阅读说明：本技术 艾替班特的合成 (Synthesis of icatibant ) 是由 瓦桑塔库玛·甘伽·拉姆 尼廷·索潘饶·帕蒂尔 文卡塔·拉加文德拉查玉鲁·帕勒 尤盖希 于 2018-09-27 设计创作，主要内容包括：本发明涉及由式(I)表示的艾替班特的有效固相合成。本发明涉及通过顺序偶联采用固相方法的用于制备艾替班特的有效方法。其涉及顺序偶联受保护的氨基酸以制备艾替班特。本发明还涉及在偶联期间使用无机盐,在Fmoc去保护步骤后用在DMF溶液中的HOBt进行洗涤以保证哌啶的完全去除并且反应进行完成,以及因此避免添加/缺失序列并且还提高方法产率。<Image he="413" wi="700" file="DDA0002511653930000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention relates to efficient solid phase synthesis of icatibant represented by formula (I). The present invention relates to an efficient process for the preparation of icatibant by sequential coupling using a solid phase process. It involves sequential coupling of protected amino acids to make icatibant. The invention also relates to the use of inorganic salts during coupling, washing with HOBt in DMF solution after the Fmoc deprotection step to ensure complete removal of piperidine and completion of the reaction, and thus avoid addition/deletion sequences and also improve process yield.)

1. A process for preparing icatibant, comprising the steps of:

loading arginine (Pbf) -OH to a resin solid support,

the unreacted functional sites are capped,

sequentially coupling the side chain protected amino acids in the presence of a coupling agent and an inorganic salt to prepare icatibant,

crude icatibant is obtained by removing the protecting groups and cleaving the peptide from the resin,

optionally purifying the crude icatibant.

2. The process for the preparation of icatibant according to claim 1, comprising the following steps,

loading arginine (Pbf) to a resin solid support in the presence of a coupling agent,

sequentially coupling amino acids with protected side chains in the presence of a coupling agent, oxymapure and an inorganic salt to prepare icatibant,

crude icatibant is obtained by removing the protecting groups and cleaving the peptide from the resin,

optionally purifying the crude icatibant.

3. The process for the preparation of icatibant according to any of the preceding claims, involving a coupling agent selected from HBTU, COMU, deptt or any combination thereof.

4. The process for the preparation of icatibant according to any preceding claim involving a coupling additive selected from the group consisting of oxymapure, HOBt or any combination thereof.

5. For the preparation ofA method of icatibant, the method comprising: deprotection of the protecting group followed by washing with 0.01-0.5M HOBt in DMF and using TFA MDC phenol M-cresol TIS H₂O is cleaved from the resin in its entirety.

Background

Icatibant is a competitive antagonist selective for the bradykinin B2 receptor, with similar affinity to bradykinin. Hereditary angioedema is caused by the loss or dysfunction of C1 esterase inhibitors, a key regulator of the factor XII/kallikrein proteolytic cascade leading to bradykinin production. Bradykinin is a vasodilator which is believed to be responsible for local swelling, inflammation and pain characteristic of HAE symptoms. Icatibant inhibits bradykinin binding to the B2 receptor and thereby treats the clinical symptoms of acute sporadic episodes of HAE.(icatibant) is a synthetic decapeptide with five non-protein amino acids.

Icatibant developed by fire Orphan therapeuticsOriginally approved in the united states in 2011 as a subcutaneous injection.

US 5,648,333B 1 discloses icatibant and a process for its preparation.

CN102532267 discloses a process for the preparation of icatibant using CTC resins, utilizing different coupling agents for coupling different amino acids.

CN103992383 discloses a process for the preparation of icatibant, which utilizes liquid phase synthesis of Boc-D-Arg-oh.2hcl, followed by coupling with the remaining fragments by solid phase synthesis using queen resin.

CN104072585 discloses a process for the preparation of icatibant using sequential coupling of amino acids using queen resin or p-hydroxymethylphenoxymethylstyrene resin as solid support.

WO2016157177 discloses a process for the preparation of icatibant in the presence of a biocompatible tertiary amine nicotinamide as catalyst.

5473/CHE/2014 discloses a process for the preparation of icatibant which uses sequential coupling of amino acids on queen resin.

Previous processes for preparing icatibant have drawbacks. Due to the technical complexity and high cost, said method is not suitable for the large-scale production of icatibant; thus, the method is not commercially viable or may continue to be problematic to implement.

During solid phase synthesis, a strong tendency of the peptide to aggregate under the conditions employed is observed. This is due to the following reasons:

the growing peptide sequence is liable to form β sheet-type structures which lead to collapse of the peptidyl resin, in which case the dispersion of reagents into the peptidyl resin is limited, the coupling and deprotection reactions will be slow and incomplete, thereby creating deletions/additions, racemic peptide impurities, leading to difficult purification and to low yields.

Therefore, there remains a need to provide an efficient process for the preparation of icatibant which is high in yield, scalable, cost-effective, environmentally friendly and commercially viable by avoiding the repetition of cumbersome and tedious purification steps.

Object of the Invention

The object of the present invention was to develop a simple, robust and commercially viable sequential process for the preparation of icatibant of formula I, which is carried out with the help of inorganic salts, new and efficient coupling conditions, deprotection and washing conditions after each amino acid in the sequence.

Summary of The Invention

One embodiment of the present invention relates to a synthetic process for the preparation of icatibant comprising the steps of:

A) loading arginine to a resin solid support in the presence of a coupling agent,

B) capping the unreacted functional sites (capping),

C) sequentially coupling the side chain protected amino acids in the presence of a coupling agent and an inorganic salt to prepare the main chain of icatibant,

D) crude icatibant is obtained by removing the protecting groups and cleaving the peptide from the resin,

E) the crude icatibant was purified.

A schematic depiction of the process is shown in fig. 1.

The method employed is solid phase peptide synthesis of icatibant by sequential methods and involves inorganic salts as well as conventional coupling agents and additives during coupling. The method provides for completion of the coupling and deprotection reactions and reduction of racemization and thereby controls the isomeric impurities in close proximity to the target molecule and thereby facilitates the purification process of the peptide.

Detailed Description

The present invention relates to an efficient process for the preparation of icatibant by sequential coupling of individual amino acids using a solid phase method. The method employed in the present invention is solid phase manual peptide synthesis using 2-chlorotrityl chloride as a solid support, using the Fmoc-/tBu method and including site-specific efficient coupling agents and inorganic salts as well as conventional coupling agents and additives used during coupling. And after deprotection of Fmoc group using 15-20% piperidine, peptidyl resin was washed with 0.01-0.5M HOBt/DMF instead of normal DMF to ensure complete removal of piperidine, which is advantageous to avoid insertion of impurities. Complete synthesis was achieved by a sequential method. The method provides for completion of the coupling and deprotection reactions and reduction of racemization and thereby controls the isomeric impurities in close proximity to the target molecule and thereby facilitates the purification process of the peptide.

The invention is represented by the following examples. These examples are for illustration only and therefore should not be considered as limiting the scope of the invention.

Abbreviations:

ACN: acetonitrile

Boc: tert-butyloxycarbonyl radical

COMU: 1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholinyl-carbenium hexafluorophosphate

CuCl₂: copper chloride

DEPBT: 3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4 (3H) -one

DIC: n, N' -diisopropylcarbodiimide

DMAP: dimethylaminopyridine compound

DMF: n, N' -dimethylformamide

DIPEA: diisopropylethylamine

Fmoc: 9-fluorenylmethoxycarbonyl

HBTU: O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate

HOBt: n-hydroxybenzotriazoles

HFIP: 1,1,1,3,3, 3-hexafluoro-2-propanol

Mtt: methyl trityl radical

MeOH: methanol

MgCl₂: magnesium chloride

NMM: n-methylmorpholine

NMP: n-methyl-2-pyrrolidone

TES: triethylsilane

TFE: trifluoroethanol

TFA: trifluoroacetic acid

Pbf: 2,2,4,6, 7-pentamethyldihydrobenzofuran

RT: at room temperature

^tBu: tert-butyl radical

And (3) TIS: tri-isopropyl silane

Trt: trityl radical

TMP: 2,4, 6-trimethylpyridine

The invention is represented by the following examples. These examples are for illustration only and therefore should not be considered as limiting the scope of the invention.

Brief Description of Drawings

FIG. 1: a flow chart of a method for solid phase synthesis of icatibant (formula-I) according to the invention.

Example 1 CTC resin with substitution 1.22mmol/g (10g) was placed in a solid phase peptide synthesis vessel, swelling of the resin was performed in DMF (80-100M L) for 1h and draining, Fmoc-arg (pbf) -OH (7.9g, 1.0 equiv) was dissolved in DMF (80-100M L) and added to the swollen resin, DIPEA, NMM and TMP (2.0-4.0 equiv) were added with stirring, preferably DIPEA. stirring was continued at room temperature for 2-3h and the reaction mixture was drained washing the resin with DMF (80-100M L), first amino acid loading evaluation was performed and found to be 0.61mmol/g, capping of the unreacted sites of the resin was performed with methanol (10 DIPEA) (4%) in DMF (86M L), capping of the resin with DMF (80-100M L) by treatment with 5-20% piperidine in DMF (10 min), and blocking of the resin with DMF (80-100M 465M) followed by removing the solution of DMF (80-100M 465.5M).

Fmoc-Oic-OH (2.0-4.0 equiv.) and HOBt (2.0-4.0 equiv.) are dissolved in DMF (80-100m L) and added to the reaction vessel the reaction mass is stirred and HBTU (2.0-4.0 equiv.) dissolved in DMF (20m L) is added DIPEA (4.0-6.0 equiv.) and the mass is stirred under nitrogen for 1.0-2.0 h the completion of the coupling is monitored by Kaiser test after the coupling is complete, the resin is washed with DMF (80-100m L.3) and used for Fmoc deblocking the remaining amino acid coupling in the MTBC sequence is carried out using HBTU/HOBt after all amino acids have been coupled according to the sequence, the Fmoc deprotection is carried out and the peptidyl-based resin is washed twice with DMF, DCM, methanol and MTBE after all amino acids have been coupled according to the sequence, the peptidyl-based resin is dried for 3min and treated with crude RRTU at RT ratio of 10.5: RRT 3.5% and the peptide-based resin is filtered off at a volume of 0.5% RRT of impurities and filtered off at room temperature of 0.0.0.0% RRT and 360.0% RRT and filtered off.