A new cysteine derivative and its use in the preparation of cyclic peptides

Abstract

The application relates to the field of medicine synthesis, and discloses a new cysteine derivative and application of the cysteine derivative in preparation of a cyclic peptide. The new cysteine derivative is used for preparing a cyclic peptide with more stable conformation.

Description

TECHNICAL FIELD

[0001] The present application relates to a new cysteine derivative and its use in the preparation of cyclic peptides. BACKGROUND

[0002] Stapled peptide is a kind of chemically modified cyclic peptide, which is developed based on the requirement of polypeptide to form alpha-helix to enter cells through cell membrane. Various life process regulations in vivo are realized through the interaction between proteins. For example, the self-assembly of viruses, the growth, division and differentiation of cells. However, the interface of protein-protein interaction is usually too large, so that it is difficult for small molecule drugs to target and locate, to achieve high efficiency and specificity to block the interaction and to show good therapeutic effect. Protein drugs cannot directly target the interaction in cells because they are difficult to pass through the cell membrane, so researchers have begun to seek a new drug molecule that can overcome the shortcomings of the two drugs and can enter the cell membrane and specifically target protein-protein interaction.

[0003] Studies have shown that polypeptides with alpha-helix structure and rich in positive charge can pass through the cell membrane. Therefore, people have developed alpha-helix structure using disulfide bond and intramolecular amide bond as a scaffold, but these scaffolds cannot exist stably in physiological environment. In 2000, Verdine et al. developed a method of using carbon-carbon bond as a scaffold to stabilize the alpha-helix structure of polypeptide, and the polypeptide obtained by this method became stapled peptide. Stapled peptide has higher alpha-helix degree, stronger affinity, can pass through the cell membrane, is difficult to be hydrolyzed by protease, has long half-life in vivo, etc.

[0004] The conventional synthesis strategy of stapled peptide is to introduce two unnatural amino acids containing alpha-methyl and alpha-alkenyl during the solid-phase synthesis of peptide chain, and then the olefin metathesis reaction (RCM) occurs between the two unnatural amino acids to form a stable alpha-helix structure conformational all-carbon scaffold, thereby synthesizing stapled peptide. Because the yield of olefin metathesis reaction is low, and hydrogenation process needs to be used, which seriously affects the product yield.

[0005] The present application provides a new cysteine derivative, which provides a new solution for stapled peptide, and can greatly improve the yield of cyclic peptide, and is more suitable for large-scale production. SUMMARY

[0006] The present application first provides a new cysteine derivative.

[0007]

[0008] m1 in structure I is an integer from 2 to 10;

[0009] m2 in structure I is an integer from 2 to 10;

[0010] R in structure I is Boc, or is Mtt, or is Dde, or is ivDde, or is nothing.

[0011] R in structure I is selected from Boc, Mtt, Dde, ivDde, preferably Dde.

[0012] Cysteine in structure I is selected from L-cysteine, D-cysteine, preferably L-cysteine.

[0013] The amino acid derivative above is used for preparing a stable conformational cyclic peptide. DETAILED DESCRIPTION

[0014] The present application discloses a new amino acid derivative and its use in preparing a cyclic peptide. Those skilled in the art can refer to the content herein and make appropriate improvements to related parameters for implementation. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method of the present application has been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the compounds and preparation methods described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0015] The Chinese names corresponding to the English abbreviations involved in the present application are shown in the following table:

[0016]

[0017] Preparation of compound 1 in Example 1

[0018]

[0019] The chemical reaction formula is as follows:

[0020]

[0021] 1. Preparation of intermediate 1

[0022] Take 0.8 mol of raw material 1 and 1 mol of raw material 2, dissolve with an appropriate amount of DMF, add 1 mol of pyridine, stir at 60°C overnight, evaporate the DMF, dissolve the residue in 2L 10% TFA / DCM solution and stir for 1 hour, evaporate the TFA / DCM solution, dissolve the residue in ethyl acetate, filter the residue, filter the filtrate and wash with saturated brine 6 times, evaporate the filtrate to obtain intermediate 1.

[0023] 2. Preparation of intermediate 2

[0024] The intermediate 1 was dissolved in appropriate amount of ethanol, 2 mol of DDE-OH was added, and the reaction was refluxed for 8 hours with stirring. The solvent was evaporated, and the intermediate 2 was obtained by dissolving the residue in 2 L of 60% TFA / DCM solution and stirring for 1 hour, and then evaporating the solvent.

[0025] 3. Preparation of the intermediate 3

[0026] The intermediate 2 was dissolved in ethyl acetate, and the filtrate was washed with saturated brine and 10% sodium carbonate solution alternately for 6 times. The intermediate 3 was obtained by evaporating the filtrate.

[0027] 4. Preparation of the intermediate 4

[0028] The intermediate 3 was dissolved in DCM, and 1 mol of bromoacetic acid and 1 mol of DIC were added. The reaction was stirred for 6 hours, and the solvent was evaporated. The residue was dissolved in DCM and evaporated again. The above operation was repeated 3 times to obtain the intermediate 4.

[0029] 5. Preparation of the intermediate 5

[0030] The intermediate 4 was dissolved in 50% aqueous tetrahydrofuran solution, and 1 mol of cysteine was added with stirring. Sodium carbonate solution was added to maintain the pH at 8.0, and the reaction was stirred for 3 hours. The pH was adjusted to 4.0 with 10% hydrochloric acid solution, and the solvent was evaporated. The residue was dissolved in ethyl acetate and washed with brine 6 times. The intermediate 5 was obtained by evaporating the solvent.

[0031] 6. Preparation of the compound 1

[0032] The intermediate 5 was dissolved in 50% aqueous tetrahydrofuran solution, and 1 mol of NaHCO3 aqueous solution was added. 1 mol of Fmoc-OSu was added with stirring, and the reaction was stirred at room temperature for 6 hours. The pH was adjusted to 3.5 with 1% brine, and the crude product was filtered. The compound 1 was recrystallized from n-hexane-ethyl acetate.

[0033] Preparation of other compounds in Example 2

[0034] The following compounds were prepared by the above method:

[0035]

[0036] Preparation of the cyclic peptide AOD243907 in Example 3

[0037]

[0038] The preparation method comprises the following steps: preparing a peptide resin by solid-phase polypeptide synthesis, obtaining a crude product by acidolysis of the peptide resin, and finally purifying the crude product to obtain a pure product.

[0039] 1. Synthesis of the peptide resin

[0040] The peptide resin is prepared by coupling the protected amino acids corresponding to the polypeptide sequence in turn using Rink Amide BHHA resin as the carrier resin through de-Fmoc protection and coupling reaction.

[0041] (1) Accessing the first protected amino acid of the main chain

[0042] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the protected amino acid DMF solution under stirring, and stir for 30 minutes at room temperature to obtain an activated protected amino acid solution, which is ready for use.

[0043] Take 0.01 mol of Rink amide MBHA resin (substitution value about 0.4 mmol / g), and deprotect it using 20% PIP / DMF solution for 25 minutes, and then wash and filter to obtain the de-Fmoc resin.

[0044] Add the activated first protected amino acid solution to the de-Fmoc resin, and couple for 60-300 minutes, and then filter and wash to obtain the resin containing one protected amino acid.

[0045] (2) Accessing the protected amino acid of the main chain

[0046] Access the corresponding protected amino acid of the polypeptide sequence in turn using the same method as described above for accessing the first protected amino acid of the main chain to obtain the resin containing the amino acid of the main chain.

[0047] The protected amino acid corresponding to the first Tyr is Boc-Tyr(tBu).

[0048] The protected amino acid corresponding to the 16th Cys is Fmoc-Cys(S-allyl acetic acid).

[0049] The protected amino acid corresponding to the 20th Cys is Compound 1.

[0050] (3) Cyclization

[0051] Take 5 mmol of tetrakis(triphenylphosphine)palladium and 50 mmol of phenylsilane, and dissolve them in an appropriate amount of dichloromethane, and then remove the Alloc and All protecting groups from the resin containing the amino acid of the main chain for 8 hours, and then filter and wash to obtain the resin with removed Alloc and All, which is ready for use.

[0052] Take 0.03 mol of HOBt, and dissolve it in an appropriate amount of DMF; take another 0.03 mol of DIC, and dissolve it in an appropriate amount of DMF; add it to the resin with removed Alloc and All under stirring, and couple for 60-300 minutes, and then filter and wash to obtain the cyclized resin.

[0053] (4) Access side chain protection amino acid or single protection fatty acid

[0054] The above cyclized resin is deprotected with 2% hydrazine hydrate / DMF solution for 10 minutes, and the deprotection is repeated twice. The Dde-deprotected resin is obtained by washing and filtering.

[0055] The first protected amino acid of the above access main chain is sequentially connected with the corresponding protected amino acid and single protected fatty acid of the side chain by the same method to obtain a peptide resin.

[0056] 2. Preparation of crude product

[0057] The above peptide resin is added with a cleavage reagent (cleavage reagent 10 mL / g resin) of TFA: water: EDT = 95:5:5 in volume ratio, stirred uniformly, and stirred at room temperature for 3 hours. The reaction mixture is filtered using a sand core funnel, the filtrate is collected, the resin is washed with a small amount of TFA for 3 times, the filtrates are combined, and then concentrated under reduced pressure. Anhydrous ether is added for precipitation, and the precipitate is washed with anhydrous ether for 3 times. The white powder obtained after drying is the crude product.

[0058] 3. Preparation of pure product

[0059] The above crude product is added with water, stirred, and adjusted to pH 8.0 with ammonia water until completely dissolved. The solution is filtered using a 0.45 μm mixed microporous filter membrane, and purified for standby use.

[0060] The purified intermediate is concentrated and filtered using a 0.45 μm filter membrane for standby use. Salt exchange is performed by high performance liquid chromatography. The mobile phase system is 1% acetic acid / water solution-acetonitrile, the chromatographic packing material for purification is 10 μm reversed-phase C18, the flow rate of the chromatographic column of 30 mm*250 mm is 20 mL / min (the flow rate can be adjusted according to different specifications of the chromatographic column), gradient elution is adopted, and the sample is loaded into the chromatographic column by circulation. The mobile phase is started to elute, the change of absorbance is observed by collecting the chromatogram, the main peak of salt exchange is collected and detected by analytical liquid phase, the main peak solution of salt exchange is combined, concentrated under reduced pressure, and the pure product acetic acid aqueous solution is obtained. The pure peptide is obtained by freeze-drying.

[0061] The purified intermediate is concentrated and filtered using a 0.45 μm filter membrane for standby use. Salt exchange is performed by high performance liquid chromatography. The mobile phase system is 1% acetic acid / water solution-acetonitrile, the chromatographic packing material for purification is 10 μm reversed-phase C18, the flow rate of the chromatographic column of 30 mm*250 mm is 20 mL / min (the flow rate can be adjusted according to different specifications of the chromatographic column), gradient elution is adopted, and the sample is loaded into the chromatographic column by circulation. The mobile phase is started to elute, the change of absorbance is observed by collecting the chromatogram, the main peak of salt exchange is collected and detected by analytical liquid phase, the main peak solution of salt exchange is combined, concentrated under reduced pressure, and the pure product acetic acid aqueous solution is obtained. The pure peptide is obtained by freeze-drying.

[0062] Example 4: Determination of the activity of cyclic peptide AOD243907 GLP-1

[0063] 1. Determination method

[0064] GLP-1R can activate intracellular adenylyl cyclase pathway, increase cAMP level, and finally lead to insulin production and release under the stimulation of its specific agonist. The stable transfected GLP-1R cell line is stimulated by the test substance, so that the intracellular cAMP level of the cell is rapidly increased. The relative light unit (RLU) after the cell is stimulated by each dose is determined by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity determination method is the commonly used GLP-1 receptor agonist activity detection method at home and abroad.

[0065] The stable GLP-1R-expressing CHO-K1 cell line is stimulated by different concentrations of agonists, and the EC50 of the agonist is calculated by determining the relative light unit of the cell after each dose stimulation. 50

[0066] 2. Determination results

[0067] The determination results are shown in the following table:

[0068] Compound GLP-1 activity

EC 50 (pmol)

[0069] Example 3 Determination of GIP activity of cyclic peptide AOD243907

[0070] 1. Determination method

[0071] GIPR can activate intracellular adenylyl cyclase pathway, increase cAMP level, and finally lead to insulin production and release under the stimulation of its specific agonist. The stable transfected GIPR cell line is stimulated by the test substance, so that the intracellular cAMP level of the cell is rapidly increased. The relative light unit (RLU) after the cell is stimulated by each dose is determined by chemiluminescence method, and then the EC50 of the agonist is calculated. This activity determination method is the commonly used GIP receptor agonist activity detection method at home and abroad.

[0072] The stable GIPR-expressing CHO-K1 cell line is stimulated by different concentrations of agonists, and the EC50 of the agonist is calculated by determining the relative light unit of the cell after each dose stimulation. 50

[0073] 2. Determination results

[0074] The determination results are shown in the following table:

[0075] Compound GIP activity

EC 50 (pmol)

Claims

1. A cysteine derivative having the structural formula I: ###0001### I wherein: ml is an integer from 2 to 10; m2 is an integer from 2 to 10; R is Boc, or is Mtt, or is Dde, or is ivDde, or is absent; and the cysteine in structural formula I is selected from L-cysteine, D-cysteine.

2. The cysteine derivative of claim 1, wherein ml is 2.

3. The cysteine derivative of claim 1, wherein m2 is 2.

4. The cysteine derivative of claim 1, wherein R is Boc.

2. The cysteine derivative according to claim 1, characterized in that, 5. The cysteine derivative of claim 1, wherein R is Mtt.

3. The cysteine derivative according to claim 1 or 2, characterized in that, 6. The cysteine derivative of claim 1, wherein R is Dde.

7. The cysteine derivative of claim 1, wherein R is ivDde.

8. The cysteine derivative of claim 1, wherein the cysteine is L-cysteine.

9. The cysteine derivative of claim 1, wherein the cysteine is D-cysteine.

10. A method for preparing

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