Methods for synthesizing peptides with sterically hindered tri-tert-butyl-tryptophan (Tbt) residues

Abstract

The present invention is directed to a method of peptide synthesis, the method comprising reacting a compound of formula (I), or a salt thereof, with a carbodiimide reagent to form an O-acylisourea intermediate; reacting the O-acylisourea intermediate with an additive to form an activated ester; and reacting the activated ester with an amino-containing moiety, which is an amino acid, peptide, or salt thereof, containing an amino group, wherein the amino group forms an amide bond with the carbonyl marked with an * in formula (I), wherein the compound of formula (I) has the structure: TIFF2026501761000047.tif61170 formula (I) where R 1 and R 2 is as defined in the present disclosure. The present invention is also directed to compounds of formula (III) or salts thereof as defined in the present disclosure.

Description

[Technical Field]

[0001] The present invention is directed to methods of peptide synthesis and methods for producing target peptides. [Background technology]

[0002] Many coupling strategies are available for the laboratory-scale production of peptides. However, most of these coupling strategies are too expensive for commercial-scale production. Coupling reactions between amino acids are almost exclusively facilitated by activation of the carboxylic acid of the incoming amino acid. For example, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HTBU) is an efficient activator that results in minimal racemization. However, the cost of HTBU is high.

[0003] It would be desirable to be able to use lower-cost activating agents without compromising yield or reaction rate. However, lower-cost activating agents are not suitable for all peptide coupling strategies. In particular, depending on the candidate amino acid to be activated, some activating agents may not allow coupling at an acceptable rate or with acceptable yield. Slow reactions may be particularly associated with undesired racemization / epimerization.

[0004] (2003) discloses a series of reactions using amide bond-forming reagents, including the reaction of the carbodiimides N,N'-diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with the additives HOPO and Oxyma. As shown in (2003) and (2004) couplings involving sterically hindered carboxylic acids can be problematic. In particular, (2003) reports that the sterically hindered carboxylic acid 2,6-dimethylbenzoic acid required restrictive conditions of prolonged reaction times at elevated temperatures (70 °C for 48 h) to provide the desired amide product in reasonable yields when using DIC / HOPO, and that all other coupling reagents tested provided inadequate yields for this sterically hindered carboxylic acid (Table 3, entries 13 and 14). Non-Patent Document 1 also reports that at a lower temperature of 20°C, the conversion of 2,6-dimethylbenzoic acid to amide using DIC / HOPO was negligible (page 4392, penultimate sentence). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Badlands et al., Tetrahedron Letters 58 (2017) 4391-4394 Summary of the Invention [Means for solving the problem]

[0006] The present inventors have surprisingly found that, despite the extremely sterically bulky side chain of the tri-tert-butyl-tryptophan (Tbt) residue in formula (I), a carbodiimide / additive approach can be used to couple amino-containing moieties, as defined herein, to compounds of formula (I) in high yields convenient for commercial processes, at relatively low temperatures, and without observing significant epimerization.

[0007] In one embodiment, the present invention provides a method for peptide synthesis. This method may be a step in the synthesis of a target peptide. The method for peptide synthesis of this embodiment comprises: reacting a compound of formula (I) or a salt thereof with a carbodiimide reagent to form an O-acylisourea intermediate; reacting the O-acylisourea intermediate with an additive to form an activated ester; reacting the activated ester with an amino-containing moiety, which is an amino acid, peptide, or salt thereof, containing an amino group, wherein the amino group forms an amide bond with the carbonyl marked with * in formula (I); Compounds of formula (I) have the structure:

[0008] [ka] Formula (I)

[0009] where R1 is a protecting group, a peptide, or an amino acid; R2 is H, an alkylsilyl group, or a protecting group.

[0010] Another aspect provides a method for amino acid coupling. In the context of this aspect, "amino acid coupling" means coupling one amino acid or peptide with another amino acid or peptide. That is, each coupling partner may be independently an amino acid or peptide. Embodiments of other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention. The method for amino acid coupling of this aspect comprises: reacting a compound of formula (I) or a salt thereof with a carbodiimide reagent to form an O-acylisourea intermediate; reacting the O-acylisourea intermediate with an additive to form an activated ester; reacting the activated ester with an amino-containing moiety, which is an amino acid, peptide, or salt thereof, containing an amino group, wherein the amino group forms an amide bond with the carbonyl marked with * in formula (I); Compounds of formula (I) have the structure:

[0011] [ka] Formula (I)

[0012] where R1 is a protecting group, a peptide, or an amino acid; R2 is H, an alkylsilyl group, or a protecting group.

[0013] In a third aspect, the present invention provides a method for producing a target peptide, the method comprising: reacting a compound of formula (I) or a salt thereof with a carbodiimide reagent to form an O-acylisourea intermediate; reacting the O-acylisourea intermediate with an additive to form an activated ester; reacting the activated ester with an amino-containing moiety, which is an amino acid, peptide, or salt thereof, containing an amino group, wherein the amino group forms an amide bond with the carbonyl marked with * in formula (I); Compounds of formula (I) have the structure:

[0014] [ka] Formula (I)

[0015] where R1 is a protecting group, a peptide, or an amino acid; R2 is H, an alkylsilyl group, or a protecting group.

[0016]

[0023] Embodiments of the other aspects of the invention described herein are subject to the third aspect of the invention mutatis mutandis. The above reaction may form a target peptide or a precursor to the target peptide. For example, a subsequent step to remove one or more protecting groups may be required to provide the target peptide. DETAILED DESCRIPTION OF THE INVENTION

[0017] Compounds of formula (I) Formula (I) contains the extremely sterically bulky tri-tert-butyl-tryptophan (Tbt) residue and has the following structure:

[0018] [ka]

[0019] The present inventors have unexpectedly found that, despite the extreme steric bulk of the Tbt side chain, the carbodiimide / additive approach can be used to couple amino acids or peptides to compounds of formula (I) in high yields and at low temperatures.

[0020] In formula (I), R1 is a protecting group (typically an amine protecting group), a peptide, or an amino acid. Optionally, the peptide or amino acid may itself contain one or more protecting groups, for example, on its N-terminal amino group.

[0021] As used herein, the term "peptide" includes peptidomimetics, although true peptides are preferred. Peptidomimetics are typically characterized by retaining the polarity, three-dimensional size, and function (biological activity) of their peptide equivalents, but their peptide bonds are often replaced with more stable bonds. "Stable" refers to greater resistance to enzymatic degradation by hydrolases. Generally, bonds that replace amide bonds (amide bond substitutes) preserve many of the properties of amide bonds, such as conformation, steric bulk, electrostatic characteristics, and hydrogen-bonding potential. Chapter 14 of "Drug Design and Development," Krogsgaard, Larsen, Liljefors, and Madsen (eds.), 1996, Horwood Academic Publishers, provides a general discussion of techniques for the design and synthesis of peptidomimetics. In the present case, because the target peptide reacts with the membrane rather than with the specific active site of the enzyme, some of the problems described for accurately mimicking affinity and efficacy or substrate function are not relevant, and peptidomimetics can be easily prepared based on a given peptide structure or motif of the required functional groups. Suitable amide bond surrogates include the following groups:N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47), retroinverse amides (Chorev, M. and Goodman, M., Accounts of Chemical Research, 1993, 26, 266), thioamides (Sherman, D.B. and Spatola, A.F., Journal of the American Chemical Society, 1994, 26, 266), etc. oc.), 1990, 112, 433), thioesters, phosphonates, ketomethylenes (Hoffman, RV and Kim, HO, J. Org. Chem., 1995, 60, 5107), hydroxymethylenes, fluorovinyls (Allmendinger, T. et al., Tetrahydron Letters Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y. and Abe, J., Chem. Pharm. Bull. 1997, 45, 13), methylenethio (Spatola, A. F., Methods Neurosci., 1993, 13, 19), alkanes (Lavielle, S. et al., Int. J. Peptide Protein Res., 1993, 42, 270), and sulfonamides (Luisi, G. et al., Tetrahedron Lett. 1993, 34, 2391).

[0022] Thus, the term "amino acid" is sometimes used conveniently herein to refer to the equivalent subunit of a peptidomimetic compound. Additionally, peptidomimetics may have groups equivalent to the R groups of an amino acid.

[0023] As discussed in the above-referenced texts, in addition to replacing amide bonds, peptidomimetics may involve replacing larger structural moieties with di- or tripeptidomimetic structures, in which case mimetic moieties containing peptide bonds, such as azole-derived mimetics, may be used as dipeptide replacements. However, peptidomimetics and peptidomimetic backbones with amide bond replacements as discussed above are preferred.

[0024] Suitable peptidomimetics include reduced peptides in which the amide bond has been reduced to a methylene amine by treatment with a reducing agent such as borane or a hydride reagent such as lithium aluminum hydride, which has the added benefit of increasing the cationicity of the overall molecule.

[0025] Other peptidomimetics include peptoids, formed, for example, by the stepwise synthesis of amide-functionalized polyglycines. Some peptidomimetic backbones are readily available from their peptide precursors, such as permethylated peptides, and suitable methods are described in Ostresh, JM, et al., Proc. Natl. Acad. Sci. USA (1994) 91, 11138-11142. Strongly basic conditions favor N-methylation over O-methylation, resulting in methylation of some or all of the nitrogen atoms of the peptide bonds and the N-terminal nitrogen. Preferred peptidomimetic backbones include polyesters, polyamines, and their derivatives, as well as substituted alkanes and alkenes. Peptidomimetics will preferably have N- and C-termini, which may be modified as discussed herein.

[0026] Preferably, the term "amino acid" as used herein refers to a proteinogenic (genetically encoded) amino acid. Preferably, the term peptide in R1 and amino-containing moieties refers to a peptide formed from a proteinogenic amino acid.

[0027] R1 typically contains 1 to 10 amino acids, preferably 1 to 5 or 1 to 3 amino acids, and most preferably 1 amino acid.

[0028] A wide selection of suitable protecting groups for amino acids is known (see, for example, Greene, TW and Wuts, PGM, Protective Groups in Organic Synthesis, 3rd ed., Wiley: New York, 1999, and Isidro-Llobet et al., Chem. Rev. 2009, 109, 6, 2455-2504).

[0029] Suitable amine-protecting groups (sometimes called amino-protecting groups) include carbobenzoxy (also known as benzyloxycarbonyl and denoted as Z or Cbz), t-butoxycarbonyl (also denoted as Boc), 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), 9-fluorenylmethoxy-carbonyl (also denoted as Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc), 2,4-dimethoxybenzyl (Dmb), 2-hydroxy-4-methoxybenzyl (Hmb), and 2-Fmoc-oxy-4-methoxybenzyl (FmocHmb). These protecting groups may themselves be R1, or one or more of these protecting groups may be present on R1 when R1 is a peptide or amino acid.

[0030] For example, suitable carboxyl protecting groups that may be used include readily cleaved ester groups such as benzyl (Bn), p-nitrobenzyl (pNb), pentachlorophenyl (PClP), pentafluorophenyl (Pfp), or t-butyl (tBu) groups, and coupling groups on solid supports, such as methyl groups attached to polystyrene. Other suitable carboxyl protecting groups include 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl ester (Dmab), allyloxycarbonyl, and 2-phenylisopropyl (2-PhiPr).

[0031] Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt), acetamidomethyl (Acm), tert-butyl (tBu), tert-butylthio (tButhio), and monomethoxytrityl (Mmt) groups.

[0032] Amine protecting groups, such as Boc, and carboxyl protecting groups, such as tBu, may be simultaneously removed by acid treatment, such as with trifluoroacetic acid. Thiol protecting groups, such as Trt, may be selectively removed using an oxidizing agent, such as iodine.

[0033] Preferably, R1 is a peptide or an amino acid, which optionally contains one or more protecting groups, for example on its N-terminal amino group. The amino acid may be the cationic amino acid AA1.

[0034] Preferably, R1 is a cationic amino acid AA1, which optionally contains one or more protecting groups, for example, on its N-terminal amino group.

[0035] AA1 is preferably lysine or arginine, but may also be histidine or any non-genetically encoded or modified amino acid that has a positive charge at pH 7.0. Suitable non-genetically encoded and modified amino acids that can provide cationic amino acids include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, and homoarginine, as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0036] Most preferably, R1 is arginine, which optionally contains one or more protecting groups, for example, at its N-terminal amino group.

[0037] In formula (I), R2 is selected from H, an alkylsilyl group, or a protecting group (typically an amine protecting group). The alkylsilyl group may be a mono(C1-C6 alkyl)silyl, di(C1-C6 alkyl)silyl, or tri(C1-C6 alkyl)silyl group. Preferably, the alkylsilyl group is a tri(C1-C6 alkyl)silyl group, more preferably a tri(C1-C3 alkyl)silyl group. Each alkyl group may be the same or different, preferably the same. When present as R2, the alkylsilyl group is preferably trimethylsilyl. The protecting group may be an amine protecting group as defined above. For example, the protecting group may be Cbz, Boc, Mtr, Troc, Dmb, Hmb, or FmocHmb.

[0038] R2 is preferably H. That is, the compound of formula (I) preferably has the following structure:

[0039] [ka]

[0040] In the process of the present invention, the compound of formula (I) may be preferably provided in the form of a salt, preferably in the form of an acid addition salt, more preferably in the form of an HCl salt. Other suitable acid addition salts are defined below in relation to the amino-containing moiety.

[0041] Amino-containing moieties The amino-containing moiety is an amino acid, a peptide, or a salt thereof. The amino acid or peptide of the amino-containing moiety may optionally contain one or more protecting groups and / or C-terminal capping groups. The amino acid or peptide of the amino-containing moiety may optionally be silylated. Suitable silylating agents are disclosed in WO 2009 / 065836. For example, suitable silylating agents are N-trialkylsilylamines or N-trialkylsilylamides, such as those selected from the group consisting of N,O-bis(trimethylsilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-trimethylsilylacetamide (MSA), N-methyl-N-trimethylsilyltrifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, and 3-(trimethylsilyl)-2-oxazolidone. Silylation may improve the solubility of the amino-containing moiety in polar organic solvents, such as polar aprotic solvents such as dimethylacetamide. During silylation, one or more functional groups with active hydrogens, such as amino, hydroxyl, mercapto, or carboxyl groups, of the amino-containing moiety react with a silylating agent, so that the silylated amino-containing moiety contains one or more silyl groups (e.g., trialkylsilyl, typically a tri(C1-C3)alkyl group, such as trimethylsilyl) attached to the functional groups.

[0042] The amino-containing moiety may preferably be provided in the form of a salt, such as an acid addition salt. Compounds having at least one basic center, such as in the side chain of the amino acid of the amino-containing moiety, may form acid addition salts. Suitable acid addition salts may be formed with strong inorganic acids, such as mineral acids, such as sulfuric acid, phosphoric acid, or hydrohalic acid; organic carboxylic acids; or organic sulfonic acids, such as (C1-C4) alkyl or aryl sulfonic acids, unsubstituted or substituted, for example, by halogens, such as methyl- or p-toluene-sulfonic acid. Examples of organic carboxylic acids include alkanecarboxylic acids of 1 to 4 carbon atoms, such as acetic acid, unsubstituted or substituted, for example, by halogens, such as chloroacetic acid; saturated or unsaturated dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, or terephthalic acid; hydroxycarboxylic acids, such as ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid; and benzoic acid.

[0043] Preferred salts of the amino-containing moiety include hydrochloride, trifluoroacetate, or acetate, most preferably hydrochloride. For example, when the amino-containing moiety is a compound of formula (VI), the amino-containing moiety may preferably be in the form of a salt, in which the side chain of AA3 is protonated.

[0044] The amino-containing moiety typically contains a reactive amino group, such as an alpha-amino group.

[0045] The amino-containing moiety typically contains 1 to 10 amino acids, preferably 1 to 5 or 1 to 3 amino acids, and most preferably 1 amino acid.

[0046] As indicated above, suitable protecting groups for amino acids are well known and the protecting groups listed above may also be used for amino-containing moieties.

[0047] Suitable C-terminal capping groups are of the formula -XYZ, where the left hyphen indicates the point of attachment to the C-terminal carbonyl carbon, and X, Y, and Z are defined with respect to formula (VI) below: In other words, when the capping group -XYZ is present, it is attached to the remaining amino-containing moiety as follows:

[0048] [ka]

[0049] Here, R represents the side chain of the C-terminal amino acid. Preferably, the entire -XYZ is a -NHCH2CH2Ph ​​group.

[0050] Preferably, the amino-containing moiety is a compound of formula (VI) or a salt thereof: AA3-XYZ (VI) wherein AA3 is a cationic amino acid, preferably lysine or arginine, but may also be histidine or any non-genetically encoded or modified amino acid that has a positive charge at pH 7.0; X is a N atom, which may be branched or unbranched C1-C 10 Alkyl or aryl groups (e.g., C4-C 10 aryl groups), such as methyl, ethyl, or phenyl, which may be substituted, but are preferably unsubstituted, and the alkyl or aryl groups may contain up to two heteroatoms selected from N, O, and S; Y is -R a -R b -, -R a -R b -R b -, and -R b -R b -R a -, wherein R a is C, O, S, or N, preferably C; R b is C;R a and R beach of which may be substituted with a C1-C4 alkyl group or unsubstituted, and preferably Y is -R a -R b -(where R a is preferably C), and preferably this group is unsubstituted, and Y is -R a -R b -R c -or-R b -R b -R a -, preferably R a and R b One or more of the following are replaced; Z is a group containing 1 to 3 cyclic groups, each of which is a cyclic group of 5 or 6 non-hydrogen atoms (preferably C atoms), and two or more cyclic groups may be fused; one or more rings may be substituted, and these substitutions may, but typically do not, include polar groups, and suitable substituents include halogen, preferably bromine or fluorine, and C1-C4 alkyl groups; the Z moiety contains up to 15 non-hydrogen atoms, preferably 5 to 12, and most preferably it is phenyl; The bond between Y and Z is the R of Y. a or R b and a non-hydrogen atom of one of the cyclic groups of Z.

[0051] The compound of formula (VI) may optionally contain one or more protecting groups and / or be silylated. The above discussion of silylation and suitable silylating agents equally applies when the amino-containing moiety is a compound of formula (VI). The protecting groups listed above may also be used when the amino-containing moiety is a compound of formula (VI).

[0052] Suitable non-genetically encoded amino acids and modified amino acids that can provide cationic amino acids include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, and homoarginine, as well as trimethylysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidoylpiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0053] Preferably, Y is -R as defined above. a -R b -, where more preferably R a and R b is unsubstituted, most preferably R a and R b are carbon atoms. In other words, Y is most preferably -CH2CH2-.

[0054] Preferably, the entire -XYZ is a -NHCH2CH2Ph ​​group.

[0055] Most preferably, AA3 is arginine.

[0056] In other preferred cases, the amino-containing moiety is an amino acid containing AA3, or a peptide containing AA3 as the N-terminal amino acid, or a salt thereof, and optionally, the amino acid or peptide contains one or more protecting groups and / or a C-terminal capping group. The definition of AA3 above in the context of formula (VI) applies equally in this case. The C-terminal capping group may have the structure -XYZ, and the preferred definitions of -XYZ given above apply equally in this case. The amino acid containing AA3 or the peptide containing AA3 as the N-terminal amino acid may optionally be silylated, and the discussion of silylation and suitable silylating agents above applies equally.

[0057] The compounds of the invention, and compounds used and made in / by the methods of the invention (e.g., compounds of Formula (I), target peptides, and amino-containing moieties), may include all enantiomeric forms, both D and L amino acids, and enantiomers resulting from chiral centers in the R groups of amino acids and the Y or Z moieties.

[0058] More preferably, the amino-containing moiety is arginine or a salt thereof, which optionally includes one or more protecting groups and / or a C-terminal capping group, in which case the C-terminal capping group may be of formula -XYZ, preferably -NHCHCHPh.

[0059] Carbodiimide Reagents and Additives Suitable carbodiimide reagents and additives for peptide coupling are disclosed in Ayman El-Faham and Fernando Alberto, Chem. Rev. 2011, 111, 6557-6602. The carbodiimide reagents and additives disclosed in Tables 1 and 2 of this document are suitable for use in the processes described herein.

[0060] The carbodiimide reagent may be a compound of formula (II) or a salt thereof:

[0061] [ka] Formula (II)

[0062] where R A and R B are each independently selected from organic groups containing 1 to 30 non-hydrogen atoms.

[0063] Preferably, R A and R B are each independently C1~C 10alkyl (preferably C1-C6 alkyl, more preferably C1-C4 alkyl), optionally mono- or di-(C1-C 10 substituted with a (C1-C6 alkyl)amino group, preferably wherein the optional substituent is a mono- or di-(C1-C6 alkyl)amino group, more preferably wherein the optional substituent is (CH3)2N-; C3-C8 cycloalkyl, preferably C5-C6 cycloalkyl; aryl, preferably 6- to 10-membered aryl, more preferably phenyl; (Aryl) C1-C 10 alkyl, preferably (6-10 membered aryl)C1-C6 alkyl, more preferably benzyl; or (C3-C8 heterocycloalkyl)C1-C 10 alkyl, wherein the heterocycloalkyl group is optionally one or more C1-C 10 Substituted with an alkyl group, preferably (C3-C6 heterocycloalkyl)C1-C4 alkyl, wherein the heterocycloalkyl group is optionally substituted with one or more C1-C4 alkyl groups, more preferably selected from the group consisting of 2-morpholinoethyl or (2,2-dimethyl-1,3-dioxolan-4-yl)methyl.

[0064] The term "alkyl" as used herein alone or as part of another group, such as (heterocycloalkyl)alkyl or (aryl)alkyl, is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C 10 Alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9, and C 10It is intended to include alkyl groups. Preferred alkyl groups are C1 to C6 alkyl groups, more preferably C1 to C4 alkyl groups. Examples of suitable alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0065] The term "alkynyl" is intended to include hydrocarbon chains of straight or branched configuration, with one or more, preferably one to three, more preferably one carbon-carbon triple bond, which may occur at any stable point along the chain. For example, "C2-C6 alkynyl" is intended to include C2, C3, C4, C5, and C6 alkynyl groups, such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0066] The term "cycloalkyl" refers to a cyclized alkyl group, including monocyclic, bicyclic, or polycyclic ring systems. "C3-C8 cycloalkyl" is intended to include C3, C4, C5, C6, C7, and C8 cycloalkyl groups, including monocyclic, bicyclic, and polycyclic rings. Examples of suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and norbornyl. Spiro and bridged cycloalkyl groups are included in the definition of "cycloalkyl."

[0067] The term cycloalkenyl refers to a non-aromatic cyclized alkenyl group containing one or more carbon-carbon double bonds. "Cycloalkenyl" includes groups such as cyclopentenyl and cyclohexenyl, as well as groups with more than one double bond, such as 1,3- and 1,4-cyclohexadienyl.

[0068] The term "aryl" refers to monocyclic or polycyclic (including bicyclic and tricyclic) aromatic hydrocarbons, including, for example, phenyl, naphthyl, anthracenyl, and phenanthranyl. In one embodiment, the term "aryl" refers to monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion (e.g., phenyl or naphthyl, including 1-naphthyl and 2-naphthyl).

[0069] The term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic or polycyclic (including bicyclic and tricyclic) aromatic hydrocarbon in which one or more carbon ring members are replaced with a heteroatom, such as O, N, or S. Typically, a heteroaryl or heteroaromatic ring contains up to four nitrogens, up to two oxygens, and up to two sulfurs. A heteroaryl preferably contains one to four heteroatoms. The term 5-10 membered heteroaryl means that there are 5 to 10 ring members, which may be selected from carbon or the heteroatoms listed above. Preferred heteroaryl / heteroaromatic rings contain 5 or 6 ring members. Examples of suitable heteroaryl groups include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4 thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane.

[0070] The term "heterocycloalkyl" refers to a saturated cyclized alkyl group in which one or more carbon ring members are replaced with a heteroatom, such as O, N, or S. Typically, the heterocycloalkyl ring contains up to four nitrogens, up to two oxygens, and up to two sulfurs. "C-C heterocycloalkyl" is intended to include C, C, C, C, and C and C heterocycloalkyl groups. Examples of heterocycloalkyl groups include oxetanyl, tetrahydrofuranyl, 1,3-dioxolanyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl.

[0071] (C3-C8 heterocycloalkyl)C1-C 10 Alkyl and (aryl) C1-C 10 In the alkyl portion, the heterocycloalkyl or aryl group is C1-C 10 Combined with alkyl groups, C1-C 10 The alkyl group is bonded to the rest of formula (II). 10 The alkyl group is preferably a C1 to C6 alkyl group, more preferably a C1 to C4 alkyl group.

[0072] Unless otherwise indicated, aryl and heterocycloalkyl groups may be attached through any available carbon or nitrogen by replacement of a hydrogen on said carbon or nitrogen.

[0073] For example, carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-cyclohexyl-N'-isopropylcarbodiimide (CIC), N-tert-butyl-N'-methylcarbodiimide, N-tert-butyl-N'-ethylcarbodiimide, N,N'-dicyclopentylcarbodiimide, 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide, N-ethyl-N'- The carbodiimide may be selected from N-phenylcarbodiimide, N-phenyl-N'-isopropylcarbodiimide, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide, N-benzyl-N'-cyclohexylcarbodiimide, or a salt thereof, such as a salt selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, sometimes referred to herein as EDC.HCl), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (CAS No. 22572-40-3), or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CAS No. 2491-17-0).

[0074] Preferred carbodiimide reagents include EDC.HCl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, more preferably EDC.HCl.

[0075] Optionally, the carbodiimide reagent may be immobilized on a solid support, such as a polymeric support, e.g., an insoluble polymeric support for solid-phase peptide synthesis (SPPS). For example, polymer-bound EDC.HCl, polymer-bound 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, or polymer-bound N-benzyl-N'-cyclohexylcarbodiimide (all of which are commercially available) may be used in the process of the invention. However, the process is preferably carried out in solution without SPPS.

[0076] The structure of the O-acylisourea intermediate is determined by the choice of carbodiimide reagent. For example, the O-acylisourea intermediate may be a compound of formula (III) or a salt thereof, where R and R are as defined above for formula (I), and R A and R B is as defined above for formula (II).

[0077] [ka] Formula (III)

[0078] In another aspect, the present invention is directed to compounds of formula (III) as defined herein. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention.

[0079] The additive reacts with the O-acylisourea intermediate to form an activated ester. The activated ester contains a good leaving group at the carbonyl marked with an * in Formula (I) / Formula (V). This means that the leaving group can effectively leave the ternary intermediate formed when the amino-containing moiety reacts with the activated ester. The leaving group is provided by the additive.

[0080] Additives (e.g., HO-R of Formula IV) CThe pKa of the compound (a) may be less than 8, for example, 3 to 7.5, 3.5 to 7, or 4 to 6.5.

[0081] The pKa values ​​in this disclosure may be as determined in water at 25°C, optionally with the addition of an organic co-solvent, such as an equal volume mixture of methanol, dioxane, and acetonitrile, when the compound (e.g., additive or acid) is poorly soluble in water. Techniques for measuring pKa values ​​are known, such as those described in Babic et al., Trends in Analytical Chemistry, Vol. 26, No. 11, 2007, pp. 1043-1061. Preferably, the pKa values ​​presented herein are determined by potentiometric titration.

[0082] The additive may be a compound of formula IV or a salt thereof: HO-R C (IV) where R C is an organic group containing 1 to 30 non-hydrogen atoms. C may be a group capable of stabilizing the negative charge of the oxygen shown in formula (IV), for example by delocalizing the charge.

[0083] R C may be a phenyl group, which is optionally substituted with one or more halogen atoms (preferably, the halogen atoms are each independently selected from F and Cl) or one or more electron-withdrawing groups such as nitro. Preferably, the phenyl group is substituted. Examples of suitable additives having this structure are pentafluorophenol, 2,3,5-trichlorophenol, and 4-nitrophenol.

[0084] Alternatively, the compound of formula (IV) may be an N-hydroxy compound, where R C is an organic group containing 1 to 30 non-hydrogen atoms and at least one nitrogen atom, and the hydroxyl group in formula (IV) is connected to R through the nitrogen atom.C For example, the N-hydroxy compound may comprise a hydroxyimino group (e.g., compounds 1-19 and 28-73 in Table 1), an N-hydroxy-triazole group (e.g., compounds 87-91 in Table 1), an N-hydroxy-tetrazole group (e.g., compound 26 in Table 1), an N-hydroxybenzimidazole group (e.g., compounds 23 and 24 in Table 1), an N-hydroxyindolin-2-one group (e.g., compound 27 in Table 1), an N-hydroxypyridinone group (e.g., compound 22 in Table 1), an N-hydroxypyrrolidine-2,5-dione group (e.g., compounds 20-21 in Table 1), a 4-aza-, 5-aza-, 6-aza-, or 7-aza-1-hydroxybenzotriazole group (e.g., compounds 80 and 82-84 in Table 1), an N-hydroxybenzotriazole group (e.g., compounds 77-79 and 81 in Table 1), or the following group:

[0085] [ka]

[0086] where Y is CH and X is N, or Y is N and X is CH, or Y is N and X is N (compounds 25, 85, and 86 in Table 1).

[0087] The additive may be a compound of formula VIII or a salt thereof:

[0088] [ka] Formula (VIII)

[0089] wherein R3 is cyano, -C(O)OR5, 5- to 10-membered heteroaryl optionally substituted with one or more C1-C6 alkyl groups, C2-C6 alkynyl, nitro, aryl (preferably 6- to 10-membered aryl, more preferably phenyl), -SO2R6, -OSO2R6, -CONR9R 10 , —C(O)—NH—OH, and C(═N—OH)R7; R4 is -C(O)R8, -CONR9R10 aryl optionally substituted with one or more halogens (aryl (preferably 6-10 membered aryl, more preferably phenyl), 5-10 membered heteroaryl optionally substituted with one or more C1-C6 alkyl groups, cyano, -C(S)NR9R 10 , nitro, C1-C6 alkyl optionally substituted with aryl (preferably 6- to 10-membered aryl, more preferably phenyl), —S(O)NR 11 R 12 , —C(O)—NH—OH, halogen (preferably Cl), —NH, and hydrogen; R5 is C1-C6 alkyl; R6 is -NH2, C1-C6 alkyl, or C1-C6 haloalkyl; R7 is halogen (preferably Cl) or C1-C6 alkyl; R8 is a C1-C6 alkoxy optionally substituted with a C3-C8 heterocycloalkyl group, wherein the C3-C8 heterocycloalkyl group is optionally substituted with one or more C1-C6 alkyl groups, or R8 is a C3-C8 heterocycloalkyl group; Each R9 and R 10 are independently selected from H or C1-C6 alkyl; Each R 11 and R 12 are independently selected from H or C1-C6 alkyl.

[0090] The additive may be a compound of formula IX:

[0091] [ka] Formula (IX)

[0092] wherein X1 and X2 are each independently selected from O or NH, and preferably at least one of X1 and X2 is NH; The dashed bond is either absent (in which case the compound of formula IX is a compound of formula IXa below) or

[0093] [ka] Formula (IXa)

[0094] or exists (in which case the compound of formula IX is a compound of formula IXb below).

[0095] [ka] Formula (IXb)

[0096] The additive may be a compound of formula X,

[0097] [ka] Formula (X)

[0098] It is optionally substituted with one or more substituents selected from C1-C6 alkyl or aryl (preferably 6-10 membered aryl, more preferably phenyl). Preferably, the substituents are on the nitrogen of the indolinone ring.

[0099] The additive may be a compound of formula XI,

[0100] [ka] Formula (XI)

[0101] where X3 and X4 are independently O and NR 19 Selected from R 19 is H or C1-C6 alkyl, preferably C1-C6 alkyl, R 13 and R 14 are independently selected from H or C1-C6 alkyl, or R 14 is absent, and R 13 is =O or =S.

[0102] The additive may be a compound of formula XII:

[0103] [ka] Formula (XII)

[0104] wherein ring A is a fused tricyclic ring containing 10-14 ring members which may be selected from carbon ring members and one or more of N, O, or S. The nitrogen of the oxime shown in formula XII is double bonded to any available carbon ring member of ring A. Ring A may be substituted with one or more substituents selected from C1-C6 alkyl, C1-C6 haloalkyl, and =N-OH. When the substituent is =N, ** When -OH, the nitrogen marked with ** is double bonded to any available carbon ring member of ring A. One or more substituents are preferably =N-OH. The compound of formula XII may be a compound of formula XIIa or XIIb,

[0105] [ka]

[0106] wherein each ring B and ring C is independently phenyl or 5-6 membered heteroaryl (preferably pyridyl). Formulas XIIa and XIIb may be substituted with one or more of the substituents set forth above for Formula XII.

[0107] The additive may be a phenol, which is optionally substituted with one or more halogen atoms (preferably, each halogen atom is independently selected from F and Cl) or one or more electron-withdrawing groups such as nitro. Preferably, the phenol is substituted. Examples of suitable additives having this structure are pentafluorophenol, 2,3,5-trichlorophenol, and 4-nitrophenol.

[0108] The additive may be a compound of formula XIII:

[0109] [ka] Formula (XIII)

[0110] Here, each R 15 and R 16 are independently H, halogen (preferably Cl), (C2-C6 alkylcarbonyl)C1-C6 alkyl (i.e., C3-C 12 alkylcarbonylalkyl), —C(O)OC1-C6 alkyl, or —C(O)C1-C6 alkyl; or R 15 and R 16 together form an aryl ring (preferably a 6- to 10-membered aryl, more preferably phenyl) or a 5- or 6-membered aromatic heterocycle containing one or more heteroatoms selected from O, N, or S, and optionally the aryl or aromatic heterocycle is substituted with one or more substituents selected from halogen, C1-C6 haloalkyl, or nitro. Preferably, R 15 and R 16 The ring formed as a whole is phenyl or pyridyl.For example, the compound of formula XIII can be 4-aza-, 5-aza-, 6-aza-, or 7-aza-1-hydroxybenzotriazole.

[0111] The additive may be a compound of formula XIV:

[0112] [ka] Formula (XIV)

[0113] where (1) Y is CH and X is N, or (2) Y is N and X is CH, or (3) Y is N and X is N.

[0114] The additive may be a compound of formula XV:

[0115] [ka] Formula (XV)

[0116] where R 17 and R 18 is H or R 17 and R 18 The overall range is C5 to C 10 Cycloalkyl or C5-C 10 It forms a cycloalkenyl ring or an aryl ring (preferably a 6- to 10-membered aryl ring, more preferably phenyl).

[0117] The additive may be a hydroxypyridine-N-oxide, preferably 2-hydroxypyridine-N-oxide.

[0118] The additive may be a hydroxytetrazole, preferably 2H-tetrazol-2-ol.

[0119] The additive may be 1H-benzo[d]imidazol-1-ol, which is optionally substituted with one or more halogen or phenyl groups. Preferably, the phenyl substituent is at the 2-position of the imidazole ring. When a halogen is present, it is preferably Cl.

[0120] The additive may be 1-hydroxyindolin-2-one, which is optionally substituted with one or more halogens or phenyl groups. When a halogen is present, it is preferably Cl. The 1-hydroxyindolin-2-one is preferably unsubstituted.

[0121] The additive may be a compound selected from the compounds listed in Table 1.

[0122] [Table 1-1]

[0123] [Table 1-2]

[0124] [Table 1-3]

[0125] [Table 1-4]

[0126] [Table 1-5]

[0127] [Table 1-6]

[0128] [Table 1-7]

[0129] [Table 1-8]

[0130] [Table 1-9]

[0131] [Table 1-10]

[0132] In some embodiments, the additive is not HOAt.

[0133] Preferably, the additive is selected from the group consisting of OxymaPure, HOBt, HOSu, HOPO, pentafluorophenol, and 6-Cl-HOBt, more preferably OxymaPure, HOBt, HOSu, and HOPO, more preferably HOPO.

[0134] In some embodiments, the carbodiimide-additive combination is not DIC-HOPO.

[0135] As shown above, the additive reacts with the O-acylisourea intermediate to provide an activated ester. When the additive is a compound of formula (IV), the activated ester is a compound of formula (V) or a salt thereof.

[0136] [ka] Formula (V)

[0137] R in formula (V) C may correspond to the non-hydroxyl moiety of the additives listed in Table 1.

[0138] Reaction conditions The reaction of the compound of formula (I) or its salt with the carbodiimide reagent, and / or the reaction of the O-acylisourea intermediate with the additive, and / or the reaction of the activated ester with the amino-containing moiety may be carried out at a temperature of −10 to 40° C., preferably 0 to 30° C., such as 0 to 25° C. Typically, these reactions are carried out sequentially in a one-pot procedure / in the same reaction vessel. In other words, typically, all reagents for all steps of the method described in claim 1 are added to form a single reaction mixture (one-pot). Thus, each of the above reactions is preferably carried out at a temperature of −10 to 40° C., preferably 0 to 30° C., such as 0 to 25° C. The reaction is typically carried out at a pressure of about 1 atm. In some embodiments, the reagents are mixed at about 2.5° C. and then heated to room temperature. Thus, the temperature may vary within the above range during the reaction.

[0139] The inventors have surprisingly found that these relatively low temperatures can be used to couple amino-containing moieties, as defined herein, to compounds of formula (I) in high yield, despite the extremely sterically bulky side chain of the Tbt residue in formula (I). It is desirable to avoid elevated temperatures in the processes described herein to reduce the risk of side reactions that lead to epimerization.

[0140] The reaction of the compound of Formula (I) or a salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety may be carried out within a total duration of less than 48 hours, preferably less than 36 hours, and more preferably less than 24 hours. In some embodiments, the total duration of the above reactions is 2 to 48 hours, preferably 4 to 36 hours, and more preferably 10 to 24 hours, such as about 18 hours or about 20 hours.

[0141] The reaction of the compound of formula (I) or a salt thereof with a carbodiimide reagent, the reaction of the O-acylisourea intermediate with an additive, and the reaction of the activated ester with an amino-containing moiety are typically carried out in a one-pot procedure, and therefore the O-acylisourea and activated ester intermediate are preferably not isolated.

[0142] The carbodiimide reagent may be added last after the compound of Formula (I) or a salt thereof, the amino-containing moiety, the additive, the solvent(s), and any optional acid or base are mixed. The reaction mixture may be cooled before the carbodiimide reagent is added.

[0143] In some embodiments, the carbodiimide reagent is added last to initiate the reaction.

[0144] acidic conditions The reaction of the compound of formula (I) or its salt with a carbodiimide reagent, the reaction of the O-acylisourea intermediate with an additive, and the reaction of the activated ester with an amino-containing moiety can be carried out in the presence of an acid or a base, or without the addition of an acid or base. Suitable bases, when used, include DIPEA, N-methylmorpholine, pyridine, trimethylamine, and 2,4,6-trimethylpyridine.

[0145] Preferably, the reaction of the compound of formula (I) or its salt with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out under acidic conditions. The inventors have surprisingly found that the addition of acid improves the conversion of the compound of formula (I) (e.g., overnight or within 4 to 36 hours, preferably 10 to 24 hours, of the total duration defined above). This was contrary to the inventors' initial expectation that the addition of acid would hinder the process by protonating the amino group of the amino-containing moiety.

[0146] Preferably, the reaction of the compound of formula (I) or its salt with a carbodiimide reagent, and / or the reaction between the O-acylisourea intermediate and / or the reaction between the additive, and / or the reaction between the activated ester and the amino-containing moiety is acid-catalyzed. The acid catalyst may increase the electrophilicity of the carbonyl marked with an * in formulas (I) and (V) (and the equivalent carbonyl in formula (III)), thereby increasing the rate of nucleophilic acyl substitution reactions at this carbonyl, such as the reaction to form an O-acylisourea.

[0147] Without wishing to be bound by theory, the inventors hypothesize that any protonation of the amino group of the amino-containing moiety may be compensated for by increasing the rate of reaction of the compound of formula (I) or its salt with a carbodiimide reagent to form an O-acylisourea. Thus, the reaction of the compound of formula (I) or its salt with a carbodiimide reagent is preferably acid-catalyzed.

[0148] Preferably, the reaction of the compound of formula (I) or a salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out at a pH of less than 6.5, preferably between 1 and 6.5, more preferably between 1.5 and 6, more preferably between 4 and 6. Additionally, these reactions may be carried out at a pH of less than 5, such as between 1 and 4.5, between 1.5 and 4, or between 2 and 3.

[0149] When the above reaction is carried out in a non-aqueous solvent, the pH is determined by taking a sample from the reaction mixture after the addition of the acid (preferably immediately after the addition of the acid), adding water (preferably in a ratio of added water to sample of the reaction mixture of 1:10 to 10:1 v / v, preferably 1:1 to 10:1 v / v, more preferably about 10:1 v / v), mixing, optionally separating the aqueous phase, and measuring the pH of the aqueous phase or the reaction mixture containing water. Optionally, a sample may also be taken from the reaction mixture before the addition of the acid for use as an in-process control. When the reaction is carried out in a non-aqueous solvent, the pH may be determined by Test Method B described in ASTM D4980.

[0150] Preferably, the reaction of the compound of Formula (I) or a salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out in the presence of at least 0.1 equivalents of acid per equivalent of Formula (I) or a salt thereof, more preferably at least 0.25 equivalents of acid, more preferably at least 0.5 equivalents of acid, and more preferably at least 0.8 equivalents of acid per equivalent of Formula (I) or a salt thereof. For example, the reaction may be carried out in the presence of 0.25 to 2, preferably 0.5 to 1.5, more preferably 0.8 to 1.2 equivalents of acid per equivalent of Formula (I) or a salt thereof. In other words, at least 0.1 equivalents of acid, preferably at least 0.25 equivalents of acid, more preferably at least 0.5 equivalents of acid, and more preferably at least 0.8 equivalents of acid may be added per equivalent of Formula (I) or a salt thereof. For example, 0.25 to 2, preferably 0.5 to 1.5, more preferably 0.8 to 1.2 equivalents of acid may be added to 1 equivalent of formula (I) or a salt thereof. Optionally, in these embodiments, the acid may be any of the acids listed below, preferably HCl (dioxane), such as 4N HCl (dioxane).

[0151] The acid may be a strong acid having a pKa of less than 1. Examples of suitable acids include hydroiodic acid, hydrobromic acid, perchloric acid (HClO), hydrochloric acid, chloric acid (HClO), sulfuric acid, and nitric acid. Preferably, the acid is hydrochloric acid, more preferably anhydrous hydrochloric acid, such as HCl (dioxane).

[0152] The reaction of the compound of Formula (I) or its salt with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety may be carried out in any suitable solvent(s). Suitable solvents include aqueous or non-aqueous solvents, with non-aqueous solvents being preferred. Examples of suitable solvents include water, dichloromethane (DCM), dimethylformamide (DMF), N,N-dimethylacetamide (DMA), acetonitrile (ACN), N-methylpyrrolidone, dimethyl sulfoxide, 2-methyltetrahydrofuran (MeTHF), dioxane, or mixtures thereof, such as a mixture of DMA, dioxane, and optionally water. The solvent may preferably comprise one or more polar aprotic solvents (such as one or more of DCM, DMF, DMA, ACN, methylpyrrolidone, dimethylsulfoxide, and MeTHF), and more preferably the solvent comprises DMA.

[0153] The amino-containing moiety is preferably added in the form of a solution. In some preferred cases, the amino-containing moiety may be added in the form of a solution containing a DMA solvent. Preferably, the solution containing the amino-containing moiety contains less than 10 wt% water, more preferably less than 7.5 wt% water, more preferably less than 5 wt% water. In some embodiments, no water solvent is added during the process (i.e., when carrying out the reaction described in claim 1), except for any water present in the solution containing the amino-containing moiety and any water accompanying the compound or formula (I) or its salt, which is typically added as a solid.

[0154] Preferably, the solvent is substantially free of water, for example the solvent in the reaction mixture, i.e. the reaction mixture as a whole, may contain less than 10 wt% water, such as less than 5 wt% water, for example less than 3 wt% or less than 1 wt% water, etc. As indicated above, relatively small amounts of water may be present in the reagents when they are added, but preferably no additional water solvent is added to the reaction mixture.

[0155] In other preferred embodiments, the solvent is substantially free of water, methanol, and ethanol, for example the solvent in the reaction mixture, i.e. the entire reaction mixture, may comprise water, methanol, and ethanol in a combined amount of less than 10 wt%, such as less than 5 wt%, for example less than 3 wt% or less than 1 wt%.

[0156] Preferably, the solvent in the reaction mixture, i.e., the entire reaction mixture, comprises at least 70 wt%, more preferably at least 80 wt%, more preferably at least 90 wt% of one or more polar aprotic solvents (such as one or more of DCM, DMF, DMA, ACN, methylpyrrolidone, dimethylsulfoxide, and MeTHF). In this embodiment, the polar aprotic solvent is preferably DMA.

[0157] The method of the present invention may further comprise one or more purification steps.

[0158] The method of the present invention may further comprise preparing a compound of formula (I), for example, R 1 When R is a peptide or amino acid, the method may further comprise coupling said peptide or amino acid to a Tbt residue. 1 Any suitable peptide coupling technique may be used to attach the group to the Tbt residue. In some cases, R is attached to the Tbt residue with, for example, pivaloyl chloride or isobutyl chloroformate. 1Compounds of formula (I) may be prepared by preactivating an amino acid or peptide and then coupling with Tbt, optionally silylated Tbt. A method for producing peptides involving silylated peptides is disclosed in WO 2009 / 065836.

[0159] The method of the present invention may further include preparing the amino-containing moiety. For example, when the amino-containing moiety includes a C-terminal capping group, such as a capping group having the formula -XYZ (e.g., -NHCHCHPh) as defined above, this process may include activating the C-terminal carboxylic acid group of the amino-containing moiety, which typically includes an amino-protecting group such as Cbz, followed by coupling with HXYZ (e.g., HNCHCHPh). Suitable activating agents for this step include pivaloyl chloride or isobutyl chloroformate.

[0160] Target peptide Target peptides according to the invention will typically have a length of up to 20 amino acids. Preferably, the target peptide is 2-10, 3-7, or 3-5, such as 3 amino acids in length.

[0161] The target peptide is preferably an antimicrobial peptide.

[0162] Preferably, the target peptide is a compound of formula (VII) or a salt thereof: AA1-AA2-AA3-XYZ (VII) where: each AA1 and AA3 is independently a cationic amino acid, preferably lysine or arginine, but may also be histidine or any non-genetically encoded or modified amino acid that has a positive charge at pH 7.0; AA2 is Tbt, i.e.

[0163] [ka]

[0164] where the wavy bond on the left indicates the attachment point to AA1 and the wavy bond on the right indicates the attachment point to AA3-XYZ; X, Y, and Z are as defined above.

[0165] Suitable non-genetically encoded or modified amino acids as AA1 and / or AA3 are set forth above.

[0166] The compound of formula (VII) is an antimicrobial peptide and is disclosed in WO 2009 / 081152.

[0167] The target peptide may include all enantiomeric forms, both D and L amino acids, as well as enantiomers resulting from chiral centers in the R groups of the amino acids and, when present, the Y or Z moieties.

[0168] Preferably, the target peptide is Arg-Tbt-Arg-NHCH2CH2Ph, i.e., the following compound:

[0169] [ka]

[0170] or a salt thereof. Most preferably, the target peptide is a compound having the structure

[0171] [ka]

[0172] (also referred to herein as AMC-109), or a salt thereof.

[0173] As shown in the examples below, protecting groups, particularly amino-protecting groups, may be used in the methods of the invention. For example, it may be necessary to remove one or more protecting groups from the reaction product of the amino-containing moiety and the activated ester to provide the target peptide. The methods of the invention may also include a step of removing any protecting groups. For example, a Cbz protecting group may be removed by hydrogenolysis with H over palladium on carbon (Pd / C). [Example]

[0174] 1.1 Preparation of intermediates Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03) As shown in the following scheme, Z-Arg-Tbt-OH (AMC-01) was prepared by activating Cbz-protected arginine with isobutylchloroformate (IBCF) followed by coupling with silylated Tbt.

[0175] [ka]

[0176] As shown in the following scheme, Z-Arg-NHEtPh (AMC-02) was prepared by activating commercially available Z-Arg-OH.HCl with IBCF and reacting the activated Cbz-protected arginine with HNEtPh, which after work-up afforded AMC-02 as a white solid in the form of the HCl salt.

[0177] [ka]

[0178] As shown in the scheme below, AMC-02 in the form of an HCl salt was then deprotected to provide H-ArgNHEtPh (AMC-03).

[0179] [ka]

[0180] The procedure for the deprotection step (Step 3) was as follows: AMC-02 (37.20 g, 75% wt.) in the form of the HCl salt, MeOH (550 mL), and water (130 mL) were introduced into a 2 L three-neck flask. The suspension was stirred under nitrogen until all AMC-02 was completely dissolved. Pd / C (10% wt., 50% wet, 2.27 g, 1.5 mol%) was added, and the N atmosphere was replaced with H (using a H generator at 0.7 bar). The deprotection conversion was followed by HPLC, and complete conversion was obtained after 3 h. The Pd / C was then filtered and washed twice with MeOH / water (8 / 2, v / v, 2 × 75 mL). The combined filtrates were concentrated under reduced pressure (bath = 55 °C, 300 mbar to 50 mbar) to give 31 g of concentrated solution. DMA (100 mL) was added and evaporation continued (Tbath = 65 °C, 25 mbar) to provide AMC-03 in the form of the HCl salt as a colorless solution (117 mL, 115 g, 17% wt by NMR, estimated 19.6 g net peptide, >99% yield).

[0181] 1.2 Coupling of Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03) to provide Z-Arg-Tbt-Arg-NHEtPh (AMC-04) A 250 mL round-bottom flask was charged with AMC-01 (20.02 g, 80% wt), followed by HOPO (2.82 g), DMA (67 mL), AMC-03 (44.10 g, 17% wt) in the colorless solution described above, and HCl 4N in dioxane (6.0 mL). The resulting solution was cooled to 2.5 ± 2.5 °C, and EDC.HCl (5.78 g) was added. The reaction mixture was warmed to room temperature and stirred for 20 h. The coupling conversion was monitored by HPLC.

[0182] The reaction mixture (138 mL) was diluted with water (276 mL) and EtOAc (276 mL). The phases were separated, and the resulting peptide aqueous phase (460 mL) was diluted with 6 N aq. HCl (31 mL, diluted from 12 N HCl). Prior to dilution with 6 N aq. HCl, the pH of the peptide aqueous phase was 5.4. NaCl (11.4 g) was added to minimize peptide loss in the aqueous phase, followed by EtOAc (276 mL). The phases were separated, and the resulting peptide organic phase (330 mL) was washed twice with aq. 2.5% NaCl (138 mL and 69 mL) and concentrated in a rotary evaporator (Tbath = 55 °C, 250 mbar, target solution weight: 50 g) to yield a solution of AMC-04 (44.21 g). The pH of the solution containing AMC-04 was measured to be 3.2. The amount of AMC-04 in solution was determined by 1H NMR, and the purity was determined by HPLC.

[0183] [Table 2]

[0184] The parameters for the HPLC method are shown below.

[0185] [Table 3]

[0186] Surprisingly, despite the extremely high steric bulk of Tbt, the carbodiimide and additive reagents enabled the coupling of Z-Arg-Tbt-OH (AMC-01) with H-Arg-NHEtPh (AMC-03) under mild reaction conditions (20 h, 2.5 °C to RT) to afford Z-Arg-Tbt-Arg-NHEtPh (AMC-04) in high yield.

[0187] 1.3 Acid / Base Effects on the Coupling of Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03) to Provide Z-Arg-Tbt-Arg-NHEtPh (AMC-04) The effect of carrying out the reaction without any acid or base (Tests 1-5), in the presence of a base (DIPEA - Tests 6-7), or in the presence of an acid (HCl 4N (dioxane) - Tests 8-9) was investigated. In the table below, the number of equivalents of the reagents is estimated relative to the number of equivalents of AMC-01.

[0188] These reactions were carried out using the same procedure as in Example 1.2, except that for runs 1-5, HCl in dioxane was not added, and for runs 6 and 7, DIPEA base was added instead of HCl in dioxane. Conversions were followed by HPLC. The abbreviation "on" stands for overnight, and "2d" stands for 2 days.

[0189] [Table 4]

[0190] We initially expected that base would increase the conversion in shorter reaction times by enhancing the reactivity of the nucleophile (AMC-03). Similarly, it was expected that the addition of acid would hinder the reaction by protonating the amino group of AMC-03. Surprisingly, the addition of acid significantly accelerated the reaction (~95% conversion after 4 h [results not shown] and complete conversion after overnight - see experiments 8-9), while the addition of base was detrimental to the coupling conversion (experiments 6-7).

Claims

1. 1. A method of peptide synthesis comprising: reacting a compound of formula (I) or a salt thereof with a carbodiimide reagent to form an O-acylisourea intermediate; reacting the O-acylisourea intermediate with an additive to form an activated ester; reacting the activated ester with an amino-containing moiety that is an amino acid, peptide, or salt thereof containing an amino group, wherein the amino group forms an amide bond with the carbonyl marked with * in formula (I); The compound of formula (I) has the following structure: 【Chemistry 1】 Formula (I) Here, R １ is a protecting group, a peptide, or an amino acid; R ２ is H, an alkylsilyl group, or a protecting group.

2. 2. The method of claim 1, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out at a temperature of −10 to 40° C., preferably 0 to 30° C.

3. 3. The method of claim 1 or claim 2, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out under acidic conditions.

4. 4. The method of any one of claims 1 to 3, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, and / or the reaction of the O-acylisourea intermediate with the additive, and / or the reaction of the activated ester with the amino-containing moiety is acid catalyzed.

5. The method of any one of claims 1 to 4, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent is acid catalyzed.

6. 6. The method of any one of claims 1 to 5, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out at a pH of less than 6.5, preferably between 1 and 6.5, more preferably between 1.5 and 6, more preferably between 4 and 6.

7. 7. The method of any one of claims 1 to 6, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out in the presence of at least 0.1 equivalents of acid, preferably at least 0.25 equivalents of acid, and more preferably at least 0.5 equivalents of acid, relative to 1 equivalent of the compound of formula (I) or salt thereof.

8. The method of any one of claims 1 to 7, wherein the amino-containing moiety is provided in the form of a salt, preferably an acid addition salt.

9. the carbodiimide reagent is a compound of formula (II) or a salt thereof; 【Chemistry 2】 Formula (II) Here, R Ａ and R Ｂ The method of any one of claims 1 to 8, wherein each is independently selected from an organic group containing 1 to 30 non-hydrogen atoms.

10. R Ａ and R Ｂ are each independently optionally mono- or di-(C １ ~C １０ C substituted with an alkyl)amino group １ ~C １０ Alkyl; C ３ ~C ８ Cycloalkyl; Aryl; (Aryl)C １ ~C １０ alkyl; and (C ３ ~C ８ Heterocycloalkyl)C １ ~C １０ alkyl, wherein said heterocycloalkyl group is optionally selected from the group consisting of one or more C １ ~C １０ The method of claim 9 , wherein the alkyl group is substituted.

11. The method of any one of claims 1 to 10, wherein the carbodiimide reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

12. the additive is a compound of formula IV or a salt thereof, HO-R Ｃ (IV) Here, R Ｃ The method of any one of claims 1 to 11, wherein is an organic group containing 1 to 30 non-hydrogen atoms.

13. (A) The compound of formula (IV) is an N-hydroxy compound, wherein R Ｃ is an organic group containing 1 to 30 non-hydrogen atoms and at least one nitrogen atom, and the hydroxyl group in formula (IV) is bonded to said R Ｃ group, optionally wherein the N-hydroxy compound comprises a hydroxyimino group, an N-hydroxy-triazole group, an N-hydroxy-tetrazole group, an N-hydroxybenzimidazole group, an N-hydroxyindolin-2-one group, an N-hydroxypyridinone group, an N-hydroxypyrrolidine-2,5-dione group, a 4-aza, 5-aza, 6-aza, or 7-aza-1-hydroxybenzotriazole group, an N-hydroxybenzotriazole group, or 【Transformation 3】 wherein Y is CH and X is N, or Y is N and X is CH, or Y is N and X is N; or (B) R Ｃ 13. The method of claim 12, wherein is a phenyl group, optionally substituted with one or more halogen atoms or one or more electron-withdrawing groups such as, for example, nitro.

14. The method of any one of claims 1 to 13, wherein the additive is selected from the compounds in Table 1.

15. The method according to any one of claims 1 to 14, wherein the additive is 2-pyridinol 1-oxide (HOPO).

16. The method of any one of claims 1 to 14, wherein the carbodiimide reagent is not N,N'-diisopropylcarbodiimide (DIC) and the additive is not HOPO.

17. R １ cationic amino acid AA optionally containing one or more protecting groups １ and preferably R １ The method of any one of claims 1 to 16, wherein is arginine optionally containing one or more protecting groups.

18. The method of any one of claims 1 to 17, wherein the amino-containing moiety comprises one or more protecting groups and / or C-terminal capping groups.

19. The amino-containing moiety is a compound of formula (IV) or a salt thereof: AA ３ -X-Y-Z (IV) where: A.A. ３ is a cationic amino acid; X is an N atom, and the N atom is a branched or unbranched C １ ~C １０ may be substituted with an alkyl or aryl group, which may contain up to two heteroatoms selected from N, O, and S; Y is -R ａ -R ｂ -, -R ａ -R ｂ -R ｂ - and -R ｂ -R ｂ -R ａ represents a group selected from: R ａ is C, O, S, or N; R ｂ is C; R ａ and R ｂ Each of these is C １ ~C ４ may be substituted or unsubstituted by alkyl groups, Z is a group containing 1 to 3 cyclic groups, each of which is a cyclic group of 5 or 6 non-hydrogen atoms, and two or more of said cyclic groups may be fused, and one or more of said cyclic groups may be substituted; said Z moiety contains up to 15 non-hydrogen atoms; The bond between Y and Z is R of Y. ａ or R ｂ and a non-hydrogen atom of said cyclic group of one of Z.

20. A.A. ３ 20. The method of claim 19, wherein is lysine and / or arginine, preferably arginine.

21. (A) X is unsubstituted, and / or (B) Y is —CH ２ -CH ２ - and / or (C) Z is phenyl.

22. 22. The method of any one of claims 1 to 21, wherein the reaction of the compound of formula (I) or salt thereof with the carbodiimide reagent, the reaction of the O-acylisourea intermediate with the additive, and the reaction of the activated ester with the amino-containing moiety are carried out in a solvent, wherein the solvent comprises water, methanol, and ethanol in a combined amount of less than 10 wt%, preferably less than 5 wt%, more preferably less than 3 wt%, more preferably less than 1 wt%.

23. A method for producing a target peptide, comprising the method of peptide synthesis according to any one of claims 1 to 22, wherein the target peptide is 【Chemistry 4】 or a salt thereof.

24. A compound of formula (III) or a salt thereof, 【Transformation 5】 Formula (III) Here, R １ and R ２ is as defined in claim 1, and R Ａ and R Ｂ A compound of formula (III) or a salt thereof, wherein:

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