阅读说明：本技术 用于wnt六肽的线性溶液相路径 (Linear solution phase route for WNT hexapeptides ) 是由 塔迪孔达·维拉巴德拉普拉塔普 卡马拉茹·拉加文德拉拉奥 丹尼斯·亨里克森 于 2019-12-02 设计创作，主要内容包括：本公开总体上涉及多肽合成领域,并且更特别地,涉及Wnt六肽Foxy-5及其受保护的衍生物和肽片段的线性溶液相合成。(The present disclosure relates generally to the field of polypeptide synthesis, and more particularly, to linear solution phase synthesis of Wnt hexapeptide, Foxy-5, and protected derivatives and peptide fragments thereof.)

1. A hexapeptide derivative having the formula:

PG-Met-Asp(OtBu)-Gly-Cys(Trt)-Glu(OtBu)-Leu-OR，

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group for nitrogen, such as Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

2. The hexapeptide derivative of claim 1 wherein PG is Fmoc.

3. The hexapeptide derivative of claim 1 or 2 having the formula:

Fmoc-Met-Asp(OtBu)-Gly-Cys(Trt)-Glu(OtBu)-Leu-OtBu(SEQ_NO 4)。

4. a process for the preparation of a hexapeptide derivative as claimed in claims 1-3 comprising the steps of:

a. provided are protected L-leucine derivatives PG-Leu-OR,

b. removing the protecting group PG from said protected L-leucine derivative and then coupling with PG-Glu-OtBu to yield the protected dipeptide PG-Glu (OtBu) -Leu-OR,

c. removing the protecting group PG from said protected dipeptide and then coupling with PG-Cys (Trt) -OH to yield the protected tripeptide PG-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. removing the protecting group PG from the protected tripeptide and then coupling with PG-Gly-OH to yield the protected tetrapeptide PG-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

e. removing the protecting group PG from the protected tetrapeptide under basic conditions, followed by removal of excess base, to produce a deblocked tetrapeptide H-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

f. coupling the deblocked tetrapeptide with PG-Asp (OtBu) under basic conditions, followed by removal of excess base to yield the protected pentapeptide PG-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

g. removing the protecting group PG from said deblocked pentapeptide and then coupling with formic acid OR an active ester thereof to yield the protected hexapeptide PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

5. The process of claim 4, wherein at least two consecutive coupling steps, such as two, three or four steps, are performed without product isolation.

6. The process of claim 4 or 5, wherein at least one intermediate purification, such as one, two, three or four times, is performed between two consecutive coupling steps, e.g. by flash chromatography, without product isolation.

7. The method of any one of claims 4-6, wherein all steps b) -g) are performed in solution.

8. A method of making Foxy-5(SEQ _ NO 1), the method comprising:

a. providing a hexapeptide derivative according to any one of claims 1, 2 or 3,

b. removing the nitrogen protecting group at the Met-terminus from the hexapeptide derivative,

c. coupling the hexapeptide obtained in step b) with formic acid OR an active ester thereof to produce a protected Foxy-5 derivative For-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. deprotecting the protected Foxy-5 derivative obtained in step c) as a whole to give Foxy-5 in crude form,

e. optionally carrying out a further purification step, and

f. optionally yielding Foxy-5 in solid form.

9. The process of any one of claims 8, wherein at least two consecutive reaction steps, such as two, three or four steps, are performed without product separation.

10. The method of any one of claims 8-9, wherein at least one intermediate purification, such as one, two or three times, is performed between two consecutive reaction steps, e.g. by flash chromatography, without product isolation.

11. The method of any one of claims 8-10, wherein the Foxy-5 in crude form is purified by chromatography, such as reverse phase chromatography.

12. The method of any one of claims 8-11, wherein Foxy-5 is isolated in solid form, may be the hexapeptide itself, or may be an acidic or basic addition salt thereof.

13. The method of any one of claims 8-12, wherein the solid form of Foxy-5 is a lyophilizate, an amorphous powder, or a crystalline compound.

Technical Field

The present disclosure relates generally to the field of polypeptide synthesis, and more particularly, to linear solution-phase methods for Wnt hexapeptide Foxy-5. The disclosure further relates to solution phase synthesis of novel Foxy-5 tripeptides, tetrapeptides, and pentapeptide fragments.

Background

Foxy-5 is a formylated, WNT 5A-derived hexapeptide and WNT5A mimetic with potential anti-metastatic activity, currently being developed as a drug candidate to prevent tumor spread in several common forms of cancer.

Foxy-5 has the amino acid sequence For-Met-Asp-Gly-Cys-Glu-Leu-OH (SEQ _ NO 1, FIG. 1).

Following intravenous administration, Foxy-5 binds and activates WNT5A receptor, primarily the Frizzled family, which activates WNT 5A-mediated signaling.

Foxy-5 was designed to compensate for the lack of protein WNT5A in tumor tissue noted in colon cancer patients to reduce the risk of metastasis. A recent sub-analysis of a retrospective study of patients with stage III colorectal cancer showed that the proportion of patients with low expression of WNT5A was significantly higher than observed in previous studies with patients with stage II colorectal cancer (CRC). Patients with CRC stage III tumors differ from those in stage II mainly by the presence of tumor cells in the lymph nodes adjacent to the primary tumor, and thus are more aggressive and progress faster. Low levels of WNT5A were observed in nearly 70% of stage III patients, compared to a proportion of about 45% in patients with a second advanced stage of tumor. This supports the hypothesis that WNT5A levels significantly affected disease progression.

Based on a completed phase 1b study for Foxy-5, pharmacokinetics and dose determination for phase 2, aimed at recording the safety profile of the drug candidate, Foxy-5 is now used in a phase 2 clinical trial study, where treatment of colon cancer patients will begin prior to surgery at the time of diagnosis. The treatment is intended to last for up to 12 weeks, or until chemotherapy is initiated.

Foxy-5 and its preparation are described in International patent No. WO 06130082A 1. To date, Active Pharmaceutical Ingredients (APIs) for preclinical and clinical studies have been produced by classical Solid Phase Peptide Synthesis (SPPS), in which Foxy-5 is produced by a linear 1+1+1+1+1+1 pathway, seeFIG. 2。

Thus, the sequence For-Met-Asp-Gly-Cys-Glu-Leu-OH was assembled on a 2-chlorotrityl resin with the C-terminal amino acid Leu using the Fmoc strategy (Fmoc ═ fluorenylmethyloxycarbonyl). The synthesis was carried out in an SPPS reactor and consisted of: alternating coupling, acetylation and N- α -deprotection procedures. The coupling was carried out in DMF (N, N-dimethylformamide) or DMF/DCM (dichloromethane) as solvent. It consists of the following if necessary: the N- α -protected amino acid derivative is coupled to the preceding amino acid in the presence of an activator and a base. Formic acid is coupled as an active ester in the absence of an activator.

If coupling is incomplete, the procedure can be continued or repeated. To avoid the formation of missing sequences as by-products, a systematic acetylation step (capping) is performed after the coupling step, or if a recoupling step is performed after the recoupling step, DMF, acetic anhydride and pyridine are used.

Acetylation was followed by an N- α -deprotection procedure consisting of: the resin was washed with DMF, the Fmoc-group was cleaved with piperidine in DMF, and then washed with DMF. In the case of incomplete cleavage, the N- α -deprotection procedure as described above may be repeated. For each individual step, solvent and/or reagents are added and the reaction mixture is stirred and then filtered to remove the solvent and/or reagents from the resin.

The coupling, acetylation and N-alpha-deprotection steps are repeated until the resin carries the complete peptide sequence For-Met-Asp-Gly-Cys-Glu-Leu-OH. After the final coupling of the formic acid active ester, no acetylation was carried out. SPPS was completed by washing the peptide resin with DMF and IPA followed by drying under reduced pressure.

Cleavage of the peptide and concomitant cleavage of the side chain protecting groups from the resin is accomplished by treating the peptide resin with TFA in the presence of a suitable scavenger (e.g., water and EDT). Subsequently, the crude peptide obtained was purified by two-dimensional preparative HPLC on a reverse phase column using ACN gradient elution (formic and acetic acid system).

The combined fractions with sufficient purity were lyophilized. In case the established purity standards are not met, the lyophilisate is analyzed by HPLC and optionally re-purified by two-dimensional preparative HPLC as outlined above.

The SPPS approach outlined above has provided sufficient material for preclinical and early clinical studies, but for further clinical studies and for ultimate commercial purposes, there is a need for a synthetic approach that is more amenable to large-scale synthesis, which can reduce the cost of goods and can provide larger batches of Foxy-5.

Therefore, there is a need for a reliable synthetic route that can provide Foxy-5 on a multi-kilogram scale for further clinical trial supply and ultimate commercial purposes.

Disclosure of Invention

The present disclosure provides a linear solution phase process For the preparation of the formylated hexapeptide known as Foxy-5 (i.e., For-Met-Asp-Gly-Cys-Glu-Leu-OH (SEQ _ NO 1)), as well as various tri-, tetra-, penta-, and hexapeptide fragments thereof, including protected derivatives thereof. The methods provided herein have many advantages over traditional solid phase synthesis, including but not limited to low raw material costs, ease of purification of process intermediates, ease of fragment assembly, high chiral purity, and adaptability to commercial scale-up, as will be described in more detail below.

It is therefore a primary object of the present invention to provide an extensible synthesis path for Foxy-5. Another objective is to identify and characterize suitable key intermediates for the scalable synthetic pathway for subsequent GMP (good manufacturing practice) production of drug substances.

Given the cost of goods and the cumbersome scalability typically associated with solid phase chemistry, emphasis has been placed on developing solution phase chemistry routes. The present disclosure provides a linear (1+1+1+1+1+1) solution phase process for the preparation of Foxy-5 or its intermediates and precursors.

In contrast to traditional, convergent hexapeptide solution-phase approaches (i.e., producing dipeptides or tripeptides alone, followed by coupling), the inventors have surprisingly found that a linear pathway (i.e., assembly of the peptide sequence of Foxy-5 by sequential solution-phase coupling of protected derivatives of the amino acids Met, Asp, Gly, Cys, Glu, and Leu) allows it to function very efficiently.

The core part of the present invention is the introduction of a formyl (For) group at the N-terminus of methionine. As will be discussed later in this application, this particular chemical step is the most difficult to achieve with good yields and high chemical and optical purity.

The inventors have now found that hexapeptide derivatives of the formula PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR can be used very well as Foxy-5 precursors, and preferably when R is selected from C₁-C₆Alkyl (e.g., methyl or t-butyl), and PG is a nitrogen base-sensitive protecting group (i.e., a protecting group that is stable under acidic conditions but cleavable from the peptide under basic/alkaline conditions). In the context of the present invention, protecting groups such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc are suitable, preferably Fmoc.

In a first aspect, the present invention therefore provides hexapeptide derivatives having the formula PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR, wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

The hexapeptide derivatives of the first aspect can be converted to the desired hexapeptide Foxy-5 by N-deprotection, followed by coupling with formic acid and finally global deprotection of the remaining protecting groups.

In a second aspect, the present invention therefore provides a method for preparing the hexapeptide Foxy-5(SEQ _ NO 1), the method comprising:

a. there is provided a hexapeptide derivative according to the first aspect,

b. removing the PG protecting group from said hexapeptide derivative,

c. coupling the product obtained in step b) with formic acid OR an active ester thereof to yield the protected Foxy-5 derivative For-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. deprotecting the protected Foxy-5 derivative obtained in step c) as a whole to give Foxy-5 in crude form,

e. optionally carrying out a further purification step, and

f. optionally, precipitating the Foxy-5 hexapeptide formed as a solid in the form of a basic or acidic salt,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

The hexapeptide derivatives of the first aspect may be prepared by any convenient method (such as solid phase synthesis or solution phase methods), and most conveniently by methods developed by the inventors set forth below.

In a third aspect, the present invention provides a process for the preparation of a hexapeptide derivative as described in the first aspect, comprising the steps of:

a. provided are protected L-leucine derivatives PG-Leu-OR,

b. removing the protecting group PG from said protected L-leucine derivative and then coupling with PG-Glu-OtBu to yield the protected dipeptide PG-Glu (OtBu) -Leu-OR,

c. removing the protecting group PG from said protected dipeptide and then coupling with PG-Cys (Trt) -OH to yield the protected tripeptide PG-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. removing the protecting group PG from the protected tripeptide and then coupling with PG-Gly-OH to yield the protected tetrapeptide PG-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

e. removing the protecting group PG from the protected tetrapeptide under basic conditions, followed by removal of excess base, to produce a deblocked tetrapeptide H-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

f. coupling the deblocked tetrapeptide with PG-Asp (OtBu) under basic conditions, followed by removal of excess base to yield the protected pentapeptide PG-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

g. removing the protecting group PG from said deblocked pentapeptide and then coupling with formic acid OR an active ester thereof to yield the protected hexapeptide PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

The above aspects of the invention (including preferred embodiments thereof) are discussed further below.

In a fourth aspect of the invention, tripeptide-based intermediates are providedINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu) with sequential coupling of protected derivatives of the amino acids Gly, Asp and Met followed by N-deprotection and coupling with formic acid to give the hexapeptide Foxy-5 in protected form.

In a fifth aspect of the invention, a method is provided for producing the hexapeptide Foxy-5 in protected form based on the sequential coupling of the tetrapeptide intermediate INTM-3(Fmoc-Gly-cys (trt) glu (OtBu) -Leu-OtBu) with protected derivatives of the amino acids Asp and Met, followed by N-deprotection and coupling with formic acid.

In a further aspect of the invention, the following peptides are provided:

Fmoc-Cys(Trt)Glu(OtBu)-Leu-OtBu

Fmoc-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 2)

Fmoc-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 3)

Fmoc-Met-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 4)

For-Met-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 5)

Cys-Glu-Leu

Gly-Cys-Glu-Leu(SEQ_NO 6)

Asp-Gly-Cys-Glu-Leu(SEQ_NO 7)

Met-Asp-Gly-Cys-Glu-Leu(SEQ_NO 8)

Drawings

FIG. 1 shows a schematic view of aThe chemical structure of Foxy-5 is shown. Foxy-5 is a linear peptide consisting of six amino acids with formylated N-termini. All optically active amino acid residues are in the L-configuration. The molecular formula of Foxy-5 is C₂₆H₄₂N₆O₁₂S₂And a molecular weight of 694.8g/mol (average mass).

FIG. 2The synthesis scheme of the SPPS pathway to Foxy-5 is shown.

FIG. 3A linear 1+1+1+1+1+1 strategy for forming Foxy-5 is illustrated.

FIG. 4Illustrating the formation of an "asparagine impurity".

FIG. 5Illustrating the role of the formyl group in the epimerisation reaction observed.

Abbreviations

Fmoc ═ fluorenylmethoxycarbonyl

Tfa ═ trifluoroacetyl group

Tsoc ═ 4-tosylethyloxycarbonyl

Mesoc ═ methylsulfonylethyloxycarbonyl

Peoc ═ 2- (triphenylphosphonyl) ethyloxycarbonyl

Cyoc ═ 2-cyano-tert-butyloxycarbonyl, and

pht ═ phthaloyl

Nsc ═ 2- (4-nitrophenylsulfonyl) ethoxycarbonyl

Boc ═ tert-butyloxycarbonyl radical

For ═ formyl

Trt ═ Trityl (Trityl)

tBu ═ tert-butyl

THF ═ tetrahydrofuran

DIPE ═ diisopropyl ether

DMF ═ N, N-dimethylformamide

TFA ═ trifluoroacetic acid

TIS-triisopropylsilane

HOBt ═ 1-hydroxybenzotriazole

DCM ═ dichloromethane

EDAC, HCl ═ 1-ethyl-3- (3' -dimethylaminopropyl) carbodiimide, HCl

DIPEA ═ diisopropylamine

DBU ═ 1, 8-diazabicyclo [5.4.0] undec-7-ene

Amino acid abbreviations:

met-methionine

Asp ═ asparagine

Gly ═ glycine

Cys ═ cysteine

Glu ═ glutamic acid

Leu ═ leucine

Foxy-5＝For-Met-Asp-Gly-Cys-Glu-Leu

Detailed Description

As described in the summary above, a linear solution phase method for assembling the Foxy-5 hexapeptide sequence was designed, which will be discussed in more detail below.

The general approach is to protect all amino acids as O-^tBu, N-Fmoc derivatives. Furthermore, the aim is to synthesize as "abbreviated" as possible, thereby avoiding the time-consuming and expensive isolation of intermediates. Preferably, each step in the sequence should be performable without product isolation, allowing only small purification steps to be performed, such as aqueous workup of organic solvent solutions and silica gel plug treatment ("flash chromatography") to remove, for example, excess base.

The linear synthesis starts in two short steps (step 1+2) with the preparation of the tripeptide INTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu) by coupling tert-butyl L-leucine ester, HCl with Fmoc-Glu (OtBu) in Dichloromethane (DCM) as reaction solvent. The reaction mixture was treated with water and brine, and the DCM solution was used directly in step 2 without isolation of the intermediate dipeptide, INTM-1(Fmoc-Glu (OtBu) Leu-OtBu).

In step 2, a solution of INTM-1 in DCM is reacted with Fmoc-Cys (Trt) -OH to obtain the desired tripeptide INTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu). The reaction proceeded well and gave a combined yield of about 68% over a two-step repetition. INTM-2 can be purified as a white solid by chromatography on silica gel (100-200). The DCM solution obtained may also beAs isProceed down and use it directly in step 3, coupling with Fmoc-Gly-OH.

In step 3, INTM-2 is Fmoc-deprotected with DBU and coupled with Fmoc-Gly-OH in DCM to give the tetrapeptide INTM-3(Fmoc-Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu). After completion of the reaction, the DCM layer was washed with water and brine. The final organic layer was then concentrated from 50 volumes to 10-15 volumes, andas isProceed down to the next stage (step 4) without separation.

In the step ofIn 4, a solution of INTM-3 in DCM was first treated with DBU to effect deprotection of the Fmoc group. After deblocking, the tetrapeptide was coupled with Fmoc-Asp-OtBu in the presence of EDC.HCl, HOBt hydrate and DIPEABefore oneThe reaction mass was passed through a silica plug to remove DBU. After completion of the reaction, the reaction mass was passed through a silica plug again to remove DIPEA still present. Removal of DBU and DIPEA as described is critical to inhibit the formation of undesirable asparagine impurities (see alsoFIG. 4). Proceeding in this manner, a yield of about 60% of INTM-4(Fmoc-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu) was observed in two steps (step 3+4) at a scale of 100-160 g. In the presence of DBU, the observed yield was only about 25%.

In step 5, INTM-4 was Fmoc-deprotected with DBU in DCM as solvent and coupled with Fmoc-methionine to give the intermediate INTM-5(Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu (SEQ _ NO 4)) in crude form. Purification was performed by column chromatography using DCM/THF as eluent. The purified product was slurried in DIPE to give a white solid.

Initially, direct coupling of INTM-4 with N-formyl-methionine (For-Met-OH) was attempted to produce the hexapeptide INTM-6, but this synthetic strategy was found to result in partial epimerization in the final product, since For-Met-OH cyclizes reversibly under the reaction conditions to produce oxazolidinones, which results in racemization of the For-Met-OH reagent. See alsoFIG. 5. Instead, using Fmoc-methionine, the Fmoc group is then DBU-deprotected and coupled with formic acid or its active ester to give the desired intermediate hexapeptide INTM-6. Global deprotection (of the Trt and O-tBu groups) here provides Foxy-5 in crude form, which may be further purified (e.g., by chromatography) and/or precipitated as a solid (e.g., an acid or basic addition salt).

In a first aspect, the present invention therefore provides hexapeptide derivatives having the formula PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR, wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base-sensitive protecting group, e.g.Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc, or Nsc.

In an embodiment of the first aspect, PG is Fmoc.

In another embodiment of the first aspect, there is provided a hexapeptide derivative having the formula: Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR, wherein R is selected from C₁-C₆Alkyl groups such as methyl or tert-butyl.

In a preferred embodiment of the first aspect, the hexapeptide derivative has the formula Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu (SEQ _ NO 4).

In a second aspect, the present invention provides a method for preparing the hexapeptide Foxy-5(SEQ _ NO 1), the method comprising:

a. there is provided a hexapeptide derivative according to the first aspect,

b. removing the PG protecting group from said hexapeptide derivative,

c. coupling the product obtained in step b) with formic acid OR an active ester thereof to yield the protected Foxy-5 derivative For-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. deprotecting the protected Foxy-5 derivative obtained in step c) as a whole to give Foxy-5 in crude form,

e. optionally carrying out a further purification step, and

f. optionally, precipitating the Foxy-5 hexapeptide formed as a solid in the form of a basic or acidic salt,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc.

In an embodiment of the second aspect, PG is Fmoc.

In another embodiment of the second aspect, the alkyl group R is tert-butyl.

In another embodiment of the second aspect, the coupling with formic acid or an active ester thereof takes place in solution.

In another embodiment of the second aspect, the crude Foxy-5 obtained is purified by chromatography (e.g. reverse phase chromatography).

In another embodiment of the second aspect, the Foxy-5 obtained is precipitated as a basic or acidic salt in solid form.

In another embodiment of the second aspect, the Foxy-5 obtained is isolated as a crystalline basic or acidic salt.

In a third aspect, the present invention provides a process for the preparation of a hexapeptide derivative as described in the first aspect, comprising the steps of:

a. provided are protected L-leucine derivatives PG-Leu-OR,

b. removing the protecting group PG from said protected L-leucine derivative and then coupling with PG-Glu-OtBu to yield the protected dipeptide PG-Glu (OtBu) -Leu-OR,

c. removing the protecting group PG from said protected dipeptide and then coupling with PG-Cys (Trt) -OH to yield the protected tripeptide PG-Cys (Trt) -Glu (OtBu) -Leu-OR,

d. removing the protecting group PG from the protected tripeptide and then coupling with PG-Gly-OH to yield the protected tetrapeptide PG-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

e. removing the protecting group PG from the protected tetrapeptide under basic conditions, followed by removal of excess base, to produce a deblocked tetrapeptide H-Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

f. coupling the deblocked tetrapeptide with PG-Asp (OtBu) under basic conditions, followed by removal of excess base to yield the protected pentapeptide PG-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

g. removing the protecting group PG from said deblocked pentapeptide and then coupling with formic acid OR an active ester thereof to yield the protected hexapeptide PG-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OR,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base sensitive protecting group, such as Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc or Nsc,

wherein R is selected from C₁-C₆Alkyl, such as methyl or tert-butyl, and PG is a base-sensitive protecting group, e.g.Fmoc, Tfa, Tsoc, Mesoc, Peoc, Cyoc, or Nsc.

In an embodiment of the third aspect, PG is Fmoc.

In an embodiment of the third aspect, all steps b) -g) are performed in solution.

In a further embodiment, the hexapeptide derivative of the first aspect is obtainable by the method of the third aspect.

In a preferred embodiment of the third aspect, the base sensitive protecting group PG is Fmoc and the alkyl group R is t-butyl.

In another embodiment of the third aspect, at least two consecutive coupling steps, such as two, three or four steps, are performed without product isolation.

In a preferred embodiment, the present invention therefore provides the following sequence of steps for the production of the hexapeptide Foxy-5 in crude form:

1. coupling Leu-OtBu with Fmoc-Glu-OtBu to produce a dipeptideINTM-1(Fmoc-Glu (OtBu) -Leu-OtBu), followed by

2. Herein coupled with Fmoc-Cys (Trt) -OH to generate tripeptidesINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu), followed by

3. This was coupled with Fmoc-Gly-OH to give a protected tetrapeptideINTM-3(Fmoc-Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ _ NO 2)), and then

4. This was coupled with Fmoc-Asp-OtBu to give a protected pentapeptideINTM-4(Fmoc-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ _ NO 3)), followed by

5. Coupling with Fmoc-Met to obtain protected hexapeptidesINTM-5(Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ _ NO 4)), followed by

6. This is reacted with formic acid to give Foxy-5 in protected form, i.e.INTM-6(For-Met-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ _ NO 5)), followed by

7. The t-Bu and Trt groups were deprotected in their entirety to give Foxy-5 in crude form, i.e., For-Met-Asp-Gly-Cys-Glu-Leu-OH (SEQ _ NO 1).

In the first placeIn a fourth aspect, tripeptide-based intermediates are providedINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu) sequential coupling with protected derivatives of the amino acids Gly, Asp and Met followed by N-deprotection and coupling with formic acid to yield the hexapeptide Foxy-5 in protected form (i.e.INTM-6(For-Met-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ. RTM. NO: 5))).

In embodiments, the intermediate is produced by solid phase synthesisINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu). In a preferred embodiment, the intermediate is produced by solution phase synthesisINTM-2。

In a fifth aspect, there is provided a sequential coupling of the tetrapeptide-based intermediate INTM-3(Fmoc-Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu) with protected derivatives of the amino acids Asp and Met followed by N-deprotection and coupling with formic acid to yield the hexapeptide Foxy-5 in protected form (i.e.INTM-6(For-Met-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu (SEQ. RTM. NO: 5))).

In embodiments, the intermediate is produced by solid phase synthesisINTM-3(Fmoc-Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu). In a preferred embodiment, the intermediate is produced by solution phase synthesisINTM-3。

In a further aspect of the invention, the following peptides are provided:

Fmoc-Cys(Trt)Glu(OtBu)-Leu-OtBu

Fmoc-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 2)

Fmoc-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 3)

Fmoc-Met-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 4)

For-Met-Asp(OtBu)-Gly-Cys(Trt)Glu(OtBu)-Leu-OtBu(SEQ_NO 5)

Cys-Glu-Leu

Gly-Cys-Glu-Leu(SEQ_NO 6)

Asp-Gly-Cys-Glu-Leu(SEQ_NO 7)

Met-Asp-Gly-Cys-Glu-Leu(SEQ_NO 8)

experiment of

Example 1 tripeptide INTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu), procedure1+2

50 g of tert-butylleucine ester in a solvent mixture of 2025ml of dichloromethane (27 vol) and 225ml of THF (3 vol) in the presence of EDAC, HCl (2.0 eq), and HOBt (2.0 eq) and DIPEA (5.0 eq)₂Coupling with 175 g (2.0 equiv.) of Fmoc-Glu- (OtBu), initially at 0 deg.C-5 deg.C for 1 hour, then at 15 deg.C-20 deg.C for 1 hour, gives the dipeptideINTM-1(Fmoc-Glu- (OtBu) -Leu-OtBu). By passing¹H NMR and mass spectroscopy confirmed identity. For the reaction with Fmoc-cys (trt) -OH in the next step, the product isolation was omitted and the dichloromethane solution was used directly after the aqueous work-up. Accordingly, the dipeptide obtained using the above methylene chloride solutionINTM-1(Fmoc-Glu- (OtBu) -Leu-OtBu). Fmoc deprotection was achieved using DBU and coupled with 204 g (1.1 eq) of Fmoc-Cys (Trt) -OH in the presence of EDAC, HCl (2.0 eq), HOBt (2.0 eq) and DIPEA (4 eq) to give a crude tripeptideINTM-2It was purified by silica gel (100-200) chromatography using EtOAc-hexane as eluent to give the tripeptide as an off-white solidINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu) (148 g, 68% overall yield by coupling reaction and 93.8% purity by HPLC).

Repeat the above stretch reaction step 1+2 from 75 g of t-butyl leucine HCl to yield 208 gINTM-2。

Example 2 tetrapeptide (Fmoc-Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu), step 3

Obtained as aboveINTM-2(Fmoc-Cys (Trt) Glu (OtBu) -Leu-OtBu) was reacted with DBU in DCM (50 vol) to effect Fmoc deprotection and in the presence of DIPEA (3 equiv.), EDC.HCl (2.0 equiv.), and HOBt (2.0 equiv.) with 1.3 equiv Fmoc-Gly-OH to give the protected tetrapeptideINTM-3(Fmoc-Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu (SEQ _ NO 2)). Good conversion was observed by TLC and DCM layer was washed with water and brine. The final organic layer was concentrated to 10-15 volumes andas isUsed in the next step without isolation.

Example 3 pentapeptide (Fmoc-Asp (OtBu) -Gly-Cys (Trt) Glu (OtBu) -Leu-OtBu), stepStep 4

Key intermediate obtained as concentrated DCM solution hereinbeforeINTM-3(Fmoc-Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu) was first reacted with DBU to effect Fmoc deprotection. The reaction mass was passed through a silica plug to remove DBU, which was found in previous experiments to induce the formation of undesirable asparagine by-products, before proceeding to the next coupling step. Removal of DBU prior to coupling with Fmoc-Asp-OtBu was effective in inhibiting asparagine formation. After silica plug treatment, DCM solution was reacted with Fmoc-Asp (OtBu) in the presence of DIPEA, EDAC, HCl (1.2 equiv.) and HOBt (1.2 equiv.) to give the protected pentapeptideINTM-4(Fmoc-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu (SEQ _ NO 3)). By passing¹H NMR and mass spectrometry confirmed the identity of the product.

From 148 grams and 190 gramsINTM-2Initially, the telescoping reaction was repeated twice (step 3+ step 4) to give 107 g and 178 g, respectivelyINTM-4(58.1% and 75.4% of theory).

Example 4 hexapeptide (Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu), procedure 5

Implementation of the pentapeptide obtained from example 3 with DBUINTM-4Deprotecting Fmoc in DIPEA (3.0 equiv.), EDC.HCl (2.0 equiv.) and HOBt.H₂Coupling with Fmoc-Met in DCM-THF (50 vol +10 vol) as solvent in the presence of O (2.0 eq.) gives the protected Foxy-5 derivative in crude formINTM-5(Fmoc-Met-Asp (OtBu) -Gly-Cys (Trt) -Glu (OtBu) -Leu-OtBu (SEQ _ NO 4)). Purification was performed by column chromatography using DCM/THF as eluent. The purified product was slurried in DIPE to give a white solid.

Purification is carried out several times under various conditions, for example precipitation with anti-solvents and chromatography. The best solution was found to be column chromatography followed by slurrying in DIPE, giving a yield of 78% and a chemical purity of 95.4% on a 25 gram scale. The reaction was repeated on an 80 gram scale to provide 74 grams of product of 94.1% chemical purity (83% yield).

Example 5-hexapeptide INTM-6(For-Met-Asp (OtBu) -Gly-Cys(Trt)-Glu(OtBu)-Leu- OtBu), step 6

The hexapeptide obtained in example 4 was achieved with DBU in DCM (50 vol.)INTM-5Deprotection of Fmoc followed by edc.hcl (4.0 eq), hobt.h₂O (4.0 equiv.) and DIPEA (4.0 equiv.) to yieldINTM-6(For-Met-Asp(OtBu)-Gly-Cys(Trt)-Glu(OtBu)-Leu-OtBu(SEQ_ID NO 5))。

The reaction (step 6) was carried out from 4 g, 18 g and 18 g, respectivelyINTM-5Three times to obtain a yield of 75% -83% and a chemical purity between 67.5% -77.2%. The reaction was repeated on a 30 gram scale with 10 equivalents of formic acid to give 17 grams of product of 88.8% chemical purity (67% yield).

Example 6-hexapeptide For-Met-Asp-Gly-Cys-Glu-Leu-OH (Foxy-5), step 7

17 g of hexapeptide obtained in example 5 were purified by column chromatography in TFA (10 vol./. i-Pr)₃SiH (TIS, 1.7 vol)/DTT (1.7 eq) in a mixture of N₂The deprotection is carried out globally by dissolution and stirring at 10 ℃ to 15 ℃ for 15 to 30min (Trt and tBu groups). Next, the reaction mixture was warmed to 25 ℃ to 30 ℃, and stirred at this temperature for 1 to 2 hours. The reaction mass was then concentrated under reduced pressure to 2 to 3 volumes. After the reaction was complete, THF (5 volumes) was added and stirring continued at 25-30 ℃ for an additional 10-15 min. MTBE (30 volumes) was then added slowly to precipitate the crude product, which was obtained as a solid in quantitative yield (12.3 g).

The crude product was finally purified by reverse phase chromatography using the following process conditions to give the desired hexapeptide For-Met-Asp-Gly-Cys-Glu-Leu-OH (Foxy-5) with a purity of 98.3%:

medium: luna C18(3) (brand: Phenomenex) pore size: 10 μm

Column id: 50mm ID X250 mm (Novasep)), flow rate: 50.0 mL/min.

Sample preparation: 100g of the sample was dissolved in 4mL of diluent and filtered through a 0.45 μm filter.

Preparing a buffer solution:

buffer-a: a0.10% + -0.01% trifluoroacetic acid buffer in water was prepared by mixing 10mL of trifluoroacetic acid into 10L of purified water.

buffer-B: a0.10% + -0.01% trifluoroacetic acid buffer in acetonitrile was prepared by mixing 5mL of trifluoroacetic acid into 5L of acetonitrile.

The operation procedure is as follows:

1. the column was equilibrated with [ buffer A: buffer B ] in a ratio (95:5) of 3-5 column volumes at a flow rate of 5.0 mL/min.

2. The sample solution was loaded onto the column.

3. The chromatography system attached to the column was programmed to provide a gradient program as follows to start the product elution.

4. The following fractions were collected: anterior, apical and caudal

Note that: due to scale dependence, elution times may vary from run to run.

5. Immediately after peak elution, the column was washed with water and acetonitrile at a ratio of 20:80 (% v/v) for 2 column volumes.

6. The fractions were sent for purity analysis.

7. The fractions were stored at-20 ℃. + -. 2 ℃.