CN113717246A - Preparation method of polypeptide heterodimer - Google Patents
Preparation method of polypeptide heterodimer Download PDFInfo
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- CN113717246A CN113717246A CN202110895474.7A CN202110895474A CN113717246A CN 113717246 A CN113717246 A CN 113717246A CN 202110895474 A CN202110895474 A CN 202110895474A CN 113717246 A CN113717246 A CN 113717246A
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Abstract
The application discloses a preparation method of polypeptide heterodimer, which comprises the following steps: preparing a compound of formula (II) or a salt thereof from a compound of formula (I) or a salt thereof; preparing a compound of formula (IV) or a salt thereof from a compound of formula (III) or a salt thereof; preparing a compound of formula (V) or a salt thereof from a compound of formula (IV) or a salt thereof; preparing a compound of formula (VI) or a salt thereof from a compound of formula (V) or a salt thereof; preparing a compound of formula (VII) or a salt thereof from a compound of formula (VI) or a salt thereof; preparing a compound of formula (VIII) or a salt thereof from a compound of formula (VII) or a salt thereof; preparing a compound of formula (IX) or a salt thereof from a compound of formula (VIII) or a salt thereof; and preparing a compound of formula (X) or a salt thereof from a compound of formula (II) or a salt thereof and a compound of formula (IX) or a salt thereof.
Description
Technical Field
The application relates to the field of polypeptide synthesis, and more particularly relates to a preparation method of a polypeptide heterodimer.
Background
Molecular imaging has gained importance in a wide range of diagnostic and therapeutic applications as a technique for the visualization, characterization and quantification of non-invasive molecular biological processes in vivo. An important issue for molecular imaging is the development of specific imaging tracers with high affinity and specificity, low non-specific uptake, sufficient retention and effective permeability. Over the past few decades, a great deal of research has been conducted on radiolabeled peptides for use in diagnosis and therapy due to their favorable properties, including extraordinary tissue permeability, rapid clearance, low immunogenicity, ease of synthesis, and low cost.
Peptides, however, also have some major drawbacks that are not negligible, such as relatively low tumor affinity and short retention time. To overcome these drawbacks, various approaches have been developed, of which heterodimeric peptides have become a very promising targeting strategy. In general, heterodimeric peptides consist of two covalently linked peptides capable of binding two different receptors simultaneously. Compared to monomeric peptides, bispecific heterodimeric peptides have a number of superior properties, including enhanced efficacy, affinity, and relatively long blood circulation time, enabling their imaging and therapeutic applications.
Disclosure of Invention
In some embodiments, the present application provides a method of making a polypeptide heterodimer that is a compound of formula (X) or a salt thereof.
In some embodiments, there is provided a method of preparing a compound, comprising: preparing a compound of formula (I) or a salt thereof; preparing a compound of formula (II) or a salt thereof from a compound of formula (I) or a salt thereof; preparing a compound of formula (III) or a salt thereof; preparing a compound of formula (IV) or a salt thereof from a compound of formula (III) or a salt thereof; preparing a compound of formula (V) or a salt thereof from a compound of formula (IV) or a salt thereof; preparing a compound of formula (VI) or a salt thereof from a compound of formula (V) or a salt thereof; preparing a compound of formula (VII) or a salt thereof from a compound of formula (VI) or a salt thereof; preparing a compound of formula (VIII) or a salt thereof from a compound of formula (VII) or a salt thereof; preparing a compound of formula (IX) or a salt thereof from a compound of formula (VIII) or a salt thereof; and preparing a compound of formula (X) or a salt thereof from a compound of formula (II) or a salt thereof and a compound of formula (IX) or a salt thereof.
In some embodiments, the compound of formula (II) or a salt thereof is prepared from the compound of formula (I) or a salt thereof by combining the compound of formula (I) or a salt thereof with BCN-PEG in acetonitrile at a pH of 7-84-NHS contact. In some embodiments, the compound of formula (IV) or a salt thereof is prepared from a compound of formula (III) or a salt thereof, comprising contacting the compound of formula (III) or a salt thereof with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a salt thereof in dichloromethane. In some embodiments, the compound of formula (V) or a salt thereof is prepared from a compound of formula (IV) or a salt thereof by contacting the compound of formula (IV) or a salt thereof with hydrazine hydrate in N, N-dimethylformamide. In some embodiments, the compound of formula (VI) or a salt thereof is prepared from the compound of formula (V) or a salt thereof by contacting the compound of formula (V) or a salt thereof with Fmoc-PEG in N, N-dimethylformamide4-propionic acid, PyBop and N, N-diisopropylethylamine. In some embodiments, the compound of formula (VII) or a salt thereof is prepared from a compound of formula (VI) or a salt thereof, comprising contacting the compound of formula (VI) or a salt thereof with piperidine in N, N-dimethylformamide. In some embodiments, a compound of formula (VIII) or a salt thereof is prepared from a compound of formula (VII) or a salt thereof, comprising reacting a compound of formula (VII) or a salt thereof with NOTA (tBu)2-N3And PyBop contact. In some embodiments, a compound of formula (IX) or a salt thereof is prepared from a compound of formula (VIII) or a salt thereof, including in trifluoroethyl fluorideIn an acid, the compound of formula (VIII) or a salt thereof, and water are contacted with triisopropylsilane.
In some embodiments, the compound of formula (III) or a salt thereof is contacted with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a salt thereof by reacting 90.3 ± 10% parts by weight of the compound of formula (III) or a salt thereof with 21.0 ± 10% parts by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a salt thereof at room temperature for 24 hours. In some embodiments, the compound of formula (IV) or a salt thereof is contacted with hydrazine hydrate by reacting 80.2 + -10% parts by weight of the compound of formula (IV) or a salt thereof with 8 + -10% parts by weight of hydrazine hydrate at 10-30 deg.C for 5 hours. In some embodiments, a compound of formula (V) or a salt thereof is combined with Fmoc-PEG4-propionic acid, PyBop and N, N-diisopropylethylamine by contacting 64.7 + -10% by weight of the compound of formula (V) or its salt with 32.0 + -10% by weight of Fmoc-PEG4Propionic acid and 34.2 + -10% PyBop by weight and 17.02 + -10% N, N-diisopropylethylamine by weight are mixed and added dropwise for reaction at 10-30 ℃ for 2 hours. In some embodiments, the contacting of the compound of formula (VI) or a salt thereof with piperidine is carried out by reacting 84.1 + -10% parts by weight of the compound of formula (VI) or a salt thereof with 41.88 + -10% parts by weight of piperidine at 10-30 deg.C for 2 hours. In some embodiments, a compound of formula (VII) or a salt thereof is reacted with NOTA (tBu)2-N3And PyBop by contacting 60.0 + -10% by weight of the compound of formula (VII) or a salt thereof with 25.2 + -10% by weight of NOTA (tBu)2-N327.3. + -. 10% by weight of PyBop at 10-30 ℃ for 2 hours. In some embodiments, the compound of formula (VIII) or a salt thereof, water, and triisopropylsilane are contacted by reacting 66.0 + -10% parts by weight of the compound of formula (VIII) or a salt thereof, 16.5 + -10% parts by weight of water, and 12.75 + -10% parts by weight of triisopropylsilane at room temperature for 2-3 hours.
In some embodiments, compounds of formula (I) or salts thereof are prepared, including: contacting the first resin with Fmoc-amino acid, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a condensed first resin; contacting the condensed first resin with piperidine to obtain deprotected first resin; contacting the deprotected first resin with trifluoroacetic acid to obtain a linear peptide of a compound of formula (I) or a salt thereof; and contacting the linear peptide of the compound of the formula (I) or the salt thereof with DMSO to obtain the compound of the formula (I) or the salt thereof. In some embodiments, a compound of formula (III), or a salt thereof, is prepared, comprising: contacting the second resin with Fmoc-amino acid, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain condensed second resin; contacting the condensed second resin with piperidine to obtain deprotected second resin; and contacting the deprotected second resin with trifluoroacetic acid to obtain the compound of formula (III) or a salt thereof.
In some embodiments, compounds of formula (I) or salts thereof are prepared, including: contacting the first resin with Fmoc-Cys (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a first condensed resin; contacting the first resin after the first condensation with piperidine to obtain a first resin after first deprotection; contacting the first resin subjected to the first deprotection with Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a second condensed first resin; contacting the first resin after the second condensation with piperidine to obtain a first resin after second deprotection; contacting the first resin subjected to second deprotection with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a first resin subjected to third condensation; contacting the first resin after the third condensation with piperidine to obtain a first resin after third deprotection; contacting the first resin subjected to third deprotection with Fmoc-Asn (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fourth condensed first resin; contacting the first resin after the fourth condensation with piperidine to obtain a first resin after fourth deprotection; contacting the fourth deprotected first resin with Fmoc-Cys (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fifth condensed first resin; contacting the fifth condensed first resin with piperidine to obtain a fifth deprotected first resin; contacting the fifth deprotected first resin with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a sixth condensed first resin; contacting the sixth condensed first resin with piperidine to obtain a sixth deprotected first resin; contacting the sixth deprotected first resin with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or a salt thereof; and contacting the linear peptide of the compound of the formula (I) or the salt thereof with DMSO to obtain the compound of the formula (I) or the salt thereof. In some embodiments, a compound of formula (III), or a salt thereof, is prepared, comprising: contacting the second resin with Fmoc-Gly-OH and N, N-diisopropylethylamine, and then contacting with methanol to obtain a first condensed second resin; contacting the second resin after the first condensation with piperidine to obtain a second resin after the first deprotection; contacting the second resin subjected to the first deprotection with Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a second resin subjected to second condensation; contacting the second resin after the second condensation with piperidine to obtain a second resin after second deprotection; contacting the second deprotected second resin with Fmoc-Lys (ivDde) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a third condensed second resin; contacting the second resin after the third condensation with piperidine to obtain a second resin after third deprotection; contacting the third deprotected second resin with Fmoc-D-Tyr (tBu) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fourth condensed second resin; contacting the fourth condensed second resin with piperidine to obtain a fourth deprotected second resin; contacting the fourth deprotected second resin with Fmoc-Asp (OtBu) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fifth condensed second resin; contacting the fifth condensed second resin with piperidine to obtain a fifth deprotected second resin; and contacting the fifth deprotected second resin with trifluoroacetic acid to obtain the compound of formula (III) or a salt thereof. In some embodiments, the first resin is Rink Amide AM resin. In some embodiments, the second resin is dichlorotrityl chloride resin. In some embodiments, the first resin is an unprotected or deprotected Rink Amide AM resin. In some embodiments, the second resin is an unprotected or deprotected dichlorotrityl chloride resin.
In some embodiments, preparing a compound of formula (X), or a salt thereof, further comprises: subjecting the crude compound of formula (X) or a salt thereof to liquid chromatography gradient elution with a mobile phase comprising: and (3) 0 minute: 0.3% aqueous acetic acid 95%, acetonitrile 5%, 5 min: 88% of 0.3% aqueous acetic acid, 12% of acetonitrile, 85 min: 0.3% aqueous acetic acid 68%, acetonitrile 32%, and 100 min: 50% of 0.3% acetic acid aqueous solution and 50% of acetonitrile. In some embodiments, preparing the compound of formula (I) or a salt thereof further comprises liquid chromatography purification. In some embodiments, preparing the compound of formula (I) or a salt thereof further comprises subjecting the crude compound of formula (I) or a salt thereof to a liquid chromatography gradient elution with a mobile phase comprising: and (3) 0 minute: 0.05% aqueous trifluoroacetic acid 98%, acetonitrile 2%, 60 min: 78% aqueous 0.05% trifluoroacetic acid, 22% acetonitrile, and 80 min: and 100% of acetonitrile. In some embodiments, preparing the compound of formula (II) or a salt thereof further comprises subjecting the crude compound of formula (II) or a salt thereof to a liquid chromatography gradient elution with a mobile phase comprising: and (3) 0 minute: 0.1% aqueous trifluoroacetic acid 95%, acetonitrile 5%, 5 min: 0.1% aqueous trifluoroacetic acid 85%, acetonitrile 15%, and 75 minutes: 50% of 0.1% trifluoroacetic acid aqueous solution and 75% of acetonitrile. In some embodiments, preparing the compound of formula (IX) or a salt thereof further comprises subjecting the crude compound of formula (IX) or a salt thereof to a liquid chromatography gradient elution with a mobile phase comprising: and (3) 0 minute: 0.1% aqueous trifluoroacetic acid 95%, acetonitrile 5%, 5 min: 0.1% aqueous trifluoroacetic acid 90%, acetonitrile 15%, and 60 min: 0.1% trifluoroacetic acid in water 70%, acetonitrile 50%. In some embodiments, preparing the compound of formula (X) or a salt thereof, further comprises subjecting the trifluoroacetate salt of the compound of formula (X) to liquid chromatography gradient elution for salt conversion, comprising: taking 100mmol/L ammonium acetate aqueous solution as a mobile phase for 30 minutes; 0.1% acetic acid in water as mobile phase for 20 minutes; and 50% aqueous 0.1% acetic acid and 50% acetonitrile as mobile phases.
In some embodiments, the present application provides the use of a compound of formula (I) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (II) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (III) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (IV) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (V) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (VI) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (VII) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the present application provides a compound of formula (VIII) or a salt thereof. In some embodiments, the present application provides the use of a compound of formula (IX) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof. In some embodiments, the compounds of formula (X) are prepared by the methods of preparation disclosed herein.
In some embodiments, the present application provides for the use of the preparation methods disclosed herein for the preparation of radiolabeled compounds for diagnosis and therapy.
The preparation method of the application provides a high-yield and effective preparation method, and the selected purification also provides a high-yield and high-purity purification method.
Drawings
Figure 1 is a reaction scheme for the preparation of NGR (compound of formula (I)) in some embodiments of the present application.
FIG. 2 is a scheme showing the reaction to NGR-PEG in some examples of the present application4-BCN (compound of formula (II)).
FIG. 3 is a reaction scheme for the preparation of FP- (ivDde) -RGD (compound of formula (III)) in some embodiments of the present application.
FIG. 4 is a drawing of preparation of N in some examples of the present application3-NOTA-PEG4Reaction scheme of-c-RGD (compound of formula (IX)).
FIG. 5 is a reaction to NGR-PEG according to some embodiments of the present application4-click-NOTA-PEG4-c-RGD (compound of formula (X)).
Detailed Description
To further clarify the technical solutions and effects adopted by the present application to achieve the intended purpose, the following detailed description is given of specific embodiments, structures, features and effects according to the present application with reference to the accompanying drawings and preferred embodiments.
Example 1
Preparation of (mono) NGR
Referring now to fig. 1, therein is shown a reaction scheme for the preparation of NGR (compound of formula (I)) in some embodiments of the present application.
1. Resin pretreatment
(1) Swelling and washing: 334.5g of Fmoc-Rink Amide AM resin was weighed into a reaction kettle, dichloromethane was added as a solvent, and stirring was carried out for 30 minutes. Discharging the liquid in the reaction kettle. The reaction mixture was washed with N, N-Dimethylformamide (DMF) 2 times, and the liquid was drained.
(2) Removing Fmoc protection: the 20% piperidine/DMF solution was added to the reaction vessel and after stirring for 30 minutes the liquid was drained. The solution was washed 6 times with DMF and drained.
2. First to sixth C-terminal amino acid couplings
(1) Condensation: weighing a proper amount of Fmoc-amino acid and 1-hydroxybenzotriazole into a 5L reaction bottle or a beaker, adding DMF, stirring to disperse, cooling to-5-5 ℃, adding 90mL of N, N' -Diisopropylcarbodiimide (DIC) under stirring, and activating for 5-10 min. Adding into a reaction kettle containing the product of the previous step (pretreatment resin or peptide resin), and stirring and reacting for 4h at the temperature of 20-30 ℃. After the reaction was completed, the stirring was stopped and the liquid was drained. The solution was washed 6 times with DMF and drained.
(2) Removing Fmoc protection: the 20% piperidine/DMF solution was added to the reaction vessel, stirred at 20-30 ℃ for 30 minutes, after which the liquid was drained. The solution was washed 6 times with DMF and drained.
The above condensation and Fmoc protection removal cycle was performed six times (first to sixth condensation). Fmoc-amino acids added in the first to sixth condensations may be Fmoc-Cys (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gly-OH, and finally H-Gly-Cys (Trt) -Asn (Trt) -Gly-Arg (Pbf) -Cys (Trt) -Rink Amide AM resin.
Specifically, in the first condensation, 351.4g of Fmoc-Cys (Trt) -OH and 82.1g of 1-hydroxybenzotriazole can be weighed into a reaction bottle or a beaker to react and condense Cys; in the second condensation, 44.1g of Fmoc-Arg (Pbf) -OH and 9.2g of 1-hydroxybenzotriazole can be weighed into a reaction flask or beaker for reaction.
After the condensation and deprotection steps, the completion of the reaction can be detected by ninhydrin detection; after the reaction is completed, the next step can be carried out. A small amount of the product was taken out into a test tube, and 2 drops of each of detection reagent A (80g of phenol dissolved in 100mL of absolute ethanol), detection reagent B (5g of ninhydrin dissolved in 100mL of absolute ethanol), and detection reagent C (pyridine) were added, heated at 100 ℃ for 5min, and then taken out for observation. Detecting after the condensation reaction, if the resin is colorless, carrying out the next procedure; detection after deprotection reaction, such as resin color development, proceeds to the next step.
(3) Obtaining a peptide resin: after the above cycle was completed with condensation and deprotection, methylene chloride was added to the reaction vessel to wash the resin 3 times, and the liquid was discharged. Wash 3 times with MeOH and drain the liquid. And (3) pumping the resin to be half-dry in the reaction kettle, taking out, airing at room temperature to constant weight and collecting. Obtaining 755.5g of peptide resin (H-Gly-Cys (Trt) -Asn (Trt) -Gly-Arg (Pbf) -Cys (Trt) -Rink Amide AM resin).
23. Cleavage to synthesize NGR linear crude peptide (H-Gly-Cys-Asn-Gly-Arg-Cys-NH)
300.0g of peptide resin was weighed into a 5L reaction flask, and cleavage reagent (trifluoroacetic acid: 1, 2-ethanedithiol: water: TIPS ═ 90:2.5:5:2.5) was added to 10ml/g of peptide resin, and the mixture was stirred at 20 to 30 ℃ for reaction for 3 hours. Filtering, adding the filtrate into 8 times of 24L methyl tert-butyl ether (the methyl tert-butyl ether is placed in-20 + -5 deg.C for 2-4 h) and stirring for 0.5-1 h. Filtering, washing a filter cake by using methyl tert-butyl ether (2L), filtering to dryness, and airing at room temperature for 15-24 h to obtain loose off-white solid powder, namely, 99.20g of crude NGR linear peptide with the purity: 70.3 percent.
Purification of crude NGR Linear peptide
The purification was performed in such a manner that the purified liquid was collected by performing liquid chromatography.
Equipment: chengdu Gelai liquid chromatograph (model: GL6000-300 mL/min). A chromatographic column: ID 100mm 450 mm; filling: Unsil-10-100C18 UItra 1200 g; length of a packing column: 250mm, product number: GL 20190215001A; flow rate: 200 mL/min; wavelength: 210nm
Sample injection pretreatment: the crude NGR linear peptide was dissolved in water under stirring and then filtered through a 0.45 μm filter to obtain a sample solution. The concentration of the sample solution was 0.04 g/mL.
Column equilibration was performed with 2% phase B until baseline stability, equilibration for 15 minutes. The sample is loaded by using a C pump, and the sample loading flow rate is 200 mL/min. And after the sample loading is finished, adding pure water to wash the sample injection pipeline for 2 min. After the sample loading is finished, gradient elution is carried out: and (3) 0 minute: phase a 98%, phase B2%, 60 min: phase a 78%, phase B22%, 80 min: 0% of phase A and 100% of phase B. Wherein phase A is 0.05% trifluoroacetic acid in water (which can be obtained by measuring 0.5mL of trifluoroacetic acid in 1L of water) and phase B is acetonitrile.
A total of 5 needles were injected, and about 20g of sample was applied to each needle.
Collecting target peaks by stages, detecting by HPLC to obtain fractions, and collecting purified solution with purity of 98.0% or more to obtain purified solution of NGR linear peptide 6.34L (containing NGR linear peptide 31.9 g). Purity: 99.0 percent; yield: 33.2 percent.
5. Formation of a disulfide bond to obtain NGR (Compound of formula (I))
1.27L DMSO was added to the purified NGR line peptide, and the reaction was carried out for 36 to 72 hours under stirring.
Samples were taken and HPLC was performed to confirm completion of the reaction. 27.54g of the compound of formula (I) are obtained after the reaction has ended.
Purity: 92.9 percent; yield: 86.6 percent. Mass spectrum 606.2 (theoretical 605.2).
(di) NGR-PEG4Preparation of-BCN
Referring to FIG. 2, in some embodiments of the present application, NGR and BCN-PEG are combined4-NHS reaction to give NGR-PEG4-BCN (compound of formula (II)).
4Synthesis of NGR-PEG-BCN (Compound of formula (II))
(1) Under stirring, NGR (compound of formula (I)) and acetonitrile (3.58L) were added;
(2) using NaHCO3Adjusting the pH value of the reaction solution to 7-8;
(3) weighing bicyclo [6.1.0]Nonoyne-PEG4-N-hydroxysuccinimide (BCN-PEG)4-NHS) (25.7g), adding acetonitrile for dilution and dissolution, then dropwise adding the mixture into the reaction solution, enabling the system to be colorless and clear, and stirring at room temperature for reaction;
(4) after 5h of reaction, samples were taken for HPLC. Purity of reaction solution: 82.4 percent
4Purification of NGR-PEG-BCN (Compound of formula (II))
The purification was performed in such a manner that the purified liquid was collected by performing liquid chromatography.
Equipment: chengdu Gelai liquid chromatograph (model: GL6000-300 mL/min). A chromatographic column: ID 100mm 450 mm; filling: Unsil-10-100C18 UItra 1200 g; length of a packing column: 250mm, product number: GL 20190215001A; flow rate: 200 mL/min; wavelength: 210nm
Sample injection pretreatment: about 10.8L of the reaction solution was concentrated to 7.2L at 35 ℃ and filtered through a 0.45 μm filter and then applied to 1.8L of the reaction solution (containing about 12.0g of NGR-PEG) 4 times4-BCN)。
Column equilibration was performed with 2% phase B until baseline stability, equilibration for 15 minutes. The sample is loaded by using a C pump, and the sample loading flow rate is 200 mL/min. A total of 5 needles were injected, and about 20g of sample was applied to each needle. And after the sample loading is finished, adding pure water to wash the sample injection pipeline for 2 min. Then a gradient elution was run: and (3) 0 minute: 95% of phase A, 5% of phase B, 5 min: 85% of phase A, 15% of phase B, 75 min: 50% of phase A and 50% of phase B. Wherein phase A is 0.1% trifluoroacetic acid in water (which can be obtained by measuring 1mL of trifluoroacetic acid in 1L of water) and phase B is acetonitrile.
(4) Collecting target peaks by stages, detecting by HPLC to obtain fractions, collecting purified solution with purity of more than or equal to 97.0% for use to obtain 5.2L of purified solution, and calibrating with reference substance to obtain INT226.0 g. Purity: 97.8 percent; yield: 59.0 percent; mass spectrum 1028.5 (theoretical value 1028.4).
(III) preparation of FP- (ivDde) -RGD
FIG. 3 is a reaction scheme for the preparation of FP- (ivDde) -RGD (compound of formula (III)) in some embodiments of the present application.
1. Resin pretreatment
285.9g of dichlorotrityl chloride resin was weighed into a reaction vessel, methylene chloride was added as a solvent, and the mixture was stirred for 30 minutes to discharge the liquid in the reaction vessel.
2. First C-terminal amino acid coupling
(1) Condensation: weighing 269.9g of Fmoc-Gly-OH into a 5L reaction bottle, adding dichloromethane, stirring to disperse the raw materials, adding the raw materials into a reaction kettle filled with pretreated resin, adding 315mL of N, N-diisopropylethylamine, controlling the temperature to be 20-30 ℃, stirring to react for 4 hours, adding 0.2L of methanol-blocked resin, reacting for 0.5 hour, discharging reaction liquid, washing with DMF for 6 times, and discharging the liquid to obtain the Fmoc-Gly-CTC resin.
(2) Removing Fmoc protection: to the reaction vessel containing the product of the previous step (pre-treatment resin or peptide resin) was added 20% piperidine/DMF solution and stirred at 20-30 ° for 30 minutes, after which the liquid was drained. The solution was washed 6 times with DMF and drained.
3. Second to fifth C-terminal amino acid couplings
(1) Condensation: weighing a proper amount of Fmoc-amino acid and 1-hydroxybenzotriazole into a 5L reaction bottle or a beaker, adding DMF, stirring for dispersing, cooling to-5-5 ℃, and adding 90mL of DIC for activating for 5-10 min under stirring. Adding into a reaction kettle containing the product of the previous step (pretreatment resin or peptide resin), and stirring and reacting for 4h at the temperature of 20-30 ℃. After the reaction was completed, the stirring was stopped and the liquid was drained. The solution was washed 6 times with DMF and drained.
(2) Removing Fmoc protection: the 20% piperidine/DMF solution was added to the reaction vessel, stirred at 20-30 ° for 30 minutes, after which the liquid was drained. The solution was washed 6 times with DMF and drained.
The condensation and Fmoc protection removal cycle is carried out four times (second to fifth condensations), wherein the Fmoc-amino acids added in the second to fifth condensations can be Fmoc-Arg (Pbf) -OH, Fmoc-Lys (ivDde) -OH, Fmoc-D-Tyr (tBu) -OH and Fmoc-Asp (OtBu) -OH respectively, and finally, Fmoc-Asp (OtBu) -D-Tyr (tBu) -Lys (ivDde) -Arg (Pbf) -Gly-CTC resin is obtained.
Specifically, in the second condensation, 333.8g of Fmoc-Arg (Pbf) -OH and 69.7g of 1-hydroxybenzotriazole can be weighed into a reaction flask or beaker for reaction.
After the condensation and deprotection steps, the completion of the reaction can be detected by ninhydrin detection; after the reaction is completed, the next step can be carried out. A small amount of the product was taken out into a test tube, and 2 drops of each of detection reagent A (80g of phenol dissolved in 100mL of absolute ethanol), detection reagent B (5g of ninhydrin dissolved in 100mL of absolute ethanol), and detection reagent C (pyridine) were added, heated at 100 ℃ for 5min, and then taken out for observation. Detecting after the condensation reaction, if the resin is colorless, carrying out the next procedure; detection after deprotection reaction, such as resin color development, proceeds to the next step.
(3) Obtaining a peptide resin: after the above cycle was completed with condensation and deprotection, methylene chloride was added to the reaction vessel to wash the resin 3 times, and the liquid was discharged. Wash 3 times with MeOH and drain the liquid. And (3) pumping the resin to be half-dry in the reaction kettle, taking out, airing at room temperature to constant weight and collecting. To obtain 476.0g of peptide resin (H-Asp (OtBu) -D-Tyr (tBu) -Lys (ivDde) -Arg (Pbf) -Gly-CTC resin).
4. Cleavage to synthesize FP- (ivDde) -RGD (H-Asp (OtBu) -D-Tyr (tBu) -Lys (ivDde) -Arg
(Pbf)-Gly-OH)
(1) Under stirring, 4000mL of dichloromethane and 20mL of trifluoroacetic acid are added into a reaction bottle;
(2) under the condition of stirring, adding 400.0g of peptide resin, reacting at room temperature for 3 hours, filtering the resin by using a sand core funnel after the reaction is finished, washing the resin by using dichloromethane, and combining the filtrates;
(3) dropwise adding N, N-diisopropylethylamine to adjust the pH value to 7-8, and concentrating under reduced pressure at 35-40 ℃ until no fraction flows out;
(4) adding 1200mL of ethyl acetate, stirring for dissolving, washing by using 1000mL of saturated saline solution, then separating liquid, adding 1000mL of saturated saline solution again into the organic phase, washing by using 1000mL of saturated saline solution, then separating liquid, adding 120.0g of anhydrous sodium sulfate into the organic phase, drying for 1 hour, concentrating the organic phase at 35-40 ℃ until no fraction flows out, adding 800mL of ethyl acetate for dissolving, dropwise adding into 8000mL of n-heptane, stirring for crystallizing for 10-20 hours;
(5) filtering, leaching a filter cake for three times by using 400mL of n-heptane, continuously pumping for 5-8 hours, and drying in vacuum at 35 ℃ until the weight is constant to obtain 181.7g of white-like solid. Purity: 91.6%, yield: 59.7%, Mass Spectrum 1027.6 (theoretical value 1027.6).
(IV) N3-NOTA-PEG4Preparation of-c-RGD
Referring to FIG. 4, in some embodiments of the present application, FP- (ivDde) -RGD (compound of formula (III)) is used for preparing N3-NOTA-PEG4Reaction scheme of-c-RGD (compound of formula (IX)).
Synthesis of FP- (ivDde) -c-RGD (Compound of formula (IV))
(1) Dichloromethane and 90.3g FP- (ivDde) -RGD (compound of formula (III)) are added into a reaction bottle, stirred and dissolved, and 21.0g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI. HCl) are added for reaction for 24 hours at room temperature;
(2) monitoring the reaction by TLC or HPLC, after the reaction is finished, condensing the system until no fraction flows out, adding 450mL of DMF into the obtained concentrate for dissolving, dropwise adding the solution into pure water to separate out white solid, and continuously stirring for crystallization for 15-20 h;
(3) filtering, leaching the filter cake with purified water for three times, putting the filter cake into an air-blast drying oven to dry for 20 hours at the temperature of 40 ℃, and collecting to obtain 85.2g of white-like solid. Purity: 85.9%, yield: 96.7%, mass spectrum 1189.6 (theoretical value 1189.6).
Synthesis of FP-c-RGD (Compound of formula (V))
(1) Adding 400mL of DMF and 8mL of hydrazine hydrate into a reaction bottle while stirring;
(2) adding 80.2g of FP- (ivDde) -c-RGD (compound of formula (IV)) into a reaction bottle, stirring to dissolve, and reacting for 5h at 10-30 ℃;
(3) monitoring the reaction by HPLC, and after the reaction is finished, dropwise adding the reaction solution into methyl tert-butyl ether/n-heptane (7:3/v: v) (8400mL) for crystallization for 5 h;
(4) filtering, washing the filter cake for three times by using n-heptane, pumping, putting the filter cake into an air-blast drying oven, and drying for 15-24 h at 35 ℃ to obtain 65.3g of off-white solid. Purity 85.3%, yield 99.2%, mass spectrum 983.5 (theory 983.5).
4Synthesis of Fmoc-NH-PEG-FP-c-RGD (Compound of formula (VI))
(1) DMF (325mL) was added to the reaction flask, followed by 64.7g of FP-c-RGD (compound of formula (V)), 32.0g of Fmoc-PEG4Propionic Acid (PA), 34.2g of PyBop, dissolved with stirring;
(2) dropwise adding N, N-diisopropylethylamine (23mL), and stirring at 10-30 ℃ for reacting for 2 h;
(3) monitoring the reaction by HPLC or TLC, after the reaction is finished, dropwise adding the reaction solution into methyl tert-butyl ether (9750mL), stirring and crystallizing for 2-5 h;
(4) filtering, washing the filter cake for 6 times by using methyl tert-butyl ether, then pumping to dry, putting the filter cake into a forced air drying oven, carrying out forced air drying at 35 ℃ for 15-24 h, and collecting to obtain 84.7g of white-like solid powder. Purity: 83.0%, yield: 88.3%, Mass Spectrum 1453.7 (theoretical 1452.7).
2 4Synthesis of NH-PEG-FP-c-RGD (Compound of formula (VII))
(1) To a reaction flask, 201.6mL of DMF and 50.4mL of piperidine were added and 84.1g of Fmoc-NH-PEG was added with stirring4adding-FP-c-RGD into a reaction bottle, stirring and dissolving, and reacting for 2h at 10-30 ℃;
(2) monitoring the reaction solution by TLC or HPLC, after the reaction is finished, dropwise adding the reaction solution into 7560mL of methyl tert-butyl ether, and continuously stirring for 1-2 h after white solid is separated out;
(3) filtering, washing the filter cake for three times by using methyl tert-butyl ether, then pumping to dry, putting the filter cake into a forced air drying oven, drying for 15-24 h at 35 ℃, and collecting to obtain 61.2g of white-like solid powder. Purity: 85.4%, yield: 86.1%, Mass Spectrum 1231.7 (theoretical 1230.7).
3 2 4Synthesis of N-NOTA (tBu) -PEG-FP-c-RGD (Compound of formula (VIII))
(1) 300mL of DMF was added to the reaction flask, followed by 60.0g of NH2-PEG4-FP-c-RGD, 25.2g of NOTA (tBu)2-N327.3g of PyBop, dissolved with stirring.
(2) Dropwise adding 19.2mL of N, N-diisopropylethylamine, and stirring at 10-30 ℃ for reaction for 5 h;
(3) monitoring the reaction by HPLC, after the reaction is finished, dropwise adding the reaction solution into 9000mL of methyl tert-butyl ether, and stirring for crystallization for 2-5 h;
(4) filtering, washing the filter cake for three times by using 300mL of methyl tert-butyl ether, draining, putting into an air-blast drying oven, carrying out air-blast drying at 35 ℃ for 15-24 h, and collecting to obtain 67.5g of white-like solid powder. Purity: 78.8%, yield: 81.6%, Mass Spectrum 1697.9 (theoretical value 1696.9).
3 4Synthesis of N-NOTA-PEG-c-RGD (Compound of formula (IX))
(1) Under the condition of stirring, 627mL of trifluoroacetic acid, 16.5mL of water and 16.5mL of triisopropylsilane are added into a reaction bottle;
(2) 66.0g of N were added3-NOTA(tBu)2-PEG4Carrying out stirring reaction for 2-3 h at room temperature on-FP-c-RGD;
(3) after the reaction is finished, dropwise adding the reaction liquid into 6600mL of methyl tert-butyl ether for crystallization for 2-5 h;
(4) filtering, washing the filter cake for three times by using methyl tert-butyl ether, then pumping, airing the filter cake for 15-24 h at room temperature, and collecting to obtain 48.9g of white-like solid powder.
Purity: 80.4%, yield: 102.9 percent.
3 4Purification of N-NOTA-PEG-c-RGD (Compound of formula (IX))
The purification was performed in such a manner that the purified liquid was collected by performing liquid chromatography.
Equipment: chengduli preparative liquid chromatograph (model: GL6000-300mL/min)
Preparing a chromatographic column: ID 100mm 450 mm; filling: Unsil-10-100C18 UItra 1200 g; length of a packing column: 250 mm; product numbering: GL 20190215001A; flow rate: 200 mL/min; wavelength: 210nm
Sample injection pretreatment: 45.2g of N3-NOTA-PEG4-c-RGD (compound of formula (IX)) was dissolved in 2250mL of water, filtered through a 0.45 μm filter and injected.
Column equilibration was performed with 5% phase B until baseline stability, equilibration for 15 minutes. The sample is loaded by using a C pump, and the sample loading flow rate is 200 mL/min. A total of 5 needles were injected, each loaded with about 9.0 g. After the end of the loading, gradient elution was run: and (3) 0 minute: 95% of phase A, 5% of phase B, 5 min: phase a 90%, phase B10%, 60 min: 70% of phase A and 30% of phase B. Wherein phase A is 0.1% trifluoroacetic acid in water (which can be obtained by measuring 1mL of trifluoroacetic acid in 1L of water) and phase B is acetonitrile.
Collecting target peaks by stages, detecting by HPLC, mixing purified solutions with purity of 98.0% or more and single impurity of less than 0.5% to obtain 11.70L of purified solution, and calibrating to contain N3-NOTA-PEG4-c-RGD (compound of formula (IX)) 16.86 g. Purity: 99.0%, yield: 37.3%, mass spectrum 1220.6 (theoretical value 1220.6).
(V) NGR-PEG4-click-NOTA-PEG4Preparation of-c-RGD
Referring now to FIG. 5, in some embodiments of the present application, the PEG is introduced via NGR-PEG4BCN (Compound of formula (II)) with N3-NOTA-PEG4c-RGD (compound of formula (IX)) to obtain NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X)).
4 4Synthesis of NGR-PEG-click-NOTA-PEG-c-RGD (Compound of formula (X))
(1) 16.00g of NGR-PEG4-a trifluoroacetic acid/acetonitrile mixture of BCN (compound of formula (II) (HPLC gradient elution purification of compound of formula (II)) with 16.20g of N3-NOTA-PEG4mixing-c-RGD (compound of formula (IX)), and reacting at 10-30 deg.C for 15-20 h.
(2) The reaction was monitored by HPLC and the final product was purified after filtration through a 0.45 μm filter.
4 4Purification of NGR-PEG-click-NOTA-PEG-c-RGD (Compound of formula (X))
The purification was performed in such a manner that the purified liquid was collected by performing liquid chromatography.
Equipment: chengduli preparative liquid chromatograph (model: GL6000-300mL/min)
Preparing a chromatographic column: ID 100mm 450 mm; filling: Unsil-10-100C18 UItra 1200 g; length of a packing column: 250 mm; product numbering: GL 20190215001A; flow rate: 200 mL/min; wavelength: 210nm
Column equilibration was performed with 5% phase B until baseline stability, equilibration for 15 minutes. The sample is loaded by using a C pump, and the sample loading flow rate is 200 mL/min. Co-sample injection of 4 needles, each needle being 2.9L (containing about NGR-PEG)4-click-NOTA-PEG4-c-RGD (compound of formula (X) 8.0 g). After the sample loading is finished, adding pure water to wash the sample injection pipeline for 2 minutes, and then operating gradient elution: and (3) 0 minute: 95% of phase A, 5% of phase B, 5 min: 88% of phase A, 12% of phase B, 85 min: phase a 68%, phase B32%, 100 min: 50% of phase A and 50% of phase B. Wherein phase A is 0.3% acetic acid water solution (obtained by adding glacial acetic acid 3mL into 1L water), and phase B is acetonitrile.
And collecting target fractions in sections, performing HPLC inspection, collecting and merging 8.20L of qualified purified liquid with the purity of more than or equal to 98 percent. Purity: 99.8 percent.
4 4NGR-PEG-click-NOTA-PEG-c-RGD (compound of formula (X)) salt transfer
The salt transfer was performed by performing liquid chromatography.
Preparing a chromatographic column: ID 100mm 450 mm; filling: Unsil-10-100C18 UItra 1200 g; length of a packing column: 250 mm; product numbering: GL 20190215001A; flow rate: 200 mL/min; wavelength: 210 nm.
Sample pretreatment: NGR-PEG4-click-NOTA-PEG48.2L of purified solution of-c-RGD (compound of formula (X)) was concentrated under reduced pressure at 30 ℃ to 6.85L and then loaded.
Mobile phase: phase A1: 0.1% aqueous acetic acid, phase a 2: 100mmol/L ammonium acetate aqueous solution, B phase: and (3) acetonitrile.
The column was equilibrated with 5% phase B and 95% phase A1 for 15 min. The sample is loaded by using a C pump, and the sample loading flow rate is 200 mL/min. After the loading, the A2 phase is used as the mobile phase for 30 minutes to carry out salt conversion, the A1 phase is used as the mobile phase for 20 minutes to wash, and finally, the A1 phase and the B phase are 50% as the mobile phases to stop after the main peak is received, so that the elution is carried out.
All target peaks were collected to obtain 3.2L of the trans-salt solution. After vacuum concentration at 30 ℃, the sample was loaded and lyophilized to obtain 23.0g of a white-like solid fluffy solid powder. Purity: 99.7%, yield: 78.0%, Mass Spectrum 2250.1 (theoretical value 2249.1).
Example 2
The synthesis of the compounds of formulae (I) to (X) is the same as in example 1, except that the A phase is 0.3% aqueous trifluoroacetic acid when the compound of formula (X) is purified.
And collecting target fractions in sections, performing HPLC inspection, and collecting and combining the fractions with the purity of more than or equal to 98 percent to obtain purified liquid with the purity of 99.3 percent.
It can be seen that the purified solution obtained in example 1 was high in purity.
The above embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto, and any insubstantial changes and substitutions made by those skilled in the art based on the present application are intended to be covered by the present application.
Claims (10)
1. A method of preparing a compound, comprising:
preparing a compound of formula (I) or a salt thereof;
preparing a compound of formula (II) or a salt thereof from a compound of formula (I) or a salt thereof;
preparing a compound of formula (III) or a salt thereof;
preparing a compound of formula (IV) or a salt thereof from a compound of formula (III) or a salt thereof;
preparing a compound of formula (V) or a salt thereof from a compound of formula (IV) or a salt thereof;
preparing a compound of formula (VI) or a salt thereof from a compound of formula (V) or a salt thereof;
preparing a compound of formula (VII) or a salt thereof from a compound of formula (VI) or a salt thereof;
preparing a compound of formula (VIII) or a salt thereof from a compound of formula (VII) or a salt thereof;
preparing a compound of formula (IX) or a salt thereof from a compound of formula (VIII) or a salt thereof; and
preparing a compound of formula (X) or a salt thereof from a compound of formula (II) or a salt thereof and a compound of formula (IX) or a salt thereof,
2. the method according to claim 1, wherein the reaction mixture,
the compound of formula (X) or a salt thereof is prepared from a compound of formula (II) or a salt thereof and a compound of formula (IX) or a salt thereof, and comprises the following steps:
reacting the compound of formula (II) or the salt thereof and the compound of formula (IX) or the salt thereof in a trifluoroacetic acid/acetonitrile mixture at 10-30 ℃ for 15-20 hours.
3. The method according to claim 1, wherein the reaction mixture,
the compound of formula (II) or a salt thereof is prepared from the compound of formula (I) or a salt thereof, and comprises the following steps:
in acetonitrile, under the condition of pH value of 7-8, the compound of formula (I) or its salt and BCN-PEG4-NHS contact; and/or
The compound of formula (IV) or a salt thereof is prepared from a compound of formula (III) or a salt thereof, and comprises the following steps:
contacting a compound of formula (III) or a salt thereof with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or a salt thereof in dichloromethane; and/or
The compound of formula (V) or a salt thereof prepared from the compound of formula (IV) or a salt thereof comprises:
contacting a compound of formula (IV) or a salt thereof with hydrazine hydrate in N, N-dimethylformamide; and/or
The compound of formula (VI) or a salt thereof prepared from the compound of formula (V) or a salt thereof comprises:
reacting a compound of formula (V) or a salt thereof with Fmoc-PEG in N, N-dimethylformamide4-propionic acid, PyBop and N, N-diisopropylethylamine; and/or
The compound of formula (VII) or the salt thereof prepared by the compound of formula (VI) or the salt thereof comprises:
contacting a compound of formula (VI) or a salt thereof with piperidine in N, N-dimethylformamide; and/or
The compound of formula (VIII) or a salt thereof prepared from the compound of formula (VII) or a salt thereof comprises:
reacting a compound of formula (VII) or a salt thereof with NOTA (tBu) in N, N-dimethylformamide2-N3And PyBop contact; and/or
The compound of formula (IX) or a salt thereof prepared from the compound of formula (VIII) or a salt thereof comprises:
a compound of formula (VIII) or a salt thereof, and water are contacted with triisopropylsilane in trifluoroacetic acid.
4. The method according to claim 3,
the compound of the formula (III) or the salt thereof is contacted with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or the salt thereof, 90.3 +/-10 weight percent of the compound of the formula (III) or the salt thereof is reacted with 21.0 +/-10 weight percent of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or the salt thereof for 24 hours at room temperature; and/or
The compound of formula (IV) or the salt thereof is contacted with hydrazine hydrate, 80.2 plus or minus 10 percent of the compound of formula (IV) or the salt thereof reacts with 8 plus or minus 10 percent of the hydrazine hydrate for 5 hours at the temperature of 10-30 ℃; and/or
The compound of the formula (V) or the salt thereof and Fmoc-PEG4-propionic acid, PyBop and N, N-diisopropylethylamine by contacting 64.7 + -10% by weightParts of a compound of formula (V) or a salt thereof and 32.0 + -10% parts by weight of Fmoc-PEG4-propionic acid, 34.2 ± 10% by weight of PyBop and 17.02 ± 10% by weight of N, N-diisopropylethylamine were added dropwise and reacted at 10-30 ℃ for 2 hours; and/or
The compound of formula (VI) or the salt thereof is contacted with piperidine, 84.1 plus or minus 10 percent of the compound of formula (VI) or the salt thereof and 41.88 plus or minus 10 percent of the piperidine react for 2 hours at the temperature of 10-30 ℃; and/or
The compound of formula (VII) or a salt thereof and NOTA (tBu)2-N3And PyBop by contacting 60.0 + -10% by weight of the compound of formula (VII) or a salt thereof with 25.2 + -10% by weight of NOTA (tBu)2-N327.3 +/-10% of PyBop by weight is reacted for 2 hours at the temperature of 10-30 ℃; and/or
The compound of formula (VIII) or the salt thereof and water are contacted with triisopropylsilane, 66.0 +/-10 weight percent of the compound of formula (VIII) or the salt thereof, 16.5 +/-10 weight percent of water and 12.75 +/-10 weight percent of triisopropylsilane are reacted for 2 to 3 hours at room temperature.
5. The method according to claim 1, wherein the reaction mixture,
the preparation of the compound of formula (I) or a salt thereof comprises:
contacting the first resin with protected amino acid, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a condensed first resin;
contacting the condensed first resin with piperidine to obtain a deprotected first resin;
contacting the deprotected first resin with trifluoroacetic acid to obtain a linear peptide of a compound of formula (I) or a salt thereof; and
contacting the linear peptide of the compound of formula (I) or the salt thereof with DMSO to obtain the compound of formula (I) or the salt thereof; and/or
The preparation of the compound of formula (III) or a salt thereof comprises:
contacting the second resin with protected amino acid, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a condensed second resin;
contacting the condensed second resin with piperidine to obtain a deprotected second resin; and
contacting the deprotected second resin with trifluoroacetic acid to obtain the compound of formula (III) or a salt thereof.
6. The method according to claim 5,
the preparation of the compound of formula (I) or a salt thereof comprises:
contacting the first resin with Fmoc-Cys (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a first condensed resin;
contacting the first resin after the first condensation with piperidine to obtain a first resin after first deprotection;
contacting the first deprotected resin with Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a second condensed resin;
contacting the second condensed first resin with piperidine to obtain a second deprotected first resin;
contacting the second deprotected first resin with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a third condensed first resin;
contacting the third condensed first resin with piperidine to obtain a third deprotected first resin;
contacting the third deprotected first resin with Fmoc-Asn (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fourth condensed first resin;
contacting the fourth condensed first resin with piperidine to obtain a fourth deprotected first resin;
contacting the fourth deprotected first resin with Fmoc-Cys (Trt) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fifth condensed first resin;
contacting the fifth condensed first resin with piperidine to obtain a fifth deprotected first resin;
contacting the fifth deprotected first resin with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a sixth condensed first resin;
contacting the sixth condensed first resin with piperidine to obtain a sixth deprotected first resin;
contacting the sixth deprotected first resin with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or a salt thereof; and
contacting the linear peptide of the compound of formula (I) or the salt thereof with DMSO to obtain the compound of formula (I) or the salt thereof; and/or
The preparation of the compound of formula (III) or a salt thereof comprises:
contacting the second resin with Fmoc-Gly-OH and N, N-diisopropylethylamine, and then contacting with methanol to obtain a first condensed second resin;
contacting the second resin after the first condensation with piperidine to obtain a second resin after first deprotection;
contacting the first deprotected second resin with Fmoc-Arg (Pbf) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a second condensed second resin;
contacting the second resin after the second condensation with piperidine to obtain a second resin after second deprotection;
contacting the second deprotected second resin with Fmoc-Lys (ivDde) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a third condensed second resin;
contacting the third condensed second resin with piperidine to obtain a third deprotected second resin;
contacting the third deprotected second resin with Fmoc-D-Tyr (tBu) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fourth condensed second resin;
contacting the fourth condensed second resin with piperidine to obtain a fourth deprotected second resin;
contacting the fourth deprotected second resin with Fmoc-Asp (OtBu) -OH, 1-hydroxybenzotriazole and N, N' -diisopropylcarbodiimide to obtain a fifth condensed second resin;
contacting the fifth condensed second resin with piperidine to obtain a fifth deprotected second resin; and
contacting the fifth deprotected second resin with trifluoroacetic acid to obtain the compound of formula (III) or a salt thereof; and/or
The first resin is Rink Amide AM resin, and/or
The second resin is a dichlorotrityl chloride resin.
7. The process of claim 1, wherein the preparation of the compound of formula (X) or a salt thereof further comprises:
subjecting the crude compound of formula (X) or a salt thereof to liquid chromatography gradient elution with a mobile phase comprising:
and (3) 0 minute: 95% of 0.3% acetic acid aqueous solution, 5% of acetonitrile,
5 minutes: 88% of 0.3% acetic acid aqueous solution, 12% of acetonitrile,
85 minutes: 68% of a 0.3% aqueous acetic acid solution, 32% of acetonitrile, and
and (3) 100 minutes: 50% of 0.3% acetic acid aqueous solution and 50% of acetonitrile.
8. The method according to claim 1, wherein the reaction mixture,
the preparation of the compound of formula (I) or the salt thereof further comprises the step of carrying out liquid chromatography gradient elution on the crude product of the compound of formula (I) or the salt thereof, wherein the mobile phase comprises:
and (3) 0 minute: 98 percent of 0.05 percent trifluoroacetic acid aqueous solution and 2 percent of acetonitrile,
and (5) 60 minutes: 78% aqueous 0.05% trifluoroacetic acid solution, 22% acetonitrile, and
80 minutes: 100% of acetonitrile; and/or
The preparation of the compound of formula (II) or the salt thereof further comprises the step of carrying out liquid chromatography gradient elution on the crude product of the compound of formula (II) or the salt thereof, wherein the mobile phase comprises:
and (3) 0 minute: 95% of 0.1% trifluoroacetic acid aqueous solution, 5% of acetonitrile,
5 minutes: 85% of 0.1% trifluoroacetic acid aqueous solution, 15% acetonitrile, and
and (3) 75 minutes: 50% of 0.1% trifluoroacetic acid aqueous solution and 75% of acetonitrile; and/or
The preparation of the compound of formula (IX) or a salt thereof further comprises subjecting the crude compound of formula (IX) or a salt thereof to liquid chromatography gradient elution, wherein the mobile phase comprises:
and (3) 0 minute: 95% of 0.1% trifluoroacetic acid aqueous solution, 5% of acetonitrile,
5 minutes: 90% aqueous 0.1% trifluoroacetic acid solution, 15% acetonitrile, and
and (5) 60 minutes: 70% of 0.1% trifluoroacetic acid aqueous solution and 50% of acetonitrile; and/or
The preparation of the compound of formula (X) or the salt thereof, further comprises the step of performing liquid chromatography gradient elution and salt transfer on the trifluoroacetate of the compound of formula (X), and comprises the following steps:
taking 100mmol/L ammonium acetate aqueous solution as a mobile phase for 30 minutes;
0.1% acetic acid in water as mobile phase for 20 minutes; and
50% aqueous 0.1% acetic acid and 50% acetonitrile were used as mobile phases.
9. Use of a compound of formula (I) or a salt thereof, for the preparation of a compound of formula (II) or a salt thereof, a compound of formula (III) or a salt thereof, a compound of formula (IV) or a salt thereof, a compound of formula (V) or a salt thereof, a compound of formula (VI) or a salt thereof, a compound of formula (VII) or a salt thereof, a compound of formula (VIII) or a salt thereof, and/or a compound of formula (IX) or a salt thereof, for the preparation of a compound of formula (X) or a salt thereof by a preparation process as claimed in any one of claims 1 to 8.
10. Use of the preparation process according to any one of claims 1 to 8 for the preparation of radiolabeled compounds for diagnosis and therapy.
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Citations (12)
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