WO2021144388A1

WO2021144388A1 - Process for the synthesis of nangibotide

Info

Publication number: WO2021144388A1
Application number: PCT/EP2021/050739
Authority: WO
Inventors: Walter Cabri; Angelo VIOLA; Antonio Ricci; Andrea ORLANDIN; Ivan DE PAOLA
Original assignee: Fresenius Kabi Ipsum S.R.L.
Priority date: 2020-01-15
Filing date: 2021-01-14
Publication date: 2021-07-22
Also published as: CN114945580A; CN114945580B

Abstract

The present invention relates to a fragment-based manufacturing process for nangibotide, a promising peptide drug active as anti-inflammatory. Also disclosed there are peptide intermediates involved in a method providing the product in high yield and purity.

Description

PROCESS FOR THE SYNTHESIS OF NANGIBOTIDE

FIELD OF THE INVENTION

The present invention relates to a manufacturing process for a peptide, in particular for nangibotide and its related intermediates.

BACKGROUND OF THE INVENTION

The peptide nangibotide (SEQ ID No 1), also known as LR12, is represented by Formula (I):

1 5 10

Leu-Gln-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-Cys-Met-NH2 (I) and is also indicated as:

LQEEDAGEYGCM.

Nangibotide is the first clinical stage agent targeting the immunoreceptor TREM-1 (triggering receptor expressed on myeloid cells-1) and is being investigated as a novel therapy for acute inflammatory disorders such as septic shock (Br J Clin Pharmacol (2018), 84, 2270-2279, 2270).

WO2011124685 by Inserm firstly disclosed the product, while no specific preparation seems to be described. There is therefore the need to provide an efficient method for manufacturing nangibotide at industrial scale. Such a need is met by the process of the present invention.

SUMMARY OF THE INVENTION

An SPPS (Solid Phase Peptide Synthesis) preparation of nangibotide has been devised and it is herewith reported. In particular, a fragment-based synthesis of nangibotide is disclosed, which leads to a crude peptide with high yield and purity.

Such synthesis provides an efficient, simple and viable process which allows to obtain nangibotide and has an important industrial application.

The present invention provides a process for the preparation of the peptide nangibotide through a convergent fragment-based condensation on solid phase.

The process for the preparation of nangibotide (I) in solid phase comprises the steps of: a) coupling an N-terminal nangibotide fragment A with a C-terminal nangibotide fragment B, wherein the reactive carboxylic acid of fragment A belongs to an achiral amino acid and wherein fragment B is attached to a resin; wherein both fragments coupled to each other result in a peptide with the amino acid sequence of nangibotide; b) deprotecting and cleaving nangibotide from the resin.

DETAILED DESCRIPTION OF THE INVENTION

The terms "peptide fragment" or "fragment" describe a peptide with a partial nangibotide amino acid sequence, that can be either free or attached to a resin at its C-terminal amino acid, and that can be independently protected or unprotected at its N-terminal alpha-amino group and at amino acids side-chains. The terms "protected fragment" or "protected peptide fragment" describe a fragment which bears independently either a terminal protecting group or side-chain protecting groups, or both of them.

A fragment can be also indicated with a specific amino acid sequence, like aa¹-aa²-...-aaⁿ wherein aa^x is the three-letter code of the amino acid in position x, and wherein the presence or absence of protecting groups, either on the side-chains or on the alpha-amino group, is undefined. The necessity of protecting side-chains of amino acids during peptide synthesis under certain conditions is known in the art and various strategies to identify suitable protection strategies have been described.

If in this description for a specific amino acid sequence (fragment) protective groups of the side-chains are specifically mentioned in brackets accompanying the three-letter code of the amino acid it is understood that the remaining amino acids which are represented in said fragment with a plain three-letter code are unprotected.

When the fragment is indicated with aa¹-aa²-...-aaⁿ-OH it is intended that the C-terminal amino acid aaⁿ has a free carboxylic acid.

When the fragment is indicated with H-aa¹-aa²-...-aaⁿ it is intended that the N-terminal amino acid aa¹ has a free alpha-amino group.

The term "resin" or "solid support", describes a functionalized insoluble polymer to which an amino acid or a peptide fragment can be attached and which is suitable for amino acids elongation until the full desired sequence is obtained. The SPPS can be defined as a process in which a peptide anchored to a resin by its C- terminal amino acid is assembled by the sequential addition of the optionally protected amino acids constituting its sequence. It comprises loading a first alpha-amino-protected amino acid, or peptide, onto a resin and is followed by the repetition of a sequence of steps referred to as a cycle, or as a step of elongation, consisting of the cleavage of the alpha- amino protecting group and the coupling of the subsequent protected amino acid.

The formation of a peptide bond between two amino acids, or between an amino acid and a peptide fragment, or between two peptide fragments, which is a coupling step, may actually involve two steps: first, the activation of the free carboxyl group, which may take any time between 5 minutes and 2 hours; then the nucleophilic attack of the amino group at the activated carboxylic group.

The cycle may be repeated sequentially until the desired sequence of the peptide is accomplished.

Finally, the peptide is deprotected and/or cleaved from the resin.

As a reference for SPPS, please see for instance Knud 1 Jensen et al. (eds.), Linkers, Resins, and General Procedures for Solid-Phase Peptide Synthesis in Peptide Synthesis and Applications, Methods in Molecular Biology, vol. 1047, Springer Science, 2013.

Because nangibotide is a peptide amide (i.e. it has an amide as C-terminal functional group), an acid sensitive resin must be used for its preparation which provides a peptide amide after cleavage, and which is selected among Rink amide, Pal and Sieber resin. More preferably, a Rink amide MBHA resin is used for the preparation of nangibotide and for the preparation of the C terminal fragment B of the present invention.

For the SPPS preparation of fragment A or of any nangibotide fragment which is cleaved from the solid support before being involved in a further coupling step, other resins can be used. Such resins are selected from 2-chlorotrityl chloride (CTC) resin and trityl chloride resin. Preferably, a CTC resin is used for the preparation of fragment A, and fragment A is cleaved from the resin before coupling with fragment B.

In a preferred embodiment of the present invention, the loading of the first C-terminal amino acid onto the resin is carried out after swelling of the resin in a suitable solvent, preferably DMF, and after either filtering the resin to remove excess solvent or adding directly to the resin the solution of the protected amino acid with an activating agent, such as a carbodiimide, optionally in the presence of a base. In case of using a trityl chloride resin, or in particular when using the CTC resin, after swelling of the resin in a suitable solvent, preferably DCM, a solution of the protected amino acid with an organic base, preferably diisopropylethylamine (DIPEA), is added.

In another preferred embodiment of the present invention, after the first C-terminal amino acid has been loaded onto the resin, an additional step to block unreacted sites is performed, often referred to as "capping", which shall avoid truncated sequences and shall prevent any side reactions.

Capping is achieved by a short treatment of the loaded resin with a large excess of a highly reactive unhindered reagent, which is chosen according to the unreacted sites to be capped. When using a Wang resin, the unreacted sites are hydroxyl groups, which are preferably capped by treatment with an acid derivative, such as an anhydride, in a basic medium, for instance with a DMF/DIEA/AC2O mixture, or with a AC2O 10% DMF solution. When using a CTC resin, the unreacted sites are chlorine atoms, which are preferably capped by treatment with an alcohol in a basic medium, for instance with a DCM/DIPEA/MeOH mixture. Then, after washing with DCM, the resin is further treated with a DCM/DIEA/AC2O mixture, to cap the hydroxyl groups possibly resulting from the chloride hydrolysis.

As an alternative to the loading of the first C-terminal amino acid, preloaded resins can be used in the preparation of peptide fragments. These are commercially available Wang/CTC resins with attached Fmoc-protected L- or D-amino acids.

In a preferred embodiment of the present invention, the loading of the first C-terminal amino acid onto the resin is determined spectrophotometrically, as described for instance in Knud J. Jensen et al. (eds.), Peptide Synthesis and Applications, Methods in Molecular Biology, vol. 1047, Springer Science, 2013.

In a preferred embodiment of the present invention, each amino acid may be protected at its alpha-amino group and/or at its side-chain functional groups.

The term "terminal protecting group" as used herein refers to a protecting group for the alpha-amino group of an amino acid or of a peptide, or a peptide fragment. The type of the terminal protecting group used is depending on the type of resin used and on the protective groups required for side-chain protection. Preferably, the terminal protecting group used for the SPPS is of the carbamate type. The most preferred protecting group is the 9-fluorenylmethoxycarbonyl (Fmoc) group, which can be removed under basic conditions. Herein, for both fragment A and fragment B, the terminal protecting group is preferably 9-fluorenylmethyloxycarbonyl (Fmoc). The amino acids side-chain functional groups are optionally protected with groups which are generally stable during coupling steps and during alpha-amino protecting group removal, and which are themselves removable in suitable conditions. Such suitable conditions are generally orthogonal to the conditions in which the alpha-amino groups are deprotected. The protecting groups of amino acids side-chain functional groups which are used herein are generally removable in acidic conditions, as orthogonal to the basic conditions generally used to deprotect Fmoc protecting groups.

In a preferred aspect of the present invention, such side-chain protecting groups are specified per individual amino acid occurring in the nangibotide sequence, as follows: the hydroxyl group of tyrosine (Tyr) is preferably protected by a group selected from the group consisting of trityl (Trt), tert-butyldimethylsilyl (TBDMS) and tert-butyl (tBu); more preferably, the tert-butyl (tBu) group is used; the carboxylic group of glutamic (Glu) or aspartic (Asp) acid is preferably protected by an ester selected from the group consisting of 2-phenylisopropyl (O-2-PhiPr), tBu (OtBu), benzyl (OBzl) and allyl ester (OAII); more preferably, the tBu ester is used; the amide group of glutamine (Gin) is preferably protected by a group selected from the group consisting of 2,4,6-trimethoxybenzyl (Tmob), benzyl (Bzl) and trityl (Trt) group; more preferably, the trityl (Trt) group is used; the sulfhydryl group of cysteine (Cys) is preferably protected by a group selected from the group consisting of 4-methoxytrityl, benzyl (Bzl), tert-butyl (tBu) and trityl (Trt) group; more preferably, the trityl (Trt) group is used.

Routinely, commercially available protected L-amino acids are used.

In a preferred aspect of present invention, the coupling steps are performed in the presence of a coupling reagent.

Preferably, the coupling reagent is selected from the group consisting of N- hydroxysuccinimide (NHS), N,N'-diisopropylcarbodiimide (DIC), N,N'- dicyclohexylcarbodiimide (DCC), (Benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(7-Aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU) and ethyl-dimethylaminopropyl carbodiimide (EDC). More preferably, the reaction is carried out in the presence of N,N'- diisopropylcarbodiimide (DIC). In a preferred aspect of present invention, the coupling steps are performed also in the presence of an additive. The presence of an additive, when used in the coupling reaction, has been described to reduce the loss of configuration at the carboxylic acid residue, to increase coupling rates and to reduce the risk of racemization.

Preferably, the additive is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N-hydroxysuccinimide (NHS), l-hydroxy-7- azabenzotriazole (HOAt), endo-N-hydroxy-5-norbornene-2, 3-dicarboxamide, 5- (Hydroxyimino)l,3-dimethylpyrimidine-2,4,6-(lH,3H,5H)-trione (Oxyma-B) and ethyl 2- cyano-2-hydroxyimino- acetate (OxymaPure). More preferably, the additive is selected from the group consisting of 1-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N-hydroxysuccinimide (NHS), l-hydroxy-7-azabenzotriazole (HOAt), endo-N-hydroxy-5- norbornene-2, 3-dicarboxamide, and ethyl 2-cyano-2-hydroxyimino- acetate (OxymaPure). Even more preferably, the reaction is carried out in the presence of 2-cyano-2- hyd roxyi mi no-acetate (Oxy ma Pu re) .

It is particularly preferred to carry out the coupling step in the presence of DIC and OxymaPure.

In a preferred aspect of present invention, the coupling steps are performed in a solvent selected from the group consisting of DMF, DCM, THF, NMP, DMA or mixtures thereof. More preferably, the coupling steps are carried out in DMF.

The coupling steps are generally carried out at a temperature between 10 and 70 °C; preferably, in between about 20 °C (usually referred to as room temperature) and about 50 °C, more preferably the temperature is in the range of 20-35 °C.

In a preferred aspect of present invention, the alpha-amino protecting groups are cleaved under basic conditions. In particular, the Fmoc protecting group is cleaved by treatment with a suitable secondary or primary amine selected from the group consisting of piperidine, pyrrolidine, piperazine, tert-butylamine and DBU, preferably with piperidine. More preferably, Fmoc deprotection is carried out by using a 20% solution of piperidine in DMF.

In a preferred aspect of the present invention, once the desired peptide fragment or final peptide has been obtained according to SPPS (stepwise or fragment based) as described above and is still attached to its solid support, the final deprotection and/or cleavage from such solid support is/are performed, either in a single step or sequentially, thus providing the crude peptide. Preferably, a single step is carried out and such step is performed by using a specific mixture individualised for the resin and the protective groups used, more preferably in acidic or slightly acidic conditions.

When a CTC resin is used, the cleavage step may be performed for instance by treatment with a mixture of HFIP:DCM (30:70 v/v) or 1-2 v/v % TFA solution in DCM. In particular, when a CTC resin is used in the preparation of fragment A or another nangibotide fragment, such cleavage does not remove the alpha-amino protecting group nor the side-chain protecting groups, thus yielding a fully protected fragment, ready to react at its free C- terminal carboxylic acid.

Optionally, scavengers are used. Scavengers are substances, like, for instance, anisole, thioanisole, triisopropylsilane (TIPS), 3,6-dioxa-l,8-octanedithiol (DODT), 1,2- ethanedithiol and phenol, capable of minimize modification or destruction of the sensitive deprotected side-chains in the cleavage environment.

The cleavage/deprotection step of the final nangibotide off the MBHA resin may be performed for instance by using a mixture of TFA/DODT/TIPS/water, for instance in a 90/4/3/3.

The crude target peptide obtained by cleavage from the resin, is optionally purified to further increase its purity.

To this aim, a solution of the peptide is loaded onto an HPLC column with a suitable stationary phase, preferably C18 or C8 modified silica, and an aqueous mobile phase comprising an organic solvent, preferably acetonitrile or methanol, is passed through the column. A gradient of the mobile phase is applied, if necessary. The peptide with desired purity is collected and optionally lyophilized.

Accordingly, the present invention provides a convergent method for the preparation of nangibotide in solid phase synthesis, wherein the method comprises the final coupling of a N-terminal fragment A with a C-terminal fragment B, followed by simultaneous or sequential deprotection and cleavage from the solid support, and optionally a final purification by chromatography.

The present invention further provides intermediate fragments A and B and methods for their preparation, preferably according to SPPS as described above.

According to the invention, the reactive carboxylic acid of fragment A belongs to an achiral amino acid. Such achiral amino acid can be selected between glycine in position 7 and glycine in position 10. Accordingly, fragment A is selected from PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-OH (Fragment 1, 1-7, SEQ ID No 2) and PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-OH (Fragment 5, 1-10, SEQ ID No 3); wherein PG is an alpha-amino protecting group or hydrogen, preferably a Fmoc group.

The N-terminal Fragment A is coupled according to the present invention to a C-terminal fragment B, in particular to a fragment B selected from H-Glu-Tyr-Gly-Cys-Met-resin (Fragment 2, 8-12, SEQ ID No 4) and H-Cys-Met-resin (Fragment 4, 11-12).

The process for the preparation of nangibotide according to the invention involves the coupling of a fragment B attached to a resin, wherein such resin is preferably a Rink amide MBHA resin.

In one embodiment, the process comprises the coupling between fragment A being PG- Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-OH (Fragment 5, 1-10) and fragment B being H- Cys-Met-resin (Fragment 4, 11-12).

Even more preferred, the process comprises the coupling between Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH and H-Cys(Trt)- Met- MBHA resin.

In a preferred embodiment, the process comprises the coupling between fragment A being PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-OH (Fragment 1, 1-7) and fragment B being H-Glu-Tyr-Gly-Cys-Met-resin (Fragment 2, 8-12).

Even more preferred, the process comprises the coupling between Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH, and H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin.

Fragments A and B are preferably prepared by SPPS, either stepwise or by a fragment- based approach.

Particularly preferred are fragments A selected from Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH and Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH.

In a preferred embodiment of the invention, the process for the preparation of nangibotide further comprises the preparation of fragment B, in particular by coupling: PG-Glu-Tyr-Gly-OH (Fragment 3, 8-10) and H-Cys-Met-resin (Fragment 4, 11-12), to obtain PG-Glu-Tyr-Gly-Cys-Met-resin, and is followed by deprotection of the PG group.

When PG is Fmoc, deprotection is performed under basic conditions as described above.

The SPPS preparation of fragment B is preferably performed by using a Rink amide MBHA resin, and preferred fragments B are selected from H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin and H-Cys(Trt)-Met-MBHA resin.

Even more preferred, the process to prepare fragment B comprises the coupling between Fmoc-Glu(OtBu)-Tyr(tBu)-Gly-OH and H-Cys(Trt)-Met-MBHA resin to obtain Fmoc-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin, and is followed by Fmoc deprotection.

In a most preferred embodiment, the process for the preparation of nangibotide according to the invention comprises the step of coupling a peptide fragment A, which is Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH or

Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH, with a peptide fragment B, which is H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin or H-Cys(Trt)-Met-MBHA resin, respectively.

A specially preferred process according to the invention is a method for the preparation of nangibotide wherein fragment A is Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)- Ala-Gly-OH, and fragment B is H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin.

Another preferred process according to the invention is a method for the preparation of nangibotide, wherein fragment A is Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)- Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH, and fragment B is H-Cys(Trt)-Met-MBHA resin.

Another embodiment of the invention is the use of a peptide fragment according to the paragraphs above for the preparation of nangibotide.

Throughout the specification the peptide sequences of fragments A and B, which are partial sequences of nangibotide, are suitable for the coupling strategy that involves coupling via a site with an achiral amino acid at the C-terminus of fragment A. ABBREVIATIONS

SPPS Solid Phase Peptide Synthesis

MBHA resin Methyl benzhydryl amide resin

CTC 2-chloro trityl chloride

Fmoc 9-Fluorenylmethyloxyca rbonyl

Boc tert-Butyloxyca rbonyl

Trt Trityl tBu Tertiary butyl

Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl

HPLC High performance liquid chromatography

DIEA/DIPEA Diisopropylethylamine

TFA Trifluoroacetic acid

AC2O Acetic anhydride

ACN Acetonitrile

DMF Dimethyl formamide

DCM Dichloromethane

MeOH Methanol

DIPE Diisopropylether

HFIP l,l,l,3,3,3-hexafluoro-2-propanol

TIPS Triisopropyl silane

DODT 3,6-dioxa-l,8-octanedithiol

DIC N,N'-diisopropylcarbodiimide DCC N,N'-dicyclohexylcarbodiimide

EDC N-(3-dimethylaminopropyl)-N'-ethylca rbodiimide

HOBt 1-Hydroxybenzotriazole

HOAt l-Hydroxy-7-azabenzotriazole

TBTU N,N,N',N'-Tetramethyl-0-(benzotriazol-l- yl)uronium tetrafluoroborate

PyBOP (Benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate

Oxyma/Oxyma Pu re Ethyl-2-cyano-2-hydroxyiminoacetate

HBTU 3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol-l-oxide hexafluorophosphate

HATU 2-(7-Aza-lH-benzotriazole-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate

Eq equivalent(s)

Cone. Concentration

Min minute(s)

EXAMPLES

Detailed experimental conditions suitable for the preparation of nangibotide according to the present invention are provided by the following examples, which are intended to be illustrative and not limiting of all possible embodiments of the invention.

Unless otherwise noted, all materials, solvents and reagents were obtained from commercial suppliers, of the best grade, and used without further purification.

Purities (%) are calculated by HPLC. Molar yields (%) are calculated considering the final moles obtained divided by the initial moles.

Example 1

Preparation of nangibotide by full SPPS (Reference)

Step 1 : Loading of the first amino acid onto the Rink Amide Resin 2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min. 2 eq Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin after 5 min. All the coupling steps were conducted in this way unless described differently. The loading step was carried out for 1.5 hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by addition of 12 mL of 20% piperidine solution in DMF for two 10 min cycles. This step was performed analogously for all the amino acid residues. The loading, calculated by UV absorption for the peptidyl resin, was 0.8 mmol/g.

Step 2: peptide elongation

For the coupling of all the amino acids involved in the synthesis of nangibotide, 3 eq of each amino acid were activated by 3 eq of DIC and OxymaPure dissolved in DMF at 0.3 M cone. At the end of the peptide elongation, a final Fmoc deprotection, as already described, was performed before moving to the cleavage step.

Step 3: Cleavage and precipitation of crude nangibotide

The cleavage of nangibotide off the resin was carried out using a solution of 16 mL of TFA/DODT/TIPS/water in 90/4/3/3 ratio cooled at 0°C. The peptidyl resin was added portionwise in 30 min keeping the internal temperature under 25°C. The cleavage was run for 3.5 hours, then the resin was filtered and washed by 10 mL of TFA for 10 min.

DIPE was used for the precipitation of the peptide, adding 12 volumes (300 mL) dropwise to the peptide TFA solution, keeping the temperature under 20°C. The suspension with nangibotide was filtered on a gooch funnel, the peptide washed again with 100 mL of DIPE and then dried under vacuum overnight. Molar yield 40%. Purity 61%.

Example 2

Preparation of nangibotide by three-fragment condensation

In the approach using three fragments, only the cysteine residue was coupled to the methionine on rink amide resin to prepare fragment 11-12, whereas protected peptide fragments 1-7 and 8-10 were synthesized using 2-CTC resin.

Step 1: Synthesis of fragment 11-12

2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min 2 eq of Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin. The loading step was carried out for 1 and half hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by addition of 12 mL of a 20% piperidine solution in DMF for two 10 min cycles. Same procedure was repeated for the coupling of Fmoc-Cys(Trt)-OH to obtain resin-attached Fmoc-deprotected fragment 11-12. The loading, calculated by UV absorption for the peptidyl resin relative to the first amino acid inserted, was 0.8 mmol/g.

Step 2: Synthesis of fragments 1-7 and 8-10

For the synthesis of both fragments the loading of 2-chloro trityl chloride resin was performed on 5 g (1.6 mmol/g) using 0.8 eq Fmoc-Gly-OH (6.40 mmol, 1.90 g) dissolved in 30 mL of DCM and addition of 3 eq DIPEA (24 mmol, 4.19 mL). The loading step was carried out for 1 hour, then the resin was washed by 30 mL DCM for three times and eventual Cl-groups were capped by two different capping solutions: first by 30 mL of methanol/DIPEA/DCM (1:2:7) and then by 30 mL AC2O/DIPEA/DCM in the same ratio. After the treatment with these solutions for 15 min and subsequent washing with DCM, the resin was washed three times with DMF, before deprotection of Fmoc and evaluation of the resin loading. Generally, this protocol gave a resin loaded with 1.1 mmol/g Fmoc-Gly-OH. The Fmoc deprotection and coupling step protocols were equally performed with all the amino acids in the respective sequences: Fmoc-Tyr(tBu)-OH and Fmoc-Glu(tBu)-OH for fragment 8-10, and Fmoc-Ala-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH twice, Fmoc-Gln(Trt)- OH and Fmoc-Leu-OH for fragment 1-7.

For each coupling, 3 eq amino acid were activated by 3 eq DIC and 3 eq OxymaPure dissolved in DMF at 0.3 M cone.

Fragment Fmoc-Glu(tBu)-Tyr(tBu)-Gly-OH (8-10) was obtained by cleavage off the resin using 6 volumes (30 mL) of a TFA 1.5 % solution in DCM, 5 times for 2 min. The final TFA solution was neutralized by 1.2 eq pyridine (15.89 mmol, 1.3 mL) diluted in 30 mL methanol. The final solution was concentrated to 50 mL under vacuum then washed by water and brine. The organic layer was dried by anhydrous sodium sulphate, filtered and further concentrated before crystallization of the tripeptide with 5 volumes of petroleum ether at 0°C. The peptide was filtered, washed by petroleum ether and dried overnight in a vacuum oven at 37°C. Molar yield 65%. Purity 90%.

Fragment Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH (1-7) was obtained by cleavage off the resin using 6 volumes (30 mL) of a TFA 1.5 % solution in DCM, 5 times for 2 min. The final TFA solution was neutralized by 1.2 eq pyridine (15.89 mmol, 1.3 mL) diluted in 30 mL methanol. The DCM was evaporated and replaced by methanol, adding and evaporating 30 mL methanol a couple of times till one third of the volume. The peptide fragment was precipitated by adding 5 volumes (150 mL) water to the methanol solution at 0°C and filtered after stirring for 30 min. The full protected heptapeptide was washed by water and dried overnight in a vacuum oven at 37°C. Molar yield 85%. Purity 89%.

Step 3: Synthesis of fragment 8-12 (Fragment condensation 1)

The fragment condensation between Fmoc-Glu(tBu)-Tyr(tBu)-Gly-OH (8-10) and H- Cys(Trt)-Met-MBHA resin (11-12) was carried out activating 2 eq (1.6 mmol, 1.12 g) of fragment 8-10 dissolved in 6 mL of DMF at 40°C by using 2 eq OxymaPure (1.6 mmol, 0.22 g) and 2 eq DIC (1.6 mmol, 0.25 mL) for 10 min. The activated ester of tripeptide 8- 10 was added to the resin-attached fragment 11-12 and stirred for 3 hours at 40°C. After filtration, the resin was washed three times by 15 mL DMF and then capped by 12 mL of AC2O 10% in DMF for 15 min. The resin was washed three timed by 12 mL DMF before deprotection of Fmoc to finally obtain resin-attached Fmoc-protected fragment 8-12. Molar yield 91%. Purity 89%.

Step 4: Synthesis of nanaibotide (Fragment condensation 2)

The fragment condensation between fragment 1-7 and H-Glu(OtBu)-Tyr(tBu)-Gly- Cys(Trt)-Met-MBHA resin (8-12) was carried out activating 1.5 eq (2.25 mmol, 2.64 g) of fragment 1-7 dissolved in 25 mL DMF at 40°C by using 2 eq OxymaPure (2.25 mmol, 0.32 g) and 2 eq DIC (2.25 mmol, 0.35 mL) for 15 min. The activated ester of fragment 1-7 was added to the resin-attached fragment 8-12 and stirred for 3.5 hours at 40°C. After filtration, the resin was washed three times by 12 mL DMF before deprotection of Fmoc with the standard procedure described above. After Fmoc deprotection, the resin was washed again by DMF and DCM and then dried at vacuum pump.

Step 5: Cleavage and precipitation of crude nanaibotide

The cleavage of nangibotide off the resin was carried out using a solution of 16 mL of TFA/DODT/TIPS/water in 90/4/3/3 ratio cooled at 0°C. The peptidyl resin was added portionwise in 30 min keeping the internal temperature under 25°C. The cleavage was run for 3.5 hours, then the resin filtered and washed by 10 mL of TFA for 10 min.

DIPE was used to precipitate the peptide, adding 12 volumes (300 mL) dropwise to the peptide TFA solution, keeping the temperature under 20°C. The suspension with nangibotide was filtered on a gooch funnel, the peptide washed again with 100 mL of DIPE and then dried at vacuum pump overnight. Molar yield 61%. Purity 73%.

Example 3

Preparation of nangibotide by two-fragment condensation In the approach using two fragments, the SPPS elongation onto MBHA resin, as described in Example 2, step 1, was continued until Glu⁸ was attached to provide fragment 8-12, then fragment 1-7, synthesized on 2-CTC resin as described in example 2, step 2, was coupled to the resin-attached fragment 8-12 as described in example 2, step 4.

Step 1: Synthesis of fragment 8-12

2 g of MBHA resin (1.0-1.3 mmol/g) was swelled using 16 mL of DMF for 30 min 2 eq of Fmoc-Met-OH (2.4 mmol, 2.67 g), 2 eq DIC (2.4 mmol, 1.136 mL) and 2 eq OxymaPure (2.4 mmol, 1.023 g) were dissolved in 8 mL of DMF at 0.3 M cone, and added to the resin. The loading step was carried out for 1 and half hour. After the loading, the resin was filtered and washed 3 times with 12 mL of DMF. The Fmoc deprotection step was carried out by addition of 12 mL of a 20% piperidine solution in DMF for two 10 min cycles. Same procedure was repeated for the coupling of Fmoc-Cys(Trt)-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Gly-OH to obtain fragment 8-12. The loading, calculated by UV absorption for the peptidyl resin relative to the first amino acid inserted, was 0.8 mmol/g. Molar yield 88%. Purity 83%.

Step 2: Synthesis of nanaibotide (Fragment condensation 2)

The final fragment condensation was performed as described in example 2, step 4.

Step 3: Cleavage and precipitation of crude nanaibotide

The cleavage of nangibotide off the resin was carried out as described in example 2, step 5. Molar yield 60%. Purity 70%.

Claims

1. Process for the preparation of nangibotide of formula I in solid phase

1 5 10

Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-Cys-Met-Nhh (I) comprising the steps of: a) coupling an N-terminal nangibotide fragment A, selected from

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-OH (Fragment 1, 1-7) and

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-OH (Fragment 5, 1-10), with a C-terminal nangibotide fragment B, selected from H-Glu-Tyr-Gly-Cys-Met-resin (Fragment 2, 8-12) and

H-Cys-Met-resin (Fragment 4, 11-12), respectively, wherein PG is an alpha-amino protecting group or hydrogen; and b) deprotecting and cleaving nangibotide from the resin.

2. The process according to claim 1, wherein the fragment A is

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-OH (Fragment 1, 1-7) and the fragment B is H-Glu-Tyr-Gly-Cys-Met-resin (Fragment 2, 8-12).

3. The process according to claim 1, wherein the fragment A is

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-OH (Fragment 5, 1-10) and the fragment B is H-Cys-Met-resin (Fragment 4, 11-12).

4. The process according to claim 2, further comprising the preparation of fragment B by coupling:

PG-Glu-Tyr-Gly-OH (Fragment 3, 8-10) and H-Cys-Met-resin (Fragment 4, 11-12), and cleaving PG.

5. The process according to any one of the previous claims, wherein the resin is a MBHA resin.

6. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of N,N'-diisopropylcarbodiimide.

7. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of ethyl-2-cyano-2-hydroxyimino acetate.

8. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of N,N'-diisopropylcarbodiimide and ethyl-2-cyano-2-hydroxyimino acetate.

9. The process according to any one of claims 1 to 4, wherein PG is Fmoc group.

10. The process according to any one of the preceding claims, wherein fragment A is Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH or

Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH, and fragment B is

H-Glu(OtBu)-Tyr(tBu)-Gly-Cys(Trt)-Met-MBHA resin or H-Cys(Trt)-Met-MBHA resin, respectively.

11. The process according to claim 4, wherein the preparation of fragment B is performed by coupling

Fmoc-Glu(OtBu)-Tyr(tBu)-Gly-OH, and H-Cys(Trt)-Met-MBHA resin, and cleaving Fmoc.

12. A peptide fragment selected from the group consisting of:

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-OH (Fragment 1, 1-7, SEQ ID No 2),

PG-Leu-GIn-Glu-Glu-Asp-Ala-Gly-Glu-Tyr-Gly-OH (Fragment 5, 1-10, SEQ ID No 3), and PG-Glu-Tyr-Gly-OH (Fragment 3, 8-10).

13. The peptide fragment according to claim 12, which is selected from the group consisting of

Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-OH,

Fmoc-Leu-Gln(Trt)-Glu(OtBu)-Glu(OtBu)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Tyr(tBu)-Gly-OH and

Fmoc-Glu(OtBu)-Tyr(tBu)-Gly-OH.

14. Use of a peptide fragment according to claim 12 or 13 for the preparation of nangibotide.