Long-acting calcitonin analog

Abstract

The present invention relates to the field of pharmaceutical synthesis and provides a long-acting calcitonin analog and the use of the long-acting calcitonin analog in the manufacture of pharmaceuticals for treating diseases. The pharmaceutical composition is used for the prevention and treatment of osteoporosis, osteoosteitis, complex regional pain syndrome, and malignant osteolysis.

Description

Cross-reference

[0001] This application claims priority based on Chinese Patent Application No. 202310299105.0, filed with the China National Intellectual Property Administration on March 24, 2023, with the invention title of "Long-acting Calcitonin Analogue", and all of its contents are incorporated herein by reference in their entirety.

Technical Field

[0002] The present invention belongs to the field of pharmaceutical technology, and specifically relates to long-acting calcitonin analogues and their uses.

Background Art

[0003] Calcitonin is one of the hormones that regulates calcium metabolism and inhibits the action of parathyroid hormone. It has the function of significantly reducing calcium loss due to highly turnover bone diseases such as osteoporosis, Paget's disease, complex regional pain syndrome (Zudetic calcitonin disease), and malignant osteolysis. The action of calcitonin on the vertebral bones in postmenopausal osteoporosis is more significant than that on the limb bones, and the action on highly turnover bone diseases is more significant than that on low turnover bone diseases. Calcitonin can inhibit the activity of osteoclasts and stimulate the formation of osteoblasts. In addition, calcitonin also inhibits bone resorption, and as a result, it reduces the pathologically elevated serum calcium concentration, decreases reabsorption in the renal tubules, increases the urinary excretion of calcium, phosphorus, and sodium, and there is no risk of the serum calcium concentration decreasing below the normal range.

[0004] Calcitonin inhibits the secretion activities of the stomach and pancreas, but does not affect gastrointestinal motility. In clinical trials, the analgesic effect of this drug on patients with specific painful bone diseases has been demonstrated.

[0005] Because calcitonin has a short half-life in the body, it needs to be administered subcutaneously to patients daily, which leads to poor medication compliance. This invention aims to provide patients with a long-acting calcitonin analog, reducing the frequency of administration and improving patient medication compliance. [Overview of the project] [Problems that the invention aims to solve]

[0006] This invention provides a long-acting calcitonin analog and its uses. [Means for solving the problem]

[0007] To achieve the above objectives, the present invention first provides a compound represented by the following structure I, a pharmaceutically acceptable salt, solvate, chelate, or non-covalent complex formed from this compound, a prodrug based on this compound, or any mixture thereof: [Structural formula I] Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-AA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2(1-7 disulfide bond) [In structural formula I, AA1 is a D-type or L-type Lys, a D-type or L-type Dap, a D-type or L-type Dab, a D-type or L-type Orn, a D-type or L-type Dah, or a D-type or L-type Dao, In structural formula I, AA2 is either NH2 or OH. In structural formula I, R represents HO2C(CH2) n1 CO-(AA3) n2 -(PEG n3 (CH2) n4 CO) n5 -or HO2C(CH2) n1 CO-(AA3) n2 -(AA4) n6- is either or does not exist. (However, n1 is an integer between 10 and 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. n6 is an integer from 1 to 10. AA3 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava. AA4 is Ala, Gly, Leu, Phe, Ser, Thr, Tyr, Asp, Glu, Gln, Lys, D-Lys, Arg, or His).

[0008] The long-acting calcitonin analogs according to the present invention include pharmaceutically acceptable salts, solvates, chelates, or non-covalent complexes, prodrugs based on this compound, or any mixtures thereof.

[0009] The present invention further provides pharmaceutical compositions of the compounds according to the present invention, and the use of pharmaceutical compositions of the compounds according to the present invention in the manufacture of pharmaceuticals for treating diseases.

[0010] Furthermore, the pharmaceutical composition is used to prevent and treat osteoporosis, degenerative osteitis, complex regional pain syndrome, and malignant osteolysis.

[0011] More details of the present invention are described below, or some of them are reflected in the embodiments of the present invention.

[0012] Unless otherwise specified, the amounts of various components and reaction conditions used herein can be interpreted as "approximate" or "approximate" in all cases. Similarly, unless otherwise specified, the numerical parameters referenced below and in the claims are approximate parameters, and different numerical values ​​may be obtained due to differences in standard errors under each experimental condition.

[0013] In this specification, if there is any discrepancy or doubt between the chemical structural formula and the chemical name of a compound, the chemical structural formula shall be used to precisely define the compound. Compounds described herein may contain one or more chiral centers and / or double bonds, as well as similar structures thereof, and may also exist as stereoisomers encompassing double bond isomers (e.g., geometric isomers), optical enantioisomers, or diastereoisomers. Therefore, any chemical structure within the scope of this specification, even if it contains some or all of the above-mentioned similar structures, includes all possible enantioisomers and diastereoisomers of the compound, as well as any pure stereoisomer (e.g., a pure geometric isomer, a pure enantioisomer, or a pure diastereoisomer), and any mixture of those isomers. These racemates and mixtures of stereoisomers can be further separated into their constituent enantioisomers or stereoisomers by separation techniques or chiral molecule synthesis techniques commonly used by those skilled in the art.

[0014] Compounds of structural formula I include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In the above cases, simple enantioisomers or diastereoisomers, such as optically active isomers, are obtained by asymmetric synthesis or racemate separation methods. Racemate separation can be achieved by various methods, such as conventional recrystallization or chromatography using separation accelerators. Compounds of structural formula I also include cis and / or trans isomers with double bonds.

[0015] The compounds according to the present invention include, but are not limited to, the compounds shown in structural formula I and all pharmaceutically usable forms thereof. The various pharmaceutically usable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures thereof. [Modes for carrying out the invention]

[0016] The present invention discloses a long-acting calcitonin analogue and its uses. Those skilled in the art can realize the present invention by referring to the content of this specification and appropriately adjusting the relevant parameters. It should be noted that all similar substitutions and modifications that are obvious to those skilled in the art are considered to be included in the present invention. Although the method according to the present invention has been described through preferred embodiments, those related can implement and apply the technology of the present invention by making corrections or appropriate changes and combinations to the compounds and their manufacturing methods described in this specification without departing from the content, spirit and scope of the present invention, which is obvious.

[0017] The Japanese names corresponding to the English abbreviations in the present invention are shown in Table 1 below. [Table 1] [Examples]

[0018] [Example 1] Preparation of the compound The preparation method includes using the solid-phase synthesis method of polypeptide, preparing a peptide resin by the solid-phase synthesis method of polypeptide, subjecting the peptide resin to acid decomposition to obtain a crude product, and finally purifying the crude product to obtain a pure product. Here, the step of preparing a peptide resin by the solid-phase synthesis method of polypeptide is to sequentially link the corresponding protected amino acids or fragments in the following sequence onto the carrier resin by the coupling solid-phase synthesis method to prepare a peptide resin.

[0019] In the above preparation method, the usage amount of the Fmoc-protected amino acid is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total molar number of the resin charged.

[0020] In the above preparation method, the substitution value of the carrier resin is 0.3 to 1.5 mmol / g of resin, preferably 0.6 to 1.0 mmol / g of resin.

[0021] In a preferred embodiment of the present invention, the coupling solid-phase synthesis method involves removing the Fmoc protecting group from the protected amino acid-resin obtained in the previous step, and then coupling it with the next protected amino acid. The deprotection time for removing the Fmoc protection is 10 to 60 minutes, preferably 15 to 25 minutes. The coupling reaction time is 60 to 300 minutes, preferably 100 to 140 minutes.

[0022] The coupling reaction described above requires the addition of a condensation reagent. The condensation reagent is one selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, hexafluorophosphate (benzotriazole-1-yloxy)tripyrrolidinophosphonium, 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate, preferably N,N-diisopropylcarbodiimide. The molar amount of the condensation reagent used is 1.2 to 6 times the total number of moles of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0023] The coupling reaction described above requires the addition of an activating reagent. The activating reagent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of activating reagent used is 1.2 to 6 times the total number of moles of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0024] In a preferred embodiment of the present invention, the reagent used to remove the Fmoc protection is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide). This mixed solution contains 10-30%(V) piperidine. The amount of reagent used to remove the Fmoc protection is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0025] Preferably, the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and the protecting groups of the side chains, followed by oxidation and cyclization to obtain the crude product.

[0026] More preferably, the acid decomposition agent used in the acid decomposition of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), and water. The mixed solvent consists of 80-95% TFA, 1-10% EDT, and the remainder being water, by volume.

[0027] More preferably, the mixed solvent consists of 89-91% TFA, 4-6% EDT, and the remainder being water by volume. Most preferably, the mixed solvent consists of 90% TFA, 5% EDT, and the remainder being water by volume.

[0028] The amount of acid decomposing agent required is 4 to 15 mL per gram of peptide resin, preferably 7 to 10 mL per gram of peptide resin.

[0029] The decomposition time using the aforementioned acid decomposing agent is 1 to 6 hours, preferably 3 to 4 hours, under room temperature conditions.

[0030] For the oxidation and cyclization described above, iodine, H2O2, or DMSO is used as the oxidizing agent, with iodine being preferred. The oxidizing agent is added by titration, and the addition is stopped when the oxidation endpoint is reached. Furthermore, the crude product is purified by high-performance liquid chromatography and freeze-dried to obtain the pure product.

[0031] 1. Synthesis of peptide resins 2. Protected amino acids corresponding to the sequence were sequentially linked to the carrier resin by Fmoc deprotection and coupling reactions to obtain a peptide resin. (1) Linking of the first protective amino acid in the main chain

[0032] 0.03 mol of the first protective amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. 0.03 mol of DIC was slowly added to the protective amino acid DMF solution while stirring, and the mixture was reacted at room temperature with stirring for 30 minutes to obtain an activated protective amino acid solution, which was prepared for use in the next step.

[0033] 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g) was deprotected with a 20% PIP / DMF solution for 25 minutes, washed, filtered, and Fmoc was removed to obtain the resin.

[0034] The activated first protective amino acid solution was added to the resin from which Fmoc had been removed, and the coupling reaction was carried out for 60 to 300 minutes. The mixture was then filtered and washed to obtain a resin containing one protective amino acid. (2) Linking of other protective amino acids in the main chain

[0035] Using the same method as the one used to link the first protective amino acid of the main chain described above, other protective amino acids corresponding to the main chain were sequentially linked to obtain a resin containing amino acids in the main chain. (3) Linking of the first protective amino acid of the side chain

[0036] 0.03 mol of the first protective amino acid of the side chain and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. Separately, 0.03 mol of DIC was slowly added to the protective amino acid DMF solution while stirring, and the mixture was reacted at room temperature with stirring for 30 minutes to obtain an activated protective amino acid solution.

[0037] 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane were dissolved in an appropriate amount of dichloromethane, deprotected for 4 hours, filtered, washed, and alloc was removed to obtain a resin.

[0038] The activated side-chain first protective amino acid solution was added to the resin from which Alloc had been removed, and the coupling reaction was carried out for 60 to 300 minutes. After filtration and washing, a resin containing the first protective amino acid of the side chain was obtained. (4) Linking of other protective amino acids in the side chain

[0039] Using the same method as described above for linking the first protective amino acid in the main chain, other protective amino acids corresponding to the side chains and monoprotective fatty acids were sequentially linked to obtain a peptide resin. 2. Preparation of the crude product

[0040] The peptide resin described above was added to a cleavage reagent in a volume ratio of TFA:water:EDT = 95:5:5 (10 mL / g of cleavage reagent in the resin), stirred well, and reacted at room temperature with stirring for 3 hours. The reaction mixture was filtered through a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrates were combined, concentrated under reduced pressure, and precipitated with anhydrous diethyl ether. The precipitate was precipitated three times with anhydrous diethyl ether, vacuum dried, and a whitish powder was obtained.

[0041] The resulting whitish powder was dissolved in a 20% aqueous acetic acid solution, and an iodine / ethanol saturated solution was added dropwise while stirring until cyclization was complete. The mixture was then concentrated under reduced pressure at 35-40°C to obtain a concentrated solution of the crude product. 3. Preparation of the pure product

[0042] The concentrated solution of the crude product described above was filtered through a 0.45 μm mixed microporous membrane to purify it and prepared for use in subsequent steps.

[0043] For purification, high-performance liquid chromatography was used, with a 10 μm reversed-phase C18 as the purification chromatography packing. A 0.1% TFA / aqueous solution - 0.1% TFA / acetonitrile solution was used as the mobile phase system, and a 30 mm × 250 mm chromatography column was set to a flow rate of 20 mL / min. Gradient elution was used, and purification was performed by cyclic sample loading. The crude product solution was injected into the chromatography column, eluted with the mobile phase, the main peak was collected, and after evaporating the acetonitrile, a purified intermediate concentrate was obtained.

[0044] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and prepared for use in subsequent steps. Salt exchange was performed by high-performance liquid chromatography, with a mobile phase system of 1% acetic acid / aqueous solution-acetonitrile, a 10 μm reverse-phase C18 chromatography packing for purification, and a flow rate of 20 mL / min on a 30 mm × 250 mm chromatography column (the flow rate can be adjusted accordingly depending on the specifications of the chromatography column). Using gradient elution and cyclic sample loading, the sample was injected into the chromatography column, eluted with the mobile phase, a chromatograph was collected, the change in absorbance was observed, the main peak of the salt exchange was collected, the purity of the liquid phase was detected, the main peak solution of the salt exchange was combined, concentrated under reduced pressure to obtain a pure aqueous acetic acid solution, and freeze-dried to obtain the pure product.

[0045] The compounds synthesized using the above method are shown in Table 2 below. [Table 2-1] [Table 2-2] [Example 2] Activity Measurement

[0046] 1.Measurement method

[0047] When Gαs-coupled GPCRs (GPCRs) are activated by their ligands, they can potentially increase intracellular cAMP levels. cAMP acts as a second messenger, mediating various cellular responses. The cAMP Hunter HEK293-CT-R Gs cell line overexpresses Gαs-coupled CT receptors, and when calcitonin (analog) binds to these overexpressed CT receptors, intracellular cAMP levels are upregulated. The bioactivity of calcitonin (analog) was evaluated by measuring the amount of intracellular cAMP produced when activated by calcitonin (analog) using the cAMP Dynamic 2 kit.

[0048] Using the HEK293 cell line, which stably expresses GLP-1R, stably transfected cells were stimulated with agonist concentrations. The biological activity of the agonists was determined by measuring the relative luminescence units of cells stimulated at each dose. 2.Measurement results

[0049] The measurement results are shown in Table 3 below. [Table 3] [Example 3] Measurement of preliminary pharmacokinetic properties

[0050] Cynomolgus monkeys were used as experimental animals, and the compound was administered subcutaneously at a dose of 0.1 mg / kg. Blood samples were collected venously before administration (0h) and at 1h, 2h, 3h, 4h, 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h after administration. Plasma samples were centrifuged. The blood drug concentration of the compound in the plasma samples was measured by liquid chromatography-mass spectrometry. The half-lives of the compound after subcutaneous (SC) administration are shown in Table 4 below. [Table 4]

[0051] The above are merely preferred embodiments of the present invention, and those skilled in the art can make various improvements and modifications without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A long-acting calcitonin analog having the following structural formula I: [Structural formula I] Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-AA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2 (1-7 disulfide bond) [In structural formula I, AA1 is a D-type or L-type Lys, a D-type or L-type Dap, a D-type or L-type Dab, a D-type or L-type Orn, a D-type or L-type Dah, or a D-type or L-type Dao, In structural formula I, AA2 is NH ２ or OH, R in Structural Formula I is HO ２ C(CH ２ ) ｎ１ CO-(AA3) ｎ２ -(PEG ｎ３ (CH ２ ) ｎ４ CO) ｎ５ -, or HO ２ C(CH ２ ) ｎ１ CO-(AA3) ｎ２ -(AA4) ｎ６ - or does not exist (However, n1 is an integer between 10 and 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, n6 is an integer from 1 to 10, AA3 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava. AA4 is Ala, Gly, Leu, Phe, Ser, Thr, Tyr, Asp, Glu, Gln, Lys, D-Lys, Arg, or His).

2. A long-acting calcitonin analog according to claim 1, comprising a pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, a prodrug based on the compound, or any mixture of the above forms.

3. A long-acting calcitonin analog according to claims 1 and 2, used for manufacturing a pharmaceutical composition for treating a disease.

4. The long-acting calcitonin analog according to claim 3, wherein the pharmaceutical composition is used to prevent and treat osteoporosis, degenerative osteitis, complex regional pain syndrome, and malignant osteolysis.

5. A pharmaceutical composition characterized by comprising a long-acting calcitonin analog as described in any one of claims 1 to 4.

6. A long-acting calcitonin analog according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5, used for manufacturing pharmaceuticals for the prevention and treatment of osteoporosis, degenerative osteitis, complex regional pain syndrome, and malignant osteolysis.

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