CN111320683B

CN111320683B - Tilappa peptide analogue

Info

Publication number: CN111320683B
Application number: CN202010247376.8A
Authority: CN
Inventors: 周述靓; 王鹏; 邓岚; 潘文强
Original assignee: Chengdu Aoda Biotechnology Co ltd
Current assignee: Chengdu Aoda Biotechnology Co ltd
Priority date: 2019-09-25
Filing date: 2020-03-31
Publication date: 2023-06-27
Anticipated expiration: 2040-03-31
Also published as: CN111320683A; CN110642935A

Abstract

The invention relates to the field of medicine synthesis, and discloses a tirapamide analogue. The tirapamide analogue is used for preparing a pharmaceutical composition for treating at least one of the following diseases, wherein the diseases comprise type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive dysfunction, atherosclerosis, myocardial infarction, coronary heart disease, cardiovascular diseases, stroke, inflammatory intestinal syndrome and/or dyspepsia or gastric ulcer, hepatic fibrosis diseases and pulmonary fibrosis diseases.

Description

Tilappa peptide analogue

Technical Field

The present invention relates to a tirapamide analogue and uses thereof, the analogue is a double-intestinal insulinotropic peptide mimic compound of agonizing human glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-l) receptor.

Background

GIP is a 42 amino acid gastrointestinal regulatory peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells and protecting pancreatic beta cells in the presence of glucose. GLP-l is a 37 amino acid peptide that stimulates insulin secretion, protects pancreatic beta cells, and inhibits glucagon secretion, gastric emptying, and food intake, resulting in weight loss. GIP and GLP-l are known as incretins; incretin receptor signaling plays a key physiologically relevant role in glucose homeostasis. In normal physiology, GIP and GLP-1 are secreted from the intestinal tract after a meal, and these incretins enhance physiological responses to food, including satiety, insulin secretion, and nutrient management.

The most common side effect of GLP-l analogues is that administration does not achieve full glycemic control and weight loss, whereas GIP alone has very modest glucose lowering capacity in type 2 diabetics. Both native GIP and GLP-l can be rapidly inactivated by the ubiquitous protease DPP IV and therefore can only be used for short-term metabolic control.

It was reported in WO 2013/164483 and WO 2014/192284 that certain GIP/GLP-1 analogs exhibit both GIP and GLP-1 activity and that the analogs can achieve better glycemic control and weight loss efficacy.

Disclosure of Invention

The invention provides a tirapamide analogue and application thereof, and the analogue is a double-incretin peptide mimic compound for exciting human glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-l) receptor.

To achieve the above object, the present invention provides a compound of the 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.

Tyr-AA1-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-AA1-Leu-Asp-Lys-Ile-Ala-Gln-Lys(R)-Ala-AA2-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-AA3

Structure I

AA in Structure I ₁ Dhthr, or Dhval;

AA in Structure I ₂ Phe, or 1-Nal, or 2-Nal;

AA in Structure I ₃ Is NH ₂ Or is OH;

r in structure I is HO ₂ C(CH ₂ ) _n1 CO-(γGlu) _n2 -(PEG _n3 (CH2) _n4 CO) _n5 -

Wherein: n1 is an integer from 10 to 20;

n2 is an integer from 1 to 5;

n3 is an integer from 1 to 30;

n4 is an integer from 1 to 5;

n5 is an integer from 1 to 5.

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.

Preferably, the pharmaceutical composition is used for the preparation of a medicament for the treatment of at least one of type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease, cardiovascular diseases, stroke, inflammatory bowel syndrome and/or dyspepsia or gastric ulcers, liver fibrosis diseases and pulmonary fibrosis diseases.

Preferably, the pharmaceutical composition is applied to the preparation of medicines for treating drug effect delay of type II diabetes and/or preventing exacerbation of type II diabetes.

Preferably, the pharmaceutical composition is used for preparing a medicament for reducing food intake, reducing beta cell apoptosis, increasing islet beta cell function, increasing beta cell mass and/or reverting sensitivity of glucose to beta cells.

The invention still further provides methods of administering the compounds to a subject to regulate blood glucose in vivo.

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, the 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.

The compound shown in the structure I provided by the invention has stable properties, is not easily degraded by dipeptidyl peptidase IV (DPP-IV) in vivo, is a GIP/GLP-I double-agonist analogue, and has remarkable blood glucose and weight reducing effects.

Detailed Description

The invention discloses a GIP/GLP-1 analogue and application thereof, and a person skilled in the art can appropriately improve related parameter realization by referring to the content of the text. 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:

english abbreviations	Chinese name	English abbreviations	Chinese name
				Fmoc	9-fluorenylmethoxycarbonyl	OtBu	Tert-butoxy radical
tBu	Tert-butyl group	Boc	Boc acid tert-butyl ester
				Trt	Trityl radical	Pbf	(2, 3-dihydro-2, 4,6, 7-pentamethylbenzofuran-5-yl) sulfonyl
Ala	Alanine (Ala)	Leu	Leucine (leucine)
				Arg	Arginine (Arg)	Lys	Lysine
Asn	Asparagine derivatives	Met	Methionine
				Asp	Aspartic acid	Phe	Phenylalanine (Phe)
Cys	Cysteine (S)	Pro	Proline (proline)
				Gln	Glutamine	Ser	Serine (serine)
Glu	Glutamic acid	Thr	Threonine (Thr)
				Gly	Glycine (Gly)	Trp	Tryptophan
His	Histidine	Tyr	Tyrosine
				Ile	Isoleucine (Ile)	Val	Valine (valine)
Dap	2, 3-diaminopropionic acid	Dab	2, 4-diaminobutyric acid
				Orn	Ornithine	Dah	2, 7-diaminoheptanoic acid
Dhthr	Dehydroxythreonine	Dhval	2, 3-Didehydrovaline

Example 1 preparation of Compound 1

Tyr-Dhthr-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhthr-Leu-Asp-Lys-Ile-Ala-Gln-Lys (AEEA-AEEA-gamma Glu-18 alkanedioic acid) -Ala-Phe-Val-Gln-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH-Ala ₂

The preparation method 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 acid or fragment in the following sequence 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 or the protected amino acid fragment is 1.2-6 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 0.2-1.0 mmol/g resin, and the preferred substitution value is 0.3-0.5 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 and simultaneously the resin and side chain protecting group are removed 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 for cleavage with acidolysis agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

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

1. Synthesis of peptide resins

The Rink Amide BHHA resin is used as carrier resin, and is coupled with the protected amino acid shown in the following table in sequence through Fmoc protection removal and coupling reaction to prepare the peptide resin. The protected amino acids corresponding to the protected amino acids used in this example are shown below:

(1) Access to backbone 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) Accessing the 2 nd to 39 th protected amino acid of the main chain

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and the corresponding 2 nd to 39 th protected amino acids are sequentially accessed to obtain the resin containing 39 amino acids of the main chain.

(3) Access to side chain 1 st protected amino acid

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

2.5mmol of tetraphenylphosphine palladium and 25mmol of phenylsilane are taken, dissolved with a proper amount of dichloromethane, deprotected for 4 hours, filtered and washed to obtain dealloc resin for later use.

Adding the 1 st protective amino acid liquid of the side chain after the activation into the dealloc-removed resin, carrying out coupling reaction for 60-300 minutes, and filtering and washing to obtain the resin containing the 1 st protective amino acid of the side chain.

(4) Accessing the 2 nd to 4 th protected amino acids of the side chain

The same method of accessing the 1 st protected amino acid of the main chain is adopted, and the 2 nd to 4 th protected amino acids and the single protected fatty acid corresponding to the side chains are sequentially accessed to obtain the 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 precipitation with anhydrous diethyl ether for 3 times, and pumping to obtain white-like powder which is a crude product.

3. Preparation of pure product

Mixing the crude product with water, adjusting pH to 8.0 with ammonia water to dissolve completely, filtering the solution 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 gradient elution, circulating sample loading method, loading in chromatographic column, starting mobile phase elution, collecting spectrum, observing change of absorbance, collecting salt-changing main peak, analyzing liquid phase to detect purity, mixing salt-changing main peak solutions, concentrating under reduced pressure to obtain pure acetic acid aqueous solution, freeze drying to obtain pure product 5.5g, purity 97.5%, and total yield 11.4%. The molecular weight was 4781.4 (100% M+H).

Example 2 preparation of Compound 2

Tyr-Dhval-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhval-Leu-Asp-Lys-Ile-Ala-Gln-Lys (AEEA-AEEA-gamma-Glu-18 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

The preparation was carried out in the same way as in example 1, using the protected amino acids as indicated in the following table:

6.1g of pure product is obtained, the purity is 96.9%, and the total yield is 12.5%. The molecular weight was 4809.5 (100% M+H).

EXAMPLE 3 preparation of Compound 3

Tyr-Dhthr-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhthr-Leu-Asp-Lys-Ile-Ala-Gln-Lys(PEG ₅ CH ₂ CO-gamma Glu-18 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

7.4g of pure product is obtained, the purity is 96.3%, and the total yield is 15.3%. The molecular weight was 4768.4 (100% M+H).

EXAMPLE 4 preparation of Compound 4

Tyr-Dhval-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhval-Leu-Asp-Lys-Ile-Ala-Gln-Lys(PEG ₅ CH ₂ CO-gamma Glu-18 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

7.1g of pure product is obtained, the purity is 96.8%, and the total yield is 14.6%. The molecular weight was 4796.5 (100% M+H).

EXAMPLE 5 preparation of Compound 5

Tyr-Dhthr-GlU-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhthr-Leu-Asp-Lys-Ile-Ala-Gln-Lys (AEEA-AEEA-gamma-Glu-20 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

7.1g of pure product is obtained, the purity is 98.3 percent, and the total yield is 14.5 percent. The molecular weight was 4809.4 (100% M+H).

EXAMPLE 6 preparation of Compound 6

Tyr-Dhthr-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhthr-Leu-Asp-Lys-Ile-Ala-Gln-Lys(PEG ₅ CH ₂ CO-gamma Glu-20 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂

6.7g of pure product is obtained, the purity is 97.4%, and the total yield is 13.8%. The molecular weight was 4796.4 (100% M+H).

Example 7 Activity assay

1. Measurement method

GLP-1R is mainly present on the surface of islet beta cells and is a G protein-coupled receptor (GPCRs). GLP-1R, 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-1 receptor agonist activity measuring method at home and abroad.

We used CHO-K1 cell lines stably expressing GLP-1R, stimulated stably transformed cells with different concentrations of agonist, and the bioactivity of the agonist was obtained by measuring the relative light units of cells after each dose of stimulation.

2. Measurement results

The measurement results are shown in the following table:

numbering of compounds	Biological Activity (%)
		Compound 1	121.72
Compound 2	66.57
		Compound 3	70.25
Compound 4	55.81
		Compound 5	117.5
Compound 6	102.6

EXAMPLE 8 determination of Primary drug substitution Properties

Each compound was divided into two dosing groups: SD rats, 4 in each group, 8 in total.

Tail vein intravenous injection group: the dose was 1mg/kg, and blood was collected from the orbital veins of rats at the time of pre-drug administration (0 h), and 30min, 1h, 2h, 4h, 8h, 24h, 48h, 96h, 144h after administration, respectively, and plasma samples were centrifuged.

Subcutaneous dosing group: the dose was 1mg/kg, and blood was collected from the orbital veins of rats at 1h, 2h, 3h, 4h, 8h, 24h, 48h, 96h, 144h, and 1h, 2h, 3h, 4h, and 144h after administration, respectively, and plasma samples were centrifuged.

Plasma concentrations of the corresponding compounds in SD rat plasma samples were determined by liquid chromatography-mass spectrometry, respectively, and after intravenous and subcutaneous administration, subcutaneous (SC) administration half-lives of the compounds SD rats were shown in the following table:

compounds of formula (I)	t _1/2 (h)
		Compound 1	9.1
Compound 2	8.7
		Compound 3	9.5
Compound 4	9.3
		Compound 5	10.2
Compound 6	11.0

Claims

1. A tirapamide analog having the structure:

Tyr-Dhthr-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Dhthr-Leu-Asp-Lys-Ile-Ala-Gln-Lys(PEG ₅ CH ₂ CO-gamma Glu-20 alkanedioic acid) -Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ₂ 。

2. A pharmaceutically acceptable salt, solvate of the tirapamide analog of claim 1.

3. A pharmaceutical composition comprising the tirapamide analogue of claim 1.

4. Use of a tirapamide analogue according to claim 1 for the preparation of a medicament for the treatment of type II diabetes.