Peptide synthesis method involving sterically hindered tri-tert-butyl tryptophan (Tbt) residues

Abstract

The invention relates to a peptide synthesis method. The peptide synthesis method comprises the following steps: reacting a compound as shown in a formula (I) or a salt thereof with a carbodiimide reagent to generate an O-acyl isourea intermediate; reacting the O-acyl isourea intermediate with an additive to generate active ester; and reacting the active ester with an amino group-containing moiety which is an amino acid, a peptide or an amino group-containing salt thereof wherein the amino group forms an amido bond with the carbonyl group marked as * in formula (I); the structure of the compound shown in the formula (I) is shown in the specification. And wherein R1 and R2 are as defined herein. The present invention also relates to a compound of formula (III) as defined in the disclosure or a salt thereof. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a peptide synthesis method and a targeting peptide preparation method. Background Art

[0002] Numerous coupling strategies are available for peptide production at the laboratory scale. However, most are too expensive for commercial-scale production. Coupling reactions between amino acids are almost exclusively facilitated by activating the carboxylic acid of the incoming amino acid. For example, O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HTBU) is a highly effective activator with minimal racemization. However, HTBU is expensive.

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

[0004] Badlands et al., in Tetrahedron Letters 58 (2017) 4391-4394, disclose a series of reactions with amide bond-forming reagents, including the carbodiimides N,N'-diisopropylcarbodiimide (DIC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) with the additives HOPO and Oxyma. As described by Badlands et al., couplings involving hindered carboxylic acids can be problematic. In particular, Badlands et al. report that the hindered carboxylic acid 2,6-dimethylbenzoic acid requires the forced conditions of extended reaction time at high temperature (70°C for 48 hours) to provide moderate yields of the desired amide product when using DIC / HOPO, and that all other coupling agents tested provide insufficient yields for this hindered carboxylic acid (Table 3, entries 13 and 14). Badlands et al. also reported that at a lower temperature of 20°C, the conversion of 2,6-dimethylbenzoic acid to the amide using DIC / HOPO was negligible (page 4392, penultimate sentence). Summary of the Invention

[0005] The present inventors have surprisingly discovered that, despite the extremely large steric bulk of the side chain of the tri-tert-butyltryptophan (Tbt) residue in formula (I), a carbodiimide / additive method can be used to couple the amino-containing moiety defined herein to the compound of formula (I) in a high yield that is advantageous for commercial processes, at relatively low temperatures, and without significant observed epimerization.

[0006] In one aspect, the present invention provides a method for peptide synthesis. This method may be a step for synthesizing a targeting peptide. The peptide synthesis method in this aspect comprises:

[0007] The compound of formula (I) or its salt reacts with a carbodiimide reagent to form an O-acylisourea intermediate;

[0008] The O-acylisourea intermediate reacts with the additive to form an active ester; and

[0009] The active ester reacts with an amino group-containing moiety, which is an amino acid, a peptide, or a salt thereof containing an amino group, wherein the amino group forms an amide bond with the carbonyl group marked with * in formula (I);

[0010] Wherein, the structure of the compound of formula (I) is:

[0011]

[0012] wherein R1 is a protecting group, a peptide or an amino acid; and

[0013] Wherein, R2 is H, alkylsilyl or protecting group.

[0014] Another aspect provides an amino acid coupling method. In the context of this aspect, "amino acid coupling" refers to the coupling of one amino acid or peptide to another amino acid or peptide. That is, each coupling partner can independently be an amino acid or peptide. The embodiments of the other aspects of the invention described herein apply mutatis mutandis to this aspect of the invention. The amino acid coupling method of this aspect includes:

[0015] The compound of formula (I) or its salt reacts with a carbodiimide reagent to form an O-acylisourea intermediate;

[0016] The O-acylisourea intermediate reacts with the additive to form an active ester; and

[0017] The active ester reacts with an amino group-containing moiety, which is an amino acid, a peptide, or a salt thereof containing an amino group, wherein the amino group forms an amide bond with the carbonyl group marked with * in formula (I);

[0018] Wherein, the structure of the compound of formula (I) is:

[0019]

[0020] wherein R1 is a protecting group, a peptide or an amino acid; and

[0021] Wherein, R2 is H, alkylsilyl or protecting group.

[0022] In a third aspect, the present invention provides a method for preparing a targeting peptide, comprising:

[0023] The compound of formula (I) or its salt reacts with a carbodiimide reagent to form an O-acylisourea intermediate;

[0024] The O-acylisourea intermediate reacts with the additive to form an active ester; and

[0025] The active ester reacts with an amino group-containing moiety, which is an amino acid, a peptide, or a salt thereof containing an amino group, wherein the amino group forms an amide bond with the carbonyl group marked with * in formula (I);

[0026] Wherein, the structure of the compound of formula (I) is:

[0027]

[0028] wherein R1 is a protecting group, a peptide or an amino acid; and

[0029] Wherein, R2 is H, alkylsilyl or protecting group.

[0030] The embodiment of other aspects of the present invention described herein is through necessary amendments, is applicable to a third aspect of the present invention.The reaction of enumerating above can generate the precursor of targeting peptide or targeting peptide.For example, the subsequent step of removing one or more protecting groups may be that targeting peptide is provided necessary. DETAILED DESCRIPTION

[0031] Compound of formula (I)

[0032] Formula (I) includes a very bulky tri-tert-butyltryptophan (Tbt) residue, the structure of which is:

[0033]

[0034] The inventors have surprisingly found that despite the extremely large steric bulk of the Tbt side chain, amino acids or peptides can be coupled to compounds of formula (I) using the carbodiimide / additive method at low temperatures and in high yields.

[0035] In formula (I), R1 is: a protecting group (usually an amine protecting group), a peptide or an amino acid. Optionally, the peptide or amino acid itself may contain one or more protecting groups, for example, on its N-terminal amino group.

[0036] As used herein, the term peptide includes peptoids, although preferred real peptides. The typical characteristics of peptoids are polarity, three-dimensional size and functionality (biological activity) that retain their peptide equivalents, but wherein peptide bonds are usually replaced by more stable connections. "Stable" refers to the enzymatic degradation that is more resistant to hydrolases. In general, the key that replaces amide bonds (amide bond substitutes) retains many properties of amide bonds, such as conformation, steric volume, electrostatic properties, the possibility of hydrogen bonds, etc. Krogsgaard, Larsen, Liljefors and Madsen (editor-in-chief) provide a general discussion of design and synthesis of peptoid technology in 1996 " Holwood College Publications " Chapter 14 " Drug Design and Development ". In this case, when the targeting peptide reacts with membrane rather than reacting with the specific active site of the enzyme, some problems of described accurate imitation affinity and efficacy or substrate function are irrelevant, and can easily prepare peptoids based on the motif of given peptide structure or required functional group. Suitable amide bond substitutes include the following groups: N-alkylation (Schmidt, R. et al., International Journal of Peptide Protein Research, 1995, 46, 47), post-inverse amide (Chorev, M and Goodman, M., Chemical Research Accounts, 1993, 26, 266), thiamine (Sherman DB and Spatola, AF, Journal of the American Chemical Society, 1990, 112, 433), thioester, phosphonate, ketone vinyl (Hoffman, RV and Kim, HO, Organic Chemistry, 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahedron Letters, 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J., Chemical and Pharmaceutical Bulletin, 1997 45, 13), methylene glycol (Spatola, AF, Methods in Neuroscience, 1993, 13, 19), alkanes (Lavielle, S. et al., International Journal of Peptide and Protein Research, 1993, 42, 270) and sulfonylamino groups (Luisi, G. et al., Tetrahedron Letters, 1993, 34, 2391).

[0037] Thus, the term "amino acid" may be conveniently used herein to refer to the equivalent subunits of peptidomimetic compounds. Furthermore, peptidomimetics may have groups that are equivalent to the R groups of amino acids.

[0038] As discussed in the aforementioned texts, as well as replacement of amide bonds, peptidomimetics can involve the replacement of larger moieties with di- or tri-peptidomimetic structures, in which case mimetic moieties involving peptide bonds, such as azole-derived mimetics, can be used as dipeptide surrogates. Therefore, however, peptidomimetics and peptidomimetic backbones in which the amide bonds have been replaced as described above are preferred.

[0039] Suitable peptidomimetics include reduced peptides in which the amide bond is reduced to a methyleneamine by treatment with a reducing agent, such as borane, or a hydriding agent, such as lithium aluminum hydride. This reduction has the added advantage of increasing the overall cationicity of the molecule.

[0040] Other peptoids include, for example, peptoids formed by the stepwise synthesis of amide-functionalized polyglycine. Some peptoid backbones are readily obtained from their peptide precursors, such as methylated peptides. Ostresh, JM et al. describe suitable methods in PNAS (1994) 91, 11138-11142. Strong base conditions will favor N-methylation over O-methylation and result in methylation of some or all of the nitrogen atoms of the peptide bonds and N-terminal nitrogen. Preferred peptoid backbones include polyesters, polyamines and their derivatives, and substituted alkanes and alkenes. Peptoids preferably have N and C termini, which can be modified as discussed herein.

[0041] Preferably, the term "amino acid" as used herein refers to proteinogenic (genetically encoded) amino acids. Preferably, the terms peptide and amino-containing moiety in R1 refer to peptides formed from proteinogenic amino acids.

[0042] R1 generally comprises 1-10 amino acids, preferably 1-5 or 1-3 amino acids, and most preferably 1 amino acid.

[0043] A wide selection of protecting groups suitable 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., Chemical Reviews, 2009, 109, 6, 2455-2504).

[0044] Suitable amine protecting groups (also known as amino protecting groups) include: carbonyl phenoxy (also known as phenoxycarbonyl, designated as Z or Cbz), tert-butyloxycarbonyl (also designated as Boc), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), 9-fluorenylmethoxycarbonyl (also known as Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc), 2,4-dimethoxyphenyl (Dmb), 2-hydroxy-4-methoxyphenyl (Hmb) and 2-Fmoc-oxy-4-methoxyphenyl (FmocHmb). These protecting groups themselves can be R1 or one or more protecting groups can be present on R1 when R1 is a peptide or amino acid.

[0045] For example, suitable carboxyl protecting groups that can be used include easily cleavable ester groups such as benzyl (Bn), p-nitrophenyl (pNb), pentachlorophenyl (PClP), pentafluorophenyl (Pfp) or tert-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-dioxanyl)-3-methylbutyl]amino}benzyl ester (Dmab), allyloxycarbonyl (Alloc) and 2-phenylisopropyl (2-PhiPr).

[0046] Thiol protecting groups include p-methoxyphenyl (Mob), tributyl (Trt), acetamidomethyl (Acm), tert-butyl (tBu), tert-butylthio (tButhio), and monomethoxytrityl (Mmt) groups.

[0047] Amine protecting groups such as Boc and carboxyl protecting groups such as tBu can be removed simultaneously by treatment with an acid, for example trifluoroacetic acid. Thiol protecting groups such as Trt can be removed selectively using an oxidizing agent such as iodine.

[0048] Preferably, R1 is a peptide or amino acid optionally including one or more protecting groups, for example on its N-terminal amino group. The amino acid may be a cationic amino acid AA1.

[0049] Preferably, R1 is a cationic amino acid AA1 optionally comprising one or more protecting groups, for example on its N-terminal amino group.

[0050] AA1 is preferably lysine or arginine, but may also be histidine or any non-genetically encoded or modified amino acid that carries a positive charge at pH 7.0. Suitable non-genetically encoded amino acids and modified amino acids that provide cationic amino acids include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminobenzoic acid, diaminopropionic acid, and homoarginine, as well as trimethyllysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidinopiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0051] Most preferably, R1 is arginine optionally including one or more protecting groups, for example on its N-terminal amino group.

[0052] In formula (I), R2 is selected from H, alkylsilyl or protecting group (usually amine protecting group). Alkylsilyl can be mono (C1-C6 alkyl)silyl, di (C1-C6 alkyl)silyl or tri (C1-C6 alkyl)silyl. Preferably, alkylsilyl is tri (C1-C6 alkyl)silyl, more preferably tri (C1-C3 alkyl)silyl. Each alkyl group can be the same or different, preferably the same. If present as R2, alkylsilyl is preferably trimethylsilyl. Protecting group can be amine protecting group as defined above. For example, protecting group is Cbz, Boc, Mtr, Troc, Dmb, Hmb or FmocHmb.

[0053] R2 is preferably H. That is, the compound of formula (I) preferably has the following structure

[0054] In the process of the present invention, the compound of formula (I) can 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 according to the amino-containing moiety.

[0055] amino-containing moiety

[0056] The amino group-containing moiety is an amino acid, a peptide, or a salt thereof. The amino acid or peptide containing the amino group may optionally contain one or more protecting groups and / or a C-terminal blocking group. The amino acid or peptide containing the amino group may optionally be silanized. Suitable silane agents are disclosed in WO 2009 / 065836. For example, suitable silane 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-oxazolidinone. Silylation can improve the solubility of the amino-containing moiety, for example, in polar organic solvents, such as polar aprotic solvents like dimethylacetamide. During the silylation process, one or more functional groups, such as amino, hydroxyl, thiol, or carboxyl groups, in the amino-containing moiety having an active hydrogen react with a silanizing agent. The silylated amino-containing moiety then includes one or more silyl groups (e.g., trialkylsilyl groups, typically tri(C1-C3)alkyl groups, such as trimethylsilyl) bonded to the functional groups.

[0057] 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 an amino acid containing the amino moiety, may form acid addition salts. Suitable acid addition salts may be formed with strong inorganic acids, such as inorganic acids such as sulfuric acid, phosphoric acid or hydrochloric acid; with organic carboxylic acids; or with organic sulfonic acids, such as (C1-C4) alkyl or aryl sulfonic acids, such as methyl or p-toluenesulfonic acid, which are unsubstituted or substituted with halogens such as chloroacetic acid; alkane carboxylic acids of 1-4 carbon atoms (e.g., acetic acid) which are unsubstituted or substituted with 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.

[0058] Preferably, the salt of the amino-containing moiety comprises 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 wherein the side chain of AA3 is protonated.

[0059] The amino-containing moiety typically includes a reactive amino group, such as an α-amino group.

[0060] The amino acid-containing portion typically includes 1-10 amino acids, preferably 1-5 or 1-3 amino acids, and most preferably 1 amino acid.

[0061] As mentioned above, suitable protecting groups for amino acids are well known, and the protecting groups listed above can also be used for amino-containing moieties.

[0062] Suitable C-terminal capping groups have the formula -XYZ, wherein the left hyphen indicates the point of attachment to the C-terminal carbonyl carbon, and X, Y, and Z are as defined below in formula (VI). In other words, the capping group -XYZ, if present, is attached to the remaining amino-containing moiety as follows:

[0063] wherein R represents the side chain of the C-terminal amino acid. Preferably, the -XYZ combination is a -NHCH2CH2Ph group.

[0064] Preferably, the amino-containing moiety is a compound of formula (VI) or a salt thereof:

[0065] AA3-XYZ(VI)

[0066] wherein AA3 is a cationic amino acid, preferably lysine or arginine, but can be histidine or any non-genetically encoded or modified amino acid that carries a positive charge at pH 7.0;

[0067] X is a nitrogen atom, which may, but preferably is not, a branched or unbranched C1-C10 Alkyl or aryl (such as C4-C 10 aryl), such as methyl, ethyl or phenyl, and the alkyl or aryl group may contain up to 2 heteroatoms selected from N, O and S;

[0068] Y represents a member selected from -R a -R b -, -R a -R b -R b -and-R b -R b -R a - a group wherein

[0069] R a is C, O, S or N, preferably C, and

[0070] R b It is C; R a and R b may be substituted or unsubstituted by C1-C4 alkyl, preferably Y is -R a -R b -(where R a Preferably C), preferably the group is unsubstituted, when Y is -R a -R b -R c - or R b -R b -R a -, then preferably one or more R a and R b is replaced; and

[0071] Z is a group comprising 1-3 cyclic groups, each cyclic group containing 5 or 6 non-hydrogen atoms (preferably C atoms), and 2 or more cyclic groups may be fused; one or more rings may be substituted, and these substitutions may, but generally do not include, polar groups; suitable substituents include halogen, preferably bromine or fluorine, and C1-C4 alkyl; the Z moiety contains up to 15 non-hydrogen atoms, preferably 5-12, and most preferably is phenyl;

[0072] The bond between Y and Z is R of Y a or R b A covalent bond to a non-hydrogen atom of one of the cyclic groups of Z.

[0073] The compound of formula (VI) optionally may contain one or more protecting groups and / or be silanized. The above discussion of silanization and suitable silanizing agents also applies to the case where the amino-containing moiety is a compound of formula (VI). When the amino-containing moiety is a compound of formula (VI), the protecting groups listed above may also be used.

[0074] 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 trimethyllysine and trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamimidinopiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0075] Preferably, Y is -R as defined above a -R b -, more preferably, wherein R a and R b is unsubstituted, most preferably, R a and R b In other words, Y is most preferably -CH2CH2-.

[0076] Preferably, the -XYZ combination is a -NHCH2CH2Ph group.

[0077] Most preferably, AA3 is arginine.

[0078] In other preferred cases, the amino-containing moiety is an amino acid comprising AA3 or a peptide comprising AA3 as the N-terminal amino acid, or a salt thereof, optionally wherein the amino acid or peptide comprises one or more protecting groups and / or a C-terminal end-blocking group. The definition of AA3 in the context of formula (VI) above also applies to this case. The C-terminal end-blocking group may have a -XYZ structure, and the respective preferred definitions of -XYZ proposed above also apply to this case. The amino acid comprising AA3 or the peptide comprising AA3 as the N-terminal amino acid may optionally be silanized, and the above discussion of silanization and suitable silane agents also applies.

[0079] The compounds of the invention and the compounds used and prepared by / with the methods of the invention (e.g., formula (I), targeting peptides and amino-containing moieties) can include all enantiomeric forms, D and L amino acids, and enantiomers arising from chiral centers within the amino acid R groups and Y or Z moieties.

[0080] More preferably, the amino-containing moiety is arginine or a salt thereof optionally comprising one or more protecting groups and / or a C-terminal blocking group. In this case, the C-terminal blocking group may have the formula -XYZ, preferably -NHCH2CH2Ph.

[0081] Carbodiimide reagents and additives

[0082] Suitable carbodiimide reagents and additives for peptide coupling are disclosed by Ayman El-Faham and Fernando Alberto, Chemical Reviews, 2011, 111, 6557-6602. The carbodiimide reagents and additives disclosed in Tables 1 and 2 of this document are suitable for use in the procedures described herein.

[0083] The carbodiimide reagent may be a compound of formula (II) or a salt thereof:

[0084]

[0085] where R A and R B Each is independently selected from an organic group containing 1 to 30 non-hydrogen atoms.

[0086] Preferably, R A and R B Each independently selected from the group consisting of:

[0087] Optional single- or double-(C1-C 10 Alkyl)amino substituted C1-C 10 Alkyl (preferably C1-C6 alkyl, more preferably C1-C4 alkyl), preferably, wherein the optional substituent is mono- or di-(C1-C6 alkyl)amino, more preferably, wherein the optional substituent is (CH3)2N-;

[0088] C3-C8 cycloalkyl, preferably C5-C6 cycloalkyl;

[0089] Aryl, preferably 6-10 membered aryl; more preferably phenyl;

[0090] (Aryl)C1-C 10 Alkyl, preferably (6-10 membered aryl) C1-C6 alkyl, more preferably phenyl; or

[0091] (C3-C8 heterocycloalkyl)C1-C 10 Alkyl, wherein the heterocycloalkyl group is optionally replaced by one or more C1-C 10 Alkyl substituted, preferably (C3-C6 heterocycloalkyl)C1-C4 alkyl, wherein the heterocycloalkyl is optionally substituted by one or more C1-C4 alkyl, more preferably morpholinylethyl or (2,2-dimethyl-1,3-dioxazol-4-yl)methyl.

[0092] 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" is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Alkyl. Preferred alkyl groups are C1-C6 alkyl groups, more preferably C1-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, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0093] The term "alkynyl" is intended to include hydrocarbon chains having one or more, preferably one to three, more preferably one carbon-carbon triple bond, in either a straight or branched configuration, 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 ethyl, propyl, butyl, pentyl, and hexyl.

[0094] 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 bornyl. The definition of "cycloalkyl" includes spiro and bridged cycloalkyl groups.

[0095] The term cycloalkenyl refers to a non-aromatic cyclized alkenyl group that includes 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.

[0096] The term "aryl" refers to monocyclic or polycyclic (including bicyclic and tricyclic) aromatic hydrocarbons, including, for example, phenyl, naphthyl, anthracenyl and phenanthryl. In one embodiment, the term "aryl" refers to monocyclic and bicyclic aromatic groups (such as phenyl or naphthyl, including 1-naphthyl and 2-naphthyl) containing 6-10 carbon atoms in the ring portion.

[0097] 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 have been replaced by a heteroatom, such as O, N or S. Typically, the heteroaryl or heteroaromatic ring contains no more than 4 nitrogen atoms, no more than 2 oxygen atoms and no more than 2 sulfur atoms. Preferably, the heteroaryl group contains 1-4 heteroatoms. The term 5-10 membered heteroaryl refers to the presence of 5-10 ring members, which can be selected from the carbon or heteroatoms set forth above. Preferred heteroaryl / heteroaromatic rings contain 5 or 6 ring members. Examples of suitable heteroaryl groups include pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolyl, isoquinolyl, thienyl, indolyl, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane.

[0098] The term "heterocycloalkyl" refers to a saturated cyclized alkyl group in which one or more carbon ring members have been replaced by a heteroatom, such as O, N, or S. Typically, the heterocycloalkyl ring contains no more than four nitrogen atoms, no more than two oxygen atoms, and no more than two sulfur atoms. "C3-C8 heterocycloalkyl" is intended to include C3, C4, C5, C6, and C7 and C8 heterocycloalkyl groups. Examples of heterocycloalkyl groups include oxyethyl, tetrahydrofuranyl, 1,3-dioxy, tetrahydropyranyl, azoridinyl, pyridinyl, piperidinyl, morpholinyl, and piperazinyl.

[0099] In (C3-C8 heterocycloalkyl) C1-C 10 Alkyl and (aryl) C1-C 10 In the alkyl part, heterocycloalkyl or aryl and C1-C 10 Alkyl linker, C1-C 10 The alkyl group is connected to the rest of the formula (II). 10 The alkyl group is preferably a C1-C6 alkyl group, more preferably a C1-C4 alkyl group.

[0100] Unless otherwise indicated, aryl and heterocycloalkyl groups may be attached through any available carbon or nitrogen by replacing a hydrogen on said carbon or nitrogen.

[0101] For example, the carbodiimide can be selected from 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-dioxazole-4-methyl)carbodiimide, N-ethyl-N'-phenylcarbodiimide, N-phenylcarbodiimide. -N'-isopropylcarbodiimide, N-cyclohexyl-N'-(morpholinoethyl)carbodiimide, N-benzyl-N'-cyclohexylcarbodiimide or a salt thereof, for example a salt selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, also referred to herein as EDC.HCl), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methyl (CAS No. 22572-40-3) or N-cyclohexyl-N'-(morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CAS No. 2491-17-0).

[0102] Preferably, the carbodiimide reagent includes EDC.HCl and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, more preferably EDC.HCl.

[0103] Optionally, the carbodiimide reagent can be immobilized on a solid support, such as a polymer support, such as an insoluble polymer support used 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 commercially available) can be used in the process of the present invention. However, the process is preferably performed in solution without involving SPPS.

[0104] The structure of the O-acylisourea intermediate is determined by the choice of the carbodiimide reagent. For example, the O-acylisourea intermediate can be a compound of formula (III) or a salt thereof, wherein R1 and R2 are as defined above for formula (I), and R A and R B As defined above in formula (II).

[0105]

[0106] In another aspect, the invention relates to a compound of formula (III) as defined herein. The embodiments described herein for the other aspects of the invention apply mutatis mutandis to this aspect of the invention.

[0107] The additive reacts with the O-acylisourea intermediate to form an active ester. The active ester contains a good leaving group on the carbonyl group marked * 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 active ester. The leaving group is provided by the additive.

[0108] Additives (e.g., Formula IV HO-R C The pKa of the compound) may be less than 8, for example from 3-7.5 or 3.5-7 or 4-6.5.

[0109] The pKa values in the present disclosure can be determined in water at 25°C. If a small amount of the compound (e.g., an additive or an acid) is soluble in water, an organic co-solvent such as an equal volume mixture of methanol, dioxane, and acetonitrile can optionally be added. Techniques for measuring pKa values are known, for example, et al., Trends in Analytical Chemistry, Vol. 26, No. 11, 2007, pp. 1043-1061. Preferably, the pKa values described herein are determined by potentiometric titration.

[0110] The additive may be a compound of formula IV or a salt thereof:

[0111] HO-R C (IV)

[0112] where R C is an organic group containing 1 to 30 non-hydrogen atoms. C It may be a group that can stabilize the negative charge on oxygen, for example by delocalization of the charge, as shown in formula (IV).

[0113] R C It can be a phenyl group optionally substituted by one or more halogen atoms (preferably, wherein the halogen atoms are each independently selected from F and Cl) or one or more electron-withdrawing groups, such as a nitro group. Preferably, the phenyl group is substituted. Examples of suitable additives with this structure are pentafluorophenol, 2,3,5-trichlorophenol and 4-nitrophenol.

[0114] Alternatively, the compound of formula (IV) may be an N-hydroxy compound, wherein R C is an organic group containing 1 to 30 non-hydrogen atoms and at least one nitrogen atom, wherein the hydroxyl group in formula (IV) is connected to R CFor example, the N-hydroxy compound may include a hydroxyimino group (such as compounds 1-19 and 28-73 of Table 1), an N-hydroxytriazolyl group (such as compounds 87-91 of Table 1), an N-hydroxytetrazolyl group (such as compound 26 of Table 1), an N-hydroxybenzimidazolyl group (such as compounds 23 and 24 of Table 1), an N-hydroxyindolin-2-one group (such as compound 27 of Table 1), an N-hydroxypyridone group (such as compound 22 of Table 1), an N-hydroxypyrrolidine-2,5-dione group (such as compounds 20-21 of Table 1), a 4-aza-, 5-aza-, 6-aza- or 7-aza-1-hydroxybenzotriazolyl group (such as compounds 80 and 82-84 of Table 1), an N-hydroxybenzotriazolyl group (such as compounds 77-79 and 81 of Table 1) or a group wherein Y is CH and X is N, or Y is N and X is CH, or Y is N and X is N (as in compounds 25, 85 and 86 in Table 1).

[0115] The additive may be a compound of formula VIII or a salt thereof,

[0116]

[0117] wherein R3 is selected from cyano, -C(O)OR5, 5-10 membered heteroaryl optionally substituted with one or more C1-C6 alkyl, C2-C6 alkynyl, nitro, aryl (preferably 6-10 membered aryl, more preferably phenyl), -SO2R6, -OSO2R6, -CONR9R 10 , -C(O)-NH-OH and C(=N-OH)R7;

[0118] R4 is selected from -C(O)R8, -CONR9R 10 , aryl optionally substituted by one or more halogens (aryl (preferably 6-10 membered aryl, more preferably phenyl), 5-10 membered heteroaryl optionally substituted by one or more C1-C6 alkyl, cyano, -C(S)NR9R 10 , nitro, C1-C6 alkyl optionally substituted by aryl (preferably 6-10 membered aryl, more preferably phenyl), -S(O)NR 11 R 12 , -C(O)-NH-OH, halogen (preferably Cl), -NH2 and hydrogen;

[0119] R5 is C1-C6 alkyl;

[0120] R6 is -NH2, C1-C6 alkyl or C1-C6 haloalkyl;

[0121] R7 is halogen (preferably Cl) or C1-C6 alkyl;

[0122] R8 is C1-C6 alkoxy optionally substituted by C3-C8 heterocycloalkyl, wherein the C3-C8 heterocycloalkyl is optionally substituted by one or more C1-C6 alkyl, or wherein R8 is C3-C8 heterocycloalkyl;

[0123] R9 and R 10 are independently selected from H or C1-C6 alkyl; and

[0124] R 11 and R 12 Each is independently selected from H or C1-C6 alkyl.

[0125] The additive may be a compound of formula IX,

[0126]

[0127] wherein X1 and X2 are independently selected from O or NH, preferably, at least one of X1 and X2 is NH, and

[0128] wherein the dotted bond is absent (in which case the compound of formula IX is a compound of formula IXa ) or is present (in which case the compound of formula IX is a compound of formula IXb ).

[0129] The additive may be a compound of formula X,

[0130]

[0131] Optionally substituted with one or more substituents selected from C1-C6 alkyl or aryl (preferably 6-10 membered aryl, more preferably phenyl). Preferably, the substituent is located on the nitrogen of the indolinone ring.

[0132] The additive may be a compound of formula XI,

[0133]

[0134] wherein X3 and X4 are independently selected from O and NR 19 ,

[0135] R 19 is H or C1-C6 alkyl, preferably C1-C6 alkyl, and

[0136] R 13 and R 14 Independently selected from H or C1-C6 alkyl, or R 14 does not exist and R 13 =O or =S.

[0137] The additive may be a compound of formula XII,

[0138]

[0139] Wherein ring A is a fused tricyclic ring comprising 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 in 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**-OH, the nitrogen marked with ** is double-bonded to any available carbon ring member of ring A. The one or more substituents are preferably =N-OH. The compound of formula XII may be a compound of formula XIIa or XIIb,

[0140]

[0141] wherein Ring B and Ring C are each independently phenyl or a 5-6 membered heteroaryl group (preferably pyridyl). Formulae XIIa and XIIb may be substituted with one or more substituents of Formula XII set forth above.

[0142] The additive can be a phenol optionally substituted with one or more halogen atoms (preferably, wherein the halogen atoms are independently selected from F and Cl) or one or more electron withdrawing groups, such as a nitro group. Preferably, the phenol is substituted. Examples of suitable additives with this structure are pentafluorophenol, 2,3,5-trichlorophenol and 4-nitrophenol.

[0143] The additive may be a compound of formula XIII,

[0144]

[0145] where R 15 and R 16 are independently selected from H, halogen (preferably Cl), (C2-C6 alkylcarbonyl) C1-C6 alkyl (i.e. C3-C 12 alkylcarbonylalkyl), -C(O)OC1-C6alkyl or -C(O)C1-C6alkyl, or wherein R 15 and R 16 are combined to form an aryl ring (preferably a 6-10 membered aryl, more preferably a phenyl) or a 5- or 6-membered heteroaromatic ring comprising one or more heteroatoms selected from O, N or S, optionally wherein the aryl or heteroaromatic ring is substituted with one or more substituents selected from halogen, C1-C6 haloalkyl or nitro. 15 and R 16 The ring formed together is phenyl or pyridyl.For example, the compound of formula XIII may be 4-aza-, 5-aza-, 6-aza- or 7-aza-1-hydroxybenzotriazole.

[0146] The additive may be a compound of formula XIV,

[0147]

[0148] wherein (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.

[0149] The additive may be a compound of formula XV,

[0150]

[0151] where R 17 and R 18 is H, or R 17 and R 18 Combined together to form C5-C 10 Cycloalkyl or C5-C 10 a cycloalkenyl ring or an aryl ring (preferably a 6-10 membered aryl ring, more preferably a phenyl ring).

[0152] The additive may be hydroxypyridine-N-oxide, preferably 2-hydroxypyridine-N-oxide.

[0153] The additive may be a hydroxytetrazole, preferably 2H-tetrazole-2-ol.

[0154] The additive may be 1H-benzo[d]imidazol-1-ol optionally substituted with one or more halogen or phenyl groups. Preferably, the phenyl substituent is present and located at position 2 of the imidazole ring. If present, the halogen is preferably Cl.

[0155] The additive may be a 1-hydroxyindole-2-one optionally substituted with one or more halogens or phenyls. If present, the halogen is preferably Cl. The 1-hydroxyindole-2-one is preferably unsubstituted. The additive may be selected from the compounds listed in Table 1:

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167] In some embodiments, the additive is not HOAt.

[0168] 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.

[0169] In some embodiments, the carbodiimide-additive combination is not DIC-HOPO.

[0170] As described above, the additive reacts with the O-acylisourea intermediate to form an active ester. When the additive is a compound of formula (IV), the active ester is a compound of formula (V) or a salt thereof:

[0171]

[0172] R in formula (V) C May correspond to the non-hydroxyl portion of the additives listed in Table 1.

[0173] Reaction conditions

[0174] The reaction between the compound of formula (I) or its salt and the carbodiimide reagent, and / or the reaction between the O-acylisourea intermediate and the additive, and / or the reaction between the active ester and the amino-containing moiety can be carried out at a temperature of from -10 to 40°C, preferably from 0 to 30°C, for example from 0 to 25°C. Typically, these reactions are carried out sequentially in a one-pot process / in the same reaction vessel. In other words, all reagents of all steps of the method described in claim 1 are typically added to form a single reaction mixture (one pot). Therefore, preferably, each of the above reactions is carried out at a temperature of -10 to 40°C, preferably at a temperature of 0 to 30°C, for example at a temperature of 0 to 25°C. The reaction is typically carried out at a pressure of about 1 standard atmosphere. In some embodiments, the reagents are mixed at about 2.5°C and then heated to room temperature. Therefore, during the reaction, the temperature can vary within the above range.

[0175] The inventors have surprisingly discovered that, despite the extremely bulky side chain of the Tbt residue in formula (I), these relatively low temperatures can be used to couple the amino-containing moieties defined herein to compounds of formula (I) in high yields. In the processes described herein, it is desirable to avoid elevated temperatures to reduce the risk of side reactions leading to epimerization.

[0176] The reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety can be carried out in 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-mentioned reaction is 2-48 hours, preferably 4-36 hours, more preferably 10-24 hours, for example, about 18 hours or about 20 hours.

[0177] Since the reactions between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety are generally carried out in one pot, it is preferred not to separate the O-acylisourea and active ester intermediate.

[0178] After the compound of formula (I) or a salt thereof, the amino-containing moiety, additives, solvent and any optional acid or base have been mixed, the carbodiimide reagent may be added last. Prior to adding the carbodiimide reagent, the reaction mixture may be cooled.

[0179] In some embodiments, the carbodiimide reagent is added last to initiate the reaction.

[0180] Acidic conditions

[0181] The reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety can be carried out in the presence of an acid or base, or in the absence of an acid or base. If used, suitable bases include: DIPEA, N-methylmorpholine, pyridine, trimethylamine and 2,4,6-collidine.

[0182] Preferably, the reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety is carried out under acidic conditions. The inventors have surprisingly found that the addition of acid can improve the conversion of the compound of formula (I) (e.g., overnight or within the total duration of 4-36 hours defined above, preferably within 10-24 hours). This is contrary to the inventors' initial expectation that the addition of acid would hinder the process due to the protonation of the amino group of the amino-containing moiety.

[0183] Preferably, the reaction between the compound of formula (I) or a salt thereof and the carbodiimide reagent, and / or the reaction between the O-acylisourea intermediate and / or the additive, and / or the reaction between the active ester and the amino-containing moiety is acid-catalyzed. Acid catalysts can enhance the electrophilicity of the carbonyl group marked * in formulas (I) and (V) (and the equivalent carbonyl group in formula (III)), thereby increasing the rate of nucleophilic acyl substitution reactions on the carbonyl group, such as the reaction to form O-acylisourea.

[0184] Without wishing to be bound by theory, the inventors hypothesize that increasing the reaction rate of the compound of formula (I) or its salt with the carbodiimide reagent to form O-acylisourea can compensate for any protonation of the amino group of the amino-containing moiety. Therefore, the reaction between the compound of formula (I) or its salt and the carbodiimide reagent is preferably acid-catalyzed.

[0185] Preferably, the reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety is carried out at a pH of less than 6.5, preferably 1-6.5, more preferably 1.5-6, and more preferably 4-6. These reactions can also be carried out at a pH of less than 5, for example 1-4.5, 1.5-4 or 2-3.

[0186] When the above reaction is carried out in a non-aqueous solvent, the pH is measured by sampling the reaction mixture after the addition of the acid (preferably immediately after the addition of the acid), adding water (preferably, wherein the ratio of water added to the reaction mixture sample is 1:10 to 10:1 v / v, preferably 1:1 to 10:1 v / v, more preferably about 10:1 v / v) and mixing; optionally, separating the aqueous phase and measuring the pH of the aqueous phase or aqueous reaction mixture. Optionally, a sample can also be taken from the reaction mixture before the addition of the acid as an in-process control. When the reaction is carried out in a non-aqueous solvent, the pH can be measured using Test Method B described in ASTM D4980.

[0187] Preferably, the reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety is carried out in the presence of at least 0.1 equivalents of acid per equivalent of formula (I) or its salt, more preferably at least 0.25 equivalents of acid, more preferably at least 0.5 equivalents of acid, more preferably at least 0.8 equivalents of acid. For example, the reaction can be carried out in the presence of 0.25-2, preferably 0.5-1.5, more preferably 0.8-1.2 equivalents of acid per equivalent of formula (I) or its salt. 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, more preferably at least 0.8 equivalents of acid can be added per equivalent of formula (I) or its salt. For example, 0.25-2, preferably 0.5-1.5, more preferably 0.8-1.2 equivalents of acid can be added per equivalent of formula (I) or its salt. Optionally, in these embodiments, the acid can be any of the acids listed below, preferably HCl(dioxane), such as 4N HCl(dioxane).

[0188] 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 (HClO4), hydrochloric acid, chloric acid (HClO3), sulfuric acid, and nitric acid. Preferably, the acid is hydrochloric acid, more preferably anhydrous hydrochloric acid, such as HCl (dioxane).

[0189] The reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety can be carried out in any suitable solvent. Suitable solvents include aqueous solvents or non-aqueous solvents, preferably non-aqueous solvents. Suitable solvent examples include water, dichloromethane (DCM), dimethylformamide (DMF), N,N-dimethylacetamide (DMA), acetonitrile (ACN), N-methylpyrrolidone, dimethyl sulfoxide, 2-methyltetrahydrofuran (MeTHF), dioxane or a mixture thereof, such as a mixture of DMA, dioxane and optionally water. The solvent may preferably include one or more polar aprotic solvents (e.g., one or more of DCM, DMF, DMA, ACN, methylpyrrolidone, dimethyl sulfoxide and MeTHF), more preferably, the solvent includes DMA.

[0190] The amino-containing moiety is preferably added as a solution. In some preferred embodiments, the amino-containing moiety can be added as a solution comprising DMA solvent. Preferably, the solution comprising the amino-containing moiety comprises less than 10 wt % water, more preferably less than 7.5 wt % water, and more preferably less than 5 wt % water. In some embodiments, no aqueous solvent is added during the process (i.e., during the reaction of claim 1), other than any water present in the solution comprising the amino-containing moiety and any water entrained in the compound or the compound of formula (I) or a salt thereof that is typically added as a solid.

[0191] Preferably, the solvent is substantially free of water, such as the solvent in the reaction mixture, and thus the reaction mixture as a whole may include less than 10 wt % water, such as less than 5 wt % water, such as less than 3 wt % water or less than 1 wt % water. As indicated above, relatively small amounts of water may be present in the reagents upon addition, but it is preferred not to add additional aqueous solvent to the reaction mixture.

[0192] In other preferred embodiments, the solvent is substantially free of water, methanol, and ethanol, such as the solvent in the reaction mixture, and thus the reaction mixture as a whole may include a total amount of less than 10 wt % water, methanol, and ethanol, such as less than 5 wt %, such as less than 3 wt % or less than 1 wt %.

[0193] Preferably, the solvent in the reaction mixture, and thus the entire reaction mixture as a whole, comprises at least 70 wt %, more preferably at least 80 wt %, more preferably at least 90 wt % of one or more polar aprotic solvents (e.g., one or more of DCM, DMF, DMA, ACN, methylpyridone, dimethyl sulfoxide, and MeTHF). In this embodiment, the polar aprotic solvent is preferably DMA.

[0194] The method of the present invention may further comprise one or more purification steps.

[0195] The method of the present invention may further comprise preparing a compound of formula (1). 1 When R is a peptide or amino acid, the method may further comprise coupling the peptide or amino acid to a Tbt residue. Any suitable peptide coupling technique may be used to couple R 1 In some cases, the Tbt residue can be activated by pre-activating R 1 Amino acids or peptides are prepared, for example, using pivaloyl chloride or isobutyl chloroformate, followed by coupling with Tbt, optionally silylated Tbt. WO2009 / 065836 discloses a peptide production method involving silylated peptides.

[0196] 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 blocking group, such as a blocking group having the formula -XYZ as defined above (e.g., -NHCH2CH2Ph), the 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, and then coupling to HXYZ (e.g., H2NCH2CH2Ph). Suitable activators for this step include pivaloyl chloride or isobutyl chloroformate.

[0197] Targeting peptides

[0198] The targeting peptide according to the present invention generally has a chain length of up to 20 amino acids. Preferably, the length of the targeting peptide is 2-10, 3-7 or 3-5, for example 3 amino acids.

[0199] The targeting peptide is preferably an antimicrobial peptide.

[0200] Preferably, the targeting peptide is a compound of formula (VII) or a salt thereof

[0201] AA1-AA2-AA3-XYZ(VII)

[0202] in:

[0203] AA1 and AA3 are each independently a cationic amino acid, preferably lysine or arginine, but may be histidine or any non-genetically encoded or modified amino acid that carries a positive charge at pH 7.0;

[0204] AA2 is Tbt, that is Wherein the left curved key represents the attachment point of AA1 and the right curved key represents the attachment point of AA3-XYZ; and

[0205] X, Y and Z are as defined above.

[0206] Non-genetically encoded or modified amino acids suitable as AA1 and / or AA3 are set forth above.

[0207] The compound of formula (VII) is an antimicrobial peptide and is disclosed in WO2009 / 081152.

[0208] The targeting peptide may include all enantiomeric forms, D and L amino acids, as well as enantiomers arising from chiral centers within the amino acid R groups and Y or Z moieties, if present.

[0209] Preferably, the targeting peptide is Arg-Tbt-Arg-NHCH2CH2Ph, i.e., compound or a salt thereof. Most preferably, the targeting peptide is a compound having the following structure:

[0210] (also referred to herein as AMC-109) or a salt thereof.

[0211] As shown in the following examples, protecting groups, particularly amino protecting groups, can be adopted in the method of the present invention.For example, it may be necessary to remove one or more protecting groups to provide the targeting peptide from the product containing the reaction between the amino moiety and the active ester.The method of the present invention may include the step of removing any protecting group.For example, the Cbz protecting group can be removed by H2 hydrogenolysis on palladium carbon (Pd / C).

[0212] Example

[0213] 1.1 Preparation of intermediates Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03)

[0214] Z-Arg-Tbt-OH (AMC-01) was prepared by activating Cbz-protected arginine using isobutyl chloroformate (IBCF) followed by coupling with silylated Tbt, as shown in the following scheme.

[0215]

[0216] As shown in the following scheme, Z-Arg-NHEtPh (AMC-02) was prepared by activating commercially available Z-Arg-OH.HCl using IBCF and reacting the activated Cbz-protected arginine with H2NEtPh, thereby providing AMC-02 as an HCl salt, which was a white solid upon inspection.

[0217]

[0218] AMC-02, in its HCl salt form, is then deprotected to provide H-ArgNHEtPh (AMC-03) as shown in the following scheme:

[0219]

[0220] The procedure for the deprotection step (step 3) is as follows. AMC-02 (37.20 g, 75% wt.) in the form of the HCl salt, MeOH (550 mL) and water (130 mL) are introduced into a 2 L three-necked flask. The suspension is stirred under nitrogen until all AMC-02 is completely dissolved. Pd / C (10% wt, 50% humidity, 2.27 g, 1.5 mol%) is added and the N atmosphere is replaced with H (using an H generator at 0.7 bar). HPLC is used to perform the deprotection conversion, and complete conversion is achieved after 3 h. The Pd / C is then filtered and washed twice with MeOH / water (8 / 2, v / v, 2 x 75 mL). The filtrates are combined and concentrated to 31 g of concentrated solution under reduced pressure (T water bath = 55°C, from 300 mbar to 50 mbar). DMA (100 mL) was added and evaporated (Twaterbath = 65 °C, 25 mbar) to provide AMC-03 as the HCl salt as a colorless solution (via TMR, 117 mL, 115 g, 17% wt, expected net peptide 19.6 g, >99% yield).

[0221] 1.2 Z-Arg-Tbt-OH (AMC-01) coupled with H-Arg-NHEtPh (AMC-03) to provide Z-Arg-Tbt-Arg- NHEtPh(AMC-04)

[0222] In a 250 mL round-bottom flask, AMC-01 (20.02 g, 80% wt) was introduced, 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 (RT) and stirred for 20 h. The coupled conversion was performed using HPLC.

[0223] The reaction mixture (138 mL) was diluted with water (276 mL) and ethyl acetate (276 mL). The phases were separated and the peptide aqueous phase (460 mL) was diluted with 6N aqueous HCl (31 mL, diluted with 12N HCl). The pH of the peptide aqueous phase was 5.4 before dilution with 6N aqueous HCl. NaCl (11.4 g) was added to minimize the loss of peptide in the aqueous phase, followed by ethyl acetate (276 mL). The phases were separated and the resulting peptide organic phase (330 mL) was washed twice with aqueous 2.5% NaCl (138 mL and 69 mL) and concentrated on a rotary evaporator (T water bath = 55°C, 250 mbar, target solution weight: 50 g) to obtain AMC-04 (44.21 g) in solution. The pH of the solution containing AMC-04 was determined to be 3.2. The amount of AMC-04 in the solution was determined by 1HNMR. Purity was determined by HPLC.

[0224] test result Quantity (net peptide) Estimated 19.0g HPLC purity 95.6% (area / area) 1H NMR content 43% wt Yield 85％

[0225] The parameters of the HPLC method are provided below:

[0226]

[0227] Surprisingly, despite the extremely large steric bulk of Tbt, carbodiimide and added reagents allowed the coupling of Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03) to provide Z Arg-Tbt-Arg-NHEtPh (AMC-04) in high yield under mild reaction conditions (20 h, 2.5 °C to RT).

[0228] 1.3 Coupling of Z-Arg-Tbt-OH (AMC-01) and H-Arg-NHEtPh (AMC-03) provides Z-Arg-Tbt-Arg- Acid-base effect during NHEtPh(AMC-04)

[0229] The effect of performing the reaction in the absence of any acid or base (runs 1-5), in the presence of a base (DIPEA - runs 6-7) or in the presence of an acid (HCl 4N (dioxane) - runs 8-9) was investigated. The equivalents of the reagents were compared to the equivalents of AMC-01 in the table below.

[0230] The reaction was carried out using a procedure similar to that of Example 1.2, except that HCl in dioxane was not added in Runs 1-5, and DIPEA base was added instead of HCl in dioxane in Runs 6 and 7. Conversion was followed by HPLC, with the abbreviations "on" representing overnight and "2d" representing 2 days.

[0231]

[0232] The inventors initially expected that the addition of a base would make the nucleophile (AMC-03) more reactive, thereby increasing conversion in a shorter reaction time. Similarly, since the amino group of AMC-03 is protonated, the addition of an acid was expected to hinder the reaction. Surprisingly, the addition of an acid accelerated the reaction (approximately 95% conversion after 4 hours [results not shown], and complete conversion overnight—see Runs 8 and 9), while the addition of a base detrimentally affected the coupling conversion (Runs 6 and 7).

Claims

1. A peptide synthesis method comprising: The compound of formula (I) or its salt reacts with a carbodiimide reagent to form an O-acylisourea intermediate; The O-acylisourea intermediate reacts with the additive to form an active ester; as well as The active ester reacts with an amino group-containing moiety, which is an amino acid, a peptide, or a salt thereof containing an amino group, wherein the amino group forms an amide bond with the carbonyl group marked with * in formula (I); Wherein, the structure of the compound of formula (I) is: wherein R1 is a protecting group, a peptide or an amino acid; and Wherein, R2 is H, alkylsilyl or protecting group.

2. The method of claim 1, wherein the reaction between the compound of formula (I) or its salt and the carbodiimide reagent, the O-acylisourea intermediate and the additive, and the active ester and the amino-containing portion is carried out at a temperature of -10-40°C, preferably at a temperature of 0-30°C.

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

4. A process as claimed in any one of the preceding claims, wherein the reaction between the compound of formula (I) or a salt thereof and the carbodiimide reagent, and / or the reaction between the O-acylisourea intermediate and the additive, and / or the reaction between the active ester and the amino-containing moiety is acid catalysed.

5. A process as claimed in any one of the preceding claims, wherein the reaction between the compound of formula (I) or a salt thereof and the carbodiimide reagent is acid catalysed.

6. A method as claimed in any one of the preceding claims, wherein the reaction between the compound of formula (I) or a salt thereof and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety is carried out at a pH of less than 6.5, preferably 1-6.5, more preferably 1.5-6, more preferably 4-6.

7. A process as claimed in any one of the preceding claims, wherein the reaction between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety is carried out in the presence of at least 0.1 equivalent of acid per equivalent of the compound of formula (I) or its salt, preferably at least 0.25 equivalent of acid, more preferably at least 0.5 equivalent of acid.

8. A method as claimed in any one of the preceding claims, wherein the amino-containing moiety is provided in the form of a salt, preferably an acid addition salt.

9. The method according to any one of the preceding claims, wherein the carbodiimide reagent is a compound of formula (II) or a salt thereof: where R A and R B Each is independently selected from an organic group containing 1 to 30 non-hydrogen atoms.

10. The method of claim 9, wherein R A and R B Each independently selected from the group consisting of: optionally mono- or di-(C1-C 10 Alkyl)amino substituted C1-C 10 Alkyl; C3-C8 cycloalkyl; aryl; (aryl) C1-C 10 Alkyl; and (C3-C8 heterocycloalkyl) C1-C 10 Alkyl, wherein the heterocycloalkyl group is optionally replaced by one or more C1-C 10 Alkyl substitution.

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

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

13. The method of claim 12, wherein: (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 wherein the hydroxyl group in the formula (IV) is connected to the R C Group connection, optionally, the N-hydroxy compound includes hydroxyimino, N-hydroxytriazole, N-hydroxytetrazolyl, N-hydroxybenzimidazole, N-hydroxyindolin-2-one, N-hydroxypyridone, N-hydroxypyrrolidine-2,5-dione, 4-aza-, 5-aza-, 6-aza- or 7-aza-1-hydroxybenzotriazole, N-hydroxybenzotriazole or group 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 is phenyl optionally substituted by one or more halogen atoms or one or more electron withdrawing groups, for example nitro.

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

15. The method of any one of the preceding claims, wherein the additive is 2-hydroxypyridine-N-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. The method of any one of the preceding claims, wherein R1 is a cationic amino acid AA1 optionally comprising one or more protecting groups, preferably, wherein R1 is arginine optionally comprising one or more protecting groups.

18. A method as claimed in any one of the preceding claims, wherein the amino-containing moiety comprises one or more protecting groups and / or C-terminal capping groups.

19. A method as claimed in any one of the preceding claims, wherein the amino-containing moiety is a compound of formula (IV) or a salt thereof AA3-XYZ(IV) in: AA3 is a cationic amino acid; X is a nitrogen atom, which can be a branched or unbranched C1-C 10 Alkyl or aryl substituted, and the alkyl or aryl group may contain up to 2 heteroatoms selected from N, O and S; Y represents a member selected from -R a -R b -, -R a -R b -R b -and-R b -R b -R a - a group wherein R a is C, O, S, or N, and R b It is C; R a and R b may be substituted or unsubstituted by C1-C4 alkyl, and Z is a group comprising 1-3 cyclic groups, each cyclic group containing 5 or 6 non-hydrogen atoms, 2 or more cyclic groups may be fused, and one or more cyclic groups may be substituted; the Z moiety contains up to 15 non-hydrogen atoms; and wherein The bond between Y and Z is R of Y a or R b A covalent bond to a non-hydrogen atom of one of the cyclic groups of Z.

20. The method of claim 19, wherein AA3 is lysine and / or arginine, preferably arginine.

21. The method of claim 19 or 20, wherein (A) X is unsubstituted; and / or (B) wherein Y is -CH2-CH2-; and / or (C) wherein Z is phenyl.

22. A method as claimed in any one of the preceding claims, wherein the reactions between the compound of formula (I) or its salt and the carbodiimide reagent, between the O-acylisourea intermediate and the additive, and between the active ester and the amino-containing moiety are carried out in a solvent comprising less than 10 wt % of water, methanol and ethanol in a total amount, preferably less than 5 wt %, more preferably less than 3 wt %, more preferably less than 1 wt %.

23. A method for preparing a targeting peptide, comprising the peptide synthesis method according to any one of the preceding claims, wherein the targeting peptide is or a salt thereof.

24. A compound of formula (III) or a salt thereof: wherein R1 and R2 are as defined in claim 1, and R A and R B As defined in claim 9 or 10.

Images