WO2018205401A1 - Preparation method for plecanatide - Google Patents

Abstract

Provided is a preparation method for plecanatide, comprising the following steps: 1) sequentially couple Fmoc-AA-OH on a solid-phase synthetic resin; 2) remove a StBu protecting group on Cys; 3) remove a Cys oxidation ring of the StBu protecting group to obtain a monodisulfide ring-containing plecanatide precursor resin; 4) crack the solid-phase synthetic resin to prepare a monodisulfide ring-containing plecanatide precursor; 5) 3-Cl-Ala at potential 7 and potential 15 in the monodisulfide ring-containing plecanatide precursor form a disulfide bond to form a ring under the action of a vulcanized agent in a liquid phase reaction to obtain plecanatide.

Description

Method for preparing pricana peptide Technical field

The invention relates to the field of medicine, in particular to a method for synthesizing pricanapeptide.

Background technique

Prikanazepine, English name is plecanatide, CAS number: 467426-54-6, is a peptide drug consisting of 16 amino acids, the peptide sequence is shown in formula I:

In the peptide sequence, the cysteine at the 4th position of the N-terminus forms a loop with the 12th cysteine, and the cysteine at the 7th position forms a loop with the cysteine at the 15th position.

On January 19, 2017, the US FDA approved the treatment of pricanaceptin for chronic idiopathic constipation in adult patients. The trade name is Trulance, and the development company is Synergy Pharmaceutical Company of the United States. Prikanazepine is a guanylate cyclase C (GC-C) receptor agonist. Its therapeutic mechanism is similar to that of natriuretic peptide guanosine, which induces liquid secretion into the gastrointestinal tract, thereby increasing gastrointestinal tract. power.

So far, there are several ways to synthesize pricanapeptide.

The preparation method of pricanapeptide was first reported by Shenzhen Hanyu Pharmaceutical (CN103694320A), and a solid phase synthetic linear polypeptide was used, which was then obtained in two steps in a solution. Since the method is carried out in two steps in the solution, the components in the solution are complicated, and the separation and purification are difficult.

Subsequently, Nanjing University of Technology reported another method for synthesizing pricanapeptide (CN104628827A). The Fmoc solid phase synthesis route was used to synthesize linear peptides, and then oxidized on the resin to form two pairs of disulfide bonds. Immediately afterwards, pricanapeptide was obtained. The yield is lower due to the directional oxidation on the solid phase to form two pairs of disulfide bonds.

Summary of the invention

It is an object of the present invention to provide a synthetic method for preparing pricanapeptide. The method is novel, the synthesis condition is mild, the process is simple, and the process is stable.

The synthetic route of the invention is as follows:

In order to solve the above technical problems, an aspect of the present invention provides a method for preparing pricanapeptide, which comprises the following steps:

1) On the solid phase synthetic resin, Fmoc-AA-OH is sequentially coupled, in order, 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 which removes the StBu protecting group into a ring to obtain a Pricana precursor resin containing a monodisulfide ring;

4) dissolving the solid phase synthetic resin to prepare a pricana precursor containing a monodisulfide ring;

5) In the liquid phase reaction, 3-Cl-Ala at the 7th and 15th positions of the Pricana precursor containing a monodisulfide ring forms a disulfide bond under the action of a vulcanizing agent to form a plexus. That peptide.

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

In the technical solution of the present invention, in step 1),

The method of coupling the 16th position Fmoc-Leu-OH is

The Fmoc-Leu-OH and the coupling agent are dissolved and activated in a solvent, and then added to the solid phase synthetic resin. After the reaction is completed, the resin is blocked to obtain a Fmoc-Leu-solid phase synthetic resin;

The method of coupling Fmoc-AA-OH other than the 16th Fmoc-Leu-OH is

i) removing the Fmoc protecting group of the Fmoc-AA-solid phase synthetic resin to completion;

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

Preferably, the reagent for removing the Fmoc protecting group in step i) is a 20% piperidine/DMF solution, preferably a mixed solution of piperidine:DMF (volume ratio) of 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 in a molar ratio: A = 1:1 - 1.5 or DIPEA: A: B = 1: 0.5 - 1: 0.5-1.

In the technical solution of the present invention, the deprotecting agent for removing the protecting group StBu in the step 2) is tributyl. a mixture of two or more of phosphine, mercaptoethanol, mercaptoethanol and N-methylmorpholine, preferably a mixture of mercaptoethanol and N-methylmorpholine, more preferably a volume ratio of mercaptoethanol to N-methylmorpholine 5:1-5;

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

The reaction temperature in the step 2) is from 0 ° C to 100 ° C, preferably from 40 ° C to 60 ° C.

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

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

In the technical solution of the present 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 solution of the present invention, in step 5), the ratio of the vulcanizing agent to the pricana precursor containing a monodisulfide ring is 1:1 to 1:10; preferably, the ratio of the two is 1:2. ;

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

In the step 5), the reaction temperature is from 25 ° C to 100 ° C, preferably at a temperature of 60 ° C.

In the technical solution of the present invention, after the step 5), the purification step 6) is further included,

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

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

Another aspect of the present invention provides a method for preparing pricanapeptide, comprising the steps of:

1) Add DMF to Wang resin with 1.0mmol/g substitution, weigh Fmoc-Leu-OH, HOBt and DMAP and dissolve in DMF, add DIC activation at 0 °C, add to Wang resin reaction column; reaction to complete The mixture was sealed with acetic anhydride and pyridine, and washed with DCM to obtain Fmoc-Leu-Wang resin;

Fmoc-AA-OH was sequentially coupled, and Fmoc-AA-OH was 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 method of coupling Fmoc-AA-OH is to remove the Fmoc protecting group with 20% piperidine/DMF solution, and weigh Fmoc-AA-OH and HOBT with DMF, add DIPCDI to activate and add solid phase synthetic resin reaction column. In the reaction, to complete;

2) adding mercaptoethanol and N-methylmorpholine to the peptide resin obtained in step 1), removing the StBu protecting group on Cys from 60 ° C to 80 ° C, and the reaction is complete;

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

4) The product obtained in step 3) was reacted to complete with TFA:H ₂ O:PhOMe: thioanisole = 90:5:4:1 (V:V) at room temperature, the resin was removed, and the mixture was precipitated with ice diethyl ether and collected. precipitation;

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

6) Using reversed-phase octadecylsilane as the stationary phase, with a volume ratio of 0.1% aqueous acetic acid/acetonitrile as mobile phase, the ratio is 70:30; prepared by gradient elution; flow rate: 70-80 ml/min; detection wavelength: 280 nm; the peak fraction of interest was collected, concentrated and lyophilized to obtain pricanapeptide.

In the technical solution of the present invention, the detection method applied in the reaction is any method known in the art to achieve this purpose, such as chromatography or chemical calibration, preferably using an agent capable of determining the end point of the reaction, preferably Ketones, when ninhydrin is used, if the resin develops color, it means that there is a free amine in the polypeptide, that is, there is no protecting group on the amine.

Beneficial effect

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

detailed description

Example 1: Preparation of 0.50 mmol Fmoc-Leu-Wang Resin

100 g of Wang resin with 1.0 mmol/g substitution was weighed into a solid phase reaction column, DMF was added, and nitrogen was bubbled for 60 minutes; Fmoc-Leu-OH 35.4 g (100 mmol) and HOBt 16.2 g (120 mmol) were weighed. , DMAP 1.2 g (10 mmol), dissolved in DMF, added 20.3 mL DIC at 0 ° C, Activate for 5 minutes and add to the reaction column. After reacting for two hours, 70 mL of acetic anhydride and 60 mL of pyridine were added, and the mixture was sealed for 24 hours, washed twice with DCM, and the resin was dried, and the resin was evaporated to obtain 130 g of Fmoc-Leu-Wang resin, and the detection degree of substitution was 0.50 mmol/g.

Example 2: Preparation of peptide resin

50 g of Fmoc-Leu-Wang resin with 0.50 mmol/g substitution prepared in Example 1 was weighed into a solid phase reaction column, 50 mL of DMF was added, and nitrogen was bubbled and swollen for 60 minutes; then deprotected with 50 mL of DBLK for 6 min + 8 min, 100 mL of DMF. Wash 6 times. 30.0 g (75 mmol) of Fmoc-3-Cl-Ala-OH and 11.7 g (75 mmol) of HOBT were weighed and dissolved in 50 mL of DMF, and after adding 13 mL (75 mmol) of DIPCDI for 3 min in an ice water bath, the mixture was added to the reaction column. The reaction was carried out for 2 hours at room temperature, and the reaction end point was detected with ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin developed color, the reaction was prolonged for 1 hour). At the end of the reaction, the resin was washed 3 times with 100 mL of DMF, deprotected by 50 ml of LDBLK for 6 min + 8 min, and washed with 100 mL of DMF for 6 times. The ninhydrin detection resin was colored. The above coupling operation was repeated, and Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(StBu)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc were sequentially coupled in sequence according to the peptide sequence. -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. Each amino acid, HOBT and DIC were each fed 75 mmol. After the coupling was completed, the resin was shrunk with 100 mL of methanol, and dried to obtain 100.5 g of a peptide resin.

Example 3: Peptide resin removal protecting group StBu

100.5 g of the peptide resin obtained in Example 2 was successively added with 500 ml of DMF, 50 mL of mercaptoethanol, and 30 mL of N-methylmorpholine, and reacted at 50 ° C for 18 hours. After completion of the reaction, the solution was drained under reduced pressure, and the resin was washed three times with 500 mL of DMF.

Example 4: Preparation of pricanapeptide precursor resin containing monodisulfide ring

The resin obtained in Example 3 was added to 500 mL of DMF, and then 40 mmol of NCS was added. After reacting for half an hour, the solution was removed, washed three times with DMF 500 mL, and 500 mL of methanol was added for 30 minutes, and methanol was removed, and dried under vacuum to obtain 91.5 g of a resin.

Example 5: Preparation of intermediate monodithiocyclic peptide

91.5 g of the resin obtained in Example 4 was added to a 1 L three-necked flask, and pre-configured TFA:H ₂ O:PhOMe: thioanisole = 90:5:4:1 (V:V) 900 mL, and reacted at room temperature. After 2 hours, the resin was filtered under reduced pressure, and the filtrate was collected. The resin was washed with a small amount of TFA and the filtrate was combined. The filtrate was slowly added to 10 L of ice diethyl ether to precipitate, centrifuged, washed 5 times with iced diethyl ether 5L, and dried under reduced pressure to give 75.3 g of crude peptide.

Example 6: Preparation of pricanatin peptide crude peptide

75.3 g of the crude intermediate peptide obtained in Example 5 was placed in a 1000 ml reaction flask, dissolved in 700 ml of a mixed solution of acetonitrile and water in a volume ratio of 5:1, and then 10.0 g of sodium thiosulfate pentahydrate was added. The mixture was heated to 60 ° C for reaction. After completion of the reaction, the solvent was evaporated under reduced pressure to give 87.2 g.

Example 7: Preparation of pricanapeptide

Example 6 was obtained by directly loading 87.2 g of a solid sample into a 10 cm x 25 cm preparative column for high-performance liquid phase purification. The reverse phase octadecylsilane was used as the stationary phase, and the volume ratio was 0.1% acetic acid aqueous solution/acetonitrile as the mobile phase, the ratio was 70:30; the gradient elution preparation; the flow rate: 70-80 ml/min; the detection wavelength: 280 nm; The peak fraction of the target was collected, concentrated and lyophilized to obtain a pure product of 25.2 g, a purity of 99.5%, and a yield of 60%.

Abbreviation and English	meaning
HOAt	1-hydroxy-7-azobenzotriazole
Fmoc	9-fluorenylmethoxycarbonyl
DIPCDI	Diisopropylcarbodiimide
HOBt	1-hydroxybenzotriazole
HATU	2-(7-Azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
HBTU	Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
DIPEA	N,N-diisopropylethylamine
EDC·HCl	1-ethyl-(3-dimethylaminopropyl)carbonyldiimide hydrochloride
PyBOP	Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate
PyAOP	(3H-1,2,3-triazolo[4,5-b]pyridin-3-yloxy)tri-1-pyrrolidinium hexafluorophosphate.
TBTU	O-benzotriazole-N,N,N',N'-tetramethylureatetrafluoroboric acid
DMF	N,N-dimethylformamide
DCM	Dichloromethane
THF	Tetrahydrofuran
TFE	Trifluoroethanol
TFA	Trifluoroacetate
TA	Benzoyl sulfide

PhOMe	Anisole
EDT	Ethylenedithiol
DMAP	4,4-dimethylaminopyridine
DBLK	20% piperidine/DMF (V/V) solution
tBu	Tert-butyl
NMP	N-methylpyrrolidone
NCS	N-chlorosuccinimide
Cla	L-3-chloroalanine L-3-Cl-Ala

Claims (10)

1. A method for preparing pricanapeptide, comprising the steps of:

   1) On the solid phase synthetic resin, Fmoc-AA-OH is sequentially coupled, and 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, to obtain a linear peptide-solid phase synthetic resin;

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

   3) oxidizing Cys which removes the StBu protecting group into a ring to obtain a Pricana precursor resin containing a monodisulfide ring;

   4) dissolving the solid phase synthetic resin to prepare a pricana precursor containing a monodisulfide ring;

   5) In the liquid phase reaction, 3-Cl-Ala at the 7th and 15th positions of the Pricana precursor containing a monodisulfide ring forms a disulfide bond under the action of a vulcanizing agent to form a plexus. That peptide.
2. The production method according to claim 1, wherein in the step 1), the solid phase synthetic resin is wang resin or 2-chloro resin, and the degree of substitution of the resin is from 0.5 to 2.0 mmol/g, preferably from 0.8 to 1.2 mmol/g.
3. The preparation method according to claim 1, wherein in the step 1),

   The method of coupling the 16th position Fmoc-Leu-OH is

   The Fmoc-Leu-OH and the coupling agent are dissolved and activated in a solvent, and then added to the solid phase synthetic resin. After the reaction is completed, the resin is blocked to obtain a Fmoc-Leu-solid phase synthetic resin;

   The method of coupling Fmoc-AA-OH other than the 16th Fmoc-Leu-OH is

   i) removing the Fmoc protecting group of the Fmoc-AA-solid phase synthetic resin to completion;

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

   Preferably, the reagent for removing the Fmoc protecting group in step i) is a 20% piperidine/DMF solution.

   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 in a molar ratio: A = 1:1 - 1.5 or DIPEA: A: B = 1: 0.5 - 1: 0.5-1.
4. The preparation method according to claim 1, wherein the deprotecting agent for removing the protecting group StBu in the step 2) is a mixture of two or more of tributylphosphine, mercaptoethanol, mercaptoethanol and N-methylmorpholine. a mixture of mercaptoethanol and N-methylmorpholine, more preferably a volume ratio of mercaptoethanol to N-methylmorpholine of 5:1-5;

   The solvent used is selected from the group consisting of DMF, NMP, dichloromethane, preferably DMF;

   The reaction temperature is from 0 ° C to 100 ° C, preferably from 40 ° C to 60 ° C.
5. The preparation method according to claim 1, wherein in the step 3), the oxidizing agent is selected from one or more of H ₂ O ₂ and NCS, preferably NCS; the solvent used is selected from the group consisting of DMF, NMP, and dichloromethane. One or more, preferably DMF.
6. The preparation method according to claim 1, wherein in the step 4), the lysate is a composition of TFA, H ₂ O, PhOMe, thioanisole; preferably, TFA: H ₂ O: PhOMe: Benzoyl sulfide = 50-120: 1-10: 1-10: 0.1-5; more preferably 80-110: 4-6: 3-5: 0.5-1.5.
7. The production method according to claim 1, wherein in the step 5), the vulcanizing agent is one or more selected from the group consisting of potassium sulfide, potassium thiocyanate, and sodium thiosulfate.
8. The preparation method according to claim 7, wherein in step 5), the ratio of the vulcanizing agent to the pricana precursor containing a monodisulfide ring is from 1:1 to 1:10; preferably, the ratio of the two is proportional Is 1:2;

   The reaction temperature is from 25 ° C to 100 ° C, preferably at a temperature of 60 ° C.
9. The preparation 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;

   Preferably, the reverse phase high pressure liquid chromatography comprises: using a reverse phase octadecylsilane as a stationary phase, a volume ratio of 0.1% aqueous acetic acid/acetonitrile as a mobile phase, and a mobile phase volume ratio of 0.1% aqueous acetic acid/acetonitrile. The ratio is preferably from 98:2 to 50:50, more preferably from 80:20 to 60:40, most preferably 70:30.
10. The preparation method according to claim 1, comprising the steps of:

    1) Add DMF to Wang resin with 1.0mmol/g substitution, weigh Fmoc-Leu-OH, HOBt and DMAP and dissolve in DMF, add DIC activation at 0 °C, add to Wang resin reaction column; reaction to complete The mixture was sealed with acetic anhydride and pyridine, and washed with DCM to obtain Fmoc-Leu-Wang resin;

    Fmoc-AA-OH was sequentially coupled, and Fmoc-AA-OH was 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 method of coupling Fmoc-AA-OH is to remove the Fmoc protecting group with 20% piperidine/DMF solution, and weigh Fmoc-AA-OH and HOBT with DMF, add DIPCDI to activate and add solid phase synthetic resin reaction column. In the reaction, to complete;

    2) adding mercaptoethanol and N-methylmorpholine to the peptide resin obtained in step 1), removing the StBu protecting group on Cys from 60 ° C to 80 ° C, and the reaction is complete;

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

    4) The product obtained in step 3) was reacted to complete with TFA:H ₂ O:PhOMe: thioanisole = 90:5:4:1 (V:V) at room temperature, the resin was removed, and the mixture was precipitated with ice diethyl ether and collected. precipitation;

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

    6) Using reversed-phase octadecylsilane as the stationary phase, with a volume ratio of 0.1% aqueous acetic acid/acetonitrile as mobile phase, the ratio is 70:30; prepared by gradient elution; flow rate: 70-80 ml/min; detection wavelength: 280 nm; the peak fraction of interest was collected, concentrated and lyophilized to obtain pricanapeptide.