CN116478269A - Long-acting tidulcin compound - Google Patents

Abstract

The invention relates to the field of medicine synthesis, and discloses a long-acting tidulcin compound. The long-acting tidulcin compound is used for preparing a pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for treating and preventing diseases such as intestinal mucosa injury, ulcerative enteritis, chronic enteritis and the like caused by short bowel syndrome and chemoradiotherapy.

Description

Long-acting tidulcin compound

Technical Field

The invention relates to a long-acting tidulcin compound and application thereof.

Background

Tidolutetin (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 intimal cells, which increases the number of these cells, which increases small intestinal absorption, reduces diarrhea.

Short bowel syndrome refers to a series of syndromes caused by serious small intestine diseases or abnormal nutrient absorption of the body due to surgical excision of most small intestine, and before tivallutide is obtained, no medicine for treating the diseases is provided, and most patients can only obtain the nutritional ingredients required by the body by means of total or partial parenteral nutrition (intravenous route), but the parenteral nutrition not only seriously affects the life quality of the patients, but also frequently causes serious complications, such as infection caused by intravenous channels, bacterial hyperproliferation in small intestine, hepatotoxicity and biliary tract diseases. Clinical trials indicate that tidolutetin can reduce the parenteral nutrition requirements of short bowel syndrome patients.

Because of the short half-life of tiltuptin in vivo, patients need to be given subcutaneously every day, and patient compliance is poor. The invention aims to provide a long-acting tidollutide compound for patients, the predicted medicine frequency is 1 time per week, the medicine compliance of the patients is improved, and the invention has good social and economic benefits.

Disclosure of Invention

The invention provides a long-acting tidulcin compound and application thereof.

To achieve the above object, the present invention provides a compound of formula (I), a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, a prodrug based on the compound, or a mixture of any of the above forms.

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

Structural formula I

AA1 in the structural formula I is Gly, or Aib, or Acpc, or Accb;

AA2 in the structural formula I is Asp, glu or Ada;

AA3 in the structural formula I is Lys, arg or His;

AA4 in the structural formula I is NH ₂ Or is OH;

n in the structural formula I is an integer of 1-10.

The long-acting tidolutetin compound comprises a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex formed, a prodrug based on the compound, or any mixture of the above forms.

The invention also provides a pharmaceutical composition comprising the compound according to the invention, and the use of the pharmaceutical composition of the compound for preparing a medicament for treating a disease.

Further, the pharmaceutical composition is used for treating and preventing diseases such as intestinal mucosa injury, ulcerative enteritis, chronic enteritis and the like caused by short bowel syndrome and chemoradiotherapy.

Further details of the invention are set forth in the accompanying drawings and the description below, or may be learned by practice of the invention.

Unless otherwise indicated, the amounts of the various components, reaction conditions, and the like, are used herein and are to be construed in any sense as "generally", "about". Accordingly, unless explicitly indicated otherwise, the numerical parameters set forth in the following claims are approximations that may vary depending upon the standard deviation employed under the particular circumstances.

Herein, when the chemical structural formula and chemical name of a compound are divergent or ambiguous, the compound is defined exactly by the chemical structural formula. The compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and stereoisomers, including isomers of double bonds (such as geometric isomers), optical enantiomers or diastereomers, may also be present. Accordingly, any chemical structure within the scope of the description herein, whether partial or whole containing such 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 mixtures of any of these isomers. These racemic isomers and mixtures of stereoisomers may also be resolved further into their constituent enantiomers or stereoisomers by methods known to those skilled in the art using continuous separation techniques or chiral molecule synthesis.

The compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds. In the above cases, single enantiomers or diastereomers, such as optical isomers, may be obtained by asymmetric synthesis or resolution of racemates. Resolution of the racemate can be accomplished in various ways, such as recrystallization with conventional resolution-aiding reagents, or by chromatographic methods. In addition, the compounds of the formula I also contain cis-and/or trans-isomers with double bonds.

The compounds of the present invention include, but are not limited to, compounds of formula I and all of their various pharmaceutically acceptable forms. Pharmaceutically useful different forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above, and mixtures of any of these forms.

Detailed Description

The invention discloses a long-acting tidulcin compound and application thereof, and a person skilled in the art can properly improve related parameters by referring to the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the process of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the compounds and methods of preparation described herein, or in appropriate combinations, without departing from the spirit and scope of the invention. The Chinese names corresponding to the English abbreviations in the invention are shown in the following table:

example 1 preparation of Compounds

The preparation method is a polypeptide solid-phase synthesis method, which comprises the following steps: preparing peptide resin by adopting a solid-phase polypeptide synthesis method, acidolysis is carried out on the peptide resin to obtain a crude product, and finally, the crude product is purified to obtain a pure product; wherein the step of preparing peptide resin by solid-phase polypeptide synthesis method comprises the steps of sequentially accessing corresponding protected amino acids in the following sequences on carrier resin by solid-phase coupling synthesis method to prepare peptide resin:

in the preparation method, the dosage of the Fmoc-protected amino acid is 1.2 to 6 times of the total mole number of the resin; preferably 2.5 to 3.5 times.

In the preparation method, the substitution value of the carrier resin is 0.3-1.5 mmol/g resin, and the preferred substitution value is 0.6-1.0 mmol/g resin.

As a preferred scheme of the invention, the solid phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is subjected to Fmoc protecting group removal and then is subjected to coupling reaction with the next protected amino acid. The deprotection time for Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

The coupling reaction needs to add a condensation reagent, wherein the condensation reagent is selected from DIC (N, N-diisopropyl carbodiimide), N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 2- (7-aza-1H-benzotriazol-1-yl) -1, 3-tetramethylurea hexafluorophosphate, benzotriazol-N, N, N ', N' -tetramethylurea hexafluorophosphate or O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate; n, N-diisopropylcarbodiimide is preferred. The molar amount of the condensing agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar amount of the amino groups in the amino resin.

The coupling reaction needs to add an activating reagent, and the activating reagent is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, and is preferably 1-hydroxybenzotriazole. The amount of the activating agent to be used is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total mole number of the amino groups in the amino resin.

As a preferred scheme of the invention, the Fmoc protection removing reagent is PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution, and the mixed solution contains 10-30% (V) of piperidine. The Fmoc-removing protective agent is used in an amount of 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

Preferably, the peptide resin is subjected to acidolysis, resin removal and side chain protecting group removal, and oxidation cyclization to obtain a crude product:

further preferably, the acidolysis agent used in acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

Still more preferably, the volume ratio of the mixed solvent is: 89-91% TFA, 4-6% EDT and the balance water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL of acidolysis agent required by each gram of peptide resin; preferably, 7 to 10mL of acidolysis agent is required per gram of peptide resin.

The time of the cleavage with the acidolysis agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

The oxidant used in the oxidation cyclization is iodine and H ₂ O ₂ Or DMSO, preferably iodine. The oxidant is added in a titration mode, and the addition is stopped when the oxidation end point is reached.

Further, purifying the crude product by high performance liquid chromatography, and lyophilizing to obtain pure product.

1. Synthesis of peptide resins

Taking carrier resin, sequentially accessing protected amino acid corresponding to a sequence through Fmoc protection removal and coupling reaction, and preparing peptide resin:

(1) Access to the 1 st protected amino acid

Taking 0.03mol of 1 st protected amino acid and 0.03mol of HOBt, and dissolving the 1 st protected amino acid and the HOBt with a proper amount of DMF; and (3) adding 0.03mol of DIC into the protected amino acid DMF solution slowly under stirring, and stirring and reacting for 30 minutes in a room temperature environment to obtain an activated protected amino acid solution for later use.

0.01mol of Rink amide MBHA resin (substitution value about 0.4 mmol/g) was taken and deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.

And adding the activated 1 st protected amino acid solution into Fmoc-removed resin, performing coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing 1 protected amino acid.

(2) Access to other protected amino acids

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and other corresponding protected amino acids of the main chain are sequentially accessed to obtain the resin containing the main chain amino acid.

(3) Finally deprotecting

Deprotection was performed for 25 min with 20% PIP/DMF solution, washed, filtered and dried in vacuo to give Fmoc-removed peptide resin.

2. Preparation of crude product

Adding a cracking reagent (10 mL/g resin) with a volume ratio of TFA to water to EDT=95 to 5 into the peptide resin, uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous diethyl ether for precipitation, washing the precipitate with anhydrous diethyl ether for 3 times, and vacuum drying to obtain a crude product.

3. Preparation of pure product

Dissolving the crude product with 10% acetic acid solution, filtering with 0.45 μm mixed microporous membrane, and purifying;

purifying by high performance liquid chromatography, wherein the chromatographic packing for purification is reverse phase C18 with the size of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column with the size of 30mm or 250mm is 20mL/min, eluting by a gradient system, circularly sampling and purifying, sampling the crude product solution into the chromatographic column, starting mobile phase eluting, collecting main peaks, evaporating acetonitrile, and obtaining purified intermediate concentrated solution;

filtering the purified intermediate concentrate with 0.45 μm filter membrane for use, changing salt by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic column flow rate of purification column is 20mL/min (corresponding flow rate can be adjusted according to chromatographic columns of different specifications) with reversed phase C18 of 10 μm and 30mm x 250 mm; adopting a gradient elution and cyclic loading method, loading in a chromatographic column, starting mobile phase elution, collecting a spectrum, observing the change of absorbance, collecting a salt-exchange main peak, analyzing the liquid phase to detect purity, combining the salt-exchange main peak solutions, concentrating under reduced pressure to obtain a pure acetic acid aqueous solution, and freeze-drying to obtain a pure product.

The following compounds were synthesized using the above procedure:

example 2 determination of Activity

1. Measurement method

GLP-2R, upon stimulation with its specific agonist, activates the intracellular adenylate cyclase pathway, elevating cAMP levels, ultimately leading to insulin production and release. The cell strain transfected with GLP-1R stably is stimulated by the to-be-detected substance, so that the intracellular cAMP level of the cell is rapidly increased, the Relative Light Unit (RLU) of the stimulated cell at each dose is measured by a chemiluminescence method, and then the EC50 of the agonist is calculated, and the activity measuring method is a current universal GLP-2 receptor agonist activity measuring method at home and abroad.

The EC of the agonist is calculated by using CHO-K1 cell lines stably expressing GLP-2R, stimulating stable transformed cells with different concentrations of the agonist and measuring the relative light units of the stimulated cells at each dose ₅₀ Values.

2. Measurement results

The measurement results are shown in the following table:

EXAMPLE 3 determination of Primary drug substitution Properties

The test animals were cynomolgus monkeys, which were given 1.5mg/kg subcutaneously, 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, 168h intravenously, and plasma samples were centrifuged, plasma samples were assayed for plasma concentration of the compounds by liquid chromatography-mass spectrometry, and the Subcutaneous (SC) administration half lives of the compounds were shown in the following table:

Claims (4)

1. a long-acting tidolin compound having structural formula i:

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

structural formula I

AA1 in the structural formula I is Gly, or Aib, or Acpc, or Accb;

AA2 in the structural formula I is Asp, glu or Ada;

AA3 in the structural formula I is Lys, arg or His;

AA4 in the structural formula I is NH ₂ Or is OH;

n in the structural formula I is an integer of 1-10.

2. The long-acting tidoluted peptide compound of claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex thereof, based on a prodrug of the compound, or a mixture of any of the foregoing forms.

3. A long acting tidoluteptin compound according to claim 1 and claim 2 for use in the preparation of a pharmaceutical composition for the treatment of a disease.

4. The pharmaceutical composition according to claim 3, which is used for the treatment and prevention of short bowel syndrome, intestinal mucosa injury caused by radiotherapy and chemotherapy, ulcerative enteritis, chronic enteritis and other diseases.