NATURAL PRODUCTS

FIELD OF THE INVENTION 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 (previously called nemaucins) 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.

However, 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. 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. Generally speaking, 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. Small to medium (less than 7 amino acids) cyclic peptides are difficult to obtain as the ground-state E geometry of the peptide bond prevents the peptide from attaining the ring-like conformation conducing to cyclization. Besides, side reactions such as oligomers formation and epimerization at the C-terminal position are major pitfalls to be avoided in peptide macrocyclization. To increase the chances of success of the synthesis, the cyclization site has thus to be carefully chosen. With respect to MDL-A-pp, three cyclization options are available, given that the cyclic part contains three residues. The first option is through cyclization between the N¾ in alpha position of Dab2 and the acidic function of Asn3. Using this strategy, the formation of aspartimide through unwanted cyclisation of Asp4 during the synthesis would not be possible (Tetrahedron 2011, 67, 8595) but a quadriorthogonal protecting group scheme would be needed and addition of Asn3 onto Asp4 would go from N- to C- terminal, favouring epimerisation. The second option is through cyclization between the NH ₂ in alpha position of Asn3 and the acidic function of the main chain of Asp4. Epimerization could occur at the C-terminal position during cyclization and the needed Alloc-Dab(Fmoc)-OH is not commercially available. Finally, 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. However, this synthetic strategy also involves some risks since unwanted cyclisation of aspartic acid Asp4 into aspartimide could occur during the synthesis.

SUMMARY OF THE INVENTION

After extensive research, the inventors have succeeded in developing a method for synthetizing the peptidic part of Madelorhabdins, in particular MDL-A-pp, which relies notably on a side-chain to side-chain cyclisation between Dab2 and Asp4. 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. In particular, an acid labile protection of lateral chains allows for concomitant TFA final cleavage of the peptide from the resin and deprotection of lateral chains. This general strategy, which can be applied to the synthesis of MLD-A-pp as well as several closely related peptides, may be carried out in the synthetic way represented hereinafter in Scheme 1 and discussed below. In the following discussion, reference is made to six amino acid residues, AA ₁ to AA ₆ , given that 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). However, as it will appear clearly to the skilled person, the above-mentioned general strategy also applies to longer peptides, comprising further amino acid residues either at the C-terminal or a at the N-terminal position of the hexapeptide AA ₆ -AA ₅ -AA ₄ -AA ₃ -AA ₂ -AA ₁ .

In the synthetic way represented in Scheme 1, the full-length linear peptide is synthesized, lateral chains of AA ₂ and AA ₄ are deprotected, cyclization is done through lactamization between the amine function of AA ₂ and acidic function of AA ₄ and the peptide is then cleaved from the resin and deprotected.

The synthesis of NOSO-74006, a particular stereoisomer of MDL-A-pp represented below, was first investigated.

The building-block needed to introduce (2S,3R)HhPro, namely (2S,3R)Fmoc- HhPro(tBu)-OH, hereinafter referred to as Target A, has, to the best of the Applicant’s knowledge, never been described before. Therefore, the invention also relates to the key building-block Fmoc-

HhPro(PG)-OH, wherein PG is an hydroxyl protecting group.

With this building-block in hand and other needed building-blocks being commercially available, the synthesis of NOSO-74006 was investigated. Fmoc-based synthesis was started on 2-chlorotrityl resin. First amino acid Target A was anchored on the resin. Coupling of amino acids Fmoc-SS-Dab(3-alloc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-D-Asp(OAll)-OH, Fmoc-His(Trt)-OH and Boc-Asp(tBu)-OH was achieved using HBTU as coupling agent.

The Fmoc-based synthesis of NOSO-74006 using the strategy described in

Scheme 1 was tested. After the chain assembly was completed, allyl and alloc protecting groups were removed, cyclization was achieved followed by cleavage and final deprotection. LC-MS analysis of the crude mixture revealed two major components in a 1:3 ratio: the major peak at 9.63 min with the expected molecular weight of 722 Da and a minor peak with a molecular weight of 607 Da corresponding to the expected compound missing the aspartic acid. After purification and separation of the two main peaks by RP-HPLC, rigorous two-dimensional NMR analysis revealed that the main peak was aspartimide NOSO-74127, represented below, which does not correspond to the desired compound.

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).

Using the synthesis strategy described above, the inventors were able to prepare compound NOSO-74006 in a reliable way.

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 ,

- x is equal to 1 or 2,

- 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 , comprising the steps of: 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; b) coupling Fmoc-AA ₂ (alloc)-OH to the anchored peptide _c obtained in step a), wherein Fmoc-AA ₂ (alloc)-OH is of the following formula (ii): wherein R ₂ and y are as defined above, and removing Fmoc from the anchored peptide obtained thereof; c) coupling Fmoc-AA ₃ -OH to the anchored peptide obtained in step b), wherein A A ₃ is an α-amino acid, preferably selected from the group consisting of Asp, Glu, Asn, Gin, Ser, Thr, Gly, Ala, Val, lie, 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, and removing Fmoc from the anchored peptide obtained thereof; d) coupling, when x = 1, Fmoc-Asp(OAll)-OH, or, when x = 2, Fmoc-Glu(OAll)-OH, to the anchored peptide obtained in step c); e) removing allyl and alloc protecting groups and forming the lactam ring between the unprotected amine and acidic functions obtained thereof; f) removing Fmoc from the anchored macrocycle-containing peptide obtained in step e); g) 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; and h) cleaving the anchored macrocycle-containing peptide obtained in step g) from the resin and optionally removing its protecting groups. DEFINITIONS

The term “amino acid” as used in the present invention 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.

The term “(C ₁ -C ₆ )alkyl", as used in the present invention, 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.

In the context of the present invention, the amine functional group of an amino acid is typically a -NH ₂ or -NHCH ₃ group or a pyrrolidine. In the context of the present invention, 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. In both cases, 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.

In the context of the present invention, 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 atom or a (C ₁ -C ₃ )alkyl group, aryl, heteroaryl, cycloalkyl and heterocycle group, said aryl, heteroaryl, cycloalkyl and heterocycle 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 -C ₃ )alkyl group. The term “(C ₂ -C ₆ )alkenyl”, as used in the present invention, 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. The term “(C ₂ -C ₆ )alkynyl”, as used in the present invention, 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.

The term “(C ₁ -C ₆ )alkoxy”, as used in the present invention, 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.

The term “(C ₁ -C ₆ )alkylamino”, as used in the present invention, 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.

The term “di(C ₁ -C ₆ )alkylamino”, as used in the present invention, 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.

The term “halogen” as used in the present invention refers to an atom of fluorine, bromine, chlorine or iodine.

The term “aryl”, as used in the present invention, 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. Advantageously, it is a phenyl group.

The term “cycloalkyl” as used in the present invention 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. The term “heterocycle” as used in the present invention 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.

The term “heteroaryl” as used in the present invention 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. In the context of the present invention, the “side chain” of an amino acid, in particular of an α-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 -C ₃ )alkyl group.

In the context of the present invention, 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 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 -C ₃ )alkyl group. Within the meaning of the present invention, the term “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. Preferably, it is a natural or non-natural α- amino acid, more preferably a natural a-amino acid.

The term “peptide” as used in the present invention 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).

The term “amino acid residue” as used in the present invention refers to an amino acid as defined above incorporated to a peptide by means of its carboxylic acid function and/or its amine function.

In the context of the present invention, 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.

The term “C-terminal position” as used in the present invention 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. The term "N-terminal position” as used in the present invention 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.

In the above definitions, the terms “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.

As previously mentioned, the method according to the invention is a solid phase peptide synthesis (SPPS). 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 μm to 1 mm, in particular between 20 μm to 600 μm. 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.

In the context of the present invention, 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. The term “trityl resin” as used in the present invention 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; and R _trt represents a chlorine or a hydrogen atom, preferably a chlorine atom.

The abbreviation “Fmoc” as used in the present description refers to the well known 9-fluorenylmethyloxycarbonyl protecting group.

The term “protecting group”, as used in the present invention, 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.

In the context of the present invention, which involves a Fmoc-based SPPS, 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:

Table 1. Suitable Fmoc-orthosonal protecting sroups

The preferred side chain protecting groups used in the context of the present invention are listed in the table below:

Table 2. Preferred Fmoc-orthosonal protecting groups

Other suitable protecting groups may be carefully selected by the person skilled in the art in view of the triorthogonal protecting group scheme involved in the method according to the invention, notably among those mentioned in “Greene’s Protective Groups In Organic Synthesis”, 4 ^th edition, 2007, John Wiley & Sons, Hoboken, New Jersey. In the present description, the notation “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”).

DETAILED DESCRIPTION AND ADDITIONAL EMBODIMENTS

Generalities

The introduction or removal of the various protecting groups used in the peptide synthesis according to the invention may be carried out by the methods and techniques well known to the person skilled in the art, notably those described in “Greene’s Protective Groups In Organic Synthesis”, 4 ^th edition, 2007, John Wiley & Sons, Hoboken, New Jersey.

In particular, 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. In the context of the present invention, particularly preferred side chain protecting groups are acid-labile protecting groups.

The term “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. Such carbodiimides are typically used together with an auxiliary nucleophile, such as hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu), ethyl cyanohydroxyiminoacetate (Oxyma) and the like.

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.

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.

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).

Step a)

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 person skilled in the art will be able to determine the reaction conditions for the coupling of peptide _c to the resin depending on the nature of the resin linker, as defined above. According to a particular embodiment of the invention, 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.

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. As it will appear clearly to the skilled person, 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. Alternatively, it is possible and preferred to 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.

According to a particular embodiment of the invention, 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.

According to another particular embodiment of the invention, AA ₁ is (2S,3R)- Fmoc-HhPro(PG)-OH, which corresponds to the following formula (ia): wherein PG is as defined above.

The invention thus also relates to a compound of the above formula (i) or (ia). According to still another particular embodiment of the method of the invention, 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.

According to yet another particular embodiment of the method of the invention, in 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.

In this particular embodiment, 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.

According to a preferred embodiment of the method of the invention, the resin is a 2-chlorortrityl chloride resin, peptide _c is Fmoc-HhPro(tBu)-OH and 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.

Once the fully protected peptide _c is anchored to the resin, 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-

AA ₂ (alloc)-OH to the anchored peptide _c obtained in step a), wherein Fmoc- AA ₂ (alloc)- OH is of the following formula (ii): wherein R ₂ represents H or CH ₃ and y is an integer comprised between 0 and 3. The abbreviation “Alloc” as used in the present description refers to the well- known allyloxycarbonyl protecting group.

According to a particular embodiment of step b) of the method of the invention, Fmoc-AA ₂ (alloc)-OH is of the following formula (iia): , wherein R ₂ and y are as defined above.

After said coupling, the Fmoc protecting group is removed from the anchored peptide obtained thereof.

Step c)

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.

Said suitable protecting groups are orthogonal to Fmoc, allyl and alloc protecting groups. In a preferred embodiment, these protecting groups are acid-labile. After the coupling of step c), the Fmoc protecting group is removed from the anchored peptide obtained thereof.

Step d)

Step d) of the method according to the invention comprises coupling of: - when the peptide to be prepared is such that, in the general formula (I) as defined above, x = 1: Fmoc-Asp(OAll)-OH, notably Fmoc-D-Asp(OAll)-OH, or

- when the peptide to be prepared is such that, in the general formula (I) as defined above, x = 2: Fmoc-Glu(OAll)-OH, notably Fmoc-D-Glu(OAll)-OH. The abbreviation “All” as used in the present description refers to the well- known allyl protecting group.

According to a particular embodiment of the method of the invention, in step b), 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)

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.

It may notably be carried out using a palladium-based catalyst, such as tetrakis(triphenylphosphine)palladium(0), optionally in the presence of 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.

Said coupling, which may be performed as described above, allows to obtain the lactam (i.e. cyclic amide) ring of the peptide of general formula (I) as defined above.

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)

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. In a preferred embodiment, 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.

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. Alternatively, it is possible and preferred to first couple 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.

According to a particular embodiment of the invention, peptide _N is a dipeptide of the following formula AA ₆ -AA ₅ , wherein AA ₆ is an amino acid residue as defined above.

According to another particular embodiment of the invention, step g) comprises the coupling of Fmoc-His(Trt)-OH followed by Fmoc removal and coupling of Boc- Asp(tBu)-OH.

According to yet another particular embodiment of the invention, step g) consists in the coupling of Fmoc-His(Trt)-OH followed by Fmoc removal and coupling of Boc- Asp(tBu)-OH.

Step h)

Step h) of the method according to the invention comprises cleaving the anchored macrocycle-containing peptide obtained in step g) from the resin.

The person skilled in the art will be able to determine the conditions to perform said cleaving, depending on the nature of the resin.

According to a particular embodiment of the invention, the resin is a 2- chlorortrityl chloride resin, and the macrocycle-containing peptide obtained in step g) is cleaved from the resin under acidic conditions.

Optionally, step h) of the method according to the invention also comprises removing the protecting groups of the peptide.

The person skilled in the art will be able to determine the conditions to remove the protecting groups depending on their nature. In a preferred embodiment, 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.

In this preferred embodiment, 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 person skilled in the art will be able to determine the reaction conditions for the cleavage of the peptide from the resin, depending on whether he or she wishes to simultaneously remove the acid-labile protecting groups of the peptide, or not. When the peptide is cleaved from the resin without removing its acid-labile protecting groups, the C-terminal acidic function of the fully protected peptide of formula (I) thus obtained may be further transformed to another chemical function.

According to a particular embodiment of the invention, the resin is a 2-chlorortrityl chloride resin. In this particular embodiment, 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. Alternatively, 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

The method according to the invention allow to prepare 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 , 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 .

In a particular embodiment of the method according to the invention, peptide _c is amino acid AA ₁ , peptide _N is a dipeptide AA ₆ -AA ₅ , and 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. In particular, AA ₁ may notably be HhPro.

In a particular embodiment of the method according to the invention, x = 1, y = 0, R ₂ represents CH ₃ , AA ₃ is Asn, and the obtained peptide is thus of the following formula (III): wherein peptide _c and peptide _N are as defined above. In particular, peptide _c may correspond to AA ₁ , which may notably be HhPro. In this particular embodiment, peptide _c may notably be amino acid AA ₁ , peptide _N may notably a dipeptide AA ₆ -AA ₅ , and the obtained peptide is thus of the following formula (IV): wherein AA ₁ , AA ₅ and AA ₆ are as defined above. In particular, AA ₁ may notably be HhPro.

In this particular embodiment, AA ₁ may notably be HhPro, AA ₅ may notably be His, AA ₆ may notably be Asp, and 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: The examples that follow illustrate the invention without limiting its scope in any way.

EXAMPLES

The following abbreviations have been used:

Ac : Acetyl (COCH ₃ )

ACN : Acetonitrile

All : Allyl

Alloc : Allyloxycarbonyl

Boc : tert-butoxycarbonyle

Bu : Butyl (CH ₂ CH ₂ CH ₂ CH ₃ )

GDI : Carbonyldiimidazole

Dab : Diaminobutyric acid

DCM : Dichloromethane

DIPEA : N,N-Diisopropylethylamine

DMF : Dimethylformamide eq. : equivalent

ESI : Electrospray ionisation

Et : Ethyl (CH ₂ CH ₃ )

Fmoc : Fluorenylmethoxycarbonyl

HBTU : Hexafluorophosphate Benzotriazole Tetramethyl Uronium

HPLC : High Performance Liquid Chromatography

IPA : Isopropanol

K-Oxyma : (Z)-ethyl 2-cyano-3 -hydroxyacrylate potassium salt

LC : Liquid Chromatography

LDA : Lithium diisopropylamide

Me : Methyl (CH ₃ )

MS : Mass Spectroscopy

NMR : Nuclear Magnetic Resonance

PyAOP : (7- Azabenzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate

Rf : Retention factor SPPS : Solid Phase Peptide Synthesis

TBME : tert-butyl methyl ether

TFA : Trifluoroacetic acid

THF : T etrahydrofuran

TIS : Triisopropylsilane

TFC : Thin-layer chromatography

Trt : Trityl uv : ultraviolet wt : weight

I - Materials

All commercially available reagents were used without further purification unless otherwise noted. Resins, HBTU and protected amino acids were purchased from Chem Impex International Inc (Wood dale, IL, USA). All other reagents and solvents, including of HPFC grade, were purchased from Sigma-Aldrich (St Louis, MO, USA).

Purification of peptides was performed on a HPFC Agilent 1260 Infinity system with a Waters Symmetry semi preparative C18 column (7 μm, 7.8 mmx300 mm). 50 μL of peptide solution (300 mg/mL) were injected, the flow rate was set up at 2.5 mL/min and UV detection was made at 230 nm. The following mobile phases were used: A: 0.1% trifluoroacetic acid in water; B: acetonitrile. The following gradient was used: 0 to 15% of B in A from 0 to 15 min.

Analytical FC-MS of peptides was performed on the same system with a Waters Symmetry analytical C18 column (5 μm, 4.6 mm × 150 mm). 10 μL 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). For 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). Spin multiplicity is described by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quadruplet, qu = quintuplet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet and br = broad. Coupling constants are expressed in Hertz (Hz). ¹ H NMR spectra were recorded at room temperature on Brüker 400 MHz NMR spectrometer. ¹³ C NMR spectra were recorded on the same instrument at 100 MHz. - Synthesis of Target A

II-1. General presentation

The imidazole-activated Boc-Pro-OH 1 was coupled with the enolate derived from allyl acetate 1A (J. Qrg. Chem. 2008, 73, 9228). Subsequent reduction with lithium borohydride produced β -hydroxy ester 3 as a mixture of C-3 diastereoisomers with a 2:1 ratio in favour of the 2S,3R isomer. Changing protecting group of the amine function from Boc to Fmoc and protecting the hydroxyl function with a tert-butyl group led to Fmoc-HhPro(tBu)-OAll 6. At this stage, diastereoisomers were separated by preparative HPLC and acidic function of the desired 2S,3R diastereoisomer was deprotected to furnish Target A.

II-2. Detailed synthesis

Preparation of compound 2 Three reactions were carried out in parallel.

To a mixture of (2S)-1-tert-butoxycarbonylpyrrolidine-2-carboxylic acid 1 (35.0 g, 162.6 mmol, 1.00 eq.) in THF (350.0 mL) was added CDI (29.0 g, 178.8 mmol, 1.1 eq.) at 25°C. n-BuLi (2.50 M, 214.6 mL, 3.30 eq.) under nitrogen was diluted with THF (350.0 mL) and cooled to 0°C with an ice bath. To the solution of n-BuLi was added dropwise diisopropylamine (59.2 g, 585.3 mmol, 82.7 mL, 3.60 eq.). After being stirred for 30 min at 0°C, the solution was diluted with THF (1050.0 mL) and cooled to -78°C. To the LDA solution was added allyl acetate 1A (56.9 g, 569.1 mmol, 3.50 eq.) at -78°C, and then the mixture was stirred at -78°C for 0.5h under N ₂ atmosphere. The

Boc-amino acid solution was cooled to -78°C and cannulated into the enolate solution under nitrogen. The reaction was allowed to stir for 1.5h at -78°C. TLC (Petroleum ether: Ethyl acetate = 5:1, starting material (Rf) = 0.13, PI (Rf) = 0.49) showed the reactant was consumed. Three reactions were combined together for work up. To the solution was added drop-wise aq. NH ₄ CI 500 mL at -60°C over a period of 30 min. The reaction mixture was warmed to 25°C, and then extracted with EtOAc (450.0 mL*4). The combined organic phases were washed with brine 500 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO ₂ , Petroleum ether/Ethyl acetate = 30/1 to 5:1). tert- butyl (2S)-2- (3 -allyloxy-3 -oxo-propanoyl)pyrrolidine-1-carboxylate 2 was obtained as a yellow oil

(92.0 g, 309.4 mmol, 63.4% yield).

¹ H NMR: CDCI ₃ , 400MHz; δ (ppm) = 6.01 - 5.84 (m, 1H), 5.35 (br d, J=17.2 Hz, 1H), 5.26 (br d, J=10.4 Hz, 1H), 4.70 - 4.59 (m, 2H), 4.46 - 4.24 (m, 1H), 3.66 - 3.40 (m, 4H), 2.28 - 1.82 (m, 4H), 1.51 - 1.41 (m, 8H)

^■ Preparation of compound 3 Two reactions were carried out in parallel.

A mixture of compound 2 (45.0 g 151.3 mmol, 1.00 eq.) in THF (450.0 mL) was cooled to -78°C under nitrogen. To this solution was added LiBH ₄ (11.5 g, 529.6 mmol, 3.50 eq.) with stirring at -78°C and then the mixture was stirred at -78°C for lh under N ₂ atmosphere. TLC (Petroleum ether: Ethyl acetate = 3:1, starting material (Rf) = 0.50, P1 (Rf) = 0.40) showed there was desired spot. Two reactions were combined together for work up. To the solution was added dropwise aq. NH ₄ CI 500.0 mL at -60°C over a period of 30 min. The reaction mixture was warmed to 25°C, and then extracted with EtOAc (500.0 mL*4). The combined organic phase was washed with brine 250.0 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO ₂ , Petroleum ether/Ethyl acetate = 30 to 5:1). Tert-butyl (2S)-2-(3 -ally loxy-1-hydroxy-3 -oxo-propyl)pyrrolidine-1-carboxylate 3 was obtained as a yellow oil (44.0 g, 146.9 mmol, 48.5% yield).

¹ H NMR: CDCl ₃ , 400MHz; δ (ppm) = 6.01 - 5.87 (m, 1H), 5.36 (quin, J=1.5 Hz, 1H), 5.33 - 5.29 (m, 1H), 5.25 (br d, J=10.4 Hz, 1H), 4.98 (br s, 1H), 4.63 (d, J=5.6 Hz, 2H), 4.50 (br s, 1H), 4.20 (br d, J=3.7 Hz, 1H), 4.07 - 3.89 (m, 1H), 3.50 (br s, 1H), 3.35 - 3.23 (m, 1H), 2.54 - 2.35 (m, 2H), 2.07 - 1.95 (m, 1H), 1.85 - 1.84 (m, 1H), 1.94 - 1.83 (m, 1H), 1.83 - 1.69 (m, 1H), 1.48 (d, J=3.3 Hz, 9H)

¹³ C NMR: CDCl ₃ , 150 MHz; δ (ppm) = 172.11, 171.13, 132.06, 118.36, 80.43, 80.12, 70.33, 65.34, 62.21, 60.38, 47.82, 47.40, 37.91, 28.45, 28.40, 27.66, 24.04, 21.04, 14.19

^■ Preparation of compound 4

To a mixture of compound 3 (44.0 g, 146.9 mmol, 1.00 eq.) in DCM (220.0 mL) was added TFA (100.5 g, 881.8 mmol, 65.2 mL, 6.00 eq.), and then the mixture was stirred at 25°C for 12h under N2 atmosphere. TLC (Petroleum ether: Ethyl acetate = 3:1, starting material (Rf) = 0.50, PI (Rf) = 0.10) showed the reactant was consumed. The solution was concentrated in vacuo. The residue was used in the next step without further purification. Allyl 3-hydroxy-3-[(2S)-pyrrolidin-2-yl]propanoate 4 was obtained as a yellow oil (46.4 g, crude, TFA). 1H NMR : CDCl ₃ , 400 MHz; δ (ppm)= 5.97 - 5.82 (m, 1H), 5.40 - 5.24 (m, 2H), 4.64 (d, J=1.2 Hz, 1H), 4.62 (d, J=1.2 Hz, 1H), 4.61 - 4.54 (m, 1H), 4.22 (dt, J=3.5, 8.1 Hz, 1H), 3.91 - 3.75 (m, 1H), 3.57 - 3.34 (m, 2H), 2.70 - 2.63 (m, 1H), 2.60 (d, J=6.5 Hz, 1H), 2.26 - 2.00 (m, 4H) ^■ Preparation of compound 5

To a mixture of (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (49.9 g, 148.1 mmol, 1.00 eq.) in dioxane (200.0 mL) was added a solution of compound 4 (46.4 g, 148.1 mmol, 1.00 eq., TFA) in aq. 10% Na ₂ CO ₃ in H ₂ O (400.0 mL) mL and dioxane (200.0 mL) at 0°C, and then the mixture was stirred at 15°C for 6h under N ₂ atmosphere. TLC (Petroleum ether: Ethyl acetate = 3:1, starting material (Rf) = 0.10, PI (Rf) = 0.50) showed the reactant was consumed. To the solution was added dropwise aq. NH4CI 300.0 mL. The reaction mixture was warmed to 25°C, and then extracted with EtOAc (150.0 mL*4). The combined organic phases were washed with brine 200.0 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate = 30/1 to 5:1). 9H-fluoren-9-ylmethyl (2S)-2-(3 -allyloxy-1-hydroxy-3 -oxo- propyl)pyrrolidine-1-carboxylate 5 was obtained as a yellow oil (47.0 g, 111.51 mmol, 75.2% yield).

¹ H NMR: CDCl ₃ , 400 MHz; δ (ppm) = 7.78 (d, J=7.3 Hz, 2H), 7.60 (br d, J=7.3 Hz, 2H), 7.46 - 7.38 (m, 2H), 7.36 - 7.29 (m, 2H), 6.01 - 5.87 (m, 1H), 5.34 (dd, J=1.3, 17.2 Hz, 1H), 5.25 (br d, J=10.1 1 Hz, 1H), 4.62 (br d, J=5.5 Hz, 3H), 4.52 - 4.34 (m, 2H), 4.25 (br t, J=6.5 Hz, 2H), 4.05 - 3.90 (m, 1H), 3.53 (br d, J=6.4 Hz, 1H), 3.45 - 3.22 (m, 1H), 2.61 - 2.36 (m, 1H), 2.61 - 2.36 (m, 1H), 2.03 - 1.70 (m, 4H)

¹³ C NMR: CDCl ₃ , 150 MHz; δ (ppm) = 172.03, 143.88, 141.31, 131.93, 127.67, 127.03, 125.01, 124.94, 119.95, 118.46, 69.53, 67.28, 65.36, 62.42, 60.35, 47.35, 47.27, 37.94, 26.99, 24.07, 14.16 ■ Preparation of compound 6

Two reactions were carried out in parallel. To a mixture of compound 5 (17.0 g, 40.3 mmol, 1.00 eq.), H ₂ SO ₄ (1.98 g, 20.1 mmol, 1.07 mL, 0.50 eq.) and about 8% of isobutylene in DCM (200.0 mL) was degassed and purged with N ₂ for 3 times, and then the mixture was stirred at 25 °C for 12h under N ₂ atmosphere. TLC (Petroleum ether: Ethyl acetate=3:1, starting material (Rf) = 0.30, PI (Rf) = 0.70) showed the reactant was consumed. Three reactions were combined together for work up. To the solution was added dropwise aq. NH ₄ CI 100 mL and then extracted with EtOAc (150.0 mL*4). The combined organic phases were washed with brine 200 mL, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO ₂ , Petroleum ether/Ethyl acetate = 30/1 to 5:1) to give 34 g of the mixture of the two diastereoi somers which was then purified by prep-HPLC. 9H-fluoren-9-ylmethyl (2S)-2-(3 -allyloxy-1-tert-butoxy-3-oxo- propyl)pyrrolidine-1-carboxylate 6A was obtained as a yellow oil (12.0 g, 25.1 mmol, 31.1% yield).

¹ H NMR: CDCl ₃ , 400MHz; δ (ppm) = 7.77 (d, J=7.5 Hz, 2H), 7.81 - 7.57 (m, 2H), 7.45 - 7.29 (m, 4H), 5.98 - 5.81 (m, 1H), 5.35 - 5.15 (m, 2H), 4.70 - 4.19 (m, 6H),

4.10 - 3.95 (m, 1H), 3.63 - 3.27 (m, 2H), 2.47 - 2.36 (m, 2H), 2.05 - 1.78 (m, 4H), 1.23 (s, 6H), 1.09 (s, 2H)

^■ Preparation of compound Target A A mixture of compound 6 A (12.0 g,25.1 mmol, 1.00 eq.), Pd(PPh ₃ ) ₄ (5.81 g, 5.03 mmol, 0.20 eq.) and phenylsilane (5.44 g, 50.2 mmol, 6.20 mL, 2.00 eq.) in DCM (60.0 mL) was degassed and purged with N ₂ for 3 times, and then the mixture was stirred at 15°C for 2h under N ₂ atmosphere. TLC (Dichloromethane : Methanol = 10:1, starting material (Rf) = 0.60, PI (Rf) = 0.15) showed the reactant was consumed. The mixture was washed with brine 50.0 mL, dried with anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO ₂ , Dichloromethane : Methanol = 20/1 to 5:1) then by prep-SFC to get the pure 3 -tert- butoxy-3-[(2S)-1-(9H-fhioren-9-ylmethoxycarbonyl)pyrrolidin- 2-yl]propanoic acid as a light yellow solid (5.00 g, 11.3 mmol, 45.0% yield, 98.9% purity).

¹ H NMR: CDCI ₃ , 400MHz; δ (ppm) = 7.78 (d, J=7.5 Hz, 2H), 7.68 - 7.58 (m, 2H), 7.45 - 7.28 (m, 4H), 4.64 - 4.42 (m, 2H), 4.41 - 4.33 (m, 1H), 4.32 - 4.19 (m, 1H), 4.05 (br d, J=4.4 Hz, 1H), 3.90 (br s, 1H), 3.64 - 3.26 (m, 2H), 2.53 - 2.20 (m, 2H), 2.05 - 1.69 (m, 4H), 1.31 - 1.02 (m, 9H)

III - Peptide synthesis

^■ General procedure for solid-phase peptide synthesis (SPPS)

All reactions were carried out in polypropylene empty reservoirs (70 mL) fitted with polyethylene frits and polytetrafluoroethylene stopcock at room temperature.

^■ Synthesis of NOSO-74006

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.

A solution of (3R)-3-tert-butoxy-3-[ 1 -Fmoc-(2S)-pyrrolidin-2-yl]propanoic acid (0.025 eq., 0.01 mmol) and Boc-Ala-OH (0.975 eq., 0.39 mmol) in anhydrous DCM (15 mL) was added to the resin. DIPEA (4.0 eq., 1.6 mmol) was added to the mixture and the reservoir was shaken for 240 min the filtered. The resin beads were rinsed with DCM (2*10 mL) and DMF (2*10 mL) and were left for drying under vacuum overnight. Loading was measured at 4.88 10 ^-3 mmol/g. A solution of MeOH, DIPEA and DCM (0.15/0.05/0.80, 10 mL) was added to the syringe and the mixture was shaken 15 minutes then filtered. The resin beads were rinsed with DMF (5*10 mL) and DCM (5*10 mL).

A solution of DMF/piperidine (80:20, 15 mL) was added to the resin, the mixture was shaken for 20 minutes then filtered under vacuum to remove the solvent. This step is repeated once.

The resin beads were rinsed with DCM (2*10 mL), DMF (2*10 mL), DCM (2*10 mL), DMF (2*10 mL).

Addition of next amino acids (Fmoc-SS'-Dab(3 -alloc)-OH, Fmoc-Asn(Trt)-OH and Fmoc-D-Asp(OAll)-OH) was achieved by repeating coupling/Fmoc deprotection cycles. Before each coupling the resin was swollen. DCM (3.0 mL) was added to immerse all the resin, the mixture was shaken for 15 min. The DCM was removed by filtration under vacuum. DMF (2.5 mL) was added to the swelled resin, the mixture was shaken for 20 seconds and the solvent was removed under vacuum. 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). 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.

For allyl and alloc removal, 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.

Addition of next amino acids (Fmoc-His(Trt)-OH and Boc-Asp(tBu)-OH) was achieved following the protocol described above.

After the last Fmoc removal, 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.

Crude peptide was purified on semi preparative HPLC. Pure fractions were combined, lyophilized and the pure peptide (1.2 mg) was analysed on analytical LC-MS (Purity >95%, 2 rotamers observed (1:1 ratio), 19% yield). ^■ Synthesis of NOSO-74007

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.

A solution of (3R)-3-tert-butoxy-3-[1-Fmoc-(2S)-pyrrolidin-2-yl]propanoic acid (0.025 eq., 0.01 mmol) and Boc-Ala-OH (0.975 eq., 0.39 mmol) in anhydrous DCM (15 mL) was added to the resin. DIPEA (4.0 eq., 1.6 mmol) was added to the mixture and the reservoir was shaken for 240 min the filtered. The resin beads were rinsed with DCM (2*10 mL) and DMF (2*10 mL) and were left for drying under vacuum overnight. Loading was measured at 5.03 10 ^-3 mmol/g. A solution of MeOH, DIPEA and DCM (0.15/0.05/0.80, 10 mL) was added to the syringe and the mixture was shaken 15 minutes then filtered. The resin beads were rinsed with DMF (5*10 mL) and DCM (5*10 mL).

A solution of DMF/piperidine (80:20, 15 mL) was added to the resin, the mixture was shaken for 20 minutes then filtered under vacuum to remove the solvent. This step is repeated once. The resin beads were rinsed with DCM (2*10 mL), DMF (2*10 mL), DCM (2*10 mL), DMF (2*10 mL).

Addition of next amino acids ((2S,3R)-Fmoc-Dab(3-alloc)-OH, Fmoc-Asn(Trt)-OH and Fmoc-D-Asp(OAll)-OH) was achieved by repeating coupling/Fmoc deprotection cycles. Before each coupling the resin was swollen. DCM (3.0 mL) was added to immerse all the resin, the mixture was shaken for 15 min. The DCM was removed by filtration under vacuum. DMF (2.5 mL) was added to the swelled resin, the mixture was shaken for 20 seconds and the solvent was removed under vacuum. 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). 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.

For allyl and alloc removal, 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.

Addition of next amino acids (Fmoc-His(Trt)-OH and Boc-Asp(tBu)-OH) was achieved following the protocol described above.

After the last Fmoc removal, 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.

Crude peptide was purified on semi preparative HPLC. Pure fractions were combined, lyophilized and the pure peptide (1.1 mg) was analysed on analytical LC-MS (Purity >95%, 17% yield).