CN112521482A

CN112521482A - Preparation method for synthesizing nesiritide by solid-liquid combination

Info

Publication number: CN112521482A
Application number: CN201910802727.4A
Authority: CN
Inventors: 陈永汉; 尹传龙; 陶安进; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2021-03-19
Anticipated expiration: 2039-08-28
Also published as: WO2021036057A1; CN112521482B

Abstract

The invention relates to a preparation method of nesiritide, which comprises the following steps: 1) synthesizing an intermediate 1 by a solid-phase synthesis method; 2) synthesizing an intermediate 2 by a solid-phase synthesis method, activating a C-terminal carboxyl group of the intermediate 2 to obtain an intermediate 3, removing a side chain protecting group from the intermediate 3 to obtain an intermediate 4, 3), and forming an intermediate 5 of an intermolecular disulfide bond by performing a disulfide bond exchange reaction on the intermediate 1 and the intermediate 4; 4) forming an amido bond by the intermediate 5 under the action of a cyclization promoting reagent to obtain an intermediate 6, and then removing an Fmoc protecting group to obtain a nesiritide crude product; 5) and purifying the nesiritide crude product to obtain the nesiritide. The invention adopts a method of combining liquid phase and solid phase, and has high preparation efficiency and high purity.

Description

Preparation method for synthesizing nesiritide by solid-liquid combination

Technical Field

The invention relates to the field of drug synthesis, in particular to a preparation method for synthesizing nesiritide by solid-liquid combination.

Background

Nesiritide is a medicine for resisting cardiac insufficiency. Is B-type natriuretic peptide synthesized by recombinant DNA technology. By binding to the natriuretic titanium receptors. Nesiritide has definite functions of expanding blood vessels and expelling natriuretic urine. Clinical research shows that the medicine is a new medicine for treating acute heart failure with obvious curative effect, fast acting and less untoward effect, and can improve the hemodynamics and clinical symptoms of heart failure patients.

Nesiritide, having the english name nesiritide, molecular weight of 3464.04, and molecular formula: ser¹-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys¹⁰-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser²⁰-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg³⁰Arg-His (Disulfide Bridge: Cys10-Cys26), which has the following structure:

as for the preparation method of nesiritide, US5114923 prepares nesiritide in a genetic engineering method, which is disadvantageous for mass production;

patent CN 101519444B reports a method for preparing nesiritide by solid-phase stepwise coupling, where the difficulty of Ser-Ser coupling in the peptide sequence is large, resulting in low yield of the final product;

patent CN 103275207B reports the preparation of nesiritide using a method of synthesizing 5 peptide fragments followed by stepwise coupling of the peptide fragments. The method needs to synthesize a plurality of fully-protected peptide fragments and carry out purification preparation, the required steps are more, and the material requirement is larger;

patent CN 104447979B reports the use of H-Arg (Boc)₂-Arg(Boc)₂-his (trt) -OtBu coupled with a fully protected nesiritide 1-29 peptide fragment to prepare nesiritide. The total protection nesiritide 1-29 peptide fragment adopts solid phase I₂Oxidation process, which results inThe fall-off of the fully protected peptide and the formation of other impurities affect the yield of the final product.

Disadvantages of the current nesiritide synthesis:

1. the peptide sequence has Ser with higher coupling difficulty¹⁹-Ser-Ser-Ser²²Fragment, adopting the conventional solid phase gradual coupling synthesis method to hardly avoid the formation of defective Ser impurities or racemic impurities;

2. the solid phase fragment synthesis method can reduce the formation of defective impurities or racemic impurities, but the peptide fragments need multiple times of feeding, so the material cost is very high;

3. by means of a solid phase I₂The oxidation process can cause the falling off of the fully protected peptide and the formation of other impurities, which affect the yield of the final product.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a solid-liquid combined synthesis method for synthesizing nesiritide, which can complete disulfide bond formation and peptide chain assembly, shorten the production time by half, reduce the formation of related defect impurities and racemization impurities, and improve the yield of final products.

One aspect of the present invention provides a method for preparing nesiritide, which comprises the following steps:

1) synthesizing an intermediate 1 by a solid-phase synthesis method, wherein the intermediate 1 is H-Ser²¹-Ser-Gly-Leu-Gly-Cys(Npys)-Lys-Val-Leu-Arg³⁰-Arg-His-OH；

2) Synthesizing an intermediate 2 by a solid-phase synthesis method, activating a C-terminal carboxyl group of the intermediate 2 to obtain an intermediate 3, removing a side chain protecting group from the intermediate 3 to obtain an intermediate 4, wherein the intermediate 2 is Fmoc-Ser (tBu) -Pro-Lys (Boc) -Met-Val-Gln (Trt) -Gly-Ser (tBu) -Gly-Cys (Trt) -Phe-Gly-Arg (Pbf) -Lys (Boc) -Met-Asp (OtBu) -Arg (Pbf) -Ile-Ser (tBu) -OH, and the intermediate 3 is Fmoc-Ser (tBu) -Gly

Intermediate 4 is

3) Intermediate 1 and intermediate 4 form an intermediate 5 of intermolecular disulfide bond through a disulfide bond exchange reaction, wherein the intermediate 5 is

4) The intermediate 5 forms amido bond under the action of a cyclization promoting reagent to obtain an intermediate 6, wherein the intermediate 6 is

Then removing the Fmoc protecting group to obtain a nesiritide crude product;

optionally, 5) purifying the nesiritide crude product to obtain nesiritide.

In the technical scheme of the invention, the cyclization promoting reagent is selected from pyridine/acetic acid solution or triethylamine/acetic acid solution.

In the technical scheme of the invention, the solid-phase synthesis method comprises the steps of coupling amino acid or coupling the amino acid and solid-phase synthesis resin under the action of a coupling agent, and removing Fmoc protecting groups.

In the technical scheme of the invention, the method for synthesizing the intermediate 1 by the solid-phase synthesis method comprises the steps of coupling solid-phase synthesis resin and Fmoc-His (Trt) -OH by a coupling agent, then removing the Fmoc protecting group, coupling amino acid Fmoc-Arg (Pbf) -OH by a coupling agent, then repeating the steps of removing Fmoc protecting group and coupling amino acid, sequentially coupling Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Npys) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH and Boc-Ser (tBu) -OH, the Fmoc protecting group was then removed and cleaved with the first cleavage solution to give intermediate 1 with the Cys side chain protected by Npys. Preferably, the first lysis solution is selected from a mixed solution of TFA, thioanisole, anisole and EDT, and more preferably, the ratio of TFA to thioanisole to EDT is 90:5: 2: 3.

In the technical scheme of the invention, the method for synthesizing the intermediate 2 by the solid-phase synthesis method comprises the steps of coupling solid-phase synthesis resin with Fmoc-Ser (tBu) -OH by a coupling agent, then removing Fmoc protecting groups, coupling amino acids Fmoc-Ser (tBu) -OH by the coupling agent, then repeatedly removing the Fmoc protecting groups, coupling amino acids, sequentially coupling Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, F, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH and Fmoc-Pro-OH, and then cleaved with a second cleavage solution to give intermediate 2. Preferably, the second cracking solution is a mixed solution of TFE and DCM, and more preferably, the ratio of TFE to DCM is 2: 8.

in the technical scheme of the invention, the activating reagent used for activating the C-terminal carboxyl group of the intermediate 2 in the step 2) is a combination of a coupling agent and salicylaldehyde, or a combination of a coupling agent and salicylaldehyde glycol acetal, or a combination of a coupling agent and salicylaldehyde dimethyl acetal. Preferably, the molar ratio of salicylaldehyde or salicylaldehyde glycol acetal or salicylaldehyde dimethyl acetal to intermediate 2 is 5:1 to 15:1, more preferably, 7.5: 1,10:1,12.5:1.

In the technical scheme of the invention, the reagents for removing the side chain protecting group from the intermediate 3 in the step 2) are TFA, TIS and H₂A mixture of O, preferably TFA, TIS and H₂The ratio of O is 80-95:3-10:3-10, more preferably TFA, TIS and H₂The ratio of O is 90:5: 5.

In the technical scheme of the invention, the conditions of the disulfide bond exchange reaction between the intermediate 1 and the intermediate 4 in the step 3) are that the intermediate 1 and the intermediate 4 are put into a solution to react to generate a disulfide bond.

In the technical scheme of the invention, the conditions for forming amide bonds in the step 4) are reaction under the conditions of pyridine and acetic acid solution, and preferably, the ratio of pyridine to acetic acid is 1: 8-15.

In the technical scheme of the invention, the condition for removing the Fmoc protecting group in the step 4) is to remove the Fmoc protecting group by using 10% -30% (20%) diethylamine in acetonitrile aqueous solution.

In the technical scheme of the invention, the reagent for removing the Fmoc protecting group in the step 1) or 2) is DBLK.

In the technical scheme of the invention, the purification method in the step 5) is high performance liquid chromatography purification.

In the technical scheme of the invention, the chromatographic column used by the high performance liquid chromatography is a C18 chromatographic column.

In the technical scheme of the invention, the steps of the high performance liquid chromatography are as follows:

i) loading crude product, gradient eluting with mobile phase A of 0.1% TFA, mobile phase B of acetonitrile and elution gradient of 21% -36%, collecting main peaks, detecting respectively, concentrating qualified fraction,

ii) sampling qualified fractions obtained in the step i), performing gradient elution, wherein a mobile phase A is 50mol/L sodium dihydrogen phosphate, the pH value is 2.5 +/-0.2, a mobile phase B is acetonitrile, the elution gradient of the phase B is 22-37%, collecting main peaks, respectively detecting, concentrating the qualified fractions for later use,

iii) sampling the qualified fraction obtained in the step i), performing gradient elution, wherein the mobile phase A is 0.1% HAC, the mobile phase B is acetonitrile, and the elution gradient of the phase B is 20% -55%, collecting a main peak part, and concentrating to obtain nesiritide.

In the technical scheme of the invention, the coupling agent is DIC, DIPCDI, DIPEA, a compound A, a compound B, DIC and compound A composition, DIC and compound A and compound B composition, DIPCDI and compound A composition or DIPEA and compound A and compound B composition, wherein the compound A is HOBt or HOAt, and the compound B is PyBOP, PyAOP, HATU, HBTU or TBTU.

The preparation method of nesiritide comprises the following steps:

A. synthesis of an intermediate 1:

using 2-CTC Resin as a carrier, firstly synthesizing Fmoc-His (Trt) -2CTC Resin, then synthesizing peptide Resin Boc-Ser (tBu) -Gly-Leu-Gly-Cys (Npys) -Lys (Boc) -Val-Leu-Arg (Pbf) -His (Trt) -2CTC Resin from C end to N end by adopting a solid phase Fmoc synthesis method, and obtaining Npys protection after crackingIntermediate 1 of Cys side chain: H-Ser²¹-Ser-Gly-Leu-Gly-Cys(Npys)-Lys-Val-Leu-Arg³⁰-Arg-His-OH

B. And (3) synthesizing an intermediate 2:

using 2-CTC Resin as a carrier, firstly synthesizing Fmoc-Ser (tBu) -2CTC Resin, then synthesizing peptide Resin Fmoc-Ser (tBu) -Pro-Lys (Boc) -Met-Val-Gln (Trt) -Gly-Ser (tBu) -Gly-Cys (Trt) -Phe-Gly-Arg (Pbf) -Lys (Boc) -Met-Asp (OtBu) -Arg (Pbf) -Ile-Ser (tBu) -2CTC Resin from C end to N end by adopting a solid phase Fmoc synthesis method, and obtaining a fully protected intermediate 2 after cracking: Fmoc-Ser (tBu) -Pro-Lys (Boc) -Met-Val-Gln (Trt) -Gly-Ser (tBu) -Gly-Cys (Trt) -Phe-Gly-Arg (Pbf) -Lys (Boc) -Met-Asp (OtBu) -Arg (Pbf) -Ile-Ser (tBu) -OH.

C. And (3) synthesizing an intermediate 4:

activating the fully-protected intermediate 2 by salicylaldehyde glycol acetal to obtain a fully-protected intermediate 3 with activated C-terminal carboxyl, and cracking to obtain an intermediate 4 with activated C-terminal carboxyl.

D. And (3) synthesizing an intermediate 5:

intermediate 1 and intermediate 4 form intermolecular disulfide bond intermediate 5 by disulfide bond exchange reaction

E. Crude peptide synthesis:

and (3) reacting the activated ester at the C end with the amino at the N end to form an amido bond under the action of a pyridine/acetic acid solution to obtain an intermediate 6, and then removing the Fmoc protecting group by using a diethylamine/acetonitrile solution to obtain a crude nesiritide product.

The meaning of english abbreviation used in the present invention.

Abbreviations and English	Means of
		Fmoc	9-fluorenylmethoxycarbonyl group
2-CTC resin	2-chlorotrityl chloride resin
		HOBt	1-hydroxybenzotriazoles
DIEA	N, N-diisopropylethylamine
		DMAP	4-dimethylaminopyridine
PyBOP	Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphates
		Boc	Tert-butyloxycarbonyl radical
tBu	Tert-butyl radical
		Trt	Trityl radical
Pbf	2,2,4,6, 7-pentamethylbenzofuran-5-sulfonyl
		DMF	N, N-dimethylformamide
DCM	Methylene dichloride
		DBLK	20% piperidine/DMF solution
TFE	Trifluoroethanol
		TFA	Trifluoroacetic acid
DIC	Diisopropylcarbodiimide

Advantageous effects

1) The invention adopts Fmoc solid phase synthesis method to synthesize two peptide fragments simultaneously, which can shorten half of the production time.

2) The intermolecular disulfide bond is formed by disulfide bond exchange, and Cys (Npys) of the peptide fragment 1 and Cys of the peptide fragment 4 form an intermolecular disulfide bond by disulfide bond exchange, whereby the accuracy of disulfide bond pairing can be improved and the generation of high polymer can be reduced.

3) The peptide sequence has Ser with higher coupling difficulty¹⁹-Ser-Ser-Ser²²Fragment, adopting the conventional solid phase gradual coupling synthesis method to hardly avoid the formation of defective Ser impurities or racemic impurities; liquid phase fragment synthesis method is adopted to synthesize nesiritide and reduce Ser¹⁹-Ser-Ser-Ser²²The difficulty of fragment coupling.

Drawings

Figure 1. nesiritide chromatogram for example 14.

Figure 2. nesiritide chromatogram of comparative experimental example 15.

Detailed Description

Example 1 Synthesis of Fmoc-His (Trt) -2CTC resin

Weighing 100g of 2-CTC resin (purchased from Tianjin Nankai Synthesis technology Co., Ltd.) with the substitution degree of 0.8mmol/g, adding the 2g of the 2-CTC resin into a solid phase reaction column, washing the resin with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 49.2g of Fmoc-His (Trt) -OH (80mmol) with 320mL of DMF, adding 27.2mL (80mmol) of DIPEA at the temperature of between 10 and 0 ℃ for activation, adding the activated product into the reaction column filled with the resin, reacting for 2 hours, and adding 40mL of anhydrous methanol for blocking for 1 hour. Washing with DMF 3 times, DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-His (Trt) -2CTC resin 140g with detection substitution of 0.452 mmol/g.

Example 2 Synthesis of intermediate 1

Fmoc-His (Trt) -2CTC44g (20mmol) with substitution 0.452mmol/g prepared in example 1 was weighed into a solid phase reaction column, washed 2 times with DMF, after swelling for 30 minutes with DMF, the Fmoc protection was removed with DBLK, then washed 4 times with DMF, washed 2 times with DCM, and the color of the resin was detected by the ninhydrin method, indicating that Fmoc was removed. 39.0g of Fmoc-Arg (Pbf) -OH (60mmol) and 9.8g of HOBt (72mmol) are dissolved in 120mL of a mixed solution of DCM and DMF with the volume ratio of 1: 1, 12.1mL of DIC (72mmol) are added at-10 ℃ to 0 ℃ for activation for 3min, and then the mixture is added into a solid phase reaction column for reaction at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method, and if the resin is colorless and transparent, indicating that the reaction is complete.

The above-mentioned steps of removing Fmoc protection and adding the corresponding amino acid for coupling were repeated to successively carry out the coupling of Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Npys) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH and Boc-Ser (tBu) -OH according to the peptide sequence of the peptide fragment 1. The resulting product was washed 3 times with DMF, 5 times with DCM, shrunk with methanol and dried overnight under vacuum to give 72.8g of peptide resin.

Adding 72.8g of peptide resin into a 1000mL reaction kettle, preparing 730mL of a cleavage reagent according to the volume ratio of TFA, phenylmethylsulfide, anisole, EDT (90: 5: 2: 3), pouring the cleavage reagent into the peptide resin, and reacting at room temperature for 2.5 hours. After the reaction, the resin was filtered and the filtrate was collected. The resin was washed with 50mL TFA, the filtrates were combined, the filtrate was precipitated by addition to 7.3L dry ether, centrifuged, washed with dry ether, and dried in vacuo to give 26.0g of a solid in 86.7% purity in 88.6% yield.

Example 3 Synthesis of Fmoc-Ser (tBu) -2CTC resin

Weighing 100g of 2-CTC resin (purchased from Tianjin Nankai Synthesis technology Co., Ltd.) with the substitution degree of 0.8mmol/g, adding the 2-CTC resin into a solid phase reaction column, washing the solid phase reaction column with DMF for 2 times, swelling the resin with DMF for 30 minutes, dissolving 46.0g of Fmoc-Ser (tBu) -OH (120mmol) with 320mL of DMF, adding 10.8mL (120mmol) of DIPEA at the temperature of between 10 and 0 ℃ for activation, adding the activated product into the reaction column filled with the resin, reacting for 2 hours, and adding 40mL of anhydrous methanol for sealing for 1 hour. Washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking and draining methanol to obtain 126g of Fmoc-Ser (tBu) -2CTC resin with detection substitution of 0.515 mmol/g.

Example 4 Synthesis of intermediate 2

38.8g (20mmol) of Fmoc-Ser (tBu) -2CTC with the substitution degree of 0.515mmol/g, prepared in example 3, was weighed and loaded into a solid phase reaction column, washed with DMF 2 times, after swelling for 30 minutes with DMF, the Fmoc protection was removed with DBLK, then washed with DMF 4 times, DCM 2 times, and the color of the resin was detected by the ninhydrin method, indicating that Fmoc was removed. Dissolving 23.0g of Fmoc-Ser (tBu) -OH (60mmol) and 9.8g of HOBt (72mmol) in 120mL of mixed solution of DCM and DMF at the volume ratio of 1: 1, adding 12.2mL of DIC (72mmol) at-10-0 ℃ for activation for 3min, adding into a solid phase reaction column, and reacting at room temperature for 2 hours. And (4) detecting and judging the reaction end point by an indetrione method, and if the resin is colorless and transparent, indicating that the reaction is complete.

The above-mentioned steps of removing Fmoc protection and adding the corresponding amino acid for coupling were repeated to sequentially complete the coupling of Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH and Fmoc-Pro-OH according to the peptide sequence of the peptide fragment 2. The resulting product was washed 3 times with DMF, 5 times with DCM, shrunk with methanol and dried overnight under vacuum to give 100.6g of peptide resin.

100g of peptide resin was charged to a 2000mL reaction kettle as TFE: DCM ═ 2: 8, preparing 1000mL of cracking reagent, pouring the cracking reagent into the peptide resin, and reacting for 2.5 hours at room temperature. After the reaction, the resin was filtered and the filtrate was collected. The resin was washed with 100mL of DCM, the filtrates were combined, the filtrate was rotary evaporated to 150mL, added to 2000mL of anhydrous ether for precipitation, centrifuged, washed with anhydrous ether, and dried in vacuo to give intermediate 2 as a solid 62.3g, 80.7% yield.

Example 5, intermediate 4 synthesis:

intermediate 2 obtained in example 4 (15.4 g, 4mmol) as a solid, PyBOP 2.5g (4.8mmol) was weighed out and dissolved in 200ml of dichloromethane, DIPEA 0.6g (4.8mmol) and salicylaldehyde 2.5g (20mmol) were added and the reaction was stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to 20ml, 100ml TFA/TIS/H was added₂O (90/5/5, v/v/v) was cleaved for 3 hours, precipitated by addition to 1000ml of anhydrous ether, centrifuged, washed with anhydrous ether, and dried under vacuum to give 9.0g of intermediate 4, 74.4% pure, 90.2% yield.

Example 6, intermediate 4 synthesis:

intermediate 2 obtained in example 4 (15.4 g, 4mmol) as a solid, PyBOP 2.5g (4.8mmol) was weighed out and dissolved in 200ml of dichloromethane, DIPEA (0.6 g, 4.8mmol) and salicylaldehyde (3.75 g, 30mmol) were added and the reaction was stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to 20ml, 100ml TFA/TIS/H was added₂O (90/5/5, v/v/v) was cleaved for 3 hours, precipitated by addition to 1000ml of anhydrous ether, centrifuged, washed with anhydrous ether, and dried under vacuum to give 9.2g of intermediate 4, 76.6% pure, 92.0% yield.

Example 7, intermediate 4 synthesis:

intermediate 2 obtained in example 4 (15.4 g, 4mmol) as a solid, PyBOP 2.5g (4.8mmol) was weighed out and dissolved in 200ml of dichloromethane, DIPEA 0.6g (4.8mmol) and salicylaldehyde 5.0g (40mmol) were added and the reaction was stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to 20ml, 100ml TFA/TIS/H was added₂O (90/5/5, v/v/v) was cleaved for 3 hours, precipitated by addition to 1000ml of anhydrous ether, centrifuged, washed with anhydrous ether, and dried under vacuum to give 9.2g of intermediate 4, 77.4% pure, 92.0% yield.

Example 8, intermediate 4 synthesis:

intermediate 2 obtained in example 4 (15.4 g, 4mmol) as a solid, PyBOP 2.5g (4.8mmol) was weighed out and dissolved in 200ml of dichloromethane, DIPEA 0.6g (4.8mmol) and salicylaldehyde acetal 5.2g (40mmol) were added and the reaction was stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to 20ml, 100ml TFA/TIS/H was added₂O (90/5/5, v/v/v) was cleaved for 3 hours, precipitated by addition to 1000ml of anhydrous ether, centrifuged, washed with anhydrous ether, and dried under vacuum to give 9.4g of intermediate 4, 76.3% pure, 94.0% yield.

Example 9, intermediate 4 synthesis:

intermediate 2 obtained in example 4 (15.4 g, 4mmol) as a solid, PyBOP 2.5g (4.8mmol) was weighed out and dissolved in 200ml of dichloromethane, DIPEA 0.6g (4.8mmol) and salicylaldehyde dimethyl acetal 5.1g (40mmol) were added and the reaction was stirred for 4 hours. The reaction mixture was concentrated under reduced pressure to 20ml, 100ml TFA/TIS/H was added₂O (90/5/5, v/v/v) was cleaved for 3 hours, precipitated by addition to 1000ml of anhydrous ether, centrifuged, washed with anhydrous ether, and dried under vacuum to give 9.5g of intermediate 4, 77.2% pure, 95.1% yield.

Example 10 crude peptide Synthesis

4.4g (3mmol) of the intermediate 1 solid obtained in example 2 was weighed and dissolved in 200ml of purified water, and 7.4g (3mmol) of the intermediate 4 solid obtained in example 5 was weighed and added to the solution, and the reaction was stirred at room temperature for 1 hour to obtain a peptide fragment 5 solution; then, 1ml of a 10mM pyridine/acetic acid solution (1/12, mol/mol) was added to the solution, and the reaction was stirred at room temperature for 4 hours to obtain an intermediate 6 solution; then, 5ml of a 10% diethylamine acetonitrile/water solution was added to the solution, and stirred at room temperature for 4 hours to obtain an aqueous solution of nesiritide crude peptide 7 with a purity of 72.2% and a yield of 65.2%.

Example 11 crude peptide Synthesis

4.4g (3mmol) of the intermediate 1 solid obtained in example 2 was weighed and dissolved in 200ml of purified water, and 7.4g (3mmol) of the intermediate 4 solid obtained in example 6 was weighed and added to the solution, and the reaction was stirred at room temperature for 1 hour to obtain a peptide fragment 5 solution; then, 1ml of a 20mM pyridine/acetic acid solution (1/12, mol/mol) was added to the solution, and the reaction was stirred at room temperature for 4 hours to obtain an intermediate 6 solution; then, 5ml of a 20% diethylamine acetonitrile aqueous solution was added to the solution, and stirred at room temperature for 4 hours to obtain an aqueous solution of nesiritide crude peptide 7 with a purity of 72.8% and a yield of 64.8%.

Example 12 crude peptide Synthesis

4.4g (3mmol) of the intermediate 1 solid obtained in example 2 was weighed and dissolved in 200ml of purified water, and 7.4g (3mmol) of the intermediate 4 solid obtained in example 7 was weighed and added to the solution, and the reaction was stirred at room temperature for 1 hour to obtain a peptide fragment 5 solution; then, 1ml of a 30mM pyridine/acetic acid solution (1/12, mol/mol) was added to the solution, and the reaction was stirred at room temperature for 4 hours to obtain an intermediate 6 solution; then, 5ml of a 30% diethylamine acetonitrile/water solution was added to the solution, and stirred at room temperature for 4 hours to obtain an aqueous solution of nesiritide crude peptide 7 with a purity of 70.9% and a yield of 63.8%.

Example 13 crude peptide Synthesis

4.4g (3mmol) of the intermediate 1 solid obtained in example 2 was weighed and dissolved in 200ml of purified water, and 7.4g (3mmol) of the intermediate 4 solid obtained in example 8 was weighed and added to the solution, and the reaction was stirred at room temperature for 1 hour to obtain a peptide fragment 5 solution; then, 1ml of a 30mM pyridine/acetic acid solution (1/12, mol/mol) was added to the solution, and the reaction was stirred at room temperature for 4 hours to obtain an intermediate 6 solution; then, 5ml of a 30% diethylamine acetonitrile/water solution was added to the solution, and stirred at room temperature for 4 hours to obtain an aqueous solution of nesiritide crude peptide 7 with a purity of 68.8% and a yield of 65.0%.

Example 14, nesiritide purification preparation:

the nesiritide crude peptide solution in example 10 was purified using high performance liquid preparative chromatography.

The first step of purification, 200ml of solution is filtered through a 0.45 mu ml filter membrane, the sample is loaded for preparation, the filler is C18, wherein the mobile phase A is 0.1% TFA, the mobile phase B is acetonitrile, the detection wavelength is 230nm, the flow rate is 220ml/min, the B phase elution gradient is 21% -36% (45min), main peaks are collected for respective detection, and qualified fractions are removed by rotary evaporation to obtain organic phases for later use.

And a second step of purification, namely preparing qualified fractions obtained in the first step by loading, wherein the prepared filler is C18, the mobile phase A is 50mol/L sodium dihydrogen phosphate, the pH value is 2.5 +/-0.2, the mobile phase B is acetonitrile, the detection wavelength is 230nm, the flow rate is 220ml/min, the elution gradient of the phase B is 22-37% (60min), collecting main peaks for respective detection, and rotationally evaporating the qualified fractions to remove organic phases for later use.

The third step is salt conversion: and (3) transferring the qualified fraction obtained in the second step into a sample, preparing a filler C18, wherein the mobile phase A is 0.1% HAC, the mobile phase B is acetonitrile, the detection wavelength is 230nm, the flow rate is 220ml/min, the elution gradient of the phase B is 20% -55% (30min), collecting the main peak part, and carrying out rotary evaporation and freeze drying to obtain 3.95g of nesiritide acetate, the purity is 99.62%, and the yield is 38.0%.

Example 15 (comparative experiment), solid phase Synthesis of Nexiridine

Using 100g of 2CTC resin (40mmol) with substitution 0.4mmol/g as starting resin, washing twice with 100mL of DMF, swelling with 100mL of DMF for 30 minutes, dissolving 24.6g of Fmoc-His (Trt) -OH (40mmol) with 250mL of DMF, activating with 13.6mL (40mmol) of DIPEA at-10 ℃ to 0 ℃, adding to the above reaction column filled with resin, reacting for 2 hours, and blocking with 20mL of anhydrous methanol for 1 hour. Washing with DMF 3 times, washing with DCM 3 times, blocking with anhydrous methanol for 30min, shrinking methanol and draining to obtain Fmoc-His (Trt) -2CTC resin 140g with detection substitution of 0.225 mmol/g.

Weighing 89g (20mmol) of Fmoc-His (Trt) -2CTC with the substitution degree of 0.225mmol/g, adding the weighed materials into a solid-phase reaction column, washing the solid-phase reaction column for 2 times by using a proper amount of DMF, swelling the solid-phase reaction column for 30 minutes by using DMF, removing Fmoc protection by using DBLK, washing the solid-phase reaction column for 4 times by using DMF and washing the solid-phase reaction column for 2 times by using DCM, and detecting the color of the resin by an ninhydrin method, wherein the resin is colored, which indicates that Fmoc. 39.0g of Fmoc-Arg (Pbf) -OH (60mmol) and 9.8g of HOBt (72mmol) are dissolved in 200mL of mixed solution of DCM and DMF with the volume ratio of 1: 1, 12.1mL of DIC (72mmol) are added at-10 ℃ -0 ℃ for activation for 3min, then the mixture is added into a solid phase reaction column and reacted for 2 hours at room temperature. And (4) detecting and judging the reaction end point by an indetrione method, and if the resin is colorless and transparent, indicating that the reaction is complete.

Repeating the above steps of removing Fmoc protection and adding the corresponding amino acid for coupling, and sequentially completing Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc, Coupling of Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH and Fmoc-Pro-OH. The resulting product was washed 3 times with DMF, 5 times with DCM, shrunk with methanol and dried overnight under vacuum to give 159.2g of peptide resin.

159.2g of peptide resin is added into a 2000mL reaction kettle, 1600mL of cleavage reagent is prepared according to the volume ratio of TFA, phenylmethylsulfide, anisole, EDT (90: 5: 2: 3), the cleavage reagent is poured into the peptide resin, and the reaction is carried out for 2.5 hours at room temperature. After the reaction, the resin was filtered and the filtrate was collected. The resin was washed with 150mL TFA, the filtrates were combined, the filtrate was precipitated by addition to 16L dry ether, centrifuged, washed with dry ether, and dried in vacuo to give 40.2g of a solid in 56.3% purity in 58.0% yield.

40.2g of the crude peptide was dissolved in 40L of purified water, the pH was adjusted to 7.0 to 8.0 with acetic acid/ammonia water, and the reaction was stirred at room temperature with oxygen for 6 hours to complete the oxidation of disulfide bonds.

The first step of purification, the solution is filtered through a 0.45 mu ml filter membrane, the sample is loaded for preparation, the filler is C18, wherein, the mobile phase A is 0.1 percent TFA, the mobile phase B is acetonitrile, the detection wavelength is 230nm, the flow rate is 220ml/min, the elution gradient of the B phase is 21 percent to 36 percent (45min), the main peaks are collected for respective detection, and the qualified fraction is removed by rotary evaporation to obtain the organic phase for later use.

The third step is salt conversion: and (3) transferring salt from the qualified fraction obtained in the second step to prepare a filler C18, wherein the mobile phase A is 0.1% HAC, the mobile phase B is acetonitrile, the detection wavelength is 230nm, the flow rate is 220ml/min, the elution gradient of the phase B is 20% -55% (30min), the main peak part is collected, and the nesiritide acetate 8.4g, the purity is 98.70%, and the yield is 11.8% is obtained by rotary evaporation and freeze-drying.

Claims

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

Intermediate 4 is

Then removing the Fmoc protecting group to obtain a nesiritide crude product;

optionally, 5) purifying the nesiritide crude product to obtain nesiritide;

wherein the cyclization promoting reagent is selected from pyridine/acetic acid solution or triethylamine/acetic acid solution.

2. The method of claim 1, wherein the intermediate 1 is synthesized by a solid phase synthesis method comprising coupling a solid phase synthesis resin with Fmoc-His (Trt) -OH via a coupling agent, then removing the Fmoc protecting group, coupling amino acid Fmoc-Arg (Pbf) -OH by a coupling agent, then repeating the steps of removing Fmoc protecting group and coupling amino acid, sequentially coupling Fmoc-Arg (Pbf) -OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Npys) -OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH and Boc-Ser (tBu) -OH, then removing the Fmoc protecting group, and cracking by using a first cracking solution to obtain an intermediate 1 of the side chain of Cys protected by Npys; preferably, the first lysis solution is selected from a mixed solution of TFA, thioanisole, anisole and EDT, and more preferably, the ratio of TFA to thioanisole to EDT is 90:5: 2: 3.

3. The method of any one of claims 1-2, wherein the intermediate 2 is synthesized by solid-phase synthesis by coupling a solid-phase synthesis resin with Fmoc-Ser (tBu) -OH by a coupling agent, followed by removal of Fmoc protecting group, coupling amino acids Fmoc-Ser (tBu) -OH by a coupling agent, followed by repetition of the step of Fmoc protecting group removal, coupling amino acids, and coupling Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Lys (Boc, Fmoc-Gln (Trt) -OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys (Boc) -OH and Fmoc-Pro-OH, and then cleaved with a second cleavage solution to obtain an intermediate 2; preferably, the second cracking solution is a mixed solution of TFE and DCM, and more preferably, the ratio of TFE to DCM is 2: 8.

4. the production process as claimed in any one of claims 1 to 3, wherein the activating agent for activating the C-terminal carboxyl group of intermediate 2 in step 2) is a combination of a coupling agent and salicylaldehyde, or a combination of a coupling agent and salicylaldehyde glycol acetal, or a combination of a coupling agent and salicylaldehyde dimethyl acetal; preferably, the molar ratio of salicylaldehyde or salicylaldehyde glycol acetal or salicylaldehyde dimethyl acetal to intermediate 2 is 5:1 to 15:1, more preferably, 7.5: 1,10:1,12.5:1.

5. The process according to any one of claims 1 to 4, wherein the reagent for deprotecting the side chain of intermediate 3 in step 2) is TFA, TIS or H₂A mixture of O, preferably TFA, TIS and H₂The ratio of O is 80-95:3-10:3-10, more preferably TFA, TIS and H₂The ratio of O is 90:5: 5.

6. The production method according to any one of claims 1 to 5, wherein the conditions for the reaction of intermediate 1 and intermediate 4 in step 3) by disulfide bond exchange are such that intermediate 1 and intermediate 4 are put in solution to react and form disulfide bonds.

7. The process according to any one of claims 1 to 6, wherein the amide bond is formed in step 4) under conditions of a pyridine and acetic acid solution, preferably, the ratio of pyridine to acetic acid is 1: 8-15.

8. The method according to any one of claims 1 to 7, wherein the Fmoc protecting group is removed in step 4) under conditions of 10% to 30% (20%) diethylamine in aqueous acetonitrile.

9. The method according to any one of claims 1 to 8, wherein the purification in step 5) is high performance liquid chromatography; preferably, the purification is by high performance liquid chromatography.

Preferably, the high performance liquid chromatography column is a C18 column.

More preferably, the high performance liquid chromatography comprises the following steps:

10. The process of any one of claims 1-9, wherein the coupling agent is DIC, DIPCDI, DIPEA, compound a, a combination of compound B, DIC and compound a, DIC and a combination of compound a and compound B, a combination of DIPCDI and compound a, or a combination of DIPEA and compound a and compound B, wherein compound a is HOBt or HOAt and compound B is PyBOP, PyAOP, HATU, HBTU or TBTU.