A long-acting tidulutide compound

Abstract

This invention relates to the field of pharmaceutical synthesis and discloses a long-acting tiduglutide compound. The long-acting tiduglutide compound of this invention is used to prepare pharmaceutical compositions for treating diseases such as short bowel syndrome, intestinal mucosal damage caused by radiotherapy and chemotherapy, ulcerative colitis, and chronic enteritis.

Description

Technical Field

[0001] This invention relates to a long-acting tiduglutide compound and its uses. Background Technology

[0002] Teduglutide is a glucagon-like peptide-2 (GLP-2) compound that reduces gastric emptying and secretion and regulates the growth, proliferation, and repair of small intestinal lining cells. Teduglutide increases the number of these cells, which in turn increases small intestinal absorption and reduces diarrhea.

[0003] Short bowel syndrome (SBS) is a syndrome caused by severe small bowel disease or surgical removal of a large portion of the small intestine, resulting in the body's inability to properly absorb nutrients. Prior to the approval of teduglutide, there were no drugs to treat this condition, and most patients relied on total or partial parenteral nutrition (intravenous) to obtain the necessary nutrients. However, parenteral nutrition not only severely impacts patients' quality of life but also frequently leads to serious complications, such as infections caused by intravenous access, small intestinal bacterial overgrowth, hepatotoxicity, and biliary tract diseases. Clinical trials have shown that teduglutide can reduce the need for parenteral nutrition in patients with SBS.

[0004] Because teduglutide has a short half-life in vivo, patients need to administer it subcutaneously daily, resulting in poor patient compliance. The purpose of this invention is to provide patients with a long-acting teduglutide compound, with a planned dosing frequency of once weekly, thereby improving patient compliance and yielding significant social and economic benefits. Summary of the Invention

[0005] This invention provides a long-acting tiduglutide compound and its uses.

[0006] To achieve the above objectives, the present invention first provides a compound represented by structural formula I, a pharmaceutically usable salt, solvate, chelate or non-covalent complex of the compound, a drug precursor based on the compound, or any mixture thereof.

[0007] His-AA1-AA2-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-AA3-Ile-Thr-Asp-(Lys)n-AA4

[0008] Structural Formula I

[0009] In structural formula I, AA1 can be Gly, Aib, Accc, or Accb;

[0010] In structural formula I, AA2 can be Asp, Glu, or Ada;

[0011] In structural formula I, AA3 can be Lys, Arg, or His;

[0012] In structural formula I, AA4 is either NH2 or OH;

[0013] In structural formula I, n is an integer from 1 to 10.

[0014] The long-acting teduglutide compound of the present invention comprises a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, a drug prodrug based on the compound, or any mixture thereof.

[0015] The present invention also provides pharmaceutical compositions comprising compounds according to the present invention, and provides pharmaceutical uses of pharmaceutical compositions comprising compounds according to the present invention for the preparation of a medicine for treating diseases.

[0016] Furthermore, the pharmaceutical composition is used for the treatment and prevention of diseases such as short bowel syndrome, intestinal mucosal damage caused by radiotherapy and chemotherapy, ulcerative colitis, and chronic enteritis.

[0017] Further details of this invention are described in detail below, or some of them may be experienced in the embodiments of this invention.

[0018] Unless otherwise specified, the quantities of different components and reaction conditions used herein are to be interpreted as "approximate" or "about". Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0019] In this document, when there is disagreement or ambiguity regarding the chemical structure and chemical name of a compound, the compound is defined precisely by its chemical structure. The compounds described herein may contain one or more chiral centers, and / or double bonds and similar structures, and may also exist as stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this description, whether partially or entirely containing similar structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer) and any mixture of these isomers. Mixtures of racemic and stereoisomers can be further separated into enantiomers or stereoisomers of their constituent components by those skilled in the art using continuous separation techniques or methods of chiral molecule synthesis.

[0020] Compounds of Formula I include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In the above cases, a single enantiomer or diastereomer, such as an optically active isomer, can be obtained by asymmetric synthesis or by racemic resolution. Racemic resolution can be achieved by various methods, such as conventional recrystallization with a resolving agent or by chromatographic methods. Additionally, compounds of Formula I also include cis and / or trans isomers with double bonds.

[0021] The compounds described in this invention include, but are not limited to, the compounds shown in structural formula I and all their various pharmaceutically usable forms. These pharmaceutically usable forms include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, drug prodrugs based on the above substances, and any mixtures of these forms. Detailed Implementation

[0022] This invention discloses a long-acting tiduglutide compound and its uses. Those skilled in the art can refer to this document and appropriately modify the relevant parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention. The Chinese names corresponding to the English abbreviations involved in this invention are shown in the table below:

[0023]

[0024] Example 1: Preparation of the compound

[0025] The preparation method is a solid-phase peptide synthesis method, including: preparing peptide resin using a solid-phase peptide synthesis method, then hydrolyzing the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of preparing peptide resin by solid-phase peptide synthesis is to sequentially insert the corresponding protected amino acids in the following sequences onto a carrier resin by a solid-phase coupling synthesis method to prepare peptide resin:

[0026] In the above preparation method, the amount of Fmoc-protected amino acid used is 1.2 to 6 times the total molar amount of the resin added; preferably 2.5 to 3.5 times.

[0027] In the above preparation method, the substitution value of the carrier resin is 0.3 to 1.5 mmol / g resin, and the preferred substitution value is 0.6 to 1.0 mmol / g resin.

[0028] As a preferred embodiment of the present invention, the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is deprotected by the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time for the Fmoc protecting group is 10-60 minutes, preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, preferably 100-140 minutes.

[0029] The coupling reaction requires the addition of a condensing agent, selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate, or O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensing agent is 1.2 to 6 times the total molar amount of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0030] The coupling reaction requires the addition of an activating agent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of activating agent used is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0031] As a preferred embodiment of the present invention, the reagent for removing Fmoc protection is a PIP / DMF (piperidine / N,N-dimethylformamide) mixed solution, wherein the mixed solution contains 10-30% (V) piperidine. The amount of the Fmoc removal reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0032] Preferably, the crude product is obtained by acid hydrolysis of the peptide resin to remove both the resin and the side chain protecting groups, followed by oxidative cyclization.

[0033] More preferably, the acid hydrolysate used during the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT) and water, with the volume ratio of the mixed solvent being: TFA 80-95%, EDT 1-10%, and the remainder being water.

[0034] More preferably, the volume ratio of the mixed solvent is: TFA 89-91%, EDT 4-6%, and the remainder is water. Most preferably, the volume ratio of the mixed solvent is: TFA 90%, EDT 5%, and the remainder is water.

[0035] The amount of acid hydrolysant used is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

[0036] The pyrolysis time using the acid hydrolysate is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

[0037] The oxidative cyclization process uses iodine, H2O2, or DMSO as the oxidant, with iodine being preferred. The oxidant is added by titration, and addition is stopped at the oxidation endpoint.

[0038] Furthermore, the crude product was purified by high performance liquid chromatography and lyophilized to obtain the pure product.

[0039] 1. Synthesis of peptide resins

[0040] Take the carrier resin, and through Fmoc deprotection and coupling reactions, sequentially insert the corresponding protective amino acids to obtain the peptide resin:

[0041] (1) Introduce the first protective amino acid

[0042] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for later use.

[0043] Take 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g), protect it with 20% PIP / DMF solution for 25 minutes, wash and filter to obtain Fmoc-free resin.

[0044] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60–300 minutes. After filtration and washing, a resin containing one protected amino acid was obtained.

[0045] (2) Add other protective amino acids

[0046] Using the same method as described above for adding the first protected amino acid to the main chain, other corresponding protected amino acids are sequentially added to the main chain to obtain a resin containing main chain amino acids.

[0047] (3) Finally, protect

[0048] The peptide resin was protected with 20% PIP / DMF solution for 25 minutes, washed, filtered, and vacuum dried to obtain Fmoc-free peptide resin.

[0049] 2. Preparation of crude product

[0050] Take the above peptide resin and add a lysis reagent with a volume ratio of TFA:water:EDT=95:5:5 (10mL / g resin of lysis reagent). Stir well and react at room temperature for 3 hours. Filter the reaction mixture using a sintered glass funnel, collect the filtrate, wash the resin three times with a small amount of TFA, combine the filtrates and concentrate under reduced pressure, add anhydrous diethyl ether to precipitate, wash the precipitate three times with anhydrous diethyl ether, and dry under vacuum to obtain the crude product.

[0051] 3. Preparation of pure products

[0052] Take the above crude product, dissolve it in 10% acetic acid solution, filter it through a 0.45μm mixed microporous membrane, and purify it for later use;

[0053] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The flow rate of the 30 mm * 250 mm column was 20 mL / min. Gradient elution was used, and the sample was injected repeatedly for purification. The crude product solution was loaded into the column, the mobile phase was started for elution, the main peak was collected, and the acetonitrile was removed by evaporation to obtain the purified intermediate concentrate.

[0054] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and set aside. High-performance liquid chromatography (HPLC) was used for salt exchange. The mobile phase system was 1% acetic acid / water solution-acetonitrile. The chromatographic packing material was a 10 μm reversed-phase C18 column (30 mm * 250 mm) with a flow rate of 20 mL / min (the flow rate can be adjusted according to different column specifications). Gradient elution and cyclic loading were used. The sample was loaded into the column, the mobile phase was started for elution, the chromatogram was collected, and the absorbance change was observed. The salt-exchanged main peak was collected and its purity was determined by analytical liquid chromatography. The salt-exchanged main peak solutions were combined, concentrated under reduced pressure to obtain pure acetic acid aqueous solution, and then freeze-dried to obtain the pure product.

[0055] The following compounds were synthesized using the method described above:

[0056]

[0057] Example 2: Determination of Activity

[0058] 1. Measurement Method

[0059] GLP-2R, upon stimulation by its specific agonist, activates the intracellular adenylate cyclase pathway, increases cAMP levels, and ultimately leads to insulin production and release. Cell lines stably transfected with GLP-1R were stimulated with the analyte, resulting in a rapid increase in intracellular cAMP levels. The relative light units (RLU) after stimulation with each dose were measured using a chemiluminescence method, and the EC50 of the agonist was calculated. This activity assay is currently a widely used method for detecting GLP-2 receptor agonist activity both domestically and internationally.

[0060] Using the CHO-K1 cell line stably expressing GLP-2R, the cells were stimulated with different concentrations of agonists. The EC50 of the agonists was calculated by measuring the relative light units after stimulation with each dose. 50 value.

[0061] 2. Measurement Results

[0062] The measurement results are shown in the table below:

[0063]

[0064] Example 3: Determination of preliminary pharmacokinetic properties

[0065] The experimental animals were cynomolgus monkeys. The drug was administered subcutaneously at a dose of 1.5 mg / kg. Blood samples were collected intravenously before administration (0 h) and at 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 18 h, 24 h, 48 h, 96 h, 144 h, and 168 h after administration. Plasma samples were separated by centrifugation, and the plasma concentration of the compound was determined using liquid chromatography-mass spectrometry (LC-MS). The half-life of the compound after subcutaneous (SC) administration is shown in the table below.

[0066]

Claims

1. A long-acting tiduglutide compound having the following structural formula or a pharmaceutically acceptable salt thereof: His-Acpc-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Lys)6-NH2.

2. A pharmaceutical composition comprising the long-acting tiduglutide compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The use of the long-acting teduglutide compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for the treatment and prevention of short bowel syndrome.

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