CN108864254B - Preparation method of polycaprolactam - Google Patents

Abstract

The invention relates to a preparation method of polycaprolactam, which comprises the following steps: 1) sequentially coupling Fmoc-AA-OH on solid-phase synthetic resin; 2) removing the StBu protecting group on Cys; 3) cys of the removed StBu protective group is oxidized into ring to obtain the prikana precursor resin containing the monothiocyclic; 4) cracking the solid phase synthetic resin to prepare a mono-dithiocyclo-containing prikana precursor; 5) in the liquid phase reaction, the 7 th and 15 th 3-Cl-Ala in the prikan precursor containing the single disulfide ring forms disulfide bond under the action of a vulcanizing agent to form ring, and the prikan peptide is obtained.

Description

Preparation method of polycaprolactam

Technical Field

The invention relates to the field of medicines, in particular to a synthetic method of polycaprolactam.

Background

Polycaprolactam, british name plecanatide, CAS number: 467426-54-6, is a polypeptide drug composed of 16 amino acids, and the peptide sequence is shown in formula I:

in the peptide sequence, the 4 th cysteine and the 12 th cysteine at the N-terminal form a ring, and the 7 th cysteine and the 15 th cysteine form a ring.

The FDA approved polycaprolactam for the treatment of chronic idiopathic constipation in adult patients in 2017, month 1, 19, under the trade name Trulance, and the research company is the Synergy pharmaceutical company in the united states. Polycaprolactam is a guanylate cyclase C (GC-C) receptor agonist that has a therapeutic mechanism similar to the natriuretic peptide guanylin, and induces secretion of fluids into the gastrointestinal tract, thereby increasing gastrointestinal motility.

To date, there are several methods for the synthesis of polycaprolactam.

The preparation method of polycaprolactam is firstly reported by Shenzhen Hanyu pharmaceutical industry (CN103694320A), and the polycaprolactam is obtained by synthesizing linear polypeptide by adopting a solid phase and then performing two steps of cyclization respectively in a solution. Because the cyclization is carried out in the solution twice, the components in the solution are complex, and the separation and purification difficulty is higher.

Subsequently, Nanjing university of industry reported another method for synthesizing polycaprolactam (CN104628827A), which employs Fmoc solid phase synthesis route, first synthesizing linear peptide, then forming two pairs of disulfide bonds by directed oxidation on resin, and finally obtaining polycaprolactam directly after cleavage and purification. The yield is low due to the formation of two disulfide bonds by directed oxidation on the solid phase.

Disclosure of Invention

The invention aims to provide a synthetic method for preparing polycaprolactam. The method is novel, mild in synthesis condition, simple in process and stable in process.

In order to solve the above technical problems, one aspect of the present invention provides a preparation method of polycaprolactam, which comprises the following steps:

1) coupling Fmoc-AA-OH on a solid-phase synthetic resin in sequence, wherein the Fmoc-AA-OH is Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH;

2) removing the StBu protecting group on Cys;

3) oxidizing Cys without StBu protecting group into ring to obtain prikana precursor resin containing monothiocyclic;

4) cracking the solid phase synthetic resin to prepare a mono-dithiocyclo-containing prikana precursor;

5) in the liquid phase reaction, the 7 th and 15 th 3-Cl-Ala in the prikan precursor containing the single disulfide ring forms disulfide bond under the action of a vulcanizing agent to form ring, and the prikan peptide is obtained.

In the technical scheme of the invention, in the step 1), the solid-phase synthetic resin is wang resin or 2-chloro resin, and the resin substitution degree is 0.5-2.0mmol/g, preferably 0.8-1.2 mmol/g.

In the technical scheme of the invention, in the step 1),

the method for coupling the 16 th Fmoc-Leu-OH comprises the following steps,

dissolving Fmoc-Leu-OH and a coupling agent in a solvent, activating, adding the mixture into solid-phase synthetic resin, and sealing the resin after complete reaction to obtain Fmoc-Leu-solid-phase synthetic resin;

the coupling method of Fmoc-AA-OH except the 16 th Fmoc-Leu-OH comprises the following steps,

i) removing the Fmoc protecting group of the Fmoc-AA-solid phase synthetic resin until the Fmoc protecting group is complete;

ii) dissolving Fmoc-AA-OH and a coupling agent in a solvent, activating, and adding into a solid phase reaction column until the reaction is complete;

preferably, the Fmoc protecting group removing reagent in step i) is a 20% piperidine/DMF solution, preferably piperidine: DMF (volume ratio) is 1: 4.

Preferably, the coupling agent in step 1) is a combination of compound A, DIPCDI and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is one or more of HOBt or HOAt, compound B is one or more of PyBOP, PyAOP, HATU, HBTU or TBTU,

more preferably, the ratio of each component in the coupling agent is DIPCDI: a: 1-1.5 or DIPEA: a: B: 1:0.5-1:0.5-1 in terms of molar ratio.

In the technical scheme of the invention, the deprotection agent for removing the protecting group StBu in the step 2) is a mixed solution of more than two of tributylphosphine, mercaptoethanol and N-methylmorpholine, preferably a mixed solution of mercaptoethanol and N-methylmorpholine, more preferably a volume ratio of mercaptoethanol to N-methylmorpholine of 5: 1-5;

the solvent used in step 2) is selected from DMF, NMP, dichloromethane, preferably DMF;

the reaction temperature in the step 2) is 0-100 ℃, preferably 40-60 ℃.

In the technical scheme of the invention, in the step 3), the oxidizing agent is selected from one or more of H2O2 and NCS, preferably NCS; the solvent is one or more selected from DMF, NMP and dichloromethane, preferably DMF.

In the technical scheme of the invention, in the step 4), the lysis solution is a composition of TFA, H2O, PhOMe and thioanisole; preferably, TFA: H2O: PhOMe: benzylsulfide ═ 50-120: 1-10: 1-10: 0.1 to 5; more preferably 80 to 110: 4-6: 3-5: 0.5-1.5.

In the technical scheme of the invention, in the step 5), the vulcanizing agent is selected from one or more of potassium sulfide, potassium thiocyanate and sodium thiosulfate.

In the technical scheme of the invention, in the step 5), the ratio of the vulcanizing agent to the mono-disulfide-ring-containing polycalathin precursor is 1:1-1: 10; preferably, the ratio of the two is 1: 2;

in the step 5), the solvent is a polar solvent, and comprises methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, 1, 4-dioxane, ethylene glycol or a mixture of the polar solvent and water in different proportions; preferably a 5:1 mixture of acetonitrile and water;

in step 5), the reaction temperature is 25 ℃ to 100 ℃, and the preferred temperature is 60 ℃.

In the technical scheme of the invention, a purification step 6) is also included after the step 5),

6) the purification step is reversed phase high pressure liquid chromatography;

preferably, the reversed-phase high-pressure liquid chromatography comprises: the reversed-phase octadecylsilane is used as a stationary phase, 0.1% aqueous acetic acid solution/acetonitrile in volume ratio is used as a mobile phase, and the ratio of the 0.1% aqueous acetic acid solution/acetonitrile in volume ratio of the mobile phase is preferably 98:2 to 50:50, more preferably 80:20 to 60:40, and most preferably 70: 30.

In another aspect, the present invention provides a method for preparing a polycaprolactam, comprising the steps of:

1) adding DMF into Wang resin with substitution degree of 1.0mmol/g, weighing Fmoc-Leu-OH, HOBt and DMAP, dissolving with DMF, adding DIC at 0 deg.C for activation, and adding into Wang resin reaction column; reacting completely, adding acetic anhydride and pyridine, mixing and sealing, and washing with DCM to obtain Fmoc-Leu-Wang resin;

coupling Fmoc-AA-OH sequentially in the order Fmoc-AA-OH being Fmoc-Gly-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH;

the Fmoc-AA-OH coupling method comprises the steps of removing an Fmoc protecting group by using 20% piperidine/DMF solution, weighing the Fmoc-AA-OH and HOBT, dissolving the Fmoc-AA-OH and the HOBT with DMF, adding DIPCDI (Dip-Chirp) for activation, adding the mixture into a solid-phase synthetic resin reaction column, and reacting completely;

2) adding mercaptoethanol and N-methylmorpholine into the peptide resin obtained in the step 1), removing the StBu protecting group on Cys at the temperature of 60-80 ℃, and reacting completely;

3) adding NCS into the product obtained in the step 2) until the reaction is complete;

4) the ratio of TFA: h₂O: PhOMe: benzylsulfide ═ 90:5:4:1(V: V) and the product obtained in the step 3) react at room temperature until the reaction is complete, the resin is removed, the precipitate is precipitated by ethyl acetate, and the precipitate is collected;

5) vulcanizing the product obtained in the step 4) by sodium thiosulfate pentahydrate at 50-70 ℃ until the reaction is complete;

6) taking reverse octadecylsilane as a stationary phase, and taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70: 30; preparing by isocratic elution; flow rate: 70-80 ml/min; detection wavelength: 280 nm; collecting the target peak fraction, concentrating and freeze-drying to obtain the polycaprolactam.

In the technical scheme of the invention, the detection method applied in the reaction is any method known in the art for achieving the purpose, such as a chromatography method or a chemical calibration method, a reagent capable of judging the reaction end point, preferably ninhydrin, is preferably used, and when ninhydrin is used, if the resin is developed, the polypeptide has free amine, namely, no protective group is arranged on the amine.

Advantageous effects

The preparation method has the advantages of simple operation, simplified process, environmental protection, high economic benefit, large-scale production and the like.

Drawings

FIG. 1 is a composite circuit diagram of the present invention.

Detailed Description

Example 1: preparation of Fmoc-Leu-Wang resin with substitution degree of 0.50mmol

Weighing 100g of Wang resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; 35.4 g (100mmol) of Fmoc-Leu-OH, 1.2 g (120mmol) of HOBt16.2 and 1.2 g (10mmol) of DMAP were weighed, dissolved in DMF, and 20.3mL of DI was added at 0 ℃ to activate for 5 minutes and then the mixture was loaded onto a reaction column. After reacting for two hours, adding 70mL of acetic anhydride and 60mL of pyridine, mixing and sealing for 24 hours, washing with DCM for three times, shrinking with methanol, and then draining the resin to obtain 130 g of Fmoc-Leu-Wang resin, wherein the detection substitution degree is 0.50 mmol/g.

Example 2: preparation of peptide resins

Weighing 50g of Fmoc-Leu-Wang resin with the substitution degree of 0.50mmol/g prepared in example 1 into a solid phase reaction column, adding 50ml DMF, and carrying out nitrogen bubbling and swelling for 60 minutes; then deprotected with 50mL DBLK for 6min +8min, washed 6 times with 100mL DMMF. 30.0g (75mmol) of Fmoc-3-Cl-Ala-OH and 11.7g (75mmol) of HOBT are weighed and dissolved in 50mL of DMF, 13mL (75mmol) of DIPCDI is added in an ice water bath for activation for 3min, the mixture is added into a reaction column and reacted for 2 h at room temperature, and the reaction endpoint is detected by ninhydrin (the reaction is stopped if the resin is colorless and transparent; the reaction is prolonged for 1 h if the resin is colored). After the reaction is finished, washing the resin for 3 times by using 100mLDMF, adding 50mLDBLK for deprotection for 6min +8min, washing the resin for 6 times by using 100mLDMF, and detecting the color of the resin by ninhydrin. The above coupling procedure was repeated to successively couple Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn Trt-OH in the order of peptide order. Each amino acid, HOBT and DIC was dosed with 75mmol of each. After the coupling was complete, the resin was shrunk with 100mL of methanol and suction dried to give 100.5 g of peptide resin.

Example 3: StBu for removing protective group of peptide resin

To 100.5 g of the peptide resin obtained in example 2 were added 500mL of DMF, 50mL of mercaptoethanol, and 30mL of methyl morpholine in this order, and the reaction was carried out at 50 ℃ for 18 hours. After the reaction was complete, the solution was drained under reduced pressure and the resin was washed 3 times with 500ml of dmf and directly to the next step without further treatment.

Example 4: preparation of Monodithiocyclo-containing Pricaritin precursor resin

To the resin obtained in example 3 was added 500mL of DMF followed by 40mmol of NCS, and after half an hour of reaction, the solution was taken out, washed three times with DMF500mL, and then 500mL of methanol was added to shrink for 30 minutes, and the methanol was taken out and dried in vacuo to give 91.5g of resin.

Example 5: preparation of intermediate mono-dithiocyclopeptide

91.5g of the resin obtained in example 4 was charged into a 1L three-necked flask, and a previously prepared TFA: H was added₂PhOMe-thioanisole-900 mL at room temperature for 2 hours, filtering the resin under reduced pressure, and collecting the filtrate. The resin was washed with a small amount of TFA and the filtrates combined. The filtrate was slowly added to 10L of ethyl acetate for precipitation, centrifuged, washed 5 times with 5L of ethyl acetate and dried under reduced pressure to give 75.3 g of crude peptide.

Example 6: preparation of crude Pricarpide

75.3 g of the intermediate crude peptide obtained in example 5 was put into a 1000ml reaction flask, and 700ml of a mixed solution of acetonitrile and water in a volume ratio of 5:1 was added to dissolve the intermediate crude peptide, followed by addition of 10.0g of sodium thiosulfate pentahydrate. The mixture was heated to 60 ℃ for reaction. After completion of the reaction, the solvent was distilled off under reduced pressure to obtain 87.2g of a solid.

Example 7: preparation of Polycanatide

87.2g of the solid obtained in example 6 was directly loaded on a 10 cm. times.25 cm preparative column for high performance liquid purification. Taking reverse octadecylsilane as a stationary phase, and taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70: 30; preparing by isocratic elution; flow rate: 70-80 ml/min; detection wavelength: 280 nm; collecting the target peak fraction, concentrating, and lyophilizing to obtain 25.2g of pure product with purity of 99.5% and yield of 60%.

Claims (27)

1. A process for preparing a polycaprolactam comprising the steps of:

1) Fmoc-AA-OH is sequentially coupled on solid-phase synthetic resin, and is Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH in sequence, Fmoc-Val-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH to obtain a linear peptide-solid phase synthetic resin;

2) removing the StBu protecting group on Cys in the linear peptide-solid phase synthetic resin;

3) oxidizing Cys without StBu protecting group into ring to obtain prikana precursor resin containing monothiocyclic;

4) cracking the solid phase synthetic resin to prepare a mono-dithiocyclo-containing prikana precursor;

5) in the liquid phase reaction, the 7 th and 15 th 3-Cl-Ala in the prikan precursor containing the single disulfide ring forms disulfide bond under the action of a vulcanizing agent to form ring, and the prikan peptide is obtained.

2. The preparation method according to claim 1, wherein, in the step 1), the solid phase synthesis resin is wang resin or 2-chloro resin, and the resin substitution degree is 0.5 to 2.0 mmol/g.

3. The production method according to claim 2, wherein the resin substitution degree is 0.8 to 1.2 mmol/g.

4. The production method according to claim 1, wherein, in step 1),

the method for coupling the 16 th Fmoc-Leu-OH comprises the following steps,

dissolving Fmoc-Leu-OH and a coupling agent in a solvent, activating, adding the mixture into solid-phase synthetic resin, and sealing the resin after complete reaction to obtain Fmoc-Leu-solid-phase synthetic resin;

the coupling method of Fmoc-AA-OH except the 16 th Fmoc-Leu-OH comprises the following steps,

i) removing the Fmoc protecting group of the Fmoc-AA-solid phase synthetic resin until the Fmoc protecting group is complete;

ii) dissolving Fmoc-AA-OH and a coupling agent in a solvent, activating, and adding into a solid-phase reaction column until the reaction is complete.

5. The method of claim 4, wherein the Fmoc protecting group removing reagent in step i) is a 20% piperidine/DMF solution.

6. The process of claim 4, wherein the coupling agent in step 1) is a combination of compound A, DIPCDI and compound A or a combination of DIPEA and compound A and compound B, wherein compound A is one or more of HOBt or HOAt, and compound B is one or more of PyBOP, PyAOP, HATU, HBTU or TBTU.

7. The method of claim 6, wherein the coupling agent comprises DIPCDI (DiPCDI) A: 1-1.5 or DIPEA: A: B: 1:0.5-1:0.5-1 in terms of molar ratio.

8. The process according to claim 1, wherein the deprotecting agent for the StBu for removing the protecting group in the step 2) is a mixture of two or more of tributylphosphine, mercaptoethanol and N-methylmorpholine; the solvent is selected from DMF, NMP, and dichloromethane;

the reaction temperature is 0-100 ℃.

9. The process according to claim 8, wherein the deprotecting agent for StBu for removing the protecting group in the step 2) is a mixture of mercaptoethanol and N-methylmorpholine.

10. The method according to claim 9, wherein the volume ratio of mercaptoethanol to N-methylmorpholine is 5: 1-5.

11. The process according to claim 8, wherein the solvent is DMF.

12. The production method according to claim 8, wherein the reaction temperature is 40 ℃ to 60 ℃.

13. The method according to claim 1, wherein in step 3), the oxidizing agent is selected from H₂O₂One or more of NCS; the solvent is one or more selected from DMF, NMP and dichloromethane.

14. The method according to claim 13, wherein in step 3), the oxidizing agent is selected from NCS.

15. The process according to claim 13, wherein the solvent is DMF.

16. The method according to claim 1, wherein the lysis solution is TFA or H in step 4)₂O, PhOMe, thiobenzol sulfide.

17. The method of claim 16, wherein the ratio of TFA: h₂O: PhOMe: benzylsulfide ═ 50-120: 1-10: 1-10: 0.1-5.

18. The method of claim 17, wherein the ratio of TFA: h₂O: PhOMe: 80-110 of benzylthion: 4-6: 3-5: 0.5-1.5.

19. The preparation method according to claim 1, wherein in the step 5), the vulcanizing agent is selected from one or more of potassium sulfide, potassium thiocyanate and sodium thiosulfate.

20. The process according to claim 19, wherein in step 5), the ratio of the vulcanizing agent to the mono-dithiocyclo-containing polycalathin precursor is 1:1 to 1: 10;

the reaction temperature is 25-100 ℃.

21. The method according to claim 20, wherein the ratio of the vulcanizing agent to the mono-dithiocyclo-containing polycalathin precursor in step 5) is 1: 2.

22. The production method according to claim 20, wherein the reaction temperature is 60 ℃.

23. The method according to claim 1, further comprising a purification step 6) after the step 5),

6) the purification step is reversed phase high pressure liquid chromatography.

24. The preparation method of claim 23, wherein the reverse phase high pressure liquid chromatography comprises: taking reverse-phase octadecylsilane as a stationary phase, taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio of the 0.1% acetic acid aqueous solution/acetonitrile in volume ratio of the mobile phase is 98:2 to 50: 50.

25. The production method according to claim 24, wherein the ratio of the mobile phase volume ratio 0.1% aqueous acetic acid solution/acetonitrile is 80:20 to 60: 40.

26. The method according to claim 25, wherein the ratio of the mobile phase volume ratio of 0.1% aqueous acetic acid/acetonitrile is 70: 30.

27. The method of claim 1, comprising the steps of:

1) adding DMF into Wang resin with substitution degree of 1.0mmol/g, weighing Fmoc-Leu-OH, HOBt and DMAP, dissolving with DMF, adding DIC at 0 deg.C for activation, and adding into Wang resin reaction column; reacting completely, adding acetic anhydride and pyridine, mixing and sealing, and washing with DCM to obtain Fmoc-Leu-Wang resin;

coupling Fmoc-AA-OH sequentially in the order Fmoc-AA-OH being Fmoc-Gly-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Val-OH, Fmoc-3-Cl-Ala-OH, Fmoc-Leu-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (StBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asn (Trt) -OH;

the Fmoc-AA-OH coupling method comprises the steps of removing an Fmoc protecting group by using 20% piperidine/DMF solution, weighing the Fmoc-AA-OH and HOBT, dissolving the Fmoc-AA-OH and the HOBT with DMF, adding DIPCDI (Dip-Chirp) for activation, adding the mixture into a solid-phase synthetic resin reaction column, and reacting completely;

2) adding mercaptoethanol and N-methylmorpholine into the peptide resin obtained in the step 1), removing the StBu protecting group on Cys at the temperature of 60-80 ℃, and reacting completely;

3) adding NCS into the product obtained in the step 2) until the reaction is complete;

4) the ratio of TFA: h₂O: PhOMe: benzylsulfide ═ 90:5:4:1(V: V) and the product obtained in the step 3) react at room temperature until the reaction is complete, the resin is removed, the precipitate is precipitated by ethyl acetate, and the precipitate is collected;

5) vulcanizing the product obtained in the step 4) by sodium thiosulfate pentahydrate at 50-70 ℃ until the reaction is complete;

6) taking reverse octadecylsilane as a stationary phase, and taking 0.1% acetic acid aqueous solution/acetonitrile in volume ratio as a mobile phase, wherein the ratio is 70: 30; preparing by isocratic elution; flow rate: 70-80 ml/min; detection wavelength: 280 nm; collecting the target peak fraction, concentrating and freeze-drying to obtain the polycaprolactam.

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