Long-acting calcitonin analogue

Description

1 / 16 Long-acting calcitonin analogue

[0001] This application claims priority over Chinese Patent Application No. 202310299105.0, filed with the China National Intellectual Property Administration on March 24, 2023, and entitled “LONG-ACTING CALCITONIN ANALOG”, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates to the field of medical technology, and in particular to a long-acting calcitonin analogue and a use thereof. BACKGROUND

[0003] Calcitonin is one of the hormones that can modulate calcium metabolism and inhibit parathyroid hormone. Calcitonin can significantly reduce bone calcium loss in high-turnover bone diseases such as osteoporosis, deformative bone disease (Paget's disease), algoneurodystrophy (Sudeck's atrophy of bone disease), and malignant osteolysis. The effect of calcitonin is more pronounced in trunk bones than in limb bones in postmenopausal osteoporosis and in high-turnover bone diseases than in low-turnover diseases. Calcitonin can inhibit osteoclast activity while stimulating osteoblast formation. Calcitonin can also inhibit osteolysis, thereby reducing a pathologically elevated blood calcium level and increasing urinary excretion of calcium, phosphorus, and sodium by reducing reabsorption in renal tubules, while the blood calcium level will not decrease to a normal range.

[0004] Calcitonin can inhibit gastric and pancreatic secretions, but it has no effect on gastrointestinal motility. Clinical trials have shown that calcitonin has an analgesic effect in some patients with painful bone disease.

[0005] Due to the short half-life of calcitonin in vivo, patients require daily subcutaneous administration, resulting in low patient adherence. Petition 870250086443, dated 09 / 24 / 2025, page 7 / 74 2 / 16 One objective of the present disclosure is to provide a long-acting calcitonin analogue for patients to reduce the frequency of administration and improve patient adherence. SUMMARY

[0006] The present disclosure provides a long-acting calcitonin analogue and a use thereof.

[0007] To achieve the aforementioned objectives, the present disclosure first provides a compound as represented by structural formula I, as well as a pharmaceutically acceptable salt, a solvate, a chelate, or a non-covalent complex thereof, a prodrug based on the compound, or any mixture of the foregoing forms: Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-HisAA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2 (a disulfide bond between positions 1 and 7) structural formula I

[0008] AA1 in structural formula I is D-Lys, L-Lys, D-Dap, L-Dap, D-Dab, L-Dab, D-Orn, L-Orn, D-Dah, L-Dah, D-Dao or L-Dao;

[0009] AA2 in structural formula I is NH2 or OH;

[0010] R in structural formula I is HO2C(CH2)mCO-(AA3)n2(PEGn3(CH2)n4CO)n5-, HO2C(CH2)n1CO-(AA3)n2-(AA4)n6-, or absence, where:

[0011] n1 is an integer in the range of 10 to 20;

[0012] n2 is an integer in the range of 1 to 5;

[0013] n3 is an integer in the range of 1 to 30;

[0014] n4 is an integer in the range of 1 to 5;

[0015] n5 is an integer in the range of 1 to 5;

[0016] n6 is an integer in the range of 1 to 10;

[0017] AA3 is yGIu, εLys, β-Ala, γ-aminobutyric acid or 5-Ava;

[0018] AA4 is Ala, Gly, Leu, Phe, Ser, Thr, Tyr, Asp, Glu, Gln, Lys, D-Lys, Arg or His.

[0019] The long-acting calcitonin analogue provided in the present Petition 870250086443, dated 09 / 24 / 2025, page 8 / 74 3 / 16 disclosure includes a pharmaceutically acceptable salt, a solvate, a chelate or a non-covalent complex thereof, a prodrug based on the compound or any mixture of the foregoing forms.

[0020] The present disclosure further provides a pharmaceutical composition comprising a compound in accordance with the present disclosure and use of the pharmaceutical composition in the manufacture of a medicament to treat a disease.

[0021] Additionally, the pharmaceutical composition is used for the prevention and treatment of osteoporosis, deforming bone disease, algoneurodystrophy and malignant osteolysis.

[0022] Further content involved in this disclosure is described in detail below. Some of the content may be covered by combining the examples in this disclosure.

[0023] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions and the like used in the present disclosure are to be understood as being modified in all cases by the term about or “approximately”. Consequently, unless otherwise indicated, the numerical parameters cited in the following description and claims are approximations that may differ due to differences in standard error found in their respective experimental conditions.

[0024] In the present disclosure, if there is a discrepancy or ambiguity between a chemical structure and a chemical name for a compound, the chemical structure shall prevail in defining the compound. The compounds described herein may contain one or more chiral centers and / or double bonds and the like, and may therefore exist as stereoisomers, including double-bond isomers (such as geometric isomers), enantiomers, or diastereomers. Consequently, any chemical structure within the scope of this disclosure that contains such similar structures in part or in whole is intended to encompass all enantiomers and diastereomers. Petition 870250086443, dated 09 / 24 / 2025, page 9 / 74 4 / 16 possible forms of the compound. This includes any simple stereoisomer (such as a simple geometric isomer, a simple enantiomer, or a simple diastereomer) as well as any mixture of these isomers. Mixtures of these racemates and stereoisomers can be further resolved into their constituent enantiomers or stereoisomers by a person skilled in the art using various separation techniques or chiral synthesis methods.

[0025] The compounds as represented by structural formula I include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In such cases, a simple enantiomer or diastereomer, including an optically active isomer, can be obtained by asymmetric synthesis or by racemate resolution. Racemate resolution can be performed by various methods, such as conventional recrystallization using a resolving agent or by chromatographic methods. Additionally, the compounds as represented by structural formula I also include cis- and / or trans-isomers with double bonds.

[0026] The compounds of the present disclosure include, but are not limited to, the compounds as represented by structural formula I and all pharmaceutically acceptable forms thereof. Pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above substances, and any mixtures of the foregoing forms. DETAILED DESCRIPTION

[0027] The present disclosure provides a long-acting calcitonin analogue and a use thereof. A person skilled in the art can, by reference to the contents of this disclosure, appropriately modify the relevant parameters to practice the present disclosure. It should be particularly noted that all similar substitutions and modifications obvious to a person skilled in the art are intended to be included in the present disclosure. Although the methods of the present disclosure have been described by preferred examples, it will be evident to those skilled in the art that they are not the most suitable for the purpose of practicing the present disclosure. Petition 870250086443, dated 09 / 24 / 2025, page 10 / 74 5 / 16 technique that modifications, or variations and suitable combinations, may be made to the compounds and preparation methods described herein without departing from the content, spirit and scope of this disclosure to realize and apply the technology of this disclosure. The full names corresponding to the English abbreviations used in this disclosure are shown in the table below: Table 1 Abbreviation s in English Full Name Abbreviation s in English Full Name Fmoc 9-fluorenylmethyloxycarbonyl OtBu tert-butoxy tBu tert-butyl Boc tert-butoxycarbonyl Trt trityl Pbf (2,3-dihydro-2,2,4,6,7pentamethylbenzofuran-5-yl)sulfonyl Ala alanine Leu leucine Arg arginine Lys lysine Asn asparagine Phe phenylalanine Asp aspartic acid Pro proline Cys cysteine ​​Ser serine Gln glutamine Thr threonine Glu glutamic acid Trp tryptophan Gly glycine Tyr tyrosine His histidine Val valine Ile isoleucine Dap 2,3-diaminopropionic acid 5-Ava 5-aminovaleric acid Dab 2,4-diaminobutyric acid Petition 870250086443, dated 09 / 24 / 2025, page 11 / 74 6 / 16 Aib aminoisobutyric acid Ornithine Ada 2aminoadipic acid Dah 2,7-diaminoheptanoic acid Apm 2aminopimelic acid Dao 2,8-diaminooctanoic acid Asu 2aminosuberic acid

[0028] Example 1: Preparation of compounds

[0029] The preparation method is solid-phase polypeptide synthesis, which comprises: preparing a peptide resin by solid-phase polypeptide synthesis, cleaving the peptide resin with an acid to obtain a crude product, and finally purifying the crude product to obtain a pure product. In which, the peptide resin preparation step by SPPS involves sequentially coupling the corresponding protected amino acids of the following sequence onto a carrier resin via a solid-phase coupling synthesis method to prepare the peptide resin:

[0030] In the preparation method mentioned above, the Fmoc-protected amino acid is used in an amount of 1.2 to 6 times the total molar amount of resin used; preferably 2.5 to 3.5 times.

[0031] In the preparation method mentioned above, the carrier resin has a substitution value of 0.3 to 1.5 mmol / g resin, preferably 0.6 to 1.0 mmol / g resin.

[0032] In a preferred embodiment of the disclosure, the solid-phase coupling synthesis method is as follows: the Fmoc protecting group is removed from the protected amino acid resin obtained from the previous step, followed by a coupling reaction with the next protected amino acid. The deprotection step of removing the Fmoc group lasted from 10 to 60 min, Petition 870250086443, dated 09 / 24 / 2025, page 12 / 74 7 / 16 preferably 15 to 25 min. The coupling reaction lasted 60 to 300 min, preferably 100 to 140 min.

[0033] The coupling reaction requires the addition of a condensation reagent. The condensation reagent is selected from the group consisting of DIC (N,N-di-isopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate and O-(benzotriazol)N,N,N',N'-tetramethyluronium tetrafluoroborate. N,N-di-isopropylcarbodiimide is preferred. The condensation reagent is used in a molar quantity of 1.2 to 6 times the total molar quantity of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0034] The coupling reaction requires the addition of an activating agent. The activating agent is selected from the group consisting of 1-hydroxybenzotriazole and N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The activating agent is used in an amount of 1.2 to 6 times the total molar quantity of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0035] In a preferred embodiment of the present disclosure, the reagent for removing the Fmoc protecting group is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide), wherein the mixed solution contains 10% to 30% (V) piperidine. The amount of Fmoc deprotecting reagent used is 5 to 15 ml per gram of amino resin, preferably 8 to 12 ml per gram of amino resin.

[0036] Preferably, the peptide resin is subjected to acidolysis to simultaneously cleave the peptide from the resin and remove protective side chain groups, followed by oxidative cyclization, to obtain the crude product.

[0037] More preferably, the acidolysis agent used in the acidolysis step of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, wherein the mixed solvent has a ratio Petition 870250086443, dated 09 / 24 / 2025, page 13 / 74 8 / 16 of the volume: 80% to 95% TFA, 1% to 10% EDT, with the remainder being water.

[0038] Even more preferably, the mixed solvent has a volume ratio of: 89% to 91% TFA, 4% to 6% EDT, with the remainder being water. Most preferably, the mixed solvent has a volume ratio of: 90% TFA, 5% EDT, with the remainder being water.

[0039] The acidolysis agent is used in an amount of 4 to 15 ml per gram of peptide resin; preferably, 7 to 10 ml per gram of peptide resin.

[0040] The cleavage step using the acidolysis agent lasts 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

[0041] For oxidative cyclization, the oxidizing agent used is selected from the group consisting of iodine, H2O2 and DMSO, preferably iodine. The oxidizing agent is added by titration until the oxidation target is reached.

[0042] Additionally, the crude product is purified by high-performance liquid chromatography and then lyophilized to obtain the pure product.

[0043] 1. Peptide resin synthesis

[0044] 2. The peptide resin was prepared by taking a carrier resin and sequentially coupling the corresponding protected amino acids of the sequence via Fmoc coupling and deprotection reactions:

[0045] (1) Coupling of the first protected amino acid of the main chain

[0046] 0.03 mol of the first protected amino acid 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 DMF solution of the protected amino acid with stirring. The reaction was carried out at room temperature with stirring for 30 min to obtain an activated protected amino acid solution, which was reserved for later use.

[0047] 0.01 mol of Rink amide MBHA resin (substitution value was approximately 0.4 mmol / g) was taken, and deprotected by treatment with Petition 870250086443, dated 09 / 24 / 2025, page 14 / 74 9 / 16 a PIP / 20% DMF solution for 25 min. The resin was washed and filtered to obtain the resin unprotected by Fmoc.

[0048] The activated protected amino acid solution was added to the unprotected resin by Fmoc, and the coupling reaction was carried out for 60 to 300 min. The resin containing a protected amino acid was obtained after filtration and washing.

[0049] (2) Coupling of other amino acids protected from the main chain

[0050] Using the same method as for coupling the first amino acid of the main chain, the other corresponding protected amino acids of the main chain were sequentially coupled to obtain a resin containing the main chain amino acids.

[0051] (3) Coupling of the first protected amino acid from the side chain

[0052] 0.03 mol of the first protected amino acid from 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 DMF solution of the protected amino acid with stirring. The reaction was carried out at room temperature with stirring for 30 min to obtain an activated protected amino acid solution.

[0053] 2.5 mmol of tetrakis(triphenylphosphine)palladium(0) and 25 mmol of phenylsilane were dissolved in an appropriate amount of dichloromethane, and a deprotection reaction was carried out for 4 h. The Alloc-deprotected resin was obtained after filtration and washing, which was reserved for later use.

[0054] The solution of the first protected amino acid side chain was added to the Alloc-unprotected resin, and the coupling reaction was carried out for 60 to 300 min. The resin containing the first protected amino acid side chain was obtained after filtration and washing.

[0055] (4) Coupling of other side-chain protected amino acids

[0056] Using the same method as for coupling the first protected amino acid of the main chain, the corresponding protected amino acids and monoprotected fatty acids of the side chain were Petition 870250086443, dated 09 / 24 / 2025, page 15 / 74 10 / 16 sequentially coupled to obtain the peptide resin.

[0057] 2. Preparation of the raw product

[0058] The peptide resin obtained above was treated with a cleavage reagent (10 ml of reagent per gram of resin) with a TFA:water:EDT volume ratio of 95:5:5. The mixture was stirred uniformly and allowed to react at room temperature for 3 h. The reaction mixture was filtered through a frit funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Anhydrous diethyl ether was added to precipitate the peptide. Then, the precipitate was washed three times with anhydrous diethyl ether and then dried under vacuum to produce a whitish powder.

[0059] The resulting whitish powder was dissolved in a 20% aqueous solution of acetic acid. A saturated iodine / ethanol solution was added dropwise with stirring until cyclization was complete. The solution was then concentrated under reduced pressure at 35–40 °C to obtain the crude product concentrate.

[0060] 3. Preparation of the pure product

[0061] The crude product concentrate obtained above was purified by filtration through a mixed microporous filter membrane of 0.45 μm and reserved for later use.

[0062] Purification was performed using high-performance liquid chromatography. The chromatographic packing material for purification was 10 μm of reversed-phase C18 material, and the mobile phase system consisted of 0.1% TFA / water and 0.1% TFA / acetonitrile. For a 30 mm*250 mm chromatography column, the flow rate was 20 ml / min. A gradient elution and cyclic loading purification was used. The crude product solution was loaded onto the column, and the mobile phase was initiated for elution. The main peak was collected, and after removing acetonitrile by evaporation, a purified intermediate concentrate was obtained.

[0063] The purified intermediate concentrate was filtered through a membrane Petition 870250086443, dated 09 / 24 / 2025, p. 16 / 74 11 / 16 of 0.45 μm and reserved for later use. Salt exchange was performed using high-performance liquid chromatography. The mobile phase system was 1% acetic acid / water-acetonitrile, and the chromatographic packing material for purification was 10 μm of C18 reversed-phase material. For a 30 mm*250 mm chromatography column, the flow rate was 20 ml / min (the flow rate can be adjusted according to different chromatography column specifications). A gradient elution and cyclic loading method was used. The sample was loaded onto the column, the mobile phase was initiated for elution, and chromatograms were collected. Changes in absorbance were observed, and the main peak of the salt exchange was collected. Purity was verified by analytical liquid chromatography.The fractions containing the main peak of salt exchange were combined, concentrated under reduced pressure to obtain an aqueous solution of purified product in acetic acid, and then lyophilized to obtain the pure product.

[0064] Using the method described above, the following compounds were synthesized: Table 2 Compound 1 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG1CH2CO-γGlu-eicosanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Compound 2 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG2CH2CO-γGlu-acid eicosanedioic)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Compound 3 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG3CH2CO-γGlu-eicosanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Petition 870250086443, dated 09 / 24 / 2025, page 17 / 74 12 / 16 Composto 4 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG4CH2CO-YGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 5 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG5CH2CO-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 6 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG6CH2CO-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 7 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(Gly-Ser-Gly-Ser-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 8 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(Gly-Gly-Ser-Gly-Ser-Gly-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GlySer-Gly-Thr-Pro-NH2 Composto 9Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γGluacid eicosanedioic)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-AsnThr-Gly-Ser-Gly-Thr-Pro-NH2 Compound 10 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(Gly-γGlu-eicosanedioic acid)-Leu- Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Petition 870250086443, dated 09 / 24 / 2025, page 18 / 74 13 / 16 Composto 11 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(Gly-Gly—YGlu-ácido eicosanodioico)Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro- NH2 Composto 12 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(Gly-Gly-Gly-YGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 13 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 14 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-Gly-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GlySer-Gly-Thr-Pro-NH2 Composto 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(Gly-Gly-Gly-Gly-Gly-Gly-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GlySer-Gly-Thr-Pro-NH2 Composto 16 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-acido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Composto 17 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(AEEA-AEEA-AEEA-γGlu-ácido eicosanodioico)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-GlySer-Gly-Thr-Pro-NH2 Petition 870250086443, de 24 / 09 / 2025, pág. 19 / 74 14 / 16 Compound 18 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(PEG5CH2CO-γGlu-octadecanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr- Gly-Ser-Gly-Thr-Pro-NH2 Compound 19 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-SerGln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-acid octadecanedioic)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-ThrGly-Ser-Gly-Thr-Pro-NH2 Compound 20 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(PEG5CH2CO-γGlu-docosanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2 Compound 21 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln-Glu-Leu-His-Lys(AEEA-AEEA-γGlu-docosanedioic acid)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- Ser-Gly-Thr-Pro-NH2

[0065] Example 2: Activity determination

[0066] 1. Determination method

[0067] After activation by a ligand, Gαs-coupled GPCRs can increase intracellular cAMP levels, and cAMP, as a second messenger, can mediate a series of cellular responses. The cAMP Hunter HEK293-CT-R Gs cell line, which overexpresses Gαs-coupled calcitonin receptors (CTRs), was used. Binding of calcitonin (or an analog thereof) to the overexpressed CTR increases intracellular cAMP levels. The amount of intracellular cAMP produced after activation by calcitonin (or an analog thereof) was detected using a Dynamic 2 cAMP Kit to assess the biological activity of calcitonin (or an analog thereof).

[0068] The HEK293 cell line, which stably expresses CT, was used. Petition 870250086443, dated 09 / 24 / 2025, page 20 / 74 15 / 16 R. Stably transfected cells were stimulated with various concentrations of agonists. The time-resolved fluorescence resonance energy signal of the cells after stimulation with different doses was measured using HTRF technology, from which the biological activity of the agonist was subsequently calculated.

[0069] 2. Determination results

[0070] The results of the determination are presented in the table below: Table 3 Compounds Result of EC50 Activity Determination (pmol) % of Relative Activity Salmon Calcitonin 9.5 100.0 Compound 1 4.6 206.5 Compound 2 4.1 231.7 Compound 3 3.7 256.8 Compound 4 3.0 316.7 Compound 5 2.8 339.3 Compound 6 5.5 172.7 Compound 7 4.4 215.9 Compound 8 3.7 256.8 Compound 9 4.1 231.7 Compound 10 4.9 193.9 Compound 11 4.3 220.9 Compound 12 3.9 243.6 Compound 13 3.5 271.4 Compound 14 3.1 306.5 Compound 15 6.5 146.2 Compound 16 2.9 327.6 Petition 870250086443, dated 09 / 24 / 2025, page 21 / 74 16 / 16 Compound 17 3.1 306.5 Compound 18 3.5 271.4 Compound 19 3.7 256.8 Compound 20 3.2 296.9 Compound 21 3.5 271.4

[0071] Example 3: Determination of preliminary pharmacokinetic properties

[0072] Macaca fascicularis were used as experimental animals. Subcutaneous administration was performed at a dose of 0.1 mg / kg. Venous blood samples were collected 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 post-administration. Plasma samples were separated by centrifugation. The plasma concentration of the compound in the plasma samples was determined using liquid chromatography-mass spectrometry. The half-life of the compound after subcutaneous (SC) administration is shown in the table below: Table 4 Compound t1 / 2 (h) Compound 5 53.6 The foregoing constitutes merely preferred embodiments of the present disclosure. It should be noted that, for a person of ordinary skill in the art, various improvements and modifications may be made without departing from the principles of the present disclosure, and such improvements and modifications should also be regarded as falling within the scope of protection of the present disclosure. Petition 870250086443, dated 09 / 24 / 2025, page 22 / 74

Claims

1 / 2 CLAIMS 1.Long-acting calcitonin analog characterized by having a structure as represented by structural formula I: Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-HisAA1(R)-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-AA2 (a disulfide bond between positions 1 and 7) structural formula I AA1 in structural formula I is D-Lys, L-Lys, D-Dap, L-Dap, D-Dab, L-Dab, DOrn, L-Orn, D-Dah, L-Dah, D-Dao or L-Dao; AA2 in structural formula I is NH2 or OH; R in structural formula I is HO2C(CH2)mCO-(AA3)n2-(PEGn3(CH2)n4CO)n5-, HO2C(CH2)n1CO-(AA3)n2-(AA4)n6-, or absence, wherein: n1 is an integer in the range of 10 to 20; n2 is an integer in the range of 1 to 5; n3 is an integer in the range of 1 to 30; n4 is an integer in the range of 1 to 5; n5 is an integer in the range of 1 to 5; n6 is an integer in the range of 1 to 10; AA3 is γGIu, 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. Long-acting calcitonin analogue according to claim 1, characterized by including a pharmaceutically acceptable salt, a solvate, a chelate, or a non-covalent complex thereof, a prodrug based on the compound, or any mixture of the foregoing forms.

3. Long-acting calcitonin analogue, according to either of claims 1 and 2, characterized by being for use in the manufacture of a pharmaceutical composition to treat a disease.

4. Long-acting calcitonin analogue according to claim 3, characterized in that the pharmaceutical composition is used for the prevention and treatment of osteoporosis, deforming bone disease, algoneurodystrophy and malignant osteolysis.

5. Pharmaceutical composition characterized by comprising the long-acting calcitonin analogue, as defined in any one of claims 1 to 4.

6. Use of the long-acting calcitonin analogue, as defined in any one of claims 1 to 4, or of the pharmaceutical composition, as defined in claim 5, characterized by being in the manufacture of a medicament for the prevention and treatment of osteoporosis, deforming bone disease, algoneurodystrophy and malignant osteolysis. Petition 870250086443, dated 09 / 24 / 2025, page 24 / 74