Preparation method of a leuprorelin-related compound

Abstract

The present invention discloses a preparation method of a leuprorelin-related compound. In this method, 2-chlorotrityl chloride resin (2-CTC resin) is used as a solid-phase carrier, which is coupled with an amino acid to prepare fragment one; then it is coupled with an amine compound to obtain fragment two, and then the side-chain protecting groups of fragment two are removed by cleavage with trifluoroacetic acid, and the leuprorelin-related compound is purified. The reaction operation of the present invention is simple, the post-treatment is convenient, the yield is high, and it has high reference value for leuprorelin. Moreover, through the preparation of the leuprorelin-related compound, the present invention provides an important basis for the scientific evaluation of the efficacy, pharmacological research, pharmacokinetics, etc. of leuprorelin, facilitating more in-depth research on its metabolic process and pharmacokinetics in the human body, and endowing it with better medicinal prospects.

Description

Technical Field

[0001] The present invention relates to a preparation method of gonadotropin drugs, and particularly to a preparation method of leuprorelin-related compounds, belonging to the field of organic synthesis. Technical Background

[0002] Leuprorelin is a highly active analogue (GnRHa) of luteinizing hormone-releasing hormone (GnRH) produced by the hypothalamus. It is a synthetic water-soluble nonapeptide with both ends closed. It was developed by a Japanese company and approved for marketing in 1994. The US FDA approved the leuprorelin sustained-release preparation product in January 2002, with the trade name "Eligard". This drug mainly treats various sex hormone-related diseases such as prostate cancer, breast cancer, and endometriosis by regulating the endocrine system.

[0003] Currently, there is no literature reporting the preparation method of this compound. This compound is the dehydration elimination product of leuprorelin, characterized by having a special amino acid dehydroalanine (Dha) in its structure and belonging to synthetic impurities. The synthesis of this compound has great reference significance for the impurity research of leuprorelin. Summary of the Invention

[0004] Object of the Invention: Aiming at the above-mentioned prior art, the object of the present invention is to provide a preparation method of leuprorelin-related compounds.

[0005] Technical Solution: The preparation method of leuprorelin-related compounds described in the present invention includes the following steps:

[0006] (1) Using 2-chlorotrityl chloride resin as a solid-phase carrier, coupling with an amino acid to prepare Fragment 1;

[0007] (2) Carboxyl liquid-phase coupling with ethylamine: Coupling the Fragment 1 obtained in step (1) with an amine compound to obtain Fragment 2 ((Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-NH-CH2-CH3));

[0008] (3) Cleaving the side-chain protecting groups of the Fragment 2 obtained in step (2) with trifluoroacetic acid and purifying to obtain leuprorelin-related compounds;

[0009] Among them, the Fragment 1 is

[0010] Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH or Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH, where the amino acid is 9-fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH) or N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH), and the amine-containing hydrochloride is ethylamine hydrochloride (NH2-CH2-CH3·HCl) or L-proline ethylamide hydrochloride (H-Pro-NHET·HCL).

[0011] Among them, in step (2), the coupling solvent for the coupling reaction is N,N-dimethylformamide (DMF), and the coupling agent for the coupling reaction is diisopropylcarbodiimide (DIC) + A or N,N-diisopropylethylamine (DIEA) + B, where A is 1-hydroxybenzotriazole (HOBT), and B is O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), or O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0012] Among them, the preferred coupling agent is DIC + HOBT, and the products of other coupling reagents are more complex.

[0013] Among them, in step (2), the temperature of the coupling reaction is 20 - 30 °C, and the time of the coupling reaction is 12 - 24 h.

[0014] Among them, after the coupling condensation in step (2) is completed, the reaction solution is added to 10 times the volume of water and then extracted with dichloromethane. After the organic layer is dried with anhydrous sodium sulfate, it is distilled under reduced pressure.

[0015] Among them, in step (3), the reagents used for protection in cleavage are trifluoroacetic acid (TFA), water, triisopropylsilane, and phenol solution. Among them, the volume ratio of trifluoroacetic acid is 80% - 90%, the volume ratio of water is 5% - 10%, the volume ratio of triisopropylsilane is 5% - 10%, and the volume ratio of phenol is 5% - 10%.

[0016] Among them, the preferred volume ratio of trifluoroacetic acid, water, triisopropylsilane, and phenol is 85:5:5:5.

[0017] Among them, in step (3), the reaction temperature for cracking is 10-20 °C, and the reaction time is 1-2 h. After the reaction is completed, the target product precipitates out with ether and is dried under vacuum.

[0018] Among them, in step (3), the purification is carried out by column chromatography. When performing column chromatography purification, the mobile phase A is 0.1% aqueous TFA solution, the mobile phase B is 0.1% acetonitrile solution of TFA, and the mobile phase gradient is 15%-45%.

[0019] Among them, the fragment one

[0020] The preparation of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH includes the following steps:

[0021] 1) Coupling 2-chlorotrityl chloride resin (2-CTC-Resin) with 9-fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH) to obtain Fmoc-Pro-Resin, and removing the 9-fluorenylmethoxycarbonyl (Fmoc) protection to obtain H-Pro-Resin;

[0022] 2) Coupling H-Pro-Resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) to obtain Fmoc-Arg(Pbf)-Pro-Resin;

[0023] 3) Sequentially condensing and coupling Fmoc-Arg(Pbf)-Pro-Resin with N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH), N-fluorenylmethoxycarbonyl-D-leucine (Fmoc-D-Leu-OH), fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH), N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), L-pyroglutamic acid (H-Pyr-OH), and then cleaving with a cleavage reagent to obtain fragment one.

[0024] Among them, in step 1), the coupling agent for coupling 2-chlorotrityl chloride resin (2-CTC-Resin) with 9-fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH) is DIEA.

[0025] Among them, in step 2), the coupling agent for coupling the H-Pro-Resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) is DIC+A or DIEA+B, where A is HOBT or 1-hydroxy-7-azabenzotriazole (HOAT), and B is HBTU, HATU, PyBOP or TBTU.

[0026] Among them, the first fragment

[0027] The preparation of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH comprises the following steps:

[0028] 1) 2-chlorotrityl chloride resin (2-CTC-Resin) is coupled with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) to obtain N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin (Fmoc-Arg(Pbf)-Resin);

[0029] 2) The N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin (Fmoc-Arg(Pbf)-Resin) obtained in step 1) is successively condensed and coupled with N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH), N-fluorenylmethoxycarbonyl-D-leucine (Fmoc-D-Leu-OH), fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH), N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), L-pyroglutamic acid (H-Pyr-OH), and then cleaved with a cleavage reagent to obtain the first fragment.

[0030] Among them, in step 1), the coupling agent for coupling the 2-chlorotrityl chloride resin (2-CTC-Resin) with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) is DIEA or NMM.

[0031] Among them, in step 2), the coupling agents for the sequential condensation coupling of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin with N-fluorenylmethoxycarbonyl-L-leucine are DIC+A or DIEA+B, where A is HOBT or HOAT, and B is HBTU, HATU, PyBOP or TBTU.

[0032] Among them, the cleavage reagent is trifluoroethanol and dichloromethane, and the volume ratio of trifluoroethanol to dichloromethane is 1:1 to 1:4.

[0033] Among them, it is preferred that the cleavage reagent is trifluoroethanol and dichloromethane, and the volume ratio of trifluoroethanol to dichloromethane is 1:2.

[0034] Among them, the dosage of the cleavage reagent is added in a corresponding volume according to 10 times the weight of 2-CTC-Resin (w:v = 1:10) to remove the resin.

[0035] Among them, in the preparation process of the present invention, 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH) is innovatively used to obtain dehydroalanine (Dha). After the condensation with the previous amino acid is completed, during the removal of Fmoc using piperidine, halogen Cl is simultaneously eliminated to generate dehydroalanine (Dha), and the target structure is obtained in one step.

[0036] The present invention uses a solid-phase bound liquid-phase synthesis preparation method to obtain this compound. This compound is the dehydration elimination product of leuprorelin, characterized by having a special amino acid dehydroalanine (Dha) in its structure and belonging to synthetic impurities. The synthesis of this compound has great reference significance for the impurity research of leuprorelin.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0038] (1) In the preparation method of the leuprorelin-related compound of the present invention, fragment one is first synthesized by solid-phase synthesis, and finally ethylamine is coupled in the liquid phase with HOBT+DIC. Among them, using 2-CTC resin effectively reduces the cost of the solid-phase resin, and selecting HOBT+DIC as the optimal coupling reagent both effectively improves the yield and reduces the cost. And through the research on the leuprorelin-related compound of the present invention, a more in-depth study on leuprorelin during the production process can be carried out, making it have better medicinal prospects.

[0039] (2) In the preparation process of the present invention, Fmoc-Ala(3-Cl)-OH was innovatively used to obtain dehydroalanine (Dha). After the condensation with the previous amino acid was completed and before the next amino acid was condensed, during the process of removing Fmoc with piperidine, the halogen Cl was simultaneously eliminated to generate dehydroalanine (Dha), and the target structure was obtained in one step. Description of the Drawings

[0040] Figure 1 It is the HPLC purification diagram of the leuprorelin-related compound in Example 1;

[0041] Figure 2 It is the mass spectrometry diagram of the leuprorelin-related compound in Example 1; Detailed Embodiments

[0042] The technical solutions of this application will be further described below in conjunction with the drawings.

[0043] The reagents used include:

[0044]

[0045]

[0046] Example 1

[0047] A preparation method of a leuprorelin-related compound, and the synthesis process is as follows:

[0048]

[0049] (1) Synthesis of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-Resin:

[0050] 1) Coupling of 2-chlorotrityl chloride resin (2-CTC-Resin) with fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH)

[0051] Add 10 g of 2-chlorotrityl chloride resin (2-CTC-Resin) with a degree of substitution of 0.4 - 0.6 mmol / g to a peptide reactor. Dissolve 3.37 g of fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH) and 3.3 mL of N,N-diisopropylethylamine (DIEA) in 100 mL of dichloromethane (DCM), then add it to the peptide reactor. After reacting for 2 hours, add 10 mL of methanol and 10 mL of N,N-diisopropylethylamine (DIEA) and continue to react for 30 min. Withdraw the reaction solution, add 50 mL of N,N-dimethylformamide (DMF) and wash twice, add 50 mL of ether for washing, and then dry in vacuo to obtain resin A1. The degree of substitution of the dried resin A1 is measured by ultraviolet spectrophotometry to be 0.5 mmol / g.

[0052] 2) Condensation of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH)

[0053] Add the dried resin A1 from step 1) to a peptide reactor, then add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After withdrawing the reaction solution, wash twice with 100 mL of DMF, wash once with 100 mL of DCM, wash twice with 100 mL of DMF. Add 6.49 g of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC), then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is based on the ninhydrin test until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, withdraw the reaction solution to obtain resin B1. Wash resin B1 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0054] 3) Condensation of N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH)

[0055] To the washed resin B1 in step 2), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 3.53 g of N-fluorenylmethyloxycarbonyl-L-leucine (Fmoc-Leu-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC), and further add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is determined by ninhydrin detection until the resin is colorless as detected by ninhydrin as the reaction end point. After detecting the completion of the reaction, pump out the reaction solution to obtain resin C1. Wash resin C1 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0056] 4) Condensation of N-fluorenylmethyloxycarbonyl-D-leucine (Fmoc-D-Leu-OH)

[0057] To the washed resin C1 in step 3), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 3.53 g of N-fluorenylmethyloxycarbonyl-D-leucine (Fmoc-D-Leu-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC), and further add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is determined by ninhydrin detection until the resin is colorless as detected by ninhydrin as the reaction end point. After detecting the completion of the reaction, pump out the reaction solution to obtain resin D1. Wash resin D1 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0058] 5) Condensation of fluorenylmethyloxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH)

[0059] To the washed resin D1 in step 4), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 4.60 g of fluorenylmethyloxycarbonyl-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC). Then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. The mixture is stirred under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin E1. Resin E1 is washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0060] 6) Condense 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH)

[0061] To the washed resin E1 in step 5), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 3.46 g of 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC). Add 100 mL of N,N-dimethylformamide (DMF). The mixture is stirred under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin F1. Resin F1 is washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0062] 7) Condense N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH)

[0063] 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution was added to the resin F1 after washing in step 6), and the reaction was carried out for 1 h to remove the Fmoc protection. After pumping out the reaction solution, it was washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. 5.27 g of N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC) were added, and then 100 mL of N,N-dimethylformamide (DMF) was added to obtain a mixture. The mixture was stirred under a nitrogen atmosphere, and the reaction was determined by ninhydrin detection until the resin was colorless by ninhydrin detection as the reaction end point. After detecting the completion of the reaction, the reaction solution was pumped out to obtain resin G1. Resin G1 was washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0064] 8) Condensation of N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH)

[0065] 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution was added to the resin G1 after washing in step 7), and the reaction was carried out for 30 min to remove the Fmoc protection. After pumping out the reaction solution, it was washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. 6.20 g of N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT), and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC) were added, and then 100 mL of N,N-dimethylformamide (DMF) was added to obtain a mixture. The mixture was stirred under a nitrogen atmosphere, and the reaction was determined by ninhydrin detection until the resin was colorless by ninhydrin detection as the reaction end point. After detecting the completion of the reaction, the reaction solution was pumped out to obtain resin H1. Resin H1 was washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0066] 9) Condensation of L-pyroglutamic acid (H-Pyr-OH)

[0067] Add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution to the washed resin H1 in step 8), react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, twice with 100 mL of DMF. Add 1.29 g of L-pyroglutamic acid (H-Pyr-OH), 1.35 g of 1-hydroxybenzotriazole (HOBT) and 1.55 mL of N,N'-diisopropylcarbodiimide (DIC), then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. The mixture is stirred under a nitrogen atmosphere, and the reaction is based on ninhydrin detection until the resin is colorless by ninhydrin detection as the reaction end point. After detecting the completion of the reaction, pump out the reaction solution to obtain resin I1. Resin I1 is washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0068] Among them, Fmoc-Ala(3-Cl)-OH was innovatively used to obtain dehydroalanine (Dha). After the condensation with the previous amino acid is completed and before condensing the next amino acid, when using piperidine to remove Fmoc, halogen Cl is simultaneously eliminated to generate dehydroalanine (Dha), and the target structure is obtained in one step.

[0069] 10) Wash the washed resin I1 twice with 100 mL of methanol (MeOH) and then dry it under vacuum. The dried resin I1 is lysed twice with 100 mL of trifluoroethanol (TFE) / dichloromethane (DCM) = 1:4 lysis reagent for 1 hour each time. Filter the resin to collect the lysis solution, and obtain 4.1 g of yellow solid by vacuum distillation: Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH.

[0070] (2) Synthesis of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-NH-CH2-CH3:

[0071] Dissolve 4.1 g of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH prepared in step (1) into 40 mL of dry DMF solution, then add 0.58 g of HOBT, 0.7 ml of DIC and 0.88 g of ethylamine hydrochloride (NH2-CH2-CH3·HCl). Stir at room temperature for 12 hours under a nitrogen atmosphere, then add 40 mL of water, extract twice with 40 mL of dichloromethane, combine the organic layers, dry the organic layer with anhydrous sodium sulfate, and obtain 3.9 g of yellow solid product by vacuum distillation:

[0072] Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-NH-CH2-CH3。

[0073] (3) Removal of side chain groups:

[0074] The yellow solid product obtained in step (2) was cleaved with a cleavage solution (trifluoroacetic acid (TFA): water (H2O): triisopropylsilane (TIS): phenol (Phenol) = 85:5:5:5, with a volume of 40 mL), and the reaction was carried out at room temperature (20 °C) for 2 h. The reaction solution was dropped into 400 mL of ice-cold diethyl ether, centrifuged and dried to obtain 3.0 g of a crude product: Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3.

[0075] (4) Purification of leuprorelin-related compounds

[0076] Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3 obtained in step (3) was dissolved in 10% acetonitrile and water and filtered.

[0077] Purification was carried out using a Hanbang UV3000 / NP7000 high-performance liquid chromatograph. Among them, the chromatographic column: DAC-50, C18, 10um, wavelength: 220 nm, mobile phase A: 0.1% aqueous TFA solution, mobile phase B: 0.1% TFA acetonitrile solution, gradient: 150%-40%, and the purification time was 40 minutes. The results are as Figure 1 shown, and 2.2 g of leuprorelin-related compounds were obtained.

[0078] The purified leuprorelin-related compounds were subjected to mass spectrometry analysis, and the results are as Figure 2 shown. After structure confirmation, the confirmed sequence was: Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3.

[0079] Example 2

[0080] A preparation method of a leuprorelin-related compound, and the synthesis process is as follows:

[0081]

[0082] (1) Synthesis of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Resin 1) Coupling of 2-chlorotrityl chloride resin (2-CTC-Resin) with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH)

[0083] Add 10 g of 2-chlorotrityl chloride resin (2-CTC-Resin) with a substitution degree of 0.4 - 0.6 mmol / g to a peptide reactor. Dissolve 9.73 g of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) and 3.3 mL of N,N-diisopropylethylamine (DIEA) in 100 mL of dichloromethane (DCM), then add it to the peptide reactor. After reacting for 2 hours, add 10 mL of methanol and 10 mL of N,N-diisopropylethylamine (DIEA) and continue to react for 30 min. Drain the reaction solution, add 50 mL of N,N-dimethylformamide (DMF) and wash twice, add 50 mL of ether for washing, and then dry in vacuo to obtain resin A2. The substitution degree of the dried resin A2 measured by ultraviolet spectrophotometry is 0.6 mmol / g.

[0084] 2) Condensation of N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH)

[0085] Add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution to the resin A2 after washing in step 1), react for 30 min to remove the Fmoc protection. Drain the reaction solution and wash twice with 100 mL of DMF, wash once with 100 mL of DCM, wash twice with 100 mL of DMF. Add 4.24 g of N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-Leu-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC), then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is determined by ninhydrin detection until the resin is colorless by ninhydrin detection as the reaction end point. After detecting the completion of the reaction, drain the reaction solution to obtain resin B2. Wash resin B2 twice with 100 mL of DMF, wash once with 100 mL of DCM, and wash twice with 100 mL of DMF.

[0086] 3) Condensation of N-fluorenylmethoxycarbonyl-D-leucine (Fmoc-D-Leu-OH)

[0087] To the washed resin B2 in step 2), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 4.24 g of N-fluorenylmethoxycarbonyl-D-leucine (Fmoc-D-Leu-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC), and further add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is determined by ninhydrin detection until the resin is colorless as detected by ninhydrin as the reaction end point. After detecting the completion of the reaction, pump out the reaction solution to obtain resin C2. Wash resin C2 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0088] 4) Condensation of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH)

[0089] To the washed resin C2 in step 3), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 4.60 g of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-O-tert-butyl-L-tyrosine (Fmoc-Tyr(tBu)-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC), and further add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is determined by ninhydrin detection until the resin is colorless as detected by ninhydrin as the reaction end point. After detecting the completion of the reaction, pump out the reaction solution to obtain resin D2. Wash resin D2 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0090] 5) Condensation of 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH)

[0091] To the washed resin D2 in step 4), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 4.2 g of 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine (Fmoc-Ala(3-Cl)-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC). Then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin E2. Wash resin E2 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0092] 6) Condensation of N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH)

[0093] To the washed resin E2 in step 5), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 1 h to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 6.30 g of N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan (Fmoc-Trp(Boc)-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC). Then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. Stir the mixture under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin F2. Wash resin F2 twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0094] 7) Condensation of N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH)

[0095] To the washed resin F2 in step 6), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 7.44 g of N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine (Fmoc-His(Trt)-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC). Then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. The mixture is stirred under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin G2. Resin G2 is washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0096] 8) Condense L-pyroglutamic acid (H-Pyr-OH)

[0097] To the washed resin G2 in step 7), add 100 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution, react for 30 min to remove the Fmoc protection. After pumping out the reaction solution, wash it twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF. Then add 1.55 g of L-pyroglutamic acid (H-Pyr-OH), 1.62 g of 1-hydroxybenzotriazole (HOBT), and 1.86 mL of N,N'-diisopropylcarbodiimide (DIC). Then add 100 mL of N,N-dimethylformamide (DMF) to obtain a mixture. The mixture is stirred under a nitrogen atmosphere, and the reaction is monitored by ninhydrin detection until the resin is colorless as detected by ninhydrin, which is the end point of the reaction. After detecting the completion of the reaction, pump out the reaction solution to obtain resin H2. Resin H2 is washed twice with 100 mL of DMF, once with 100 mL of DCM, and twice with 100 mL of DMF.

[0098] Among them, Fmoc-Ala(3-Cl)-OH is innovatively used to obtain dehydroalanine (Dha). After the condensation with the previous amino acid is completed, during the removal of Fmoc using piperidine, the halogen Cl is simultaneously eliminated to generate dehydroalanine (Dha), and the target structure is obtained in one step.

[0099] 9) Wash the resin H2 after step 8) with 100 mL of methanol twice. The resin H2 after vacuum drying is lysed twice with 100 mL of trifluoroethanol (TFE) / dichloromethane (DCM) = 1:4 lysis reagent for 1 hour each time. Filter the resin to collect the lysis solution, and obtain 4.3 g of yellow solid by vacuum distillation: Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH.

[0100] (2) Synthesis of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-NH-CH2-CH3

[0101] Dissolve 4.3 g of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH obtained in step (1) in 40 mL of DMF solution, add 0.5 g (L-proline ethylamide hydrochloride) H-Pro-NHET·HCL, then add 0.5 g of HOBT and 0.2 ml of DIC. After stirring at room temperature for 12 hours, add 40 mL of water, extract twice with 40 mL of dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, and obtain 3.9 g of yellow solid product by vacuum distillation:

[0102] Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-NH-CH2-CH3.

[0103] (3) Removal of side chain groups:

[0104] Lyse the yellow solid product in step (2) with a lysis solution (trifluoroacetic acid (TFA): water (H2O): triisopropylsilane (TIS): phenol (Phenol) = 85:5:5:5, volume 40 mL), react at room temperature (20 °C) for 2 h, drop the reaction solution into 400 mL of ice ether, centrifuge and dry to obtain 3.3 g of crude product: Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3.

[0105] (4) Purification of leuprorelin related compounds:

[0106] Dissolve Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3 obtained in step (3) in 10% acetonitrile and water, and filter.

[0107] Purification was carried out using a Hanbang UV3000 / NP7000 high performance liquid chromatograph. Among them, the chromatographic column: DAC-50, C18, 10um, wavelength: 220nm, mobile phase A: 0.1% aqueous TFA solution, mobile phase B: 0.1% TFA acetonitrile solution, gradient: 150%-40%, purification time was 40 minutes, and 2.6 g of leuprorelin related compound was obtained.

[0108] The purified leuprorelin related compound was subjected to mass spectrometry analysis. After structure confirmation, the sequence was confirmed as: Pyr-His-Trp-Dha-Tyr-D-Leu-Leu-Arg-Pro-NH-CH2-CH3.

Claims

1. A preparation method of a leuprorelin-related compound, characterized in that, It includes the following steps: (1) Using 2-chlorotrityl chloride resin as a solid-phase carrier, coupling with an amino acid to prepare Fragment 1; (2) Carboxyl liquid-phase coupling with ethylamine: Coupling the Fragment 1 obtained in step (1) with an amine compound to obtain Fragment 2; (3) Cleaving the side-chain protecting groups of the Fragment 2 obtained in step (2) with trifluoroacetic acid and purifying to obtain a leuprorelin-related compound; Wherein, the Fragment 1 is Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH or Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH, the amino acid is 9-fluorenylmethoxycarbonyl-L-proline or N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, and the amine compound is ethylamine hydrochloride or L-proline ethylamide hydrochloride; Wherein, the Fragment 1 The preparation of Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-Pro-OH includes the following steps: A1) Coupling 2-chlorotrityl chloride resin with 9-fluorenylmethoxycarbonyl-L-proline to obtain Fmoc-Pro-Resin, and removing the 9-fluorenylmethoxycarbonyl protection to obtain H-Pro-Resin; A2) Coupling H-Pro-Resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine to obtain Fmoc-Arg(Pbf)-Pro-Resin; A3) Sequentially condensing and coupling Fmoc-Arg(Pbf)-Pro-Resin with N-fluorenylmethoxycarbonyl-L-leucine, N-fluorenylmethoxycarbonyl-D-leucine, fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine, 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine, N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan, N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine, L-pyroglutamic acid, and then cleaving with a cleavage reagent to obtain Fragment 1; The Fragment 1 obtained in step A3) is coupled with ethylamine hydrochloride to obtain Fragment 2; The preparation of the Fragment 1 Pyr-His(Trt)-Trp(Boc)-Dha-Tyr(tBu)-D-Leu-Leu-Arg(Pbf)-OH includes the following steps: B1) Coupling 2-chlorotrityl chloride resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine to obtain a modified N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin; B2) The modified N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin obtained in step 1) is successively condensed and coupled with N-fluorenylmethoxycarbonyl-L-leucine, N-fluorenylmethoxycarbonyl-D-leucine, fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine, 3-chloro-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine, N-alpha-fluorenylmethoxycarbonyl-N-in-tert-butoxycarbonyl-L-tryptophan, N-(9-fluorenylmethoxycarbonyl)-N'-trityl-L-histidine, and L-pyroglutamic acid, and then cleaved with a cleavage reagent to obtain Fragment 1. Fragment 1 obtained in step B2) is coupled with L-proline ethylamide hydrochloride to obtain Fragment 2.

2. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, In step (2), the coupling solvent for the coupling reaction is N,N-dimethylformamide, and the coupling agent for the coupling reaction is DIC+A or DIEA+B, where A is HOBT and B is HBTU, HATU, PyBop, or TBTU.

3. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, In step (2), the temperature of the coupling reaction is 20-30 °C, and the time of the coupling reaction is 12-24 h.

4. The preparation method of the leuprorelin-related compound according to claim 1, wherein, In step (3), the purification is carried out by column chromatography. When performing column chromatography purification, the mobile phase A is 0.1% aqueous TFA solution, the mobile phase B is 0.1% TFA acetonitrile solution, and the flow gradient is 15%-45%.

5. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, In step A1), the coupling agent for the coupling of 2-chlorotrityl chloride resin with 9-fluorenylmethoxycarbonyl-L-proline is DIEA.

6. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, In step A2), the coupling agent for the coupling of H-Pro-Resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine is DIC+A or DIEA+B, where A is HOBT or HOAT and B is HBTU, HATU, PyBOP, or TBTU.

7. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, In step B1), the coupling agent for the coupling of 2-chlorotrityl chloride resin with N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine is DIEA or NMM. In step B2), the coupling agent for the successive condensation and coupling of N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine resin with N-fluorenylmethoxycarbonyl-L-leucine is DIC+A or DIEA+B, where A is HOBT or HOAT and B is HBTU, HATU, PyBOP, or TBTU.

8. The preparation method of the leuprorelin-related compound according to claim 1, characterized in that, The cleavage reagent is trifluoroethanol and dichloromethane, and the volume ratio of trifluoroethanol to dichloromethane is 1:1-1:4.

Images