CN110317130B - Compound and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of polypeptide synthesis, in particular to a compound and a preparation method and application thereof. The synthetic reaction vector 1/2/3 of the present invention is used for the liquid phase synthesis of angiotensin II. The reaction carrier is adopted to protect the C end, and simultaneously, the total protection peptide in an organic solvent such as CHCl can be effectively increased3The solubility of the total protection peptide in aqueous solution is reduced; the reaction carrier 1/2/3 is used as a raw material, and the steps of purification in each step of the traditional liquid phase synthesis can be reduced by a liquid phase synthesis method, so that the production efficiency is improved; the synthesis method provided by the invention can effectively reduce the dosage of organic solvents (DMF, DCM) in solid phase synthesis, and achieves the effect of waste reduction.
Description
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a compound and a preparation method and application thereof.
Background
Angiotensin is a peptide substance with strong functions of contracting blood vessels and stimulating adrenal cortex to secrete aldosterone, and is involved in the regulation of blood pressure and body fluid. Can be divided into angiotensin I-VII.
Angiotensin ii is the most important component of angiotensin. Angiotensin receptors are present on the vascular smooth muscle, adrenal cortico zonal cells, and cells of several parts of the brain, heart and kidney organs of the human body. Angiotensin ii binds to angiotensin receptors, causing corresponding physiological effects. Angiotensin acts on vascular smooth muscle, and can contract systemic arteriole and increase arterial blood pressure. Angiotensin ii is one of the most potent vasoactive substances known. Acting on peripheral blood vessels to contract veins and increase the amount of blood returning to the heart, acting on the central nervous system to cause thirst.
Angiotensin II agent LJPC-501 from La Jolla Pharmaceutical is being developed for the treatment of Catecholamine Resistant Hypotension (CRH) patients.
The structure of angiotensin II is shown in formula I:
regarding the preparation method of angiotensin ii, the conventional synthesis method of short peptides can also adopt the Fmoc solid phase synthesis method. Disadvantages of solid synthesis: a solid carrier is needed and the reaction is ensured to be complete through excessive feeding, so that the cost is high; the resin needs to be repeatedly washed in the synthesis process, and a large amount of organic solvents such as DMF and DCM are needed; the synthesis scale of the solid-phase synthesis method is greatly influenced by equipment and is difficult to amplify; disadvantages of conventional liquid phase synthesis: after the liquid phase reaction is completed, the required compound and the by-product are all in the reaction mixture, and after each step of synthesis is completed, purification is needed, and then the next step of synthesis is performed, so that the operation is complicated, and the yield is relatively low.
Therefore, the preparation method of the angiotensin II, which is simple and convenient to operate and high in yield, has important practical significance.
Disclosure of Invention
In view of the above, the present invention provides compounds, and methods for their preparation and use. The synthetic reaction vector 1/2/3 of the present invention is used for the liquid phase synthesis of angiotensin II. The reaction carrier is adopted to protect the C end, and simultaneously, the total protection peptide in an organic solvent such as CHCl can be effectively increased3The solubility of the total protection peptide in aqueous solution is reduced; the reaction carrier 1/2/3 is used as raw material, and the liquid phase synthesis method can reduce the steps of purification in each step of the traditional liquid phase synthesis and improve the production efficiencyRate; the synthesis method provided by the invention can effectively reduce the dosage of organic solvents (DMF, DCM) in solid phase synthesis, and achieves the effect of waste reduction.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a compound, which is characterized in that the structure is shown as formula II:
wherein R is1、R2Independently selected from H or
In some embodiments of the invention, the compounds have the structure shown in formula IV (reaction support 1), formula V (reaction support 2), formula VI (reaction support 3):
the invention also provides a preparation method of the compound, which comprises the steps of carrying out halogenation reaction on the compound shown in the formula III, carrying out reaction with p-hydroxy-benzene alkyl acid ester to generate ether, and carrying out reduction reaction to obtain the compound.
Wherein R is1、R2Independently selected from H or
In some embodiments of the invention, the compound of formula III comprises a compound of formula VII (starting material 1, preparation reaction support 1), formula VIII (starting material 2, preparation reaction support 2), or formula IX (starting material 3, preparation reaction support 3).
In some embodiments of the invention, the reagent used for the halogenation reaction is one or both of HCl or HBr. HBr is preferred.
In some embodiments of the present invention, the reaction temperature of the halogenation reaction is 90 to 110 ℃, the reaction time is 12 to 24 hours, the catalyst is one or a mixture of more than two of concentrated sulfuric acid and zinc chloride, and the reaction solvent is one or a mixture of more than two of water and acetic acid.
In some embodiments of the invention, the paraben is one or a mixture of more than two of methylparaben, methylparaben. Methyl paraben is preferred.
In some embodiments of the present invention, the reaction temperature for the reaction with the p-hydroxyalkanoate to generate the ether is 90 to 110 ℃, the reaction time is 12 to 24 hours, the catalyst is one or a mixture of more than two of potassium carbonate, sodium carbonate, potassium hydroxide or sodium hydroxide, and the reaction solvent is one or a mixture of more than two of DMF, tetrahydrofuran or acetone.
In some embodiments of the present invention, the reducing agent used in the reduction reaction is one or a mixture of two of diisobutylaluminum hydride, aluminum lithium hydride, and sodium borohydride. Diisobutylaluminum hydride is preferred.
In some embodiments of the invention, the reaction temperature of the reduction reaction is-5 to 5 ℃, the reaction time is 12 to 24 hours, and the reaction solvent is one or a mixture of more than two of tetrahydrofuran, dioxane and toluene.
The invention also provides the application of the compound or the compound prepared by the preparation method in polypeptide synthesis.
The invention also provides the application of the compound or the compound prepared by the preparation method in the preparation of angiotensin II.
The invention also provides a preparation method of the angiotensin II, which takes the compound or the compound prepared by the preparation method as a raw material to synthesize the full-protection peptide by liquid phase, and the full-protection peptide is obtained by cracking and purifying.
In some embodiments of the invention, the solvent used in the process for the preparation of angiotensin ii is one or both of chloroform or dichloromethane, preferably chloroform.
In some embodiments of the invention, the coupling agent in the process for the preparation of angiotensin ii is HOBt/DIC, HOBt/edc.hcl, preferably HOBt/edc.hcl.
In some embodiments of the present invention, in the method for preparing angiotensin ii, the cleavage solution is a mixed solvent of TFA and water, the mixed solvent is 80-95% by volume of TFA and 5-20% by volume of water, wherein the preferred ratio is TFA: H2O ═ 95: 5.
in some embodiments of the invention, methods for the preparation of angiotensin II-
The invention provides a compound, which has a structure shown in a formula II and has the following beneficial effects:
1. reaction vector 1/2/3 synthesized in the present invention is used for the liquid phase synthesis of angiotensin II. The reaction carrier is adopted to protect the C end, and simultaneously, the total protection peptide in an organic solvent such as CHCl can be effectively increased3The solubility of the total protection peptide in aqueous solution is reduced;
2. by adopting a novel liquid phase synthesis method, the steps of purifying each step in the traditional liquid phase synthesis can be reduced, and the production efficiency is improved;
3. the new synthesis method can effectively reduce the dosage of organic solvents (DMF, DCM) in solid phase synthesis, and achieve the effect of waste reduction.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a scheme for the synthesis of a compound of formula IV (reaction support 1);
FIG. 2 shows a scheme for the synthesis of a compound of formula V (reaction support 2);
FIG. 3 shows a scheme for the synthesis of a compound of formula VI (reaction support 3);
FIG. 4 shows a reaction scheme for synthesizing a fully protected peptide from a compound of formula IV (reaction support 1);
FIG. 5 shows a reaction scheme for cleavage of a fully protected peptide to give a crude peptide;
FIG. 6 shows an HPLC chromatogram of a crude angiotensin II peptide prepared in example 13;
FIG. 7 shows an HPLC chromatogram of a crude angiotensin II peptide prepared in example 14;
FIG. 8 shows an HPLC chromatogram of a crude angiotensin II peptide prepared in example 15;
FIG. 9 shows an HPLC chromatogram of angiotensin II fine peptide prepared in example 16;
FIG. 10 shows a mass spectrum of angiotensin II fine peptide prepared in example 16;
FIG. 11 shows an HPLC chromatogram of angiotensin II fine peptide prepared in example 17;
FIG. 12 shows an HPLC chromatogram of angiotensin II fine peptide prepared in example 18;
FIG. 13 shows an HPLC chromatogram of angiotensin II refined peptide prepared in example 19 amplification experiment;
FIG. 14 shows a mass spectrum of angiotensin II fine peptide prepared in example 19 amplification experiment;
FIG. 15 shows an HPLC chromatogram of the solid-phase synthesis of angiotensin II fine peptide in comparative example 1;
fig. 16 shows an HPLC profile of the liquid phase synthesis of angiotensin ii smart peptide in comparative example 2.
Detailed Description
The invention discloses a compound, a preparation method and application thereof, and can be realized by appropriately improving process parameters by one skilled in the art with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The compound, the preparation method and the raw materials and reagents used in the application of the compound are all available in the market.
The invention is further illustrated by the following examples:
example 1 Synthesis of reaction Carrier 1
13.6g of neopentyl alcohol (100mmol) are weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid is slowly added dropwise, the mixture is heated to 100 ℃ and reacted for 18 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added for washing twice, and 100ml of the solution was washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off by rotary evaporation, and purified by silica gel column to give 25.85g of intermediate 1a with a yield of 67.5%.
23.0g of intermediate 1a (60mmol), 44.1g of methyl p-hydroxyphenylate (290mmol) and 39.7g of potassium carbonate (290mmol) are dissolved in 200ml of DMF and heated to 100 ℃ to react 12. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing once with 200ml of 1M hydrochloric acid solution, and washing twice with 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated to dryness to give 31.5g of intermediate 1b in 85.0% yield.
31.5g of intermediate 1b (51mmol) are dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 0 ℃, 245ml of 1M diisobutylaluminum hydride/toluene solution is slowly added dropwise under the protection of nitrogen, the reaction is carried out at room temperature for 12 hours, and then the reaction is quenched with 100ml of 0.2M hydrochloric acid solution. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 25.18g of reaction carrier 1, yield 88.2%, MS 560.50.
1H NMR(400MHz,CDCl3)δ7.04–6.94(m,8H),6.93–6.79(m,8H),4.61(s,8H),4.16(s,8H),1.38(s,4H).
Example 2 Synthesis of reaction Carrier 1
13.6g of neopentyl alcohol (100mmol) are weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid is slowly added dropwise, and the mixture is heated to 90 ℃ and reacted for 24 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added for washing twice, and 100ml of saturated brine was added for washing twice. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off by rotary evaporation, and purified by silica gel column to give 25.10g of intermediate 1a with a yield of 65.5%.
23.0g of intermediate 1a (60mmol), 44.1g of methyl p-hydroxyphenyl ate (290mmol) and 39.7g of potassium carbonate (290mmol) were dissolved in 200ml of DMF solution, and the mixture was heated to 90 ℃ for 24 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing once with 200ml of 1M hydrochloric acid solution, and washing twice with 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated to dryness to give 30.5g of intermediate 1b, with a yield of 82.3%.
30.5g of the intermediate 1b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 5 ℃, 245ml of 1M diisobutylaluminum hydride/toluene solution is slowly dropped under the protection of nitrogen, the reaction is carried out for 8 hours at room temperature, and then 100ml of 0.2M hydrochloric acid solution is used for quenching the reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 24.9g of reaction carrier 1, yield 87.2%, MS 560.58.
1H NMR(400MHz,CDCl3)δ7.05–6.93(m,8H),6.94–6.79(m,8H),4.61(s,8H),4.17(s,8H),1.39(s,4H).
Example 3 Synthesis of reaction Carrier 1
13.6g of neopentyl alcohol (100mmol) are weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid is slowly added dropwise, and the mixture is heated to 110 ℃ and reacted for 12 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added for washing twice, and 100ml of the solution was washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off by rotary evaporation, and purified by silica gel column to give 26.10g of intermediate 1a with a yield of 68.1%.
23.0g of intermediate 1a (60mmol), 44.1g of methyl p-hydroxyphenyl ate (290mmol) and 39.7g of potassium carbonate (290mmol) were dissolved in 200ml of DMF solution, and heated to 110 ℃ for 8 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing once with 200ml of 1M hydrochloric acid solution, and washing twice with 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated to dryness to give 30.9g of intermediate 1b in 83.4% yield.
30.9g of the intermediate 1b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to-5 ℃, 245ml of 1M diisobutylaluminum hydride/toluene solution is slowly dropped under the protection of nitrogen, the reaction is carried out for 24 hours at room temperature, and then 100ml of 0.2M hydrochloric acid solution is used for quenching the reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 25.5g of reaction carrier 1, yield 89.3%, MS 560.48.
1H NMR(400MHz,CDCl3)δ7.04–6.93(m,8H),6.92–6.78(m,8H),4.62(s,8H),4.18(s,8H),1.38(s,4H).
Example 4 Synthesis of reaction Carrier 2
10.6g of 2-hydroxymethyl-1, 3-propanediol (100mmol) were weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 100 ℃ and reacted for 18 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added to the organic phase for washing twice, 100ml of saturated saline was washed twice with water, anhydrous sodium sulfate was added for drying, the organic solvent was evaporated by rotary evaporation after filtration, and purification was carried out by a silica gel column to obtain 27.0g of intermediate 1b with a yield of 92.0%.
23.5g of intermediate 2a (80mmol), 44.1g of methyl p-hydroxyphenyl ate (290mmol) and 39.7g of potassium carbonate (290mmol) were dissolved in 200ml of DMF solution, heated to 100 ℃ and reacted for 18 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and after filtration, the organic solvent was evaporated to dryness by rotary evaporation to obtain 33.1g of intermediate 2b with a yield of 89.0%.
33.1g of the intermediate 2b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 0 ℃, 255ml of 1M diisobutylaluminum hydride/toluene solution is slowly dropped under the protection of nitrogen, the reaction is carried out for 18 hours at room temperature, and then 100ml of 0.2M hydrochloric acid solution is used for quenching the reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 27.66g of reaction carrier 2, 91.8% yield, MS 424.45.
1HNMR(400MHz,CDCl3)δ7.08–6.93(m,6H),6.93–6.77(m,6H),4.61(s,6H),3.91(d,J＝14.24Hz,6H),3.09–3.04(m,1H),1.39(s,3H).
Example 5 Synthesis of reaction Carrier 2
10.6g of 2-hydroxymethyl-1, 3-propanediol (100mmol) were weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 90 ℃ for reaction for 24 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added to the organic phase for washing twice, 100ml of saturated saline was washed twice with water, anhydrous sodium sulfate was added for drying, the organic solvent was evaporated by rotary evaporation after filtration, and purification was carried out by a silica gel column to obtain 26.2g of intermediate 1b with a yield of 89.2%.
23.5g of intermediate 2a (80mmol), 44.1g of methyl p-hydroxyphenyl ate (290mmol) and 39.7g of potassium carbonate (290mmol) were dissolved in 200ml of DMF solution, heated to 90 ℃ and reacted for 24 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and after filtration, the organic solvent was evaporated to dryness by rotary evaporation to obtain 32.1g of intermediate 2b with a yield of 86.3%.
33.1g of the intermediate 2b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 5 ℃, 255ml of 1M diisobutylaluminum hydride/toluene solution is slowly dropped under the protection of nitrogen, the reaction is carried out for 12 hours at room temperature, and then 100ml of 0.2M hydrochloric acid solution is used for quenching the reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 26.6g of reaction carrier 2, yield 88.4%, MS 424.48.
1H NMR(400MHz,CDCl3)δ7.09–6.94(m,6H),6.93–6.78(m,6H),4.62(s,6H),3.912(d,J＝14.3Hz,6H),3.10–3.04(m,1H),1.38(s,3H).
Example 6 Synthesis of reaction Carrier 2
10.6g of 2-hydroxymethyl-1, 3-propanediol (100mmol) were weighed into 200ml of hydrobromic acid solution (40%), 0.5ml of concentrated sulfuric acid was slowly added dropwise, and the mixture was heated to 110 ℃ for reaction for 12 hours. The reaction solution was cooled to room temperature, 200ml of n-hexane was added for extraction, 100ml of 5% sodium bicarbonate solution was added to the organic phase for washing twice, 100ml of saturated saline was washed twice with water, anhydrous sodium sulfate was added for drying, the organic solvent was evaporated by rotary evaporation after filtration, and purification was carried out by a silica gel column to obtain 26.1g of intermediate 1b with a yield of 88.9%.
23.5g of intermediate 2a (80mmol), 44.1g of methyl p-hydroxyphenyl ate (290mmol) and 39.7g of potassium carbonate (290mmol) were dissolved in 200ml of DMF solution, and the mixture was heated to 110 ℃ for 12 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and after filtration, the organic solvent was evaporated to dryness by rotary evaporation to obtain 31.8g of intermediate 2b with a yield of 85.5%.
31.8g of the intermediate 2b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to-5 ℃, 255ml of 1M diisobutylaluminum hydride/toluene solution is slowly dropped under the protection of nitrogen, the reaction is carried out for 24 hours at room temperature, and then 100ml of 0.2M hydrochloric acid solution is used for quenching the reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 26.7g of reaction carrier 2, yield 88.8%, MS 424.35.
1H NMR(400MHz,CDCl3)δ7.08–6.93(m,6H),6.93–6.77(m,6H),4.61(s,6H),3.91(d,J＝14.2Hz,6H),3.09–3.04(m,1H),1.39(s,3H).
Example 7 Synthesis of reaction Carrier 3
16.2g of intermediate 3a (80mmol), 29.2g of methyl p-hydroxyphenyl ate (192mmol) and 26.5g of potassium carbonate (192mmol) were weighed out and dissolved in 200ml of DMF solution, and heated to 100 ℃ for 18 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and after filtration, the organic solvent was evaporated to dryness by rotary evaporation to obtain 23.2g of intermediate 2b with a yield of 92.0%.
23.2g of intermediate 3b are dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 0 ℃, 175ml of 1M diisobutylaluminum hydride/toluene solution is slowly added dropwise under the protection of nitrogen, the reaction solution is quenched at room temperature for 18 hours, and then 100ml of 0.2M hydrochloric acid solution is used for quenching reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated to dryness to give 19.7g of reaction carrier 3, yield 93.8%, MS 288.35.
1H NMR(400MHz,CDCl3)δ7.08–6.91(m,4H),6.91–6.77(m,4H),4.61(s,4H),4.29(s,4H),2.25(s,2H),1.38(s,2H).
Example 8 Synthesis of reaction Carrier 3
16.2g of intermediate 3a (80mmol), 29.2g of methyl p-hydroxyphenyl ate (192mmol) and 26.5g of potassium carbonate (192mmol) were weighed out and dissolved in 200ml of DMF solution, and heated to 90 ℃ for reaction for 24 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and the organic solvent was evaporated off by rotary evaporation after filtration to give 22.5g of intermediate 2b with a yield of 89.2%.
22.5g of the intermediate 3b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to-5 ℃, 175ml of 1M diisobutylaluminum hydride/toluene solution is slowly added dropwise under the protection of nitrogen, the reaction solution is quenched at room temperature for 24 hours, and then 100ml of 0.2M hydrochloric acid solution is used for quenching reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 18.9g of reaction carrier 3, yield 91.4%, MS 288.45.
1H NMR(400MHz,CDCl3)δ7.08–6.92(m,4H),6.91–6.76(m,4H),4.61(s,4H),4.28(s,4H),2.26(s,2H),1.39(s,2H).
Example 9 Synthesis of reaction Carrier 3
16.2g of intermediate 3a (80mmol), 29.2g of methyl p-hydroxyphenyl ate (192mmol) and 26.5g of potassium carbonate (192mmol) were weighed out and dissolved in 200ml of DMF solution, and heated to 110 ℃ for 12 hours. Cooling the reaction solution to room temperature, adding 400ml of n-hexane for extraction, washing the organic phase once by 200ml of 1M hydrochloric acid solution, and washing twice by adding 200ml of 5% sodium bicarbonate solution; the reaction solution was washed once with 200ml of saturated saline. Anhydrous sodium sulfate was added for drying, and the organic solvent was evaporated off by rotary evaporation after filtration to give 22.6g of intermediate 2b with a yield of 89.6%.
22.6g of intermediate 3b is dissolved in 500ml of tetrahydrofuran, the reaction solution is cooled to 5 ℃, 175ml of 1M diisobutylaluminum hydride/toluene solution is slowly added dropwise under the protection of nitrogen, the reaction solution is quenched at room temperature for 12 hours, and then 100ml of 0.2M hydrochloric acid solution is used for quenching reaction. Adding 800ml ethyl acetate to extract twice, washing once by 300ml 1M hydrochloric acid solution, and adding 200ml 5% sodium bicarbonate solution to wash twice; the reaction solution was washed once with 200ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered and the organic solvent was evaporated off to dryness to give 18.7g of reaction carrier 3, yield 90.4%, MS 288.52.
1H NMR(400MHz,CDCl3)δ7.08–6.93(m,4H),6.92–6.77(m,4H),4.63(s,4H),4.27(s,4H),2.27(s,2H),1.39(s,2H).
Example 10 Synthesis of angiotensin II full protective peptide
5.60g of reaction carrier 1(10mmol) from example 1 were weighed out, dissolved in 200ml of chloroform, and 20.12g of Fmoc-Phe-OH (52mmol), 10.72g of EDC.HCl (56mmol) and 1.20g of DMAP (5.2mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, and 15.6g of 2-mercaptosuccinic acid (104mmol) and 31.6g of DBU (208mmol) were added to the organic phase in an ice bath, followed by stirring and reacting for 3 hours to obtain intermediate 1 c. Then washed 1 times with 50ml of 10% aqueous sodium bicarbonate (containing 20% DMF) and twice with saturated brine to give intermediate 1 d.
17.52g Fmoc-Pro-OH (52mmol), 10.72g EDC.HCl (56mmol) and 7.02g HOBt (52mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, 15.6g of 2-mercaptosuccinic acid (104mmol) and 31.6g of DBU (208mmol) were added to the organic phase under ice bath, and the mixture was stirred for reaction for 3 hours, washed 1 time with 50ml of 10% sodium bicarbonate DMF/water solution (1/4) and twice with 10% NaCl DMF/water solution (1/4). The subsequent Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH and Fmoc-Asp (OtBu) -OH were coupled in the same manner, and the dosage of all amino acids was 5.2eq, and the reaction time was 3 hours. After all the amino acids have been coupled, half of the solvent is evaporated off under reduced pressure, then precipitated by addition to 200ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. 53.0g of crude peptide of the side chain full-protection angiotensin II is obtained, and the yield is 75.2%.
Example 11 Synthesis of angiotensin II full protective peptide
4.24g of reaction carrier 2(10mmol) from example 2 were weighed out, dissolved in 200ml of chloroform, and 15.09g of Fmoc-Phe-OH (39mmol), 8.04g of EDC.HCl (42mmol) and 0.90g of DMAP (3.9mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, and 11.72g of 2-mercaptosuccinic acid (78mmol) and 23.72g of DBU (156mmol) were added to the organic phase in an ice bath and stirred for reaction for 3 hours to obtain intermediate 1 c. Then washed 1 time with 50ml 10% aqueous sodium bicarbonate (containing 20% DMF) and twice with saturated brine to give intermediate 2 d.
13.14g Fmoc-Pro-OH (39mmol), 8.04g EDC.HCl (42mmol) and 5.27g HOBt (39mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, and 11.72g of 2-mercaptosuccinic acid (78mmol) and 23.72g of DBU (156mmol) were added to the organic phase under ice bath, and the mixture was stirred for reaction for 3 hours, then washed 1 time with 50ml of 10% sodium bicarbonate in DMF/water solution (1/4) and twice with 10% NaCl in DMF/water solution (1/4). The subsequent Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH and Fmoc-Asp (OtBu) -OH were coupled in the same manner, and the dosage of all amino acids was 3.9eq, and the reaction time was 3 hours. After all the amino acids have been coupled, half of the solvent is evaporated off under reduced pressure, then precipitated by addition to 200ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. 42.2g of crude peptide of the side chain full-protection angiotensin II is obtained, and the yield is 79.8%.
EXAMPLE 12 Synthesis of angiotensin II full protective peptide
2.88g of reaction carrier 3(10mmol) of example 3 were weighed out and dissolved in 200ml of chloroform, 10.06g of Fmoc-Phe-OH (26mmol), 5.36g of EDC.HCl (28mmol) and 0.60g of DMAP (2.6mmol) were added under ice-cooling, and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, and 7.80g of 2-mercaptosuccinic acid (52mmol) and 15.81g of DBU (104mmol) were added to the organic phase in an ice bath, followed by stirring and reacting for 3 hours to obtain intermediate 3 c. Then washed 1 time with 50ml 10% aqueous sodium bicarbonate (containing 20% DMF) and twice with saturated brine to give intermediate 3 d.
8.76g Fmoc-Pro-OH (26mmol), 5.36g EDC.HCl (28mmol) and 3.51g HOBt (26mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 50ml of saturated brine was added and washed once, and 7.80g of 2-mercaptosuccinic acid (52mmol) and 15.81g of DBU (104mmol) were added to the organic phase under ice bath, and the mixture was stirred for reaction for 3 hours, then washed 1 time with 50ml of 10% sodium bicarbonate DMF/water solution (1/4), and twice with 10% NaCl DMF/water solution (1/4). The subsequent Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH and Fmoc-Asp (OtBu) -OH were coupled in the same manner, and the dosage of all amino acids was 5.2eq, and the reaction time was 3 hours. After all the amino acids have been coupled, half of the solvent is evaporated off under reduced pressure, then precipitated by addition to 200ml of acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. 30.1g of crude peptide of the side chain full-protection angiotensin II is obtained, and the yield is 84.9%.
Example 13 cleavage of crude angiotensin II peptide
53.0g of crude side chain-fully-protected angiotensin II peptide from example 4 was placed in a 1000m1 reaction flask and 530mL of the prepared cleavage reagent (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction, the reaction mixture was poured into 5.3L of ethyl glacial ether to wash out a large amount of white precipitate, which was centrifuged, washed, dried and weighed to obtain 39.8g of crude angiotensin ii peptide with yield 95.1% and HPLC purity 80.7% (see fig. 6), MS 1046.50.
Example 14 cleavage of crude angiotensin II peptide
42.2g of the crude side chain-fully protected angiotensin II peptide from example 5 were placed in a 500m1 reaction flask and 425mL of the formulated cleavage reagent (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction is finished, pouring the reaction solution into 4.3L of glacial ethyl ether, washing out a large amount of white precipitate, centrifuging, washing, drying and weighing to obtain the blood vessel29.6g of crude tensin II peptide, 94.5% yield, 81.2% HPLC purity (see FIG. 7), MS 1046.20.
Example 15 cleavage of crude angiotensin II peptide
30.1g of crude side chain-fully-protected angiotensin II peptide from example 6 was placed in a 500m1 reaction flask and 301mL of the prepared cleavage reagent (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction, the reaction mixture was poured into 3.1L of ethyl glacial ether to wash out a large amount of white precipitate, which was centrifuged, washed, dried and weighed to obtain 20.3g of crude angiotensin ii peptide with yield 96.1% and HPLC purity 81.7% (see fig. 8), MS 1046.18.
Example 16 preparation of angiotensin II
39.8g of crude angiotensin ii peptide from example 7 was purified by HPLC, concentrated and lyophilized to give 21.2g of refined angiotensin ii peptide with HPLC purity of 99.5% (see fig. 9), total yield of 50.7%, MS1046.45 (see fig. 10).
Example 17 preparation of angiotensin II
29.6g of crude angiotensin ii peptide from example 8 was purified by HPLC, concentrated and lyophilized to give 16.7g of refined angiotensin ii peptide with HPLC purity of 99.5% (see fig. 11), overall yield 53.4%, MS 1046.18.
Example 18 preparation of angiotensin II
20.3g of crude angiotensin ii peptide from example 9 was purified by HPLC, concentrated and lyophilized to give angiotensin ii refined peptide 10.4g with HPLC purity of 99.6% (see fig. 12), total yield 49.7%, MS 1046.21.
Example 19 amplification of angiotensin II
56.0g of reaction support 1(100mmol) were dissolved in 2L of chloroform, and 201.2g of Fmoc-Phe-OH (520mmol), 107.2g of EDC.HCl (560mmol) and 12.0g of DMAP (52mmol) were added under ice-cooling, and the reaction was stirred at room temperature for 3 hours. After the reaction, 500ml of saturated brine was added and washed once, and 156.2g of 2-mercaptosuccinic acid (1.04mol) and 316.0g of DBU (2.08mol) were added to the organic phase in an ice bath, followed by stirring and reacting for 3 hours to obtain an intermediate 1 c. Then washed 1 times with 500ml of 10% aqueous sodium bicarbonate (containing 20% DMF) and twice with 500ml of saturated brine to give intermediate 1 d.
175.2g Fmoc-Pro-OH (520mmol), 107.2g EDC.HCl (560mmol) and 70.2g HOBt (520mmol) were added under ice-bath and the reaction was stirred at room temperature for 3 hours. After the reaction, 500ml of saturated brine was added and washed once, and 156.2g of 2-mercaptosuccinic acid (1.04mol) and 316.2g of DBU (2.08mol) were added to the organic phase under ice bath, and the mixture was stirred and reacted for 3 hours, followed by washing 1 time with 500ml of a 10% aqueous DMF solution (1/4) and two times with 500m of a 10% aqueous DMF solution (1/4). The subsequent Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH and Fmoc-Asp (OtBu) -OH were coupled in the same manner, and the dosage of all amino acids was 5.2eq, and the reaction time was 3 hours. After all the amino acids were coupled, half of the solvent was evaporated under reduced pressure, then precipitated by adding to 2L acetonitrile, filtered, washed twice with acetonitrile and dried in vacuo for 12 hours. 509.1g of crude peptide of angiotensin II with fully protected side chain is obtained, and the yield is 72.1%.
509.1g of crude peptide of side chain-protected angiotensin II was put into a 6L reaction flask, and 5.1L of the prepared cleavage reagent (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction, the reaction solution was poured into 51L of ethyl glacial ether, and a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain crude angiotensin ii peptide 397.8g, yield 95.1%, HPLC purity 75.7%, MS 1046.50.
397.8g of crude angiotensin ii peptide from example 7 was purified by HPLC, concentrated and lyophilized to obtain 206.2g of refined angiotensin ii peptide with HPLC purity of 99.5% (see fig. 13), total yield of 49.3%, MS1046.62 (see fig. 14).
Comparative example 1 solid phase Synthesis of angiotensin II
Using 200g of Wang resin (100mmol) as a real resin with a degree of substitution of 0.5mmol/g, washed twice with 1L of DMF, swollen with 1L of DMF for 30 minutes, 153.8g of Fmoc-Phe-OH (400mmol), 145.9g of HOBt (420mmol) and 9.0g of DMAP (40mmol) were weighed out, and the reaction was stirred at room temperature for 3 hours. Blocking was performed by adding 1L of acetic anhydride/pyridine (1:1) solution for 6 hours. The blocking solution was pipetted and washed 3 times with 1L DCM and 3 times with 1L DMF. Fmoc was removed twice 10 minutes each time by adding 1L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 1L DMF after removal. 168.7g (500mmol) of Fmoc-Pro-OH and 81.0g (500mmol) of HOBt were weighed out, dissolved in 0.5L of LDMF and 0.5L of DCM, activated for 5 minutes by adding 108.3ml (600mmol) of DIC in an ice-water bath, and the mixture was charged into a reaction column and reacted at room temperature for 2 hours. The subsequent Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Arg (Pbf) -OH and Fmoc-Asp (OtBu) -OH were coupled in the same manner, the charge factor of all amino acids was 500mmol, the reaction time was 2 hours, and 1L of DMF was washed 6 times. Fmoc was removed twice 10 minutes each time by adding 1L of 20% piperidine/DMF (V/V) solution and the resin was washed 6 times with 1L DMF after removal. After the peptide resin was coupled, it was shrunk 3 times with 1L of methanol for 10 minutes each. Then, vacuum drying was carried out to obtain 365.1g of a peptide resin.
365.1g of angiotensin II peptide resin was added to a 10L reaction flask, and 3.65L of the prepared cleavage reagent (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction, the reaction solution was poured into 36.5L of ethyl glacial ether, a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain 98.0g of crude angiotensin ii peptide, yield 93.6%, HPLC purity 70.7%, MS 1046.18.
The 196g crude angiotensin ii peptide was purified by HPLC, concentrated and lyophilized to obtain 48.8g refined angiotensin ii peptide with HPLC purity of 99.6% (see fig. 15), total yield of 46.7%, MS 1046.18.
Comparative example 2
21.6g Boc-Pro-OH (100mmol) were dissolved in 200ml THF, 36.3g H-Phe-OMe.HCl (100mmol),13.8g DIC (110mmol), 14.9g HOBt (110mmol) were added in succession, 10.1g NMM (100mmol) were added dropwise with stirring, stirring at room temperature for 1.5 h and the reaction was followed by TCL. After the reaction, the solid was filtered off, THF was rotary-distilled off under reduced pressure, 300ml of ethyl acetate was added to dissolve the solid, the ethyl acetate solution was washed with a 10% citric acid solution, a saturated sodium bicarbonate solution and a saturated sodium chloride solution, respectively, the organic layer was separated, and dried over anhydrous sodium sulfate. Filtering out the drying agent, evaporating ethyl acetate under reduced pressure, adding 400ml of petroleum ether, freezing to wash out solids, filtering and collecting the solids, and drying under reduced pressure. 39.3g of Boc-Pro-Phe-OMe.HCl were obtained in 75.1% yield. 39.3g were reacted with 200ml of 4N HCl/dioxane solution at room temperature for 1.5 hours to precipitate a solid, which was collected by filtration and dried under vacuum to give 31.5g of H-Pro-Phe-OMe.HCl.
The subsequent Boc-His (Trt) -OH, Boc-Ile-OH, Boc-Tyr (tBu) -OH, Boc-Val-OH, Boc-Arg (Pbf) -OH and Boc-Asp (OtBu) -OH were coupled in the same manner, at a feed scale of 100mmol, to give 116.6g of the fully protected peptide in 64.8% yield.
116.6g of the angiotensin II pan-protected peptide described above was added to a 250m1 reaction flask and the prepared cleavage reagent 1.2L (TFA: H)2O ═ 9:1) was poured into the above reaction flask, and reacted at room temperature for 2 hours. After the reaction, the reaction solution was poured into 12.0L of ethyl glacial ether, and a large amount of white precipitate was washed out, centrifuged, washed, dried and weighed to obtain 67.9g of crude angiotensin ii peptide with yield of 65.1%, HPLC purity of 72.9%, MS 1046.28.
67.9g of crude angiotensin ii peptide was purified by HPLC, concentrated and lyophilized to obtain 42.6g of refined angiotensin ii peptide with HPLC purity of 99.6% (see fig. 16), total yield of 40.7%, MS 1046.26.
Example 20
Experimental group 1: crude and fine peptides obtained in example 19;
control group 1: crude and fine peptides obtained in comparative example 1;
control group 2: crude and fine peptides obtained in comparative example 2;
the results of the comparison are shown in Table 1.
TABLE 1
Note:*shows a significant difference (P < 0) compared to control 1.05)；**Shows a very significant difference (P < 0.01) compared with the control group 1;
#shows a significant difference (P < 0.05) compared with the control group 2;##it shows a very significant difference (P < 0.01) compared to control 2.
The results in table 1 show that the new carrier synthesized by the method has 2-4 reaction sites, and the actual synthesis scale is higher under the same synthesis scale, so that more products are obtained; compared with solid phase synthesis, the newly synthesized carrier is adopted to synthesize the polypeptide, so that the use amount of organic solvents DMF and DCM can be effectively reduced; compared with liquid phase synthesis, the new carrier is adopted to synthesize the polypeptide, so that the synthesis steps can be reduced, the dosage of an organic solvent can be reduced, and the total yield of the product can be improved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. The application of the compound in the preparation of angiotensin II;
the structure of the compound is shown as follows:
2. the preparation method of the angiotensin II is characterized in that a compound is used as a raw material, a full-protection peptide is synthesized in a liquid phase, and the full-protection peptide is obtained by cracking and purifying;
the structure of the compound is shown as follows:
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