78 mg, 100%) as a yellow oil, which was used in the next step without further purification.

PF1022A

The crude mixture from the previous reaction (78 mg, 0.0777 mmol) was dissolved in CH₂Cl₂ (16 ml, 0.005 M). Added to the reaction mixtureN,N-diisopropylethylamine (66µl, 0.389 mmol) and PyBOP (81 mg, 0.155 mmol) at room temperature. After stirring for 48 hours, the reaction mixture was quenched with saturated aqueous NaHCO₃ (16 ml) at 0°C, and this mixture was extracted with CHCl₃ (20 ml × 2). The combined organic layers were washed with 10% aqueous NaHSO₄ (60 ml) and saline solution (60 ml), dried over Na₂SO₄, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl₃: MeOH = 400 : 1 - 40 : 1) to obtain PF1022A (48 mg, 65%) as a colorless solid.

All physical data for synthetic PF1022A are the same as those for authentic PF1022A.

5. Preparation of emodepside (method 1; see reaction schemes in Fig. 6, NMR spectrum in Fig. 7 and LC-UV spectrum in Fig. 8)

5-1. Synthesis technique

N -Fmoc -N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-O-TAGa (11)

To a stirred solution of compound 1 (259 mg, 0.233 mmol) in CH ₂ Cl ₂ (4.7 ml) was added a 0.2 M solution in toluene of compound 3 (0.122 ml, 0.244 mmol), N,N- diisopropylethylamine (0.12 ml, 0.698 mmol) and PyBroP (163 mg, 0.349 mmol) at room temperature. After stirring for 40 hours, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 11 (395 mg, 100%) as a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.74 (m, 2H), 7.57 (m, 2H), 7.42-7.27 (complex m, 4H), 7.05 (m, 2H), 6.70 (d, J = 8.6 Hz, 2H), 6.48 (s, 2H), 5.40 (dd, J = 6.9, 8.4 Hz, 3/10H, rotamer), 5.35-5.27 (complex m, 17/10H), 5.10-4.97 (complex m, 4H), 4.72-4.12 (complex m, 3H), 3.93 (m, 6H), 3.75 (m, 4H), 3.00-2.79 (complex m, 12H), 1.80-1.60 (complex m, 9H), 1.49-1.42 (complex m, 9H), 1.27 (complex m, 87H), 0.93-0.80 (complex m, 21H).

HRMS (FAB, NBA matrix) m/z : 1717.2701 [(M+Na) ⁺ , calculated for C ₁₀₆ H ₁₇₁ N ₃ O ₁₃ Na: 1717.2710]

N -Fmoc -N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-OH (12)

Following the procedure described for the general TAGa cleavage procedure, the compoundeleven(210 mg, 0.124 mmol) was converted to compound12(100 mg, ~0.124 mmol) in the form of a brown oil. The crude product was used in the next step without further purification.

N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-O-TAGa (13)

Following the procedure described for the general Fmoc deprotection procedure, the compoundeleven (158 mg, 0.0934 mmol) was converted to13(138 mg, 100%) in the form of a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.15 (m, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.49 (s, 2H), 5.47 (dd, J = 6.9, 8.0 Hz, 1H), 5.32 (dd, J = 4.9, 11.2 Hz, 1H), 5.09-5.00 (complex m, 3H), 3.94 (m, 6H), 3.85 (m, 4H), 3.27 (t, J = 6.9 Hz, 1H), 3.13-2.79 (complex m, 9H), 2.29 (s, 3H), 1.81-1.25 (complex m, 105H), 1.01-0.78 (complex m, 21H).

HRMS (FAB, NBA matrix)m/z: 1473.2214 [(M+H)⁺, calculated for C₉₁H₁₆₂N₃O_eleven : 1473.2209]

N- Fmoc -N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-O-TAGa (14)

To a stirred solution of compound 13 (138 mg, 0.934 mmol) in CH ₂ Cl ₂ (1.9 ml) was added 12 (100 mg, ~0.124 mmol), N,N -diisopropylethylamine (48 μl , 0.280 mmol) and PyBroP (65 mg, 0.140 mmol) at room temperature. After stirring for 18 hours, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 14 (211 mg, 100%) as a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (m, 2H), 7.58 (m, 2H), 7.42-7.28 (complex m, 4H), 7.09 (m, 4H), 6.77 (m, 4H) , 6.48 (s, 2H), 5.45-5.15 (complex m, 6H), 5.06-4.97 (complex m, 4H), 4.73-4.12 (complex m, 3H), 3.95-3.73 (complex m, 14H), 3.15- 2.73 (complex m, 24H), 1.80-1.25 (complex m, 114H), 0.96-0.77 (complex m, 33H).

HRMS (FAB, NBA matrix)m/z: 2276.5962 [(M+Na)⁺, calculated for C₁₃₆H₂₁₆N₆O₂₀Na : 2276.5967]

N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-O-TAGa

Following the procedure described for the general Fmoc deprotection procedure, the compound14 (211 mg, 0.0934 mmol) was converted to the corresponding amine (178 mg, 94%) in the form of a colorless powder.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.15 (m, 4H), 6.82 (m, 4H), 6.49 (s, 2H), 5.14 (t, J = 7.45 Hz, 1H), 5.44-5.09 ( complex m, 5H), 5.07-5.00 (complex m, 3H), 3.93 (m, 6H), 3.85 (m, 8H), 3.32 (m, 1H), 3.17-2.74 (complex m, 21H), 2.33 (s , 3H), 1.81-1.64 (complex m, 12H), 1.55-1.25 (complex m, 102H), 1.03-0.77 (complex m, 33H).

HR-MS (FAB, NBA matrix+NaI)m/z: 2032.5488 [(M+H)⁺, calculated for C₁₂₁H₂₀₇N₆O₁₈ : 2032.5467]

N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac-OH

Following the procedure described for the general TAGa cleavage procedure, the compound N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-LacO-TAGa (178 mg, 0.0876 mmol) was converted to the corresponding carboxylic acid (~0.0876 mmol) as a crude oil, which was used in the next step without further purification.

Emodepside (high dilution condition using PyBOP)

To the crude carboxylic acid (~0.0876 mmol) in CH ₂ Cl ₂ (18 ml, 0.005 M) was added N,N- diisopropylethylamine (0.10 ml, 0.613 mmol) and PyBOP (91 mg, 0.175 mmol) at room temperature. After stirring for 19 hours, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (18 ml) at 0°C, and the mixture was extracted with CHCl ₃ (20 ml x 3). The combined organic layers were washed with 10% aqueous NaHSO ₄ (60 ml) and brine (60 ml), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl ₃ : MeOH = 400: 1 - 100: 1) to give emodepside (45 mg, 46% for 2 steps) as a light yellow solid.

melting point:98-103°C

[α] _D ²⁴ : -40.3 ( c 0.67, CHCl ₃ )

IR (clean) : 2954, 2862, 1743, 1659, 1520, 1458, 1412, 1265, 1234, 1188, 1119, 1072, 1026, 926, 810.

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.14-7.11 (complex m, 4H), 6.83-6.79 (complex m), 5.64-5.54 (complex m), 5.52-5.43, 5.43-5.39, 5.33, 5.18 , 5.06 and 4.46 (5 m, 6H), 3.85 (apparently t, 8H), 3.15-3.04 (complex m, 8H), 3.04-2.92 (complex m, 4H), 3.00, 2.82, 2.79, 2.72 and 2.71 (5 s, 12H), 1.82-1.24 (complex m, 18H), 1.03-0.79 (complex m, 30H).

¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 171.6, 171.2, 171.0, 170.9, 170.3, 170.2, 170.1, 169.8, 169.7, 150.2, 130.4, 130.2, 115.7, 115.6 , 71.2, 70.8, 68.5, 66.8, 66.7 , 57.1, 54.0, 53.9, 49.3, 49.2, 38.0, 37.5, 37.1, 36.9, 36.8, 36.7, 36.6, 36.1, 31.1, 30.6, 30.4, 29.7, 29.6, 29.3, 25.0, 2 4.8, 24.6, 24.5, 24.5, 24.1 , 23.6, 23.5, 23.4, 23.3, 23.3, 23.1, 22.6, 21.6, 21.5, 21.1, 21.1, 21.0, 20.8, 17.1, 15.7.

HRMS (ESI)m/z: 1141.6404 [(M+Na)⁺, calculated for C₆₀H₉₀N₆O₁₄ Na: 1141.6413]

*References ( Eur. J. Org. Chem. , 2012 , 1546-1553)

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.17-7.09 (m, 4 H, Ar-H), 6.86-6.77 (m, 4 H, Ar-H), 5.67-5.54 (m, 2 H, CαH-Lac), 5.53-5.38, 5.34, 5.19, 5.08 and 4.47 (5 m, 6 H, CαH-Leu, CαH-morphPhLac), 3.88-3.81 (pseudo-t, 8 H, OCH ₂ morpholine), 3.15- 3.08 (m, 8 H, N-CH ₂ -morpholine), 3.07-2.85 (m, 4 H, CβH ₂ -morphPhLac), 3.00, 2.83, 2.80, 2.74 and 2.73 (5 s, 12 H, NCH ₃ ), 1.83-1.20 (m, 18 H, CβH ₂ -Leu, CγH-Leu, CH ₃ -Lac), 1.05-0.77 (m, 30 H, CδH ₃ -Leu, CβH ₃ -Lac).

¹³ C-NMR (100 MHz, CDCl ₃ ) δ: 171.7, 171.2, 171.0, 170.6, 170.4, 170.2, 169.8, 141.7, 130.6, 130.4, 116.9, 116.1, 71.3, 70.8, 68.6, 66.9, 66.6, 66.5, 57.1 , 54.0, 49.9, 38.1, 37.5, 37.2, 36.7, 36.2, 31.2, 30.5, 29.4, 24.9, 24.7, 24.2, 23.6, 23.5, 23.5, 23.4, 21.2, 21.1, 20.9, 1 7.1, 15.8.

Emodepside (slow addition condition using T3P)

TAGa Cleavage: TFA (1.8 mL) was added to a stirred solution of the linear compound with TAG (179.0 mg, 0.088 mmol) in DCM (1.8 mL) at room temperature. After stirring for 6 hours at room temperature, the solution was concentrated in vacuo. The resulting mixture was dissolved in toluene (10 ml) and concentrated under reduced pressure 3 times to remove excess TFA. The crude residue was dissolved in CH ₂ Cl ₂ (1.0 ml), then recrystallized to eliminate TAGa substances by adding MeOH (8.0 ml) at room temperature. The precipitates were filtered through a pad of Celite ^® and washed with MeOH (20 ml). The combined filtrates were concentrated in vacuo. 4 M HCl/dioxane (0.05 M for product) was added to the resulting product, then diluted with toluene (10 mL) and concentrated to give the TFA salt-free product compound. To remove excess HCl from the crude product, the product was again diluted in toluene (10 ml) and concentrated under reduced pressure 2 times.

Cyclization: To a stirred solution of T3P ^® (50% in EtOAc, 110 µl, 0.187 mmol) in DIPEA (110 µl, 0.187 mmol) was added dropwise the crude linear compound (0.088 mmol) in DCM (1.8 ml, 0.05 M including rinse) for 2.5 hours at room temperature. After stirring for 20 hours at room temperature, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (3.0 ml), extracted with CHCl ₃ (2.0 ml × 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (CHCl ₃ /MeOH = 100/1) to obtain emodepside (86.4 mg, 88%) in amorphous form. Analytical data were identical to those for the authentic compound.

6. Preparation of emodepside (method 2; see reaction scheme in Fig. 9)

N -Fmoc -N -MeLeu-D-morphPhLac-O-TAGa

To a stirred solution of compound HO-TAGa (381 mg, 0.417 mmol) in CH ₂ Cl ₂ (8.4 ml) was added a 0.2 M solution in toluene of substance 3 (2.71 ml, 0.542 mmol), 4-dimethylaminopyridine (2.5 mg, 20.8 μmol) and N,N' -dicyclohexylcarbodiimide (129 mg, 0.626 mmol) at room temperature under N ₂ atmosphere. After stirring for 1 hour, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain the compound N -Fmoc -N -MeLeu-D-morphPhLac-O-TAGa (628 mg, 100%) as a colorless powder.

melting point : 46-47°C

[α] _D ²⁷ = -3.1 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.78 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.57 (m, 2H), 7.39 (m, 2H) , 7.27 (m, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.67 (m, 2H), 6.49 (2 s, 4:3 rotamer, 2H ), 5.17 (2 dd, rotamer, J = 4.0 Hz, 8.0 Hz, 1H), 5.08-4.98 (complex m, 3H), 4.67-4.13 (complex m, 3H), 3.93 (m, 6H), 3.73 (m , 4H), 3.08 (dd, J = 4.0 Hz, 14.6 Hz, 1H), 3.07-2.95 (complex m, 5H), 2.80 (rotamer 4:3, 3H), 1.76 (m, 6H), 1.64- 1.53 (complex m, 3H), 1.45 (m, 6H), 1.28 (complex m, 84H), 0.93-0.86 (complex m, 14H), 0.76 (d, J = 6.3 Hz, 1H).

HRMS (FAB, NBA matrix)m/z: 1495.1583 (M⁺, calculated for C₉₆H₁₅₄N₂O₁₀ : 1495.1604)

N -MeLeu-D-morphPhLac-O-TAGa (15)

Following the procedure described for the general Fmoc deprotection procedure, the compound N -Fmoc -N -MeLeu-D-morphPhLac-O-TAGa (628 mg, 0.417 mmol) was converted to the compound15 (530 mg, 100%) in the form of a colorless powder.

melting point : 49-50°C

[α] _D ²⁷ = +5.7 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.08 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.51 (s, 2H), 5.24 (dd, J = 4.0, 9.7 Hz, 1H), 5.07 (q, J = 12.0 Hz, 2H), 3.94 (m, 6H), 3.85 (m, 4H), 3.18 (m, 2H), 3.10 (m, 4H), 3.00 ( d, J = 10.3, 14.3 Hz, 1H), 2.21 (s, 3H), 1.76 (m, 6H), 1.47 (m, 6H), 1.34-1.25 (complex m, 87H), 0.88 (t, J = 6.9 Hz, 9H), 0.80 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.9 Hz, 3H).

HRMS (FAB, NBA matrix)m/z : 1274.0986 [(M+H)⁺, calculated for C₈₁H₁₄₅N₂O₈ : 1274. 1001]

N -Fmoc -N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-O-TAGa (16)

To the mixed solution of the compound15 (530 mg, 0.417 mmol) in CH₂Cl₂(8.4 ml) added 0.2 M solution in toluenesubstances 1(0.22 ml, 0.44 mmol),N,N-diisopropylethylamine (0.212 ml, 1.25 mmol) and PyBroP (291 mg, 0.62 mmol) at room temperature. After stirring for 16 hours, the reaction mixture was crystallized using the procedure described in the synthesis N -Fmoc -N -MeLeu-D-Lac-O-TAGa, with receiving connection16(670 mg, 95%) as a colorless powder.

melting point : 48-49°C

[α] _D ²⁷ = -12.4 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.76 (m, 2H), 7.61 (m, 2H), 7.38 (m, 2H), 7.30 (m, 2H), 7.02 (m, 2H), 6.74 ( m, 2H), 6.49 (2 s, rotamer, 2H), 5.38-5.22 (complex m, 2H), 5.15-4.98 (complex m, 4H), 4.47 (complex m, 3H), 3.94 (m, 6H), 3.81 (m, 4H), 3.12-2.78 (complex m, 12H), 1.82-1.56 (complex m, 9H), 1.53-1.40 (complex m, 8H), 1.34-1.26 (complex m, 88H), 0.98-0.75 (complex m, 21H).

HRMS (FAB _, NBA matrix) m/z : 1694.2828 (M ⁺ _, calculated for _{C106H171N3O13} : _1694.2812 )

N -Fmoc- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-OH (17)

Following the procedure described for the general TAGa cleavage procedure, compound 16 (376 mg, 0.222 mmol) was converted to compound 17 . In the case of this substrate, the reaction required more time than in the case of the general TAGa elimination condition (about 1 h). The reaction of compound 16 in 50% TFA/CH ₂ Cl ₂ at room temperature required stirring for 8 hours to consume all the starting material, giving product 17 (178 mg, 0.222 mmol) as a crude oil, which was used in the next step without further cleaning.

N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-O-TAGa (18)

Following the procedure described for the general Fmoc deprotection procedure, the compound16(290 mg, 0.171 mmol) was converted to compound18 (251 mg, 100%) in the form of a colorless powder.

melting point : 43-45°C

[α] _D ²⁵ = -2.8 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.07 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.6 Hz, 2H), 6.49 (s, 2H), 5.46 (q, J = 6.9 Hz, 1H), 5.33 (dd, J = 5.2, 10.9 Hz, 1H), 5.17 (dd, J = 5.2, 7.5 Hz, 1H), 5.06 (m, 2H), 3.95 (m, 6H), 3.84 ( m, 4H), 3.33 (t, J = 7.5 Hz, 1H), 3.11-3.06 (complex m, 6H), 2.82 (2 s, rotamer 4:1, 3H), 2.40 (s, rotamer, 3H), 1.82 -1.59 (complex m, 10H), 1.52-1.43 (complex m, 8H), 1.34-1.25 (complex m, 87H), 0.97-0.86 (complex m, 21H).

HRMS (FAB, NBA matrix + NaI)m/z: 1473.2222 [(M+H)⁺, calculated for C₉₁H₁₆₂N₃O_eleven : 1473.2209]

N -Fmoc- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-O-TAGa (19)

To a stirred solution of compound 18 (251 mg, 0.171 mmol) in CH ₂ Cl ₂ (3.4 ml) was added 17 (178 mg, 0.222 mmol), N,N- diisopropylethylamine (87 μl , 0.513 mmol) and PyBroP (120 mg, 0.257 mmol) at room temperature. After stirring for 46 h, the reaction mixture was crystallized using the procedure described in the synthesis of N -Fmoc -N -MeLeu-D-Lac-O-TAGa to obtain compound 19 (350 mg, 91%) as a colorless powder.

melting point : 50-52°C

[α] _D ²⁵ = -25.5 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.75 (m, 2H), 7.62 (m, 2H), 7.38 (m, 2H), 7.30 (m, 2H), 7.06 (m, 4H), 6.77 ( m, 4H), 6.49 (2 s, rotamer, 2H), 5.42-4.98 (complex m, 10H), 4.73-4.20 (complex m, 3H), 3.94 (m, 6H), 3.84 (m, 8H), 3.17 -2.66 (complex m, 24H), 1.80-1.64 (complex m, 14H), 1.46-1.25 (complex m, 100H), 1.00-0.77 (complex m, 33H).

HRMS (FAB, NBA matrix)m/z: 2276.5940 [(M+Na)⁺, calculated for C₁₃₆H₂₁₆N₆O₂₀Na : 2276.5967]

N- MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-O-TAGa

Following the procedure described for the general Fmoc deprotection procedure, the compound19 (114 mg, 0.0505 mmol) was converted to the corresponding amine (103 mg, 100%) in the form of a colorless powder.

melting point : 45-47°C

[α] _D ²⁵ = -19.6 ( c 1.0, CHCl ₃ )

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 7.76 (m, 4H), 6.79 (m, 4H), 6.48 (s, 2H), 5.50-4.96 (complex m, 9H), 3.94 (m, 6H) , 3.83 (m, 8H), 3.30 (m, 1H), 3.16-2.74 (complex m, 21H), 2.38 (m, 3H), 1.80-1.55 (complex m, 9H), 1.47-1.24 (complex m, 105H ), 1.00-0.81 (complex m, 33H).

HRMS (FAB, NBA matrix)m/z: 2032.5468 [(M+H)⁺, calculated for C₁₂₁H₂₀₇N₆O₁₈ : 2032.5467]

N- MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morphPhLac-OH

Following the procedure described for the general TAGa cleavage procedure, the compound, N- MeLeu-D-Lac- N -MeLeu-D-morphPhLac- N -MeLeu-D-Lac- N -MeLeu-D-morph-PhLac-O-TAGa ( 102 mg, 0.0502 mmol) was converted to the corresponding carboxylic acid. In the case of this substrate, the reaction required more time than in the case of the general TAGa elimination condition (about 1 h). The reaction in 50% TFA/CH ₂ Cl ₂ at room temperature needed to be stirred for 5 hours to consume all the starting material and give the desired product (~0.0502 mmol) as a crude oil, which was used in the next step without further purification.

Emodepside

N,N- diisopropylethylamine (60 μL , 0.351 mmol) and PyBOP (52 mg, 0.175 mmol) were added to the crude carboxylic acid (~0.0502 mmol) in CH ₂ Cl ₂ (10 ml, 0.005 M) at room temperature. After stirring for 44 hours, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (10 ml) at 0°C, and the mixture was extracted with CHCl ₃ (20 ml x 3). The combined organic layers were washed with 10% aqueous NaHSO ₄ (60 ml) and brine (60 ml), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (CHCl ₃ : MeOH = 400: 1 - 100: 1) to give emodepside (25 mg, 44% for 2 steps) as a light brown solid.

All physical data for the synthetic emodepside are the same as those for the authentic compound.

Comparative example 1

TAG, similar to TAGa but with _C12 instead of _C18 alkyl chains (C12-TAG), was prepared and coupled to N -Fmoc- N -MeLeu-D-Lac-OH (substance 1) according to the following reaction scheme:

Purification by silica gel chromatography was necessary after each reaction step. Compounds 2, C12-TAG and compound 3 do not crystallize in methanol. This is in contrast, for example, to the procedure for the synthesis of N -Fmoc- N -MeLeu-D-Lac-O-TAGa (part 2.1 above). The C12-TAG in this comparative example is not suitable for the intended synthesis using the TAG group, and further functionalization of compound 3 was not further explored.

Comparative example 2

Commercially available C1-TAG was coupled to N -Fmoc- N -MeLeu-D-Lac-OH (substance 1) according to the following reaction scheme:

Purification by column chromatography was necessary after the reaction step. Compounds C1-TAG and 6 do not crystallize in methanol. The C1-TAG in this comparative example is not suitable for the intended synthesis using the TAG group, and further functionalization of compound 6 was not further explored.

Claims (122)

1. A method for the synthesis of cyclic depsipeptides of general formula (I) on an industrial scale from depsipeptides of general formula (II): where B represents an amine protecting group and A represents a carboxylic acid protecting group, wherein the method includes the stages: - removing the protecting group of the amine group, which is protected by group B, thus obtaining an unprotected amine group; - removing the protecting group of the carboxylic acid which is protected by group A, thus obtaining an unprotected carboxylic acid group; - condensation of an unprotected amine group and an unprotected carboxylic acid group using a coupling agent, wherein the coupling agent is added slowly, preferably dropwise, to the reaction solution, thereby obtaining cyclic depsipeptide (I), wherein R2 and R8 each independently represent hydrogen, straight or branched C1-C8 alkyl, in particular methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, hexyl, isohexyl, sec-hexyl, heptyl, isoheptyl, sec-heptyl, tert-heptyl, octyl, isooctyl, sec-octyl, straight or branched halogenated C1-C8 alkyl, in particular fluorinated sec-butyl, hydroxy- C1-C6-alkyl, in particular hydroxymethyl, 1-hydroxyethyl, C1-C4-alkanoyloxy-C1-C6-alkyl, in particular acetoxymethyl, 1-acetoxyethyl, C1-C4-alkoxy-C1-C6-alkyl, in particular methoxymethyl, 1-methoxyethyl, aryl-C1-C4-alkyloxy-C1-C6-alkyl, in particular benzyloxymethyl, 1-benzyloxyethyl, mercapto-C1-C6-alkyl, in particular mercaptomethyl, C1-C4-alkylthio-C1-C6-alkyl, in particular methylthioethyl, C1-C4-alkylsulfinyl-C1-C6-alkyl, in particular methylsulfinylethyl, C1-C4-alkylsulfonyl-C1-C6-alkyl, in particular methylsulfonylethyl, carboxy-C1-C6-alkyl, in particular carboxymethyl, carboxyethyl, C1-C4-alkoxycarbonyl-C1-C6-alkyl, in particular methoxycarbonylmethyl, ethoxycarbonylethyl, C1-C4-arylalkoxycarbonyl-C1-C6-alkyl, in particular benzyloxycarbonylmethyl, carbamoyl-C1-C6-alkyl, in particular carbamoylmethyl, carbamoylethyl, amino- C1-C6-alkyl, in particular aminopropyl, aminobutyl, C1-C4-alkylamino-C1-C6-alkyl, in particular methylaminopropyl, methylaminobutyl, C1-C4-dialkylamino-C1-C6-alkyl, in particular dimethylaminopropyl, dimethylaminobutyl, guanidino- C1-C6-alkyl, in particular guanidinopropyl, C1-C4-alkoxycarbonylamino-C1-C6-alkyl, in particular tert-butoxycarbonylaminopropyl, tert-butoxycarbonylaminobutyl, 9-fluorenylmethoxycarbonyl(Fmoc)amino-C1-C6-alkyl, in particular 9- fluorenylmethoxycarbonyl(Fmoc)aminopropyl, 9-fluorenylmethoxycarbonyl(Fmoc)aminobutyl, C2-C8-alkenyl, in particular vinyl, allyl, butenyl, C3-C7-cycloalkyl, in particular cyclopentyl, cyclohexyl, cycloheptyl, C3-C7-cycloalkyl-C1- C4-alkyl, in particular cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, benzyl, substituted benzyl, phenyl, phenyl-C1-C4-alkyl, in particular phenylmethyl, which may be optionally substituted by radicals from the group consisting of halogen, in particular fluorine, chlorine, bromine or iodine, where x is 1, y is 1, R1, R4, R7 and R10 are methyl, R6 and R12 are methyl, R5 and R11 are, independently of each other, straight or branched C1-C4 alkyl or straight or branched halogenated C1-C4 alkyl and R3 and R9 are, independently of each other, benzyl or p-morpholino-substituted benzyl and wherein the coupling agent is T3P® (propylphosphonic acid anhydride, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide, PPACA). 2. The method according to claim 1, wherein cyclic depsipeptides of general formula (I) are synthesized from depsipeptides of general formula (IIa): where Y is an amine protecting group and X is a carboxylic acid protecting group, wherein the method includes the stages: - removing the protecting group of the amine group, which is protected by the Y group, in the presence of an acid, thereby obtaining an unprotected amine group; - removing the protecting group of the carboxylic acid group, which is protected by group X, by hydrogenolysis, thereby obtaining an unprotected carboxylic acid group; - condensation of an unprotected amine group and an unprotected carboxylic acid group, thereby obtaining a cyclic depsipeptide (I). 3. The method according to claim 1 or 2, wherein X represents a substituted or unsubstituted -CH ₂ -aryl group. 4. Method according to any one of paragraphs. 1-3, wherein X is selected from the group of benzyl (Bn), 4-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DPMB), 4-phenylbenzyl (PPB), 2-naphthylmethyl (Nap), benzyloxymethyl acetal ( BOM). 5. Method according to any one of paragraphs. 1-4, wherein Y represents tert-butyloxycarbonyl (BOC). 6. Method according to any one of paragraphs. 1-5, where the depsipeptide of general formula (IIa) is obtained from precursors of general formulas (IV) and (III): through: - in the precursor (IV), removing the protecting group of the amine group, which is protected by the PG2 group, in the presence of a base, thus obtaining an unprotected amine group; - in the precursor (III), removing the protecting group of the carboxylic acid group, which is protected by the PG3 group, in the presence of an acid, thus obtaining an unprotected carboxylic acid group; - condensation of an unprotected amine group and an unprotected carboxylic acid group, thus obtaining depsipeptide (II); where R1-R12, X, Y, x and y have the meanings as defined in any of paragraphs. 1-3, PG2 represents a protecting group of an amine group and PG3 represents a protecting group of a carboxylic acid group. 7. Method according to any one of paragraphs. 1-6, wherein the precursor of general formula (IV) is obtained from the precursors of general formulas (VI) and (V): through: - in the precursor (VI), removing the protecting group of the amine group, which is protected by the PG4 group, in the presence of a base, thus obtaining an unprotected amine group; - condensation of the unprotected amine group of the precursor (VI) and the carboxylic acid group of the precursor (V), thus obtaining the precursor (IV); where R7-R12, X and x are as defined above, PG2 is as defined above, and PG4 is a protecting group of an amine group, and the deprotection and condensation methods may be the same as those specified for the reaction of the compounds (II)-(I) above. 8. Method according to any one of paragraphs. 1-7, where the precursor of the general formula (III) is obtained from the precursors of the general formulas (VIII) and (VII): through: - in the precursor (VII), removing the protecting group of the amine group, which is protected by the PG5 group, in the presence of a base, thus obtaining an unprotected amine group; - condensation of the unprotected amine group of the precursor (VII) and the carboxylic acid group of the precursor (VIII), thus obtaining the precursor (III); where R1-R6 and y have the meanings as defined in any of paragraphs. 1-3, PG3 has the meaning as defined in paragraph 3, PG1 and PG5 are protecting groups of the amine group. 9. Method according to any one of paragraphs. 1-8, where the precursor of general formula (VI) is obtained by esterification of the precursor of general formula (IX) with X-LG: wherein R10-R12 and X are as defined in claim 1 or 2, PG4 is as defined in claim 7, and LG is a leaving group. 10. Method according to any one of paragraphs. 1-9, where the precursor of general formula (VII) is obtained by esterification of the precursor of general formula (X) with PG3-OH: where R4-R6 and y have the meanings as defined in clause 1, PG3 has the meaning as defined in clause 6, and PG5 has the meaning as defined in clause 8. 11. Method according to any one of paragraphs. 1-10, where PG3 represents X. 12. Method according to any one of paragraphs. 1-11, where the precursors (III) and (IV) are identical. 13. Method according to any one of paragraphs. 1-12, where R3 and R9 are identical, R2 and R8 are identical, R5 and R11 are identical. 14. The method according to claim 1, including the stage of synthesis of cyclic depsipeptides of general formula (I) from depsipeptides of general formula (IIb): where PG1 represents an amine protecting group and TAG represents a carboxylic acid protecting group, wherein the method includes the stages: - removing the protecting group of the amine group, which is protected by the PG1 group, in the presence of a base, thereby obtaining an unprotected amine group; - removing the protecting group of the carboxylic acid group, which is protected by the TAG group, in the presence of an acid, thereby obtaining an unprotected carboxylic acid group; - condensation of an unprotected amine group and an unprotected carboxylic acid group, thereby obtaining a cyclic depsipeptide (I); characterized in that the TAG group is: or , wherein m is ≥15 to ≤25, p is ≥8 to ≤18, and q is ≥15 to ≤25. 15. The method according to claim 14, where PG1 is 9-fluorenylmethoxycarbonyl (Fmoc), tert-butylcarbamate (Boc), benzylcarbamate (Z), acetamide, trifluoroacetamide, phthalimide, benzyl (Bn), triphenylmethyl (Tr), benzylidene or p -toluenesulfonamide (Ts). 16. Method according to claim 14 or 15, where the depsipeptide of general formula (IIb) is obtained from precursors of general formulas (IVb) and (IIIb): through: - in the precursor (IVb), removing the protecting group of the amine group, which is protected by the PG2 group, in the presence of a base, thus obtaining an unprotected amine group; - in the precursor (IIIb), removing the protecting group of the carboxylic acid group, which is protected by the PG3 group, in the presence of an acid, thus obtaining an unprotected carboxylic acid group; - condensation of an unprotected amine group and an unprotected carboxylic acid group, thereby obtaining depsipeptide (IIb); where R1-R12, x and y have the meanings as defined in clause 1, PG1 has the meaning as defined in clause 8, TAG has the meaning as defined in clause 14, and PG2 and PG3 have the meanings as defined in clause clause 6. 17. Method according to any one of paragraphs. 14-16, wherein the precursor of general formula (IVb) is obtained from the precursors of general formulas (VIb) and (Vb): through: - in the precursor (VIb), removing the protecting group of the amine group, which is protected by the PG4 group, in the presence of a base, thus obtaining an unprotected amine group; - condensation of the unprotected amine group of the precursor (VIb) and the carboxylic acid group of the precursor (Vb), thereby obtaining the precursor (IVb); where R7-R12 and x have the meanings as defined in clause 1, TAG has the meaning as defined in clause 14, PG2 has the meaning as defined in clause 6, and PG4 has the meaning as defined in clause 7. 18. Method according to any one of paragraphs. 14-17, where the precursor of general formula (IIIb) is obtained from the precursors of general formulas (VIIIb) and (VIIb): through: - in the precursor (VIIb), removing the protecting group of the amine group, which is protected by the PG5 group, in the presence of a base, thus obtaining an unprotected amine group; - condensation of the unprotected amine group of the precursor (VIIb) and the carboxylic acid group of the precursor (VIIIb), thereby obtaining the precursor (IIIb); where R1-R6 and y have the meanings as defined in clause 1, PG1 and PG5 have the meanings as defined in clause 8, and PG3 have the meanings as defined in clause 6. 19. Method according to any one of paragraphs. 14-18, where the precursor of the general formula (VIb) is obtained by esterification of the precursor of the general formula (IXb) with TAG-OH: where R10-R12 have the meanings as defined in clause 1, TAG has the meaning as defined in clause 14, and PG4 has the meaning as defined in clause 7. 20. Method according to any one of paragraphs. 14-19, where the precursor of the general formula (VIIb) is obtained by esterification of the precursor of the general formula (Xb) with PG3-OH: where R4-R6 and y have the meanings as defined in clause 1, PG3 has the meaning as defined in clause 6, and PG5 has the meaning as defined in clause 8. 21. Method according to any one of paragraphs. 14-20, where PG3 is a TAG as defined in claim 14. 22. Method according to any one of paragraphs. 14-21, wherein at least one of the reaction steps leading to the production of the TAG-bearing molecule is accompanied by precipitation of the crude reaction product in methanol, thereby purifying the crude reaction product. 23. Method according to any one of paragraphs. 14-21, wherein at least one of the reaction steps in which the TAG-protected carboxylic acid groups are deprotected is accompanied by precipitation of the separated TAG-OH in methanol and removal of the precipitate by filtration, thereby purifying the crude reaction product. 24. Method according to any one of paragraphs. 1-23, where depsipeptide (II) is selected from one of the general formulas (II-1)-(II-6b): , where B is an amine protecting group, A is a carboxylic acid protecting group, Y is an amine protecting group removed by acid, X is a carboxylic acid protecting group removed by hydrogenolysis, PG1 is an amine protecting group group and TAG has the meaning as defined in paragraph 14. 25. Method for the synthesis of cyclic depsipeptides of general formula (I) on an industrial scale from depsipeptides of general formula (IIc): including stages providing a mixture of compound (IIc) and base in a solvent having an E _T (30) value of ≥30 to ≤43, slowly, preferably dropwise, adding a solution of the binding agent in the solvent to form cyclic depsipeptide (I), wherein R2 and R8 each independently represent hydrogen, straight or branched C1-C8 alkyl, in particular methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, hexyl, isohexyl, sec-hexyl, heptyl, isoheptyl, sec-heptyl, tert-heptyl, octyl, isooctyl, sec-octyl, straight or branched halogenated C1-C8 alkyl, in particular fluorinated sec-butyl, hydroxy- C1-C6-alkyl, in particular hydroxymethyl, 1-hydroxyethyl, C1-C4-alkanoyloxy-C1-C6-alkyl, in particular acetoxymethyl, 1-acetoxyethyl, C1-C4-alkoxy-C1-C6-alkyl, in particular methoxymethyl, 1-methoxyethyl, aryl-C1-C4-alkyloxy-C1-C6-alkyl, in particular benzyloxymethyl, 1-benzyloxyethyl, mercapto-C1-C6-alkyl, in particular mercaptomethyl, C1-C4-alkylthio-C1-C6-alkyl, in particular methylthioethyl, C1-C4-alkylsulfinyl-C1-C6-alkyl, in particular methylsulfinylethyl, C1-C4-alkylsulfonyl-C1-C6-alkyl, in particular methylsulfonylethyl, carboxy-C1-C6-alkyl, in particular carboxymethyl, carboxyethyl, C1-C4-alkoxycarbonyl-C1-C6-alkyl, in particular methoxycarbonylmethyl, ethoxycarbonylethyl, C1-C4-arylalkoxycarbonyl-C1-C6-alkyl, in particular benzyloxycarbonylmethyl, carbamoyl-C1-C6-alkyl, in particular carbamoylmethyl, carbamoylethyl, amino- C1-C6-alkyl, in particular aminopropyl, aminobutyl, C1-C4-alkylamino-C1-C6-alkyl, in particular methylaminopropyl, methylaminobutyl, C1-C4-dialkylamino-C1-C6-alkyl, in particular dimethylaminopropyl, dimethylaminobutyl, guanidino- C1-C6-alkyl, in particular guanidinopropyl, C1-C4-alkoxycarbonylamino-C1-C6-alkyl, in particular tert-butoxycarbonylaminopropyl, tert-butoxycarbonylaminobutyl, 9-fluorenylmethoxycarbonyl(Fmoc)amino-C1-C6-alkyl, in particular 9- fluorenylmethoxycarbonyl(Fmoc)aminopropyl, 9-fluorenylmethoxycarbonyl(Fmoc)aminobutyl, C2-C8-alkenyl, in particular vinyl, allyl, butenyl, C3-C7-cycloalkyl, in particular cyclopentyl, cyclohexyl, cycloheptyl, C3-C7-cycloalkyl-C1- C4-alkyl, in particular cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, benzyl, substituted benzyl, phenyl, phenyl-C1-C4-alkyl, in particular phenylmethyl, which may be optionally substituted by radicals from the group consisting of halogen, in particular fluorine, chlorine, bromine or iodine, where x is 1, y is 1, R1, R4, R7 and R10 are methyl, R6 and R12 are methyl, R5 and R11 are, independently of each other, straight or branched C1-C4 alkyl or straight or branched halogenated C1-C4 alkyl and R3 and R9 are, independently of each other, benzyl or substituted benzyl and wherein the coupling agent is T3P® (propylphosphonic acid anhydride, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide, PPACA). 26. The method according to claim 25, wherein the E _T (30) value of the solvent is from ≥34 to ≤39. 27. The method according to claim 25 or 26, wherein the addition of the binding agent solution is carried out at a rate of ≤2 (mol)% per minute. 28. Method according to any one of paragraphs. 25-27, and the ratio of base to compound (IIb) before the reaction (in mol:mol) is from ≥2:1 to ≤10:1. 29. Method according to any one of paragraphs. 25-28, and the ratio of the coupling agent to the compound (IIb) before the reaction (in mol:mol) is from ≥1:1 to ≤5:1. 30. Method according to any one of paragraphs. 25-29, and during the addition of the binding agent the temperature is maintained at ≤25°C. 31. A linear depsipeptide selected from one of the general formulas (II-1)-(II-6), or a pharmaceutically or veterinarily acceptable salt thereof: where B is an amine protecting group, A is a carboxylic acid protecting group, Y is an amine protecting group removed by acid, and X is a carboxylic acid protecting group removed by hydrogenolysis, PG1 is a protecting group amine group and TAG has the meaning as defined in paragraph 14.