WO2021148594A1 - Synthèse chimique de la partie peptidique de produits naturels bioactifs - Google Patents

Synthèse chimique de la partie peptidique de produits naturels bioactifs Download PDF

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Publication number
WO2021148594A1
WO2021148594A1 PCT/EP2021/051435 EP2021051435W WO2021148594A1 WO 2021148594 A1 WO2021148594 A1 WO 2021148594A1 EP 2021051435 W EP2021051435 W EP 2021051435W WO 2021148594 A1 WO2021148594 A1 WO 2021148594A1
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peptide
fmoc
resin
amino acid
anchored
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PCT/EP2021/051435
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English (en)
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Emilie RACINE
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Nosopharm
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • the present invention relates to a method for synthesizing peptides comprising a macrocycle which contains three amino acid residues. It also relates to specific peptides, which have been prepared by the method according to the invention, and key synthesis intermediates. BACKGROUND OF THE INVENTION
  • Madelorhabdins are a family of antibacterial peptides isolated from a Xenorhabdus cabanillasii strain and first described by M. Gualtieri et al. (WO 2012/085177 Al). They are made of a peptidic and a C-terminal part which terminates in a polyagmatine chain.
  • Madelorhabdins are produced by bacterial fermentation. They are active against a broad range of Gram-negative and Gram-positive bacterial strains, including resistant ones, and could thus be useful for the treatment of microbial infections, and notably severe infections that are multidrug-resistant. In view of their antibiotic properties, there exists a strong interest in producing Madelorhabdins by chemical synthesis for industrial development in order to improve yields, reproducibility and costs. Besides, the development of a method for synthesizing the peptidic part of Madelorhabdins as well as structurally related peptides would allow to thoroughly study their antibacterial activities.
  • the peptidic structure of Madelorhabdins is highly complex: it comprises six amino acids, among which two are non-proteinogenic, namely a proline derivative at the C-terminal position and a diaminobutyric acid derivative (Dab2).
  • the peptidic part of Madelorhabin A is represented below and referred to hereinafter as MDL-A-pp.
  • MDL-A-pp The formation of the 11 -membered ring formed by the three amino acids at the centre of the molecule (Dab2, Asn3 and Asp4) is particularly challenging.
  • success or failure of the macrocyclization of a peptide relies on the ability of the linear precursor to conformationally pre-organize its reactive ends in close spatial proximity before ring closure.
  • the third option is through cyclization between the NH 2 of the lateral chain of Dab2 and the acidic function of the lateral chain of Asn3.
  • C-terminal position cannot epimerize as there is no chiral centre in alpha position of the acidic function and the steric hindrance around the amine and acidic function is lower than for the two other strategies.
  • this synthetic strategy also involves some risks since unwanted cyclisation of aspartic acid Asp4 into aspartimide could occur during the synthesis.
  • the method according to the invention is a solid phase peptide synthesis (SPPS) using a Fmoc-strategy and a polymer-supported cyclization. It involves a triorthogonal protecting group scheme with orthogonal allyl-based protection for both amino and carboxylic moieties involved in the cyclization, Fmoc protection of N-terminal amine and suitable protection of lateral chains.
  • SPPS solid phase peptide synthesis
  • MLD-A-pp is a hexapeptide, wherein AA 1 , which is the amino acid located at the C-terminal position, is a hydroxylated homologated proline (HhProl), AA 2 is a diaminobutyric acid derivative (Dab2), AA 3 is aspargine (Asn3), AA 4 is aspartic acid (Asp4), AA 5 is histidine (His5) and AA 6 , which is the amino acid located at the N- terminal position, is aspartic acid (Asp6).
  • HhProl hydroxylated homologated proline
  • AA 2 is a diaminobutyric acid derivative
  • AA 3 is aspargine (Asn3)
  • AA 4 is aspartic acid (Asp4)
  • AA 5 is histidine (His5)
  • AA 6 which is the amino acid located at the N- terminal position, is aspartic acid (Asp6).
  • the inventors thus tested another synthetic way, in which the peptide synthesis is stopped after the introduction of AA 4 , allyl/alloc protecting groups of the lateral chains of AA 2 and AA 4 are deprotected and cyclization is done through lactamization between the amine and acidic function. Once the peptide cyclized, AA 5 and AA 6 are added before final cleavage and deprotection (Scheme 2).
  • the present invention thus relates to a method for preparing a peptide of following general formula (I): wherein:
  • R 2 represents H or CH 3 ,
  • R 3 represents H or the side chain of amino acid AA 3 ,
  • - y is an integer comprised between 0 and 3
  • - peptide c is amino acid AA 1 or a peptide comprising at its C-terminal position amino acid AA 1
  • - peptide N is amino acid AA 5 or a peptide comprising at its C-terminal position amino acid AA 5
  • a) anchoring peptide c to a resin via the carboxylic acid function of AA 1 wherein the N-terminal position of peptide c is Fmoc protected and the side chains of peptide c , if present and if necessary, are protected by suitable protecting groups, and removing Fmoc from the anchored peptide c obtained thereof
  • amino acid refers to an organic compound containing a carboxylic acid functional group and an amine functional group, namely a -COOH group and a -NRR’ group, wherein R and R’ are, independently of each other, a hydrogen atom or a (C 1 -C 6 )alkyl group, preferably a hydrogen atom or a (C 1 -C 3 )alkyl group, or together form, with the nitrogen atom that bears them, a saturated monocycle comprising preferably 5 or 6 atoms in the ring, including the nitrogen that bears R and R’, the remainder being carbon atoms.
  • (C 1 -C 6 )alkyl refers to a straight or branched monovalent saturated hydrocarbon chain containing from 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec- butyl, t-butyl, n-pentyl, n-hexyl, and the like.
  • the amine functional group of an amino acid is typically a -NH 2 or -NHCH 3 group or a pyrrolidine.
  • the amine and the carboxylic acid groups of an amino acid may be attached to the same carbon atom, such an amino acid being referred to as a-amino acid, or to distinct carbon atoms.
  • the shortest chain of atoms, notably of carbon atoms, that bears the amine and the carboxylic acid groups is referred to as the main chain, and the chain of atoms, notably of carbon atoms that is branched to the main chain, if any, as the “side chain” of the amino acid.
  • the “side chain” of an amino acid, in particular of an a-amino acid is typically a (C 1 -C 6 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )thioalkoxy , (C 1 -C 6 )alky lamino, di(C 1 -C 6 )alkylamino group, optionally substituted by one or more groups selected from a halogen atom, -OR, -NRR’, -SR, -SeH, -C(O)OR, -C(O)NRR’, -C(O)R, -NH-C(NH)-NRR’, wherein R and R’ are, independently of each other, a hydrogen atom or a (C 1 -C 6 )alkyl group, preferably a hydrogen
  • (C 2 -C 6 )alkenyl refers to a straight or branched monovalent unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one double bond including, but not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl and the like.
  • (C 2 -C 6 )alkynyl refers to a straight or branched monovalent unsaturated hydrocarbon chain containing from 2 to 6 carbon atoms and comprising at least one triple bond including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
  • (C 1 -C 6 )alkoxy refers to a (C 1 - C 6 )alkyl group as defined above bound to the molecule via an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec- butoxy, t-butoxy, n-pentoxy, n-hexoxy, and the like.
  • (C 1 -C 6 )alkylamino refers to an -NHAlk group with Alk representing a (C 1 -C 6 )alkyl group as defined above, including, but not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, n- butylamino, iso-butylamino, sec-butylamino, t-butylamino, n-pentylamino, n- hexylamino, and the like.
  • di(C 1 -C 6 )alkylamino refers to an - NAlk 1 Alk 2 group with Alk 1 and Alk 2 representing, independently of one another, a (C 1 - C 6 )alkyl group as defined above, including, but not limited to, dimethylamino, diethylamino, ethylmethylamino and the like.
  • halogen refers to an atom of fluorine, bromine, chlorine or iodine.
  • aryl refers to an aromatic hydrocarbon group comprising preferably 6 to 10 carbon atoms and comprising one or more fused rings, such as, for example, a phenyl or naphtyl group.
  • aryl is a phenyl group.
  • cycloalkyl refers to a saturated hydrocarbon ring comprising from 3 to 7, advantageously from 5 to 7, carbon atoms including, but not limited to, cyclohexyl, cyclopentyl, cyclopropyl, cycloheptyl and the like.
  • heterocycle refers to a saturated or unsaturated non- aromatic monocycle or polycycle, comprising fused, bridged or spiro rings, preferably fused rings, advantageously comprising 3 to 10, notably 3 to 6, atoms in each ring, in which the atoms of the ring(s) comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms.
  • heteroaryl refers to an aromatic heterocycle as defined above. It can be more particularly an aromatic monocyclic, bicyclic or tricyclic heterocycle, each cycle comprising 5 or 6 members, such as a pyrrole, a furane, a thiophene, a thiazole, an isothiazole, an oxazole, an isoxazole, an imidazole, a pyrazole, a triazole, a tetrazole, a pyridine, a pyrimidine, an indole, a benzofurane, a benzothiophene, a benzothiazole, a benzoxazole, a benzimidazole, an indazole, a benzotriazole, a quinoline, an isoquinoline, a cinnoline, a quinazoline, a quinoxaline, a carbazole.
  • the “side chain” of an amino acid, in particular of an ⁇ -amino acid is preferably a (C 1 -C 6 )alkyl group, optionally substituted by one or more groups selected from a halogen atom, -OR, -NRR’, -SR, -SeH, -C(O)OR, -C(O)NRR’, -C(O)R, -NH-C(NH)-NRR’, aryl and heteroaryl group, said aryl and heteroaryl groups being optionally substituted by one or more groups selected from a halogen atom, -OR, -NRR’, -SR, -C(O)OR, -C(O)NRR’, -C(O)R, wherein R and R’ are, independently of each other, a hydrogen atom or a (C 1 -C 6 )alkyl group, preferably a hydrogen atom or a (C 1 -
  • the “main chain” of an amino acid is typically a straight divalent saturated hydrocarbon chain containing from 1 to 6 carbon atoms, bearing at one end the carboxylic acid functional group of the amino acid and at the other end its amine functional group, said hydrocarbon chain being optionally further substituted by a side chain as defined above and optionally by one or more groups selected from a halogen atom, -OH, -SH, -C(O)OAlk with Alk representing a (C 1 -C 6 )alkyl group as defined above, -C(O)NRR’, -C(O)R, wherein R and R’ are, independently of each other, a hydrogen atom or a (C 1 -C 6 )alkyl group, preferably a hydrogen atom or a (C 1 -C 3 )alkyl group, aryl, heteroaryl, cycloalkyl and heterocycle group, said aryl, heteroaryl, cycloalkyl and heterocycle
  • amino acid is thus intended to refer to natural a-amino acids, namely Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamine (Gin), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (lie), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Selenocysteine (Sec), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) and Valine (Val), in the D or L form, as well as non-natural amino acids.
  • it is a natural or non-natural ⁇ - amino acid, more preferably a natural a-amino acid.
  • peptide refers to a linear chain of amino acids as defined above linked together by means of a peptide bond (i.e. by means of an amide function C(O)-N).
  • amino acid residue refers to an amino acid as defined above incorporated to a peptide by means of its carboxylic acid function and/or its amine function.
  • the “peptide” may consist in 2 to 10 amino acid residues as defined above (and preferably natural a-amino acids), notably 2 to 6 amino acid residues. In particular, it is a dipeptide, tripeptide or tetrapeptide.
  • C-terminal position refers to the free carboxylic acid function at one end of the peptide and by extension to the amino acid residue to which it belongs.
  • N-terminal position refers to the free amine function at the other end of the peptide and by extension to the amino acid residue to which it belongs.
  • free carboxylic acid and “free amine” functions refer to said functions which are not involved in a peptide bond together with another amino acid residue.
  • the method according to the invention is a solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • the term “resin” as used in the present invention refers to the solid phase material to which the (amino-protected) amino acid to be located at the C-terminal position of the targeted peptide is anchored, at the beginning of the peptide synthesis. It thus refers to the polymer beads and the linker attached to it; the C-terminal amino acid residue being covalently bound to said linker at the initial stage of the synthesis.
  • the polymer beads of the resin are typically made of a polymer-matrix and have a particle size comprised between 10 ⁇ m to 1 mm, in particular between 20 ⁇ m to 600 ⁇ m.
  • Said polymer-matrix typically comprises at least a linear or branched, optionally crosslinked, polymer or copolymer selected from the group consisting of polystyrene (PS), polyacrylic acid (PA), polyethylene glycol (PEG) and composites thereof.
  • the linker of the resin may be any linker commonly used in the field of Fmoc-based SPPS. It may notably be a a trityl linker, a Rink amide linker, a 4-hydroxymethylphenoxyacetyl (HMPA) linker or a Sieber amide linker, preferably a trityl linker.
  • trityl resin refers in particular to a resin of the following formula (R): wherein:
  • PB represents a polymer bead
  • - R trt represents a chlorine atom, a -NH- (C 1 -C 6 )alkyI-NH 2 group, a -O-(C 1 -C 6 )alkyl- NH 2 group or a Fmoc protected amino acid residue, preferably a chlorine atom
  • R trt represents a chlorine or a hydrogen atom, preferably a chlorine atom.
  • Fluorenylmethyloxycarbonyl protecting group refers to the well known 9-fluorenylmethyloxycarbonyl protecting group.
  • protecting group refers to a chemical group which selectively blocks a reactive site in a multifunctional compound so as to allow selectively performing a chemical reaction on another unprotected reactive site.
  • the protecting groups of the amino acid side chains are orthogonal to the Fmoc-protecting group, meaning that the Fmoc-protected amine function may be specifically deprotected without affecting the other functions protected by said protecting groups.
  • the side chain protecting groups that may typically be used in the context of the present invention depending on the functional group to be protected are summarized in the table below:
  • Fmoc-AA(PG)-OH refers to an amino acid AA having its amine functional group protected by Fmoc, its side chain protected by a protecting group “PG”, and its carboxylic acid functional group “free”, i.e. unprotected (“-OH”).
  • the Fmoc protecting group may be removed in steps a), b), c), f) and g) by using a solution of piperidine in N,N-dimethylformamide.
  • particularly preferred side chain protecting groups are acid-labile protecting groups.
  • acid-labile protecting group refers to a protecting group as defined above, which can be removed under acidic conditions, for example by using trifluoracetic acid.
  • the coupling reaction occurring in steps b), c), d) and g), which aims at forming a peptide bond between an amine function and a carboxylic acid function, is well known to the person skilled in the art who will be able to determine the reaction conditions thereof.
  • Such an amide bond formation is typically carried out in the presence of a peptide coupling reagent, which allows the activation of the carboxylic acid.
  • Said coupling agent may be notably a carbodiimide, an iminium/uranium salt, a phosphonium salt or propanephosphonic acid anhydride (T3P).
  • Carbodiimide coupling reagents include notably dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC), N-(3- Dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, ED AC, EDCI) and the like.
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC N-(3- Dimethylaminopropyl)-N’-ethylcarbodiimide
  • EDC N-(3- Dimethylaminopropyl)-N’-ethylcarbodiimide
  • auxiliary nucleophile such as hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), ethyl cyanohydroxyiminoacetate (Oxyma) and the
  • Iminium/uranium or phosphonium type coupling reagents are typically salts of a non-nucleophilic anion, such as tetrafluoroborate anion, hexafluorophosphate anion, and the like.
  • Phosphonium reagents include notably benzotriazol-1-yloxy- tris(dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy- tripyrrolidino phosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy)- tripyrrolidino phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidino phosphonium hexafluorophosphate (PyBrOP), ethyl cyano(hydroxyimino)acetato-02)- tri-(1-pyrrolidinyl) phosphonium hexafluorophosphate and the like.
  • BOP benzotriazol-1-yloxy- tris(dimethylamino) phosphonium hexafluorophosphate
  • PyBOP benzo
  • Iminium/uranium salt reagents include notably 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyl uranium hexafluorophosphate / tetrafluoroborate (HBTU / TBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl uranium hexafluorophosphate / tetrafluoroborate (HATU / TATU), O-( 1H-6-chlorobenzotriazole-1-yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophosphate / tetrafluoroborate (HCTU / TCTU) and the like.
  • HBTU / TBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyl uranium hexafluorophosphate
  • Such iminium/uranium or phosphonium type coupling reagents are typically used together with a tertiary amine base, such as N, N-diisopropylethylamine (DIPEA or DIEA) and N-methylmorpholine (NMM).
  • DIPEA or DIEA N, N-diisopropylethylamine
  • NMM N-methylmorpholine
  • Step a) of the method according to the invention comprises anchoring peptide c to a resin via the carboxylic acid function of AA 1 , the N-terminal position of peptide c being Fmoc protected and the side chains of peptide c , if present and if necessary, being protected by suitable protecting groups as defined above.
  • Said suitable protecting groups are orthogonal to Fmoc, allyl and alloc protecting groups. In a preferred embodiment, these protecting groups are acid-labile.
  • the resin is a 2- chlorortrityl chloride resin, and peptide c may be anchored to it by a well-known nucleophilic substitution, typically in the presence of a base, notably a tertiary amine base, such as DIPEA.
  • Peptide c is amino acid AA 1 or a peptide comprising at its C-terminal position amino acid AA 1 , AA 1 being an amino acid as defined above.
  • peptide c When peptide c is a peptide, it is as defined above.
  • Peptide c may thus consist in 2 to 10 amino acid residues as defined above, notably 2 to 6 amino acid residues. In particular, it is a dipeptide, tripeptide or tetrapeptide.
  • anchoring peptide c to the resin may be performed in two ways. In the first way, peptide c is directly anchored in its entirety to the resin.
  • first anchor AA 1 more precisely Fmoc-AA 1 (PG)-OH, wherein PG is a suitable protecting group, remove Fmoc, and subsequently incorporate the following amino acid residues of peptide c (C-terminal to N-terminal) by repeating coupling / Fmoc removal cycles.
  • PG Fmoc-AA 1
  • AA 1 is Fmoc- HhPro(PG)-OH, which corresponds to the following formula (i): wherein PG represents a hydroxyl protecting group, notably selected from the group consisting of tert-butyl, cyclohexyl and trityl, preferably it is tert-butyl.
  • AA 1 is (2S,3R)- Fmoc-HhPro(PG)-OH, which corresponds to the following formula (ia): wherein PG is as defined above.
  • peptide c is AA 1 , preferably Fmoc-HhPro(PG)-OH, notably (2S,3R)-Fmoc-HhPro(PG)- OH, wherein PG represents an hydroxyl protecting group, notably selected from the group consisting of tert-butyl, cyclohexyl and trityl, advantageously PG is tert-butyl.
  • step a) peptide c is anchored to the resin together with Boc-AA res -OH, wherein AA res is an amino acid residue selected from the group consisting of Ala, Gly, Val, Leu and Ile, preferably AA res is Ala.
  • the skilled person will be able to finely tune the loading of peptide c on the resin by varying the introduced amounts of peptide c / Fmoc- AA 1 (PG)-OH and Boc-AA res -OH, the latter allowing to render a portion of the active sites of the resin unreactive.
  • the resin is a 2-chlorortrityl chloride resin
  • peptide c is Fmoc-HhPro(tBu)-OH
  • the loading of peptide c on the resin is comprised between 0.004 mmol/g and 0.20 mmol/g, notably between 0.004 mmol/g and 0.10 mmol/g, preferably between 0.004 mmol/g and 0.080 mmol/g.
  • the Fmoc protecting group is removed from its N-terminal position.
  • Step b) Step b) of the method according to the invention comprises coupling Fmoc-
  • Fmoc-AA 2 (alloc)-OH is of the following formula (iia): , wherein R 2 and y are as defined above.
  • the Fmoc protecting group is removed from the anchored peptide obtained thereof.
  • Step c) of the method according to the invention comprises coupling Fmoc- AA 3 - OH to the anchored peptide obtained in step b), wherein AA 3 is an «-amino acid, preferably selected from the group consisting of Asp, Glu, Asn, Gin, Ser, Thr, Gly, Ala, Val, Ile, Leu, Phe, Trp, Tyr, Cys, Met, Lys, His and Arg, and wherein, when AA 3 is not Gly, its side chain R 3 , if necessary, is protected by a suitable protecting group as defined above.
  • AA 3 is an «-amino acid, preferably selected from the group consisting of Asp, Glu, Asn, Gin, Ser, Thr, Gly, Ala, Val, Ile, Leu, Phe, Trp, Tyr, Cys, Met, Lys, His and Arg, and wherein, when AA 3 is not Gly, its side chain R 3 , if necessary, is protected by a suitable protecting
  • Said suitable protecting groups are orthogonal to Fmoc, allyl and alloc protecting groups.
  • these protecting groups are acid-labile.
  • Fmoc-AA 2 (alloc)-OH is of the formula (iia) as defined above, and step d) consists in the coupling of Fmoc-D-Asp(OAll)-OH or Fmoc-D-Glu(OAll)-OH.
  • Step e) of the method according to the invention comprises removing the allyl and alloc protecting groups from the anchored peptide obtained in step d).
  • Such deprotection reaction may be performed using the reaction conditions well-known to the person skilled in the art.
  • a palladium-based catalyst such as tetrakis(triphenylphosphine)palladium(0)
  • an auxiliary nucleophile notably phenylsilane, thiosalicylic acid or trimethylamine borane.
  • the resulting anchored peptide thus comprises a free amine and a free carboxylic acid functions, which are then coupled to form a peptide bond.
  • Step f) Step f) of the method according to the invention consists in removing Fmoc from the anchored macrocycle-containing peptide obtained in step e).
  • Step g) of the method according to the invention comprises coupling peptide N to the anchored peptide obtained in step f) via the carboxylic acid function of AA 5 , wherein the N-terminal position and the side chains of peptide N , if present and if necessary, are protected by suitable protecting groups as defined above.
  • Said suitable protecting groups are orthogonal to Fmoc.
  • these protecting groups are acid-labile.
  • Peptide N is amino acid AA 5 or a peptide comprising at its C-terminal position amino acid AA 5 , AA 5 being an amino acid as defined above.
  • peptide N When peptide N is a peptide, it is as defined above. Peptide N may thus consist in 2 to 10 amino acid residues as defined above, notably 2 to 6 amino acid residues. In particular, it is a dipeptide, tripeptide or tetrapeptide. As it will appear clearly to the skilled person, coupling peptide N to the anchored peptide obtained in step f) may be performed in two ways. In the first way, peptide N is directly coupled in its entirety to the anchored peptide.
  • AA 5 more precisely Fmoc-AA 5 (PG)-OH, wherein PG is a suitable protecting group, remove Fmoc, and subsequently incorporate the following amino acid residues of peptide N (C-terminal to N-terminal) by repeating coupling/Fmoc removal cycles.
  • PG Fmoc-AA 5
  • peptide N is a dipeptide of the following formula AA 6 -AA 5 , wherein AA 6 is an amino acid residue as defined above.
  • step g) comprises the coupling of Fmoc-His(Trt)-OH followed by Fmoc removal and coupling of Boc- Asp(tBu)-OH.
  • step g) consists in the coupling of Fmoc-His(Trt)-OH followed by Fmoc removal and coupling of Boc- Asp(tBu)-OH.
  • Step h) of the method according to the invention comprises cleaving the anchored macrocycle-containing peptide obtained in step g) from the resin.
  • the resin is a 2- chlorortrityl chloride resin
  • the macrocycle-containing peptide obtained in step g) is cleaved from the resin under acidic conditions.
  • step h) of the method according to the invention also comprises removing the protecting groups of the peptide.
  • the protecting groups of the side chains and of the N-terminal position of the macrocycle-containing peptide obtained in step g) are acid- labile, and can thus be removed under acidic conditions.
  • the macrocycle-containing peptide obtained in step g) may also be cleaved from the resin under acidic conditions, and said cleaving and the removal of the protecting groups may be performed simultaneously.
  • the resin is a 2-chlorortrityl chloride resin.
  • the peptide may be cleaved from the resin without removing its acid-labile protecting groups typically by using a mixture of trifluoroacetic acid (TFA, typically between 0.5 to 5% v/v, in particular between 1 to 2% v/v) and dichloromethane or of hexafluoroisopropanol (HFIP, typically between 10 to 30% v/v, notably 20% v/v) and dichloromethane.
  • TFA trifluoroacetic acid
  • HFIP hexafluoroisopropanol
  • the peptide may be cleaved from the resin and its acid-labile protecting groups may be removed simultaneously typically by using a mixture of TFA / triisopropylsilane (typically between 5 to 10% v/v, notably 7.5% v/v) / water (typically between 5 to 10% v/v, notably 7.5% v/v).
  • Peptide prepared by the method according to the invention is a mixture of TFA / triisopropylsilane (typically between 5 to 10% v/v, notably 7.5% v/v) / water (typically between 5 to 10% v/v, notably 7.5% v/v).
  • R 2 represents H or CH 3 ,
  • R 3 represents H or the side chain of amino acid AA 3 , x is equal to 1 or 2, y is an integer comprised between 0 and 3,
  • - peptide c is amino acid AA 1 as defined above or a peptide as defined above comprising at its C-terminal position amino acid AA 1 ,
  • - peptide N is amino acid AA 5 as defined above or a peptide as defined above comprising at its C-terminal position amino acid AA 5 .
  • peptide c is amino acid AA 1
  • peptide N is a dipeptide AA 6 -AA 5
  • the obtained peptide is thus of the following formula (II): wherein AA 1 , AA 5 , AA 6 , R 2 , R 3 , x and y are as defined above.
  • AA 1 may notably be HhPro.
  • peptide c may correspond to AA 1 , which may notably be HhPro.
  • peptide c may notably be amino acid AA 1
  • peptide N may notably a dipeptide AA 6 -AA 5
  • the obtained peptide is thus of the following formula (IV): wherein AA 1 , AA 5 and AA 6 are as defined above.
  • AA 1 may notably be HhPro.
  • AA 1 may notably be HhPro
  • AA 5 may notably be His
  • AA 6 may notably be Asp
  • the obtained peptide is thus of the following formula (V):
  • the invention thus also relates to a compound of the above formula (V), which may notably be one of the following compounds:
  • V a compound of the above formula (V)
  • the examples that follow illustrate the invention without limiting its scope in any way.
  • Trt Trityl uv : ultraviolet wt : weight
  • Analytical FC-MS of peptides was performed on the same system with a Waters Symmetry analytical C18 column (5 ⁇ m, 4.6 mm ⁇ 150 mm). 10 ⁇ L were injected, the flow rate was set up at 0.7 mL/min and UV detection was made at 230 nm.
  • the following mobile phases were used: A: 0.2% heptafluorobutyric acid in water; B: acetonitrile. The following gradient was used: 20 to 50% of B in A from 0 to 15 min.
  • ESI-FC-MS data were obtained in the positive mode on an Agilent 1260 Infinity system (Agilent 6120 Quadrupole LC/MS, 1260 Quaternary Pump, 1260 ALS, 1260 TCC, 1260 DAD VL, 1260 FC-AS).
  • NMR nuclear magnetic resonance
  • spectra chemical shifts are expressed in parts per million (ppm) with the solvent resonance as the internal standard ( 1 H NMR: CDCl 3 : 7.26 ppm; 13 C NMR: CDCft: 77.2 ppm).
  • 2-Chlorotrityl chloride resin (900 mg, 1.6 mmol/g, 1.4 mmol) was swelled in DCM (15 mL) for 30 min at rt. DCM was removed by filtration.
  • the resin beads were rinsed with DCM (2*10 mL), DMF (2*10 mL), DCM (2*10 mL), DMF (2*10 mL).
  • the reaction vessel was filled with DMF (4.0 mL) and shaken for 20 seconds. DIPEA (4.0 eq.) was added and the mixture was shaken until dissolution of the amino acid. HBTU (2.9 eq.) was added, the reaction vessel was closed with a cap and the system shaken for 120 min. The mixture was filtered under vacuum and the coupling was repeated once. The mixture was filtered under vacuum then washed with DMF (5*2.5 mL), MeOH (2.5 mL), DCM (5*2.5 mL), DMF (2*2.5 mL). A solution of DMF/piperidine (80:20, 4.0 mL) was added to the resin; the mixture was shaken for 20 min then filtered under vacuum to remove the solvent. This step was repeated once.
  • the mixture was filtered under vacuum then washed with DMF (5*2.5 mL), MeOH (2.5 mL), DCM (5*2.5 mL).
  • the resin was dried 15 min under vacuum. The resin was stored at room temperature during week days or at 4°C for the week-end.
  • the reservoir was shielded from light by aluminum foil and the reaction was set up and ran under nitrogen atmosphere.
  • a solution of phenylsilane (30 eq.) and Pd(PPh 3 ) 4 (0.3 eq.) in dry DCM (10 mL ) was added to the reservoir and the mixture was shaken for 15 min. The solvent was removed, and the procedure was repeated once.
  • the resin was washed with DCM (2*5 mL), and DMF (2*5 mL).
  • a solution of K-Oxyma (5.0 eq.) and PyAOP (5.0 eq.) in anhydrous DMF (1.5 mL) was added to the reservoir and the mixture was stirred for 2 h at rt. The mixture was filtered and this step was repeated once. The mixture was filtered under vacuum then washed with DMF (5*2.5 mL), MeOH (2.5 mL), DCM (5*2.5 mL) and dried under vacuum overnight.
  • the resin was rinsed carefully with DCM (5*2.5 mL) and dried overnight (18h) at room temperature.
  • 4.0 mL of cleavage cocktail were freshly prepared (TFA/ H 2 O/TIS, 85/7.5/7.5) and added to the resin.
  • the mixture was shaken for 3h and the solution was added dropwise to tubes containing 30 mL of cold (0°C) TBME. This step was repeated once, the cold mixture was stirred at 0°C for 30 min and the tubes were centrifuged (2,200 g, 5 min). The supernatant was discarded, the solid was washed with cold TBME (10 mL) and then centrifuged again (2,200 g, 5 min).
  • the crude product obtained was air-dried (3h) at room temperature, dissolved in water, combined with crude product from two similar reactions and freeze dried.
  • 2-Chlorotrityl chloride resin (900 mg, 1.6 mmol/g, 1.4 mmol) was swelled in DCM (15 mL) for 30 min at rt. DCM was removed by filtration.
  • the Fmoc-protected amino-acid (3.0 eq.) was added.
  • the reaction vessel was filled with DMF (4.0 mL) and shaken for 20 seconds.
  • DIPEA (4.0 eq.) was added and the mixture was shaken until dissolution of the amino acid.
  • HBTU 2.9 eq. was added, the reaction vessel was closed with a cap and the system shaken for 120 min. The mixture was filtered under vacuum and the coupling was repeated once. The mixture was filtered under vacuum then washed with DMF (5*2.5 mL), MeOH (2.5 mL), DCM (5*2.5 mL), DMF (2*2.5 mL).
  • the reservoir was shielded from light by aluminum foil and the reaction was set up and ran under nitrogen atmosphere.
  • a solution of phenylsilane (30 eq.) and Pd(PPh 3 ) 4 (0.3 eq.) in dry DCM (10 mL ) was added to the reservoir and the mixture was shaken for 15 min. The solvent was removed, and the procedure was repeated once.
  • the resin was washed with DCM (2*5 mL), and DMF (2*5 mL).
  • a solution of K-Oxyma (5.0 eq.) and PyAOP (5.0 eq.) in anhydrous DMF (1.5 mL) was added to the reservoir and the mixture was stirred for 2h at rt. The mixture was filtered and this step was repeated once.
  • the mixture was filtered under vacuum then washed with DMF (5*2.5 mL), MeOH (2.5 mL), DCM (5*2.5 mL) and dried under vacuum overnight.

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Abstract

La présente invention concerne un procédé de préparation d'un peptide de formule générale suivante (I). L'invention concerne également des peptides spécifiques, qui ont été préparés par le procédé selon l'invention, et des intermédiaires de synthèse clés.
PCT/EP2021/051435 2020-01-23 2021-01-22 Synthèse chimique de la partie peptidique de produits naturels bioactifs WO2021148594A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006032457A1 (fr) * 2004-09-20 2006-03-30 Lonza Ag Cyclisation de peptides
WO2012085177A1 (fr) 2010-12-22 2012-06-28 Nosopharm Némaucine, un antibiotique produit par xenorhabdus cabanillasii entomopathogène
CN109928908A (zh) * 2019-03-08 2019-06-25 联宁（苏州）生物制药有限公司 一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006032457A1 (fr) * 2004-09-20 2006-03-30 Lonza Ag Cyclisation de peptides
WO2012085177A1 (fr) 2010-12-22 2012-06-28 Nosopharm Némaucine, un antibiotique produit par xenorhabdus cabanillasii entomopathogène
CN109928908A (zh) * 2019-03-08 2019-06-25 联宁（苏州）生物制药有限公司 一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Greene's Protective Groups In Organic Synthesis", 2007, JOHN WILEY & SONS
CHRISTOPHER G. NELSON ET AL: "Samarium Iodide-Mediated Reformatsky Reactions for the Stereoselective Preparation of [beta]-Hydroxy-[gamma]-amino Acids: Synthesis of Isostatine and Dolaisoleucine", JOURNAL OF ORGANIC CHEMISTRY, vol. 77, no. 1, 14 December 2011 (2011-12-14), US, pages 733 - 738, XP055711082, ISSN: 0022-3263, DOI: 10.1021/jo202091r *
GUEREIRO P ET AL: "ASYMMETRIC SYNTHESIS OF HYDROXYLATED PYRROLIZIDINE, INDOLIZIDINE AND (+)-ALPHA-CONHYDRINE VIA RUTHENIUM-CATALYZED HYDROGENATION", CHIRALITY, WILEY-LISS, NEW YORK, US, vol. 12, no. 5/06, 1 January 2000 (2000-01-01), pages 408 - 410, XP001105953, ISSN: 0899-0042, DOI: 10.1002/(SICI)1520-636X(2000)12:5/6<408::AID-CHIR20>3.0.CO;2-G *
J. ORG. CHEM., vol. 73, 2008, pages 9228
TETRAHEDRON, vol. 67, 2011, pages 8595

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