Polypeptide compound and preparation method therefor, pharmaceutical composition, and use thereof

Abstract

A polypeptide compound, a pharmaceutical composition thereof, and a use thereof. A polypeptide compound, the polypeptide compound having a structural formula (XX). The polypeptide compound exhibits good osteogenic activity in vitro, and has relatively good pharmacokinetic properties, indicating that the polypeptide compound has potential application value in the treatment of osteoporosis.

Description

A polypeptide compound, its preparation method, pharmaceutical composition and uses Technical Field

[0001] This application relates to the technical field of polypeptide compound drugs, and more particularly to a polypeptide compound, its preparation method, pharmaceutical composition and use. Background Technology

[0002] Osteoporosis (OP) is a systemic bone disease caused by various factors, resulting in decreased bone density, decreased bone quality, destruction of bone microstructure, and increased bone fragility, making fractures more likely. Osteoporosis generally occurs in postmenopausal women and elderly men, seriously affecting patients' health and quality of life, even shortening lifespan and increasing the burden on families, making it a major social concern. Bone tissue is constantly undergoing bone remodeling / reconstitution, a process where old bone is replaced by new bone. The cellular basis of bone remodeling / reconstitution is the balance between the "coupled" processes of osteoblast (OB) bone formation and osteoclast (OC) bone resorption. This balance is essential for maintaining normal bone mass and the integrity of bone structure. The development of osteoporosis is primarily the result of an imbalance in the bone remodeling / reconstitution process constituted by osteoblastic bone formation and osteoclast-mediated bone resorption.

[0003] Currently, most treatments for osteoporosis involve inhibiting bone resorption, such as bisphosphonates and monoclonal antibody drugs like denosumab. Although various osteoporosis treatments that inhibit osteoclast formation are available clinically, most drugs have limited effectiveness against osteoporosis, and some have significant side effects and adverse reactions. For example, bisphosphonates have low bioavailability, are prone to gastrointestinal reactions, and long-term use can lead to esophageal cancer and osteonecrosis of the mandible; denosumab's use is also limited due to its high price.

[0004] Currently available drugs promoting bone formation include recombinant human parathyroid hormones such as hPTH(1-34) (teriparatide) and PTHrp (albalotide), and the sclerostin monoclonal antibody Evenity (romosozumab). These drugs have all been approved by the FDA for the treatment of osteosarcoma (OP). However, as hormonal drugs, clinical evidence suggests that long-term use of teriparatide and abalotide carries a risk of inducing osteosarcoma, and their use is not recommended for patients with a history of radiation exposure or primary or secondary hyperparathyroidism. Evenity (romosozumab) is a fully humanized monoclonal antibody that works by inhibiting the activity of sclerostin. In the ARCH trial, Evenity showed stronger efficacy compared to the existing OP drug alendronate, with reduced rates of vertebral and non-vertebral fractures. However, the Evenity (romosozumab) group showed a certain risk of cardiovascular side effects.

[0005] Therefore, there is an urgent need to find new osteogenic anabolic drugs for the treatment of osteoporosis and to promote fracture healing. Summary of the Invention

[0006] The purpose of this application is to provide a polypeptide compound, a pharmaceutical composition, and its use. The polypeptide compound has the structural formula XX. The polypeptide compound exhibits good osteogenic activity in vitro and has good pharmacokinetic characteristics, and has great application value in bone repair and osteoporosis treatment.

[0007] To solve the above-mentioned technical problems, this application is implemented as follows:

[0008] The primary objective of this application is to provide a polypeptide compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a tautomer thereof, or a polymorph thereof, or a solvate thereof, or an N-oxide thereof, or an isotopically labeled compound thereof, or a metabolite thereof, or a prodrug thereof, comprising the following structural formula (XX);

[0009] In the structural formula (XX), R 1 Independently selected from H, halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl; R 2 Independently selected from C1-C6 alkyl, H, halogen, C1-C6 alkoxy, and C1-C6 haloalkyl; Y 1 -Y 4 X is selected from amino acid residues; X is selected from CH3, COOH, SO3H, or OH.

[0010] m, n, q, and w are each individually selected from any value between 0 and 6;

[0011] The subscript 'o' is selected from any value between 1 and 5;

[0012] The subscript r is selected from any value between 0 and 3;

[0013] The subscript p is selected from any value between 10 and 20.

[0014] In some embodiments, R 1 Independently selected from H, halogens, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl;

[0015] In some embodiments, R 2 Independently selected from C1-C6 alkyl, H, halogen, C1-C6 alkoxy and C1-C6 haloalkyl;

[0016] In some embodiments, Y 4 Selected from citrulline residues, N6-(3-(pyridin-3-yl)propionyl)-lysine residues, high-citrulline residues, leucine residues, (N-methyl)leucine residues, isoleucine residues, (N-methyl)isoleucine residues, and high-phenylalanine residues;

[0017] Y 4 Selected from citrulline residues or N6-(3-(pyridin-3-yl)propionyl)-lysine residues, high-citrulline residues, leucine residues, (N-methyl)leucine residues, isoleucine residues, oroleucine residues, (N-methyl)isoleucine residues, and high-phenylalanine acid residues; Y 3 Selected from glutamic acid residues or glutamine residues, aspartic acid residues or asparagine residues; Y 2 Selected from serine residues, threonine residues, tyrosine residues, or cysteine ​​residues; Y 1 Selected from valine residues, isoleucine residues, leucine residues, methionine residues, or oroleucine;

[0018] In some embodiments, Y 3 Selected from glutamic acid residues, glutamine residues, aspartic acid residues, or asparagine residues;

[0019] In some embodiments, Y 2 Selected from serine residues, threonine residues, tyrosine residues, or cysteine ​​residues;

[0020] In some embodiments, Y 1 Selected from valine residues, isoleucine residues, leucine residues, methionine residues, or oroleucine;

[0021] In some embodiments, o = 4;

[0022] In some embodiments, r = 1 or 2;

[0023] In some embodiments, -Y 4 -YY 2 -Y 1 -Selected from -Cit-Glu-Ser-Val-;

[0024] In some embodiments, -Y 4 -Y 3 -Y 2 -Y 1 -(AEEA)n- is selected from -Cit-Glu-Ser-Val-(AEEA)n-, m=0-2, n=0-2, o=4, r=2; the compound of formula (XX) has the structure of the following structural formula (Ⅰ);

[0025] In structural formula (Ⅰ), n is any value of 0, 1, 2, 3, 4, 5, 6; m is any value of 0, 1, 2, 3, 4, 5, 6; p is any value of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; X is selected from CH3, COOH, SO3H or OH.

[0026] A second objective of this application is to provide a pharmaceutical composition comprising the above-described polypeptide compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its tautomer, or its polymorph, or its solvate, or its N-oxide, or its isotope-labeled compound, or its metabolite, or its prodrug, and one or more pharmaceutically acceptable excipients or carriers.

[0027] A third objective of this application is to provide the use of the above-mentioned polypeptide compound in the preparation of a medicament for activating cell signaling in osteoprogenitor cells, thereby promoting bone formation.

[0028] A fourth objective of this application is to provide the use of the above-mentioned polypeptide compound in the preparation of a medicament for the prevention, treatment or relief of osteoporosis.

[0029] The beneficial effects of this application are as follows:

[0030] This application presents polypeptide compounds prepared through extensive screening experiments. Experiments have demonstrated that these polypeptide compounds have significant osteoblast-inducing activity, including increased ALP activity and calcification as shown by Alizarin Red staining, exhibiting good osteogenic activity and favorable pharmacokinetic characteristics, indicating their potential application value in bone repair and osteoporosis treatment.

[0031] In rat administration studies, peptide compound (III) exhibited a superior biological half-life compared to drug YLL3 in patent WO2020018941A1, demonstrating better therapeutic effects in osteoporosis, improved fracture healing, in vivo stability, and potential sustained-release efficacy. Compared to existing treatments, peptide compound (III) offers fewer toxic side effects and higher therapeutic efficiency, thereby improving patient outcomes and quality of life. Attached Figure Description

[0032] Figure 1 shows the chemical structural formula of the polypeptide compound XX described in this application;

[0033] Figure 2 is a process flow diagram of the preparation of target peptide II in Example 2 of this application;

[0034] Figure 3 is the mass spectrum of target peptide II in Example 2 of this application;

[0035] Figure 4 is an HPLC chromatogram of the target peptide II in Example 2 of this application;

[0036] Figure 5 is a process flow diagram of the target peptide III in Example 3 of this application;

[0037] Figure 6 is the mass spectrum of target peptide III in Example 3 of this application;

[0038] Figure 7 shows the HPLC of the target peptide III in Example 3 of this application;

[0039] Figure 8 is a process flow diagram of the target peptide IV or its salt in Example 4 of this application;

[0040] Figure 9 shows the MS of the target peptide IV or its salt in Example 4 of this application;

[0041] Figure 10 shows the HPLC of the target peptide IV or its salt in Example 4 of this application;

[0042] Figure 11 is a process flow diagram of the target peptide V or its salt in Example 5 of this application;

[0043] Figure 12 shows the MS of the target peptide V or its salt in Example 5 of this application;

[0044] Figure 13 shows the HPLC of the target peptide V or its salt in Example 5 of this application;

[0045] Figure 14 shows the osteogenic induction ALP activity (OD) of each target peptide and YLL3 in Example 6 of this application;

[0046] Figure 15 shows the osteogenic induction alizarin red (OD) staining of each target peptide and YLL3 in Example 6 of this application;

[0047] Figure 16 shows the individual blood drug concentration data (unit: ng / mL) of target peptide III in rats at different time points after subcutaneous administration in Example 7 of this application;

[0048] Figure 17 is a pharmacokinetic curve of subcutaneous administration of target peptide III to rats in Example 7 of this application;

[0049] Figure 18 shows the individual blood drug concentration data of YLL3 rats at different time points after subcutaneous administration in Example 7 of this application;

[0050] Figure 19 is a pharmacokinetic curve of subcutaneous administration of the drug to YLL3 rats in Example 7 of this application;

[0051] Figure 20 shows the individual data of blood drug concentration at different time points after intravenous administration of target peptide III to rats in Example 7 of this application;

[0052] Figure 21 is a pharmacokinetic curve of intravenous administration of target peptide III to rats in Example 7 of this application;

[0053] Figure 22 shows the individual blood drug concentration data of YLL3 rats at different time points after intravenous administration in Example 7 of this application;

[0054] Figure 23 is a pharmacokinetic curve of intravenous administration to YLL3 rats in Example 7 of this application;

[0055] Figure 24 is a bar chart of serum bone turnover index in osteoporotic rats in Example 8 of this application;

[0056] Figure 25 shows a bar chart and 3D diagram of bone-related indicators in osteoporotic rats in Example 8 of this application;

[0057] Figure 26 is a bar chart of the spinal biomechanical data of osteoporotic rats in Example 8 of this application;

[0058] Figure 27 is a bar chart of callus volume data from Embodiment 9 of this application;

[0059] Figure 28 is an X-ray image of new bone tissue formation in Embodiment 9 of this application;

[0060] Figure 29 shows a tissue section observed under a microscope in Example 9 of this application. Detailed Implementation

[0061] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. In this application, the meanings or references of some English / chemical formula abbreviations are shown in the table below:

[0062] The compounds of the present invention can be synthesized by a variety of methods known to those skilled in the art (see Richard C. Larock's *Comprehensive Organic Transformations*, 1989) or by suitable combinations of generally well-known synthetic methods. The techniques available for synthesizing the compounds of the present invention will be readily apparent to those skilled in the art and are readily available. The following discussion is provided to illustrate some of the various methods that can be used to prepare the compounds of the present invention. However, this discussion is not intended to limit the scope of reactions or reaction sequences that can be used to prepare the compounds of the present invention. Those skilled in the art will understand that other methods for preparing the compounds can be used in the present invention.

[0063] Explanation of some terms:

[0064] Polypeptide compounds are compounds composed of multiple amino acids linked by peptide bonds. Their molecular weight is smaller than that of proteins but larger than that of dipeptides and tripeptides, and they generally have biological activity. In this invention, polypeptide compounds specifically refer to peptide compounds with specific structural formulas and sequences.

[0065] Solid-phase synthesis is a method for the stepwise synthesis of biomolecules such as peptides or oligonucleotides on a solid support. In peptide synthesis, resins (such as Rink amide MBHA resin) are commonly used as solid supports. The target peptide sequence is constructed stepwise by repeatedly adding protected amino acid monomers and removing protecting groups.

[0066] Rink amide MBHA resin is a type of resin used for solid-phase peptide synthesis. It possesses a specific chemical structure that stably links and supports peptide chains during synthesis. MBHAresin (4-methyldiphenylmethylamine resin) is the carrier component, while rink amide acts as a linker arm, used to release the peptide from the resin after synthesis.

[0067] Fmoc removal reagents remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group from amino acids during peptide synthesis, preventing unwanted reactions with other amino acids. Fmoc removal reagents are used to remove the Fmoc protecting group during synthesis, exposing the amino group of the amino acid for condensation with the next amino acid.

[0068] AEEA (Aminoethyl Ethylene Amine) may exist as a linker or a specific functional group in the structure of a polypeptide compound to connect other chemical parts or to endow the polypeptide with specific biological activities.

[0069] In peptide synthesis, some amino acids, in addition to the amino acid sequence on the main chain, also possess side chain groups. Side chain fragments are synthesized as fragments of certain amino acids with complex side chains beforehand, especially in solid-phase synthesis, to simplify operations and improve efficiency, and then introduced during the main chain synthesis process.

[0070] Pharmacokinetics is the science that studies the absorption, distribution, metabolism, and excretion of drugs in organisms and their changes over time. Pharmacokinetic characteristics typically include parameters such as half-life, clearance rate, and bioavailability, and are of great significance for evaluating the clinical efficacy and safety of drugs.

[0071] When substituent groups are specified by their conventional chemical formula written from left to right, they also cover chemically identical substituents produced by writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0072] As used herein, the term “halogenated” or “halogen” itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom.

[0073] As used herein, the term "alkyl" refers to a straight-chain or branched saturated aliphatic group having a specified number of carbon atoms. For example, C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, etc.

[0074] As used herein, the term "haloalkyl" refers to an alkyl group as defined above, wherein some or all of its hydrogen atoms are replaced by halogen atoms. For example, haloalkyl groups include trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluorine" defines a compound or group having at least two available fluorine-substituted hydrogen atoms. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0075] As used herein, the term "alkoxy" refers to an alkyl group containing an oxygen atom, such as methoxy, ethoxy, etc.

[0076] As used herein, the term “amino acid” refers to naturally occurring, non-natural, and synthetic amino acids, as well as amino acid analogs and amino acid simulants that function in a manner similar to that of naturally occurring amino acids.

[0077] As used herein, the term "naturally occurring amino acid" refers to those amino acids encoded by the genetic code, as well as those that have been modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0078] As used herein, the term "non-natural amino acid" includes, but is not limited to, L- or D-configured amino acid analogs, amino acid mimics, synthetic amino acids, and N-amino acids that function in a manner similar to naturally occurring amino acids. 6 - Modified lysine and N-methyl amino acids. Non-natural amino acids are not encoded by the genetic code and may, but do not necessarily, have the same basic structure as naturally occurring amino acids.

[0079] As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure (i.e., the α-carbon, carboxyl, amino, and R groups attached to hydrogen) as naturally occurring amino acids, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.

[0080] As used herein, the term "amino acid mimic" refers to a compound having a structure different from the general chemical structure of an amino acid but functioning in a manner similar to that of naturally occurring amino acids. Suitable amino acid mimics include, but are not limited to, β-amino acids and γ-amino acids. In β-amino acids, the amino group is bonded to the β-carbon atom of the carboxyl group, such that there are two carbon atoms between the amino and carboxyl groups. In γ-amino acids, the amino group is bonded to the γ-carbon atom of the carboxyl group, such that there are three carbon atoms between the amino and carboxyl groups. Suitable R groups for β-amino or γ-amino acids include, but are not limited to, side chains present in naturally occurring and non-natural amino acids.

[0081] Regarding amino acid sequences, those skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence (that alters, adds, or deletes a single amino acid or a small subset of amino acids in the coding sequence) is a “variant of a conserved modification” where the alteration results in the substitution of the amino acid with a chemically similar amino acid (i.e., hydrophobic, hydrophilic, positively charged, neutral, or negatively charged). This chemically similar amino acid includes, but is not limited to, naturally occurring amino acids such as L-amino acids, stereoisomers of naturally occurring amino acids such as D-amino acids, and non-natural amino acids such as amino acid analogs, amino acid mimics, synthetic amino acids, N-substituted glycine, and N-methyl amino acids. Exemplary hydrophobic amino acids include valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. Exemplary aromatic amino acids include phenylalanine, tyrosine, and tryptophan. Exemplary aliphatic amino acids include serine and threonine. Exemplary basic amino acids include lysine, arginine, and histidine. Exemplary amino acids with carboxylate side chains include aspartate and glutamate. Exemplary amino acids having formamide side chains include asparagine and glutamine. A table of conserved substitutions for functionally similar amino acids is well known in the art. Such conserved variants are complementary to the polymorphs, interspecific homologs, and alleles of the present invention, and do not exclude the polymorphs, interspecific homologs, and alleles of the present invention.

[0082] Providing a table of conservative substitutions for functionally similar amino acids is well known in the art. For example, substitutions can be made where an aliphatic amino acid (e.g., G, A, I, L, or V) is replaced by another member of the group. Similarly, aliphatic polar uncharged groups, such as C, S, T, M, N, or Q, can be replaced by another member of the group; basic residues such as K, R, or H can be substituted for each other. In some embodiments, amino acids with acidic side chains, such as E or D, can be replaced by their uncharged counterparts, such as Q or N, respectively; or vice versa. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for each other:

[0083] 1) Alanine (A), glycine (G);

[0084] 2) Aspartic acid (D), glutamic acid (E);

[0085] 3) Asparagine (N), glutamine (Q);

[0086] 4) Arginine (R), Lysine (K);

[0087] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0088] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0089] 7) Serine (S), Threonine (T);

[0090] 8) Cysteine ​​(C), Methionine (M)

[0091] As used herein, the term "salt" refers to the acidic or basic salt of a compound used in the methods of the present invention. Illustrative examples of pharmaceutically acceptable salts are inorganic acid (hydrochloric acid, hydrobromic acid, phosphate, etc.) salts, organic carboxylic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, organic sulfonic acid (methanesulfonic acid) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts. It is understood that pharmaceutically acceptable salts are non-toxic. Further information regarding suitable pharmaceutically acceptable salts may be incorporated herein by reference.

[0092] As used herein, the term "hydrate" refers to a compound that is complexed with at least one water molecule. The compounds of the present invention can be complexed with 1 to 10 water molecules.

[0093] As used herein, the terms "pharmaceutical-acceptable excipient" and "pharmaceutical-acceptable carrier" refer to substances that facilitate the administration and absorption of an active agent by an individual. "Pharmaceutical-acceptable excipient" means an excipient that can be included in the compositions of the present invention and will not produce significant harmful toxicological effects on a patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, aqueous saline solutions, lactated Ringer's solution, conventional sucrose, conventional glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, and colorants, etc. Those skilled in the art will recognize that other pharmaceutical excipients may be used in the present invention.

[0094] As used in this article, the term "prodrug" refers to a compound obtained by chemically modifying a drug, which is inactive or has low activity in vitro, but releases an active drug in vivo through enzymatic or non-enzymatic conversion to exert its pharmacological effect.

[0095] As used herein, the term "polymorph" refers to the crystalline form of a compound (or its salts, hydrates, or solvates) that exhibits a specific crystalline packing. All polymorphs have the same elemental composition. Different polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can cause one polymorph to become dominant.

[0096] As used herein, the term "osteoporosis" refers to a condition that results in increased bone fragility and / or decreased or reduced bone mineral density due to any known or unknown cause or condition. "Low bone mass" or "osteopenia" is a condition, not a disease, that can develop into osteoporosis once bone mineral density continues to decrease over time. Low bone mass is characterized by a T-score of -1 to -2.15. Osteoporosis is characterized by a T-score less than -2.15. Among the specific causes or conditions targeted by the method of the present invention are primary osteoporosis associated with menopause (natural, premature, or surgical), aging, or both, and secondary osteoporosis or secondary low bone mass associated with medical conditions (e.g., Paget's disease, chronic kidney disease, amenorrhea due to eating disorders, transplantation, hyperthyroidism, hyperparathyroidism) or the use of certain medications (e.g., various cancer chemotherapy, gonadotropin-releasing hormone agonists, medroxyprogesterone acetate for fertility control, corticosteroids, anticonvulsants, etc.).

[0097] Currently, osteoporosis (OP) is a systemic bone disease caused by decreased bone density, decreased bone quality, destruction of bone microstructure, and increased bone fragility, primarily affecting postmenopausal women and elderly men. Existing treatments mainly focus on inhibiting bone resorption, such as bisphosphonates and the monoclonal antibody denosumab. However, these drugs have limited efficacy and some have significant toxic side effects, such as gastrointestinal reactions, esophageal cancer, and risks of osteonecrosis of the mandible. While bone-forming drugs such as teriparatide, abalotene, and Evenity are effective, their use and safety are limited as they are hormonal drugs.

[0098] To solve the above-mentioned technical problems, this application provides a polypeptide compound comprising the following structural formula XX;

[0099] Where R 1 Independently selected from H, halogens, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl;

[0100] R 2 Independently selected from C1-C6 alkyl, H, halogen, C1-C6 alkoxy and C1-C6 haloalkyl;

[0101] Y 1 -Y 4 Selected independently from amino acid residues;

[0102] X is selected from CH3, COOH, SO3H, or OH;

[0103] The subscripts m, n, q, and w are each selected individually from any value between 0 and 6;

[0104] The subscript 'o' is selected from any value between 1 and 4;

[0105] The subscript r is selected from any value between 0 and 2;

[0106] The subscript p is selected from any value between 10 and 20.

[0107] In some embodiments, -Y 4 -YY 2 -Y 1 -Selected from -Cit-Glu-Ser-Val-;

[0108] In some embodiments, -Y 4 -Y 3 -Y 2 -Y 1 -(AEEA)n- is selected from -Cit-Glu-Ser-Val-(AEEA)n-, m=0-2, n=0-2, o=4, r=2;

[0109] In some embodiments, the polypeptide compound includes compounds with the following structural formulas:

[0110] In structural formula I, n is any value of 0, 1, 2, 3, 4, 5, 6; m is any value of 0, 1, 2, 3, 4, 5, 6; p is any value of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19; X is selected from CH3, COOH, SO3H or OH.

[0111] In some embodiments, the polypeptide compound includes compounds with the following structural formulas:

[0112] In some embodiments, polypeptide compounds are prepared by using a standard solid-phase synthesis method on Rink amide MBHA resin.

[0113] Specifically, the standard solid-phase synthesis method, also known as solid-phase peptide synthesis (SPPS), is a technique for synthesizing peptides by progressively linking amino acids onto a solid support. Its core principle is to covalently link the first amino acid (or its protected form) to an insoluble solid support (such as resin), and then, through repeated deprotection and coupling reactions, link subsequent amino acids one by one until the desired peptide sequence is synthesized. This method simplifies the peptide synthesis process and improves synthesis efficiency and product purity by avoiding the multiple separation and purification steps in traditional liquid-phase synthesis.

[0114] More specifically, the preparation method of the polypeptide compound includes the following steps:

[0115] S1. Resin Preparation

[0116] Rink amide MBHA resin with a degree of substitution of 0.2–0.75 mmol / g was selected, preferably 0.73 mmol / g. The MBHA resin was pretreated, such as by washing and drying, to ensure its suitability for subsequent peptide synthesis.

[0117] S2. Condensation of amino acids and side chain fragments

[0118] (1) Synthesis of the main chain peptide:

[0119] Following the superscript number order of the amino acid sequence, amino acids and / or side chain fragments are sequentially condensed onto the resin.

[0120] The amino acids and side chain fragments used include: Fmoc-RinkLinker, Fmoc-Lys(Mtt)-OH, Fmoc-AEEA-OH, Fmoc-Val-OH, and Fmoc-Ser(O t Bu)-OH, Fmoc-Glu(O t Bu)-OH.H2O, Fmoc-Cit-OH, {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid, etc.

[0121] The condensation reagent combinations used in the condensation reaction include: DIC / HOBt, DIC / 6-Cl-HOBt, DIC / Oxyma, HATU / DIPEA, HATU / DIPEA / HOBt, HBTU / DIPEA, HBTU / DIPEA / HOBt, PyBOP / DIPEA, PyBOP / DIPEA / HOBt, etc., with DIC / HOBt being preferred.

[0122] The reaction solvent is one or a combination of DCM, DMF, NMP, and DMSO, with DCM / DMF being preferred.

[0123] The Fmoc protecting group is removed using a 15%–25% v / v piperidine or 4-methylpiperidine / DMF solution.

[0124] (2) Synthesis of side chain peptide fragments:

[0125] Side chain fragments include Fmoc-AEEA-OH and Fmoc-Glu-O t Bu, dodecanoic acid, hexadecanoic acid, octadecanedioic acid monotert-butyl ester, eicosanedioic acid monotert-butyl ester, etc.

[0126] Synthesize according to the order marked in the upper right corner of the side chain fragment, from side 1 to side 3, to obtain the corresponding side chain peptide fragment, such as side chain peptide fragment - (AEEA). m 侧1 -γGlu 侧2 -Eicosanedioic acid 侧3 -(AEEA) m 侧1 -γGlu 侧2 -Octadecanedioic acid 侧3 -(AEEA) m 侧1 -γGlu 侧2 -Hexadecanoic acid 侧3 -(AEEA) m 侧1 -γGlu 侧2 -Ladecanoic acid 侧3 .

[0127] (3) Condensation of main chain peptide and side chain peptide fragments

[0128] The main chain peptide resin is condensed with the side chain peptide fragment, and the target peptide resin is obtained by washing and drying.

[0129] The peptide sequence of the target peptide resin is: 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl

[0130] 7 -Cit 6 -Glu 5 -Ser 4 -Val 3 -(AEEA) n 2 -Lys 1 [(AEEA) m 侧1 -γGlu 侧2 -X 侧3]-NH2.

[0131] (4) Cleavage and purification of the target peptide

[0132] The target peptide resin was lysed using a lysis reagent (TFA:H2O:EDT:TIS = 94:2:2:2, V:V:V:V) to obtain the target peptide lysate.

[0133] After filtration and vacuum concentration, the lysate is precipitated using an ether solvent (such as methyl tert-butyl ether) at a temperature below 10±3℃, with a volume of 8 to 10 times that of the concentrate.

[0134] The precipitate was subjected to centrifugation, repeated washing, further centrifugation, and vacuum drying to obtain the crude target peptide.

[0135] The crude target peptide was dissolved in 50 mmol / L disodium hydrogen phosphate (pH=7.5) aqueous solution and purified by high performance liquid chromatography.

[0136] The stationary phase was C18 alkyl-bonded silica gel. The column temperature was set to 40℃, and the detection wavelength was set to 214 nm. Mobile phase A was 35 mmol / L disodium hydrogen phosphate (pH = 7.0) aqueous solution, and mobile phase B was acetonitrile. Gradient elution purification was performed, with the organic phase increasing from 30% to 40%.

[0137] After purification, the salt was replaced with 0.2 mmol / L sodium hydroxide and then lyophilized to obtain a high-purity target peptide or its salt as a finished product.

[0138] On the one hand, this application provides a pharmaceutical composition comprising the peptide compound described above or a pharmaceutically acceptable salt thereof, or a solvate, hydrate thereof, or prodrug thereof.

[0139] Specifically, acceptable salts for pharmaceutical use include salts that form with bases, i.e., cationic salts, such as alkali metal salts and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, and magnesium salts, as well as ammonium salts, such as dimethylammonium, trimethylammonium, diethylammonium, and tri-(hydroxymethyl)-methyl-ammonium. Alkali metal salts or alkaline earth metal salts are preferred, and alkali metal salts, such as sodium, magnesium, calcium, lithium, and potassium salts, are more preferred.

[0140] Specifically, prodrugs are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted into the compounds of the present invention in an in vitro environment using chemical or biochemical methods.

[0141] Another aspect is a fourth objective of this application, which is to provide the use of the aforementioned polypeptide compound in the preparation of a medicament for activating cell signaling in osteoprogenitor cells, thereby promoting bone formation.

[0142] In another aspect, this application provides the use of the above-mentioned polypeptide compound in the preparation of a medicament for the prevention, treatment or relief of osteoporosis.

[0143] The following examples and other details further illustrate a polypeptide compound of this application.

[0144] Example 1

[0145] A polypeptide compound with the following structural formula: target peptide II:

[0146] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl-Cit-Glu-Ser-Val-Lys(γGlu-eicosanoic acid)-NH2

[0147] Target peptide II

[0148] As shown in Figure 2, the preparation method of target peptide II includes the following steps:

[0149] The target peptide II has a linear backbone containing 6 amino acid residues, with an amide group at the C-terminus. The ε-amino group at the 6th Lys position of the linear peptide is branched (γGlu, eicosanoic acid). Solid-phase synthesis (linear peptide synthesis, branched condensation) was employed, followed by cleavage to obtain the crude peptide. Finally, the crude peptide was separated, purified, and lyophilized to obtain the final target peptide II.

[0150] 1. Peptide resin synthesis

[0151] MBHAResin (S = 0.73 mmol / g) was used, with conventional Fmoc protection of amino acids at a dosage of 2.5-4.0 times the normal amount. DIC / HOBt was used as the condensing agent. MBHAResin lever arm modification was performed first, followed by sequential condensation from the C-terminus to the N-terminus of the peptide backbone (numbers 1 to 6 marked in the upper right corner of the peptide sequence). After the backbone synthesis was completed, the synthesis sequence was: 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl 6 -Cit 5 -Glu 4 -Ser 3 -Val 2 -Lys 1 -(γGlu 侧1 -Eicosanedioic acid 侧2 )-NH2, then after removing Mtt from the ε-amino group of Fmoc-Lys(Mtt)-OH at position 1, it sequentially condenses from side 1 to side 2 (γGlu, monotert-butyl eicosanoate).

[0152] Protecting amino acids used in the main chain: Fmoc-RinkLinker, Fmoc-Lys(Mtt)-OH, Fmoc-Val-OH, Fmoc-Ser( t Bu)-OH, Fmoc-Glu(O t Bu)-OH.H2O, Fmoc-Cit-OH, and {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid are coupled one by one using conventional SPPS coupling.

[0153] After the main chain peptide condensation is complete, the Mtt protecting group on the ε-amino group of Fmoc-Lys(Mtt)-OH at the 1-position is removed using hexafluoroisopropanol (HFIP), followed by branched chain condensation of Fmoc-Glu-O. t Bu and tert-butyl eicosanoate, after washing and drying, yielded the peptide resin {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid-Cit-Glu(O) t Bu)-Ser( t Bu)-Val-Lys-(γGlu(aO t Bu)-Eicosanedioic acid monotert-butyl ester)-Rink Linker-MBHAResin.

[0154] 2. Peptide resin cleavage

[0155] Prepare the pyrolysis reagent with the following formula: trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), and 1,2-ethylenedithiol (EDT) in a volume ratio of TFA:H2O:EDT:TIS = 94:2:2:2 (V:V:V:V). Pre-cool to 10±3℃, and slowly add the resin to the pyrolysis solution while stirring. Continue stirring until the reaction system temperature stabilizes, and control the temperature at 25±3℃. Continue stirring for 3 hours.

[0156] The lysate was filtered and concentrated to a viscous state. It was then precipitated with 8 to 10 times the volume of the concentrated lysate using methyl tert-butyl ether (MTBE). The precipitate was filtered off, washed with TFA, and dried under reduced pressure at room temperature to obtain crude target peptide II.

[0157] 3. Purification of crude target peptide II

[0158] The crude target peptide II was dissolved in 50 mmol / L disodium hydrogen phosphate (pH = 7.5) aqueous solution, and then purified by high performance liquid chromatography (HPLC) using a C18 silica gel matrix and a 35 mmol / L disodium hydrogen phosphate (pH = 7.0) aqueous solution system. The organic phase was purified by gradient elution from 30% to 40%. The product was then replaced with 0.2 mmol / L sodium hydroxide and lyophilized to obtain the target peptide II or its sodium salt (product purity 94.2%, molecular weight MS: m / z = 1337.2, calculated value: 1337.62). Figure 3 shows the mass spectrum of the target peptide II in Example 2, and Figure 4 shows the HPLC chromatogram of the target peptide II in Example 2.

[0159] Example 2

[0160] A polypeptide compound with the following structural formula: Target peptide III:

[0161] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl-Cit-Glu-Ser-Val-AEEA-Lys(γGlu-eicosanoic acid)-NH2

[0162] Target peptide III

[0163] As shown in Figure 5, the preparation method of target peptide III includes the following steps:

[0164] The main chain of this peptide contains 7 condensed residues, with an amide group at the C-terminus. The ε-amino group at the 7th Lys position of the linear peptide is branched (γGlu, eicosanoic acid). Solid-phase synthesis (linear peptide synthesis, branched condensation) was employed, followed by cleavage to obtain crude peptide III. Finally, after purification and drying, the target peptide III product was obtained.

[0165] 1. Peptide resin synthesis

[0166] MBHAResin (S = 0.73 mmol / g) was used, with conventional Fmoc protection of amino acids at a dosage of 2.5-4.0 times the normal amount. DIC / HOBt was used as the condensing agent. MBHAResin lever arm modification was performed first, followed by sequential condensation from the C-terminus to the N-terminus of the peptide backbone (numbers 1 to 7 in the upper right corner of the sequence). After the backbone synthesis was completed, the synthesis sequence was as follows:

[0167] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl 7 -Cit 6 -Glu 5 -Ser 4 -Val 3 -AEEA 2 -Lys 1 (γGlu 侧1 -Eicosanedioic acid侧2 )-NH2, then after removing Mtt from the ε-amino group of Fmoc-Lys(Mtt)-OH at position 1, it sequentially condenses from side 1 to side 2 (γGlu, monotert-butyl eicosanoate).

[0168] The protective amino acids used are: Fmoc-Rink Linker, Fmoc-Lys(Mtt)-OH, Fmoc-AEEA-OH, Fmoc-Val-OH, and Fmoc-Ser( t Bu)-OH, Fmoc-Glu(O t Bu)-OH.H2O, Fmoc-Cit-OH, {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid.

[0169] After the main-chain peptide condensation is complete, deprotection is performed using hexafluoroisopropanol (HFIP), followed by branched-chain condensation of Fmoc-Glu(O) t Bu)-OH and eicosanoic acid monotert-butyl ester, after washing and drying, yielded the peptide resin {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid-Cit-Glu(O) t Bu)-Ser( t Bu)-Val-AEEA-Lys-(γGlu(O t Bu)-Eicosanedioic acid monotert-butyl ester)-Rink Linker-MBHAResin.

[0170] 2. Peptide resin cleavage

[0171] Prepare the pyrolysis reagent with the following formula: trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), and 1,2-ethylenedithiol (EDT) in a volume ratio of TFA:H2O:EDT:TIS = 94:2:2:2 (V:V:V:V). Pre-cool to 10±3℃, and slowly add the resin to the pyrolysis solution while stirring. Continue stirring until the reaction system temperature stabilizes, and control the temperature at 25±3℃. Continue stirring for 3 hours.

[0172] The lysate was filtered and concentrated to a viscous state. It was then precipitated with 8 to 10 times the volume of the concentrated lysate using methyl tert-butyl ether (MTBE). The precipitate was filtered off, washed with TFA, and dried under reduced pressure at room temperature to obtain crude target peptide III.

[0173] 3. Purification of target peptide III

[0174] The crude target peptide III was dissolved in 50 mmol / L disodium hydrogen phosphate (pH = 7.5) aqueous solution, and then purified by high performance liquid chromatography (HPLC) using a C18 silica gel matrix and a 35 mmol / L disodium hydrogen phosphate (pH = 7.0) aqueous solution system. The organic phase was purified by gradient elution from 30% to 40%. The solution was then replaced with 0.2 mmol / L sodium hydroxide and lyophilized to obtain the target peptide III or its sodium salt product (purity of target peptide III was 99.3%, molecular weight was MS: m / z = 1482.2, calculated value: 1482.76). Figure 6 shows the mass spectrum of target peptide III in Example 3, and Figure 7 shows the HPLC of target peptide III in Example 3.

[0175] Example 3

[0176] A polypeptide compound with the following structural formula: target peptide IV:

[0177] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl-Cit-Glu-Ser-Val-Lys - (AEEA-γGlu-eicosanedioic acid)-NH2

[0178] Target peptide IV

[0179] As shown in Figure 8, the preparation method of target peptide IV or its salt includes the following steps:

[0180] The main chain of this peptide contains 6 condensed residues, with an amide group at the C-terminus. The ε-amino group at the 6th Lys position of the linear peptide is branched (AEEA, γGlu, eicosanoic acid). Solid-phase synthesis (linear peptide synthesis, branched condensation) was employed, followed by cleavage to obtain crude peptide IV. Finally, the target peptide IV product was obtained through purification and drying.

[0181] 1. Peptide resin synthesis

[0182] MBHAResin (S = 0.73 mmol / g) was used, with conventional Fmoc protection of amino acids at a dosage of 2.5-4.0 times the normal amount. DIC / HOBt was used as the condensing agent. First, MBHAResin lever arm modification was performed, followed by sequential condensation from the C-terminus to the N-terminus of the peptide backbone (numbers 1 to 6 in the upper right corner of the peptide sequence). After the backbone synthesis was complete, the synthesis sequence was as follows:

[0183] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl 6 -Cit 5 -Glu 4 -Ser 3 -Val 2 -Lys 1 (AEEA 侧1 -γGlu侧2 -Eicosanedioic acid 侧3 )-NH2, then after removing Mtt from the ε-amino group of Fmoc-Lys(Mtt)-OH at position 1, it sequentially condenses from side 1 to side 3 (AEEA, γGlu, eicosanoic acid monotert-butyl).

[0184] The protective amino acids used are: Fmoc-RinkLinker, Fmoc-Lys(Mtt)-OH, Fmoc-Val-OH, and Fmoc-Ser( t Bu)-OH, Fmoc-Glu(O t Bu)-OH.H2O, Fmoc-Cit-OH, {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid.

[0185] After the main-chain peptide condensation is complete, deprotection is performed using hexafluoroisopropanol (HFIP), followed by branched-chain condensation of Fmoc-AEEA-OH and Fmoc-Glu(O) t Bu)-OH and eicosanoic acid monotert-butyl ester, after washing and drying, yielded the peptide resin {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid-Cit-Glu(O) t Bu)-Ser( t Bu)-Val-Lys-(AEEA-γGlu(O t Bu)-Eicosanedioic acid monotert-butyl ester)-RinkLinker-MBHAResin.

[0186] 2. Peptide resin cleavage

[0187] Prepare the pyrolysis reagent with the following formula: trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), and 1,2-ethylenedithiol (EDT) in a volume ratio of TFA:H2O:EDT:TIS = 94:2:2:2 (V:V:V:V). Pre-cool to 10±3℃, and slowly add the resin to the pyrolysis solution while stirring. Continue stirring until the reaction system temperature stabilizes, and control the temperature at 25±3℃. Continue stirring for 3 hours.

[0188] The lysate was filtered and concentrated to a viscous state. It was then precipitated with 8 to 10 times the volume of the concentrated lysate using methyl tert-butyl ether (MTBE). The precipitate was filtered off, washed with TFA, and dried under reduced pressure at room temperature to obtain crude target peptide IV.

[0189] 3. Purification of target peptide IV

[0190] The crude target peptide IV was dissolved in 50 mmol / L disodium hydrogen phosphate (pH=7.5) aqueous solution, and then purified by high performance liquid chromatography (HPLC) using C18 silica gel matrix packing material and a 35 mmol / L disodium hydrogen phosphate (pH=7.0) aqueous solution system. The organic phase was increased from 30% to 40% for gradient elution purification. Then, the salt was replaced with 0.2 mmol / L sodium hydroxide, and the product was lyophilized to obtain the target peptide IV or its sodium salt (purity of target peptide IV was 98.7%, molecular weight was MS: m / z=1482.2, calculated value: 1482.76). Figure 9 shows the MS of target peptide IV or its salt in Example 4; Figure 10 shows the HPLC of target peptide IV or its salt in Example 4.

[0191] Example 4

[0192] A polypeptide compound with the following structural formula as target peptide V:

[0193] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]acetyl-Cit-Glu-Ser-Val-AEEA-Lys - (AEEA-γGlu-eicosanedioic acid)-NH2

[0194] Target peptide V

[0195] As shown in Figure 11, the preparation method of target peptide V or its salt includes the following steps:

[0196] The main chain of this peptide sequence is a straight peptide containing 7 amino acid residues, with an amide group at the C-terminus. The ε-amino group at the 7th Lys position of the straight peptide is branched (AEEA, γGlu, eicosanoic acid). Solid-phase synthesis (straight peptide synthesis, branched condensation) was used, followed by cleavage to obtain crude peptide V. Finally, after purification and drying, the target peptide V or its sodium salt product was obtained.

[0197] 1. Peptide resin synthesis

[0198] MBHAResin (S = 0.73 mmol / g) was used, with conventional Fmoc protection of amino acids at a dosage of 2.5-4.0 times the normal amount. DIC / HOBt was used as the condensing agent. First, MBHAResin lever arm modification was performed, followed by sequential condensation from the C-terminus to the N-terminus of the peptide backbone (numbers 1 to 7 in the upper right corner of the peptide sequence). After the backbone synthesis was complete, the synthesis sequence was as follows:

[0199] 2-[4-[(2-methylphenylaminocarbonyl)-amino]-phenyl]-acetyl 7 -Cit 6 -Glu 5 -Ser 4 -Val 3 -AEEA 2 -Lys1 (AEEA 侧1 -γGlu 侧2 -Eicosanedioic acid 侧 3 )-NH2, then after removing Mtt from the ε-amino group of Fmoc-Lys(Mtt)-OH at position 1, it sequentially condenses from side 1 to side 3 (AEEA, γGlu, eicosanoic acid monotert-butyl).

[0200] The protective amino acids used are: Fmoc-Rink Linker, Fmoc-Lys(Mtt)-OH, Fmoc-AEEA-OH, Fmoc-Val-OH, and Fmoc-Ser( t Bu)-OH, Fmoc-Glu(O t Bu)-OH.H2O, Fmoc-Cit-OH, {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid.

[0201] After the main-chain peptide condensation is complete, deprotection is performed using hexafluoroisopropanol (HFIP), followed by branched-chain condensation of Fmoc-AEEA-OH and Fmoc-Glu(O) t Bu)-OH and eicosanoic acid monotert-butyl ester, after washing and drying, yielded the peptide resin {2[4-(2-methylphenylaminocarbonyl)amino]-phenyl}-acetic acid-Cit-Glu(O) t Bu)-Ser( t Bu)-Val-AEEA-Lys-(AEEA-γGlu(O t Bu)-Eicosanedioic acid monotert-butyl ester)-Rink Linker-MBHA Resin.

[0202] 2. Peptide resin cleavage

[0203] Prepare the pyrolysis reagent with the following formula: trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), and 1,2-ethylenedithiol (EDT) in a volume ratio of TFA:H2O:EDT:TIS = 94:2:2:2 (V:V:V:V). Pre-cool to 10±3℃, and slowly add the resin to the pyrolysis solution while stirring. Continue stirring until the reaction system temperature stabilizes, and control the temperature at 25±3℃. Continue stirring for 3 hours.

[0204] The lysate was filtered and concentrated to a viscous state. It was then precipitated with 8 to 10 times the volume of the concentrated lysate using methyl tert-butyl ether (MTBE). The precipitate was filtered off, washed with TFA, and dried under reduced pressure at room temperature to obtain crude target peptide V.

[0205] 3. Purification of target peptide V

[0206] The crude target peptide V was dissolved in a 50 mmol / L disodium hydrogen phosphate (pH = 7.5) aqueous solution, and then purified by high performance liquid chromatography (HPLC) using a C18 silica gel matrix and a 35 mmol / L disodium hydrogen phosphate (pH = 7.0) aqueous solution. The organic phase was purified by gradient elution, increasing from 30% to 40%. The solution was then replaced with 0.2 mmol / L sodium hydroxide and lyophilized to obtain the target peptide V or its sodium salt (purity of target peptide V was 98.6%, molecular weight: MS: m / z = 1627.2, calculated value: 1627.94). Figure 12 shows the MS of the target peptide V or its salt in Example 5, and Figure 13 shows the HPLC of the target peptide V or its salt in Example 5.

[0207] Example 5

[0208] A polypeptide compound comprising a target peptide with the following structural formula:

[0209] The following compounds were prepared using a method similar to that used to prepare the compounds in Example 1, and the final product or its sodium salt was obtained by preparative column chromatography purification.

[0210] Example 6

[0211] Osteogenic induction activity of compound (XX) and YLL3

[0212] 1. Experimental Materials and Instruments

[0213] Reagents: α-MEM medium, PBS, and trypsin were purchased from Gibco; fetal bovine serum (FBS) was purchased from Procell; vitamin C and sodium β-glycerophosphate, which are osteogenic induction components, were purchased from Peiyu and Aladdin, respectively; ALP detection kit and penicillin-streptomycin and alizarin red staining solutions were purchased from Beyotime.

[0214] Cells: MC3T3-E1, purchased from IMMOCELL, cultured in complete α-MEM medium, i.e., α-MEM + 10% FBS + 1% penicillin and streptomycin.

[0215] Instruments: CO2 incubator and biosafety cabinet, microplate reader purchased from Thermofisher, USA, constant temperature water bath purchased from BKMAMLAB, China, inverted microscope purchased from Mingmei, China, centrifuge purchased from Xiangyi, China, pipette purchased from Dalong, China.

[0216] 2. Cell preparation and culture

[0217] Thaw cryovials containing 1 mL of cell suspension rapidly by shaking in a 37°C water bath. Add 4 mL of complete α-MEM medium and mix thoroughly. Centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1-2 mL of complete α-MEM medium, and mix well by pipetting. Then, transfer all cell suspensions to culture dishes containing an appropriate amount of complete α-MEM medium and incubate overnight. Change the medium the next day and check the cell density.

[0218] 3. In vitro osteoblast calcification detection using compounds induced osteoblast formation

[0219] After the cells were expanded to a sufficient quantity, they were seeded into 96-well plates. When the cell density reached 60%–100%, the blank control group (BK group) was cultured in complete α-MEM medium without osteogenic induction components, the negative control group (NC group) was cultured in complete α-MEM medium containing osteogenic induction components, and the other groups were cultured in three concentrations (10-10) of this series of peptides. -6 mol / L, 10 -8 mol / L, 10 -10 The culture medium was filled with complete α-MEM medium containing osteogenic induction components (mol / L), and the medium was changed every 2-3 days. ALP activity was detected on day 7, and the degree of mineralization was detected on day 28 (Alizarin Red staining).

[0220] 4. ALP activity detection

[0221] Cells were washed with PBS and then lysed with cell lysis buffer. After lysis, the supernatant was mixed with substrate and buffer in a new well plate (100 μL / well) and incubated at 37°C for 10 min.

[0222] The standard stock solution dosages per 100 μL were 4, 8, 16, 24, 32, and 40 μL, respectively.

[0223] Add 100 μL of reaction stop solution to each well to terminate the reaction.

[0224] OD values ​​were measured at 405 nm. One unit of enzyme activity (U) is defined as the amount of ALP required per minute to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 μmol of p-nitrophenol in diethanolamine (DEA) buffer at pH 9.8 and 37°C.

[0225] ALP calculation: Based on the definition of enzyme activity, the ALP activity in the sample is calculated and corrected by the ratio of lysis volume to the volume of substrate involved in the reaction.

[0226] 5. Alizarin Red staining and detection

[0227] Mix 10 mL of fixative solution with 40 mL of anhydrous ethanol in advance, wash three times with PBS, add the fixative solution, and incubate at room temperature for 20 minutes to fix the cells. After washing the cells with PBS, add Alizarin Red S staining solution, evenly cover the cells, and incubate at room temperature for 30 minutes to stain. Wash with Wahaha water until no further destaining occurs. Read the OD value at a wavelength of 560 nm.

[0228] The results showed that this series of polypeptide compounds had good osteogenic activity in vitro.

[0229] Example 7

[0230] Pharmacokinetics of SD rats after subcutaneous and intravenous administration

[0231] The blood drug concentrations of peptide III and the prodrug YLL3 (see patent: WO2020018941A1) in rats after subcutaneous and intravenous administration were determined by LC / MS, and their biological half-lives were obtained. The biological half-lives of peptide III after subcutaneous and intravenous administration were both longer than those of YLL3.

[0232] (1) Experimental materials and instruments

[0233] Animals: Male SD rats were purchased from Spiford (Beijing) Biotechnology Co., Ltd.;

[0234] Reagents: Acetonitrile, methanol, and isopropanol were purchased from Merck; formic acid was purchased from Fisher.

[0235] Equipment: Centrifuge purchased from Thermo; LC-MS / MS purchased from AB SCIEX; chromatographic column purchased from ACE; ultrapure water system purchased from Millipore; Vortex purchased from Beijing Beifang Tongzheng Biotechnology Development Co., Ltd.

[0236] (2) Experimental methods

[0237] ① Drug preparation

[0238] Accurately weigh a certain amount of the drug, dissolve it fully in DMSO, then add an appropriate amount of PEG400, stir thoroughly, and dilute to the specified volume with physiological saline (the solvent formulation for polypeptide III is 5% DMSO + 10% PEG400 + 85% physiological saline; the solvent formulation for YLL3 is 5% DMSO + 5% PEG400 + 90% physiological saline), to obtain a clear solution. Filter the resulting preparation through a 0.22 μm filter membrane.

[0239] ② Animal administration and blood collection

[0240] Polypeptide III

[0241] Eight male SD rats were randomly divided into two groups of four each. The subcutaneous administration group and the intravenous administration group received a single dose (one injection only), both at a dose of 0.220 mg / kg. Blood samples were collected from the jugular vein before administration and at 5±1 min, 15±1 min, 30±1 min, 2 h±5 min, 4 h±5 min, 6 h±10 min, 24 h±10 min, 48 h±15 min, 72 h±15 min, 96 h±15 min, 120 h±15 min, 168 h±30 min, 240 h±15 min, and 336 h±15 min, with a maximum blood volume of 0.25 mL per sample.

[0242] YLL3

[0243] Four male SD rats were randomly divided into two groups of two each. The subcutaneous administration group and the intravenous administration group received a single injection (0.220 mg / kg) of medication. In the intravenous administration group, blood was collected from the jugular vein before administration and at 0.5±0.1 min, 1±0.1 min, 2 min±0.5 min, 5 min±1 min, 15 min±15 min, and 30 min±1 min after administration. In the subcutaneous administration group, blood was collected at 5 min±1 min, 15 min±15 min, and 30 min±1 min before administration. The maximum blood volume collected each time was 0.25 mL.

[0244] ④ Plasma separation and pretreatment

[0245] After blood collection, the blood collection tube containing the anticoagulant was inverted several times to ensure thorough mixing, and then placed on moist ice. Within 60 minutes of blood collection, the blood was centrifuged at 2000g for 10 minutes at 2–8°C to obtain a plasma sample. The plasma sample was transferred to cryovials and stored at -60 to -90°C until analysis. LC-MS / MS was used for detection.

[0246] (3) Experimental Results

[0247] 1. Subcutaneous administration

[0248] 1.1 The half-lives of peptide III (target peptide III) after subcutaneous administration to rats were 6.9, 7.2, 7.3 and 7.1 hours, respectively, with a mean ± standard deviation of 7.1 ± 0.2 hours.

[0249] Individual pharmacokinetic data are shown in Table 1; individual blood drug concentration data at each time point are shown in Figure 16; and drug-time curves are shown in Figure 17.

[0250] Table 1 Individual data on pharmacokinetic parameters of peptide III administered subcutaneously to rats

[0251] 1.2 YLL3 was administered subcutaneously once, and the blood concentration in rats was BQL (<0.100 ng / mL) 30 min later, with a half-life of less than 2 minutes.

[0252] Individual pharmacokinetic data could not be calculated; individual blood drug concentration data at each time point are shown in Figure 18, and drug-time curves are shown in Figure 19.

[0253] When prepared using 5% DMSO + 10% PEG400 + 85% physiological saline and administered subcutaneously to rats, the half-life of peptide III (7.1 ± 0.2 hours) was significantly better than that of YLL3 (less than 2 minutes).

[0254] 2. Intravenous administration

[0255] 2.1 The half-lives of peptide III (target peptide III) in rats after intravenous administration were 5.7, 6.1, 5.3 and 8.0 hours, respectively, with a mean ± standard deviation of 6.3 ± 1.2 hours.

[0256] Individual pharmacokinetic data are shown in Table 2; individual blood drug concentration data at each time point are shown in Figure 20; and drug-time curves are shown in Figure 21.

[0257] Table 2 Individual data on pharmacokinetic parameters of peptide III administered intravenously to rats.

[0258] 2.2 The half-life of YLL3 in rats after a single intravenous administration was 1.52 and 1.71 minutes, with a mean ± standard deviation of 1.62 ± 0.13 minutes.

[0259] 2.3 Individual pharmacokinetic data are shown in Table 3; individual blood drug concentration data at each time point are shown in Figure 22, and drug-time curves are shown in Figure 23.

[0260] Table 3 Individual data of pharmacokinetic parameters of YLL3 rats after intravenous administration

[0261] When prepared by a single intravenous administration of 5% DMSO, 10% PEG400, and 85% saline to rats, the half-life of peptide III (6.3 ± 1.2 hours) was significantly better than that of YLL3 (1.62 ± 0.13 minutes).

[0262] Example 8

[0263] Pharmacodynamic study of osteoporosis in SD rats

[0264] (1) Modeling methods

[0265] After 10 weeks of acclimatization, the experimental animals were randomly divided into 9 groups. All animals in the OVX group underwent ovariectomy, including bilateral oophorectomy, removal of the ovarian cyst and part of the fallopian tubes, and were fed a normal diet. The sham surgery involved only laparotomy without removal of the ovaries; subcutaneous drug administration began 3 days post-surgery.

[0266] (2) Administration method

[0267] SD rats were randomly divided into four groups: Sham group (sham surgery, n=8), OVX group (solvent group, administered solvent, referred to as OVX group, n=8), Polypeptide III-25μg group (low-dose group, n=8), Polypeptide III-75μg group (medium-dose group, n=8), and Polypeptide III-225μg group (high-dose group, n=8). A PTH-25μg group (rhPTH, recombinant human parathyroid hormone, positive control group, n=8) was also included. In the Sham group (sham surgery group), rats underwent laparotomy without ovarian removal. The OVX group (solvent group) received subcutaneous saline once a week for 6 weeks. The Polypeptide III-25μg group (low-dose group), Polypeptide III-75μg group (medium-dose group), and Polypeptide III-225μg group (high-dose group) received subcutaneous injections of Polypeptide III at 25μg / kg, 75μg / kg, and 225μg / kg, respectively, once a week for 6 weeks.

[0268] All experimental animals were weighed weekly before administration. Seven days after the end of the administration schedule, they were dissected and weighed again before dissection, followed by uterine removal and weighing. Blood samples were collected, and serum was separated. Serum levels of bone alkaline phosphatase (BALP) and type 1 collagen C-terminal peptide (CTX-1) were measured using ELISA. Simultaneously, the right femur and tibia were subjected to microCT scans to measure bone volume fraction (BV / TV), bone mineral density (BMD), tissue volume (TV), cortical bone thickness (Ct.Th), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), cortical bone mineral density (Ct-BMD), cortical bone porosity (Ct-Po), cortical bone pore volume (Ct-Po.V), and the ratio of cortical bone area to total area (Ct-B.Ar / T.Ar). The mechanical properties of the rat spine were tested using a mechanical testing device (compression device) to measure Maximum Load and Maximum Stress.

[0269] As shown in Figure 24A, compared with the OVX group, the serum BALP level of peptide III-75 μg was significantly increased (P < 0.05), indicating higher osteogenic-related activity; as shown in Figure 24B, the serum CTX-1 level of peptide III-225 μg was significantly decreased (P < 0.05), indicating lower osteoclast-related activity. This suggests that peptide III improves the osteogenic effect and inhibits osteoclastogenesis in bone metabolism.

[0270] In Figure 25, compared with the OVX group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; the peptide III-75μg group showed significant improvements in knee joint bone-related parameters compared to the OVX group: bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) were all significantly increased, while cortical bone porosity (Ct-Porosity) and cortical bone pore volume (Ct-Porosity Volume) were significantly decreased. This indicates that peptide III-75μg has a potential role in osteoporosis repair.

[0271] In Figure 26, compared with the OVX group (*P<0.05, **P<0.01, ***P<0.001), the peptide III-75μg group showed better endurance in mechanical properties, with significant increases in both Maximum Load and Maximum Stress. This indicates that peptide III-75μg has a potential role in osteoporosis repair.

[0272] result:

[0273] 1. Weight: There was no significant difference between the groups;

[0274] 2. Uterine weight: all model groups were significantly lower than the normal group;

[0275] 3. Serum bone turnover markers: Serum BALP levels were significantly elevated with peptide III-75 μg (P < 0.05), while serum CTX-1 levels were significantly decreased with peptide III-225 μg (P < 0.05).

[0276] 4. Micro CT showed that compared with the OVX group, the peptide III-75μg group showed significant improvement in knee joint bone-related parameters: bone mineral density (BMD, P<0.05), bone volume fraction (BV / TV, P<0.05), trabecular bone number (Tb.N, P<0.05), and trabecular bone thickness (Tb.Th, P<0.05) were all significantly increased, while cortical bone porosity (Ct-Porosity) and cortical bone pore volume (Ct-PorosityVolume) were significantly decreased (P<0.05).

[0277] 5. Spinal biomechanics test: Compared with the OVX group, the polypeptide III-75μg group showed better endurance in terms of biomechanical indicators, with significant increases in Maximum Load (maximum endurance, P<0.01) and Maximum Stress (maximum endurance pressure, P<0.05).

[0278] in conclusion:

[0279] Subcutaneous injection of peptide III 75 μg / week / rat for 6 weeks in OVX osteoporotic rats can alleviate uterine atrophy, increase osteogenic activity-related BALP and reduce osteoclast activity-related CTX-1 serum levels, improve knee joint bone-related indicators and spinal biomechanical indicators, and has a significant therapeutic effect on OVX osteoporotic rats.

[0280] Example 9

[0281] Pharmacodynamic study of nonhealing fractures in SD rats

[0282] 1. Experimental Objective

[0283] By establishing a critical bone defect model in SD rats, the therapeutic effect of peptide III on bone defects was investigated.

[0284] 2. Experimental Materials

[0285] Laboratory animals, surgical instruments, internal fixation PEEK plates, screws, etc.

[0286] 3. Experimental Design

[0287] A rat model of critical bone defect was established. The material was placed on the day of surgery, and the material was taken and fixed 12 weeks later for X-ray examination. Calcein was injected once at -8 / -9 days before the material was taken, and Alizarin Red (fluorescent dose 20 mg / kg) was injected once at -1 / -2 days before the material was taken.

[0288] 4. Surgical Method

[0289] Twenty-four SD rats were anesthetized via intraperitoneal injection of sodium pentobarbital. After anesthesia took effect, the rats were placed in a lateral decubitus position. The right hind limb was routinely disinfected and draped with sterile towels. A 3cm incision was made along the femur on the ventral side of the thigh, and the muscle was bluntly dissected to expose the femur. A 6-well PEEK plate was placed on the femur, and holes were drilled in the femur using an electric drill. A screw was then drilled into the holes. A custom-made fixator was placed on the screw, and a wire saw was used to cut approximately 6mm of the femur through the fixator and removed. A 7*15mm sterile collagen sponge was compressed to a size of 6mm using sterile forceps. The drug was dissolved in 200ul of sterile water, loaded into the sponge, and inserted into the femoral notch. Each layer was sutured sequentially. After the rats regained consciousness, they were placed in cages for feeding. Penicillin was injected subcutaneously for 3 days, and samples were collected 2 months later. X-ray examination was used to determine the formation of nonunion.

[0290] 5. Grouping (8 animals per group):

[0291] Group A: Bone defect surgery + bone material + solvent control group;

[0292] Group B: Bone defect surgery + bone material + YLL3 (dosage: 50ug / animal);

[0293] Group C: Bone defect surgery + bone material + polypeptide III (dosage: 50ug / animal).

[0294] 6. Evaluation:

[0295] Body weight: During weeks 1-4, rat body weight was measured weekly; after week 4, it was measured every two weeks.

[0296] Plasma: Plasma was collected from rats via orbital blood collection. Plasma collection time points were: postoperative, 24 hours, day 3, week 1, and week 2.

[0297] Bone tissue: Bone tissue from the surgical side was collected, fixed in 4% paraformaldehyde for 24 hours, then transferred to 70% ethanol and sent to LBRC to measure bone volume, bone mass, bone morphology, and trabecular bone structure to assess bone formation efficiency.

[0298] Rat fracture nonunion model experiment: In a rat fracture nonunion model with a 6cm gap, the fracture was stabilized using a transmedullary screw. The fracture gap was filled with collagen sponge loaded with PBS, or with a single instillation of 1mg of YLL3 or polypeptide III collagen sponge, respectively. Each group had n=8 rats. All mice received subcutaneous injections (sc) of calciferol (10mg / kg) and alizarin red (20mg / kg) to label active bone-forming surfaces 8 days and 1 day before euthanasia. Rats were euthanized 12 weeks post-surgery.

[0299] Experimental Results: The bar graph of callus volume is shown in Figure 27, and the X-ray image of new bone formation is shown in Figure 28. It can be seen that compared with the placebo group, single-dose peptide III (1 mg) treatment increased the formation of new bone tissue in the nonunion fracture model (blue arrows point to new bone formation, bridging the fracture gap). Microscopic observation of tissue sections is shown in Figure 29. It can be seen that the amount of calciferol and alizarin red markers on the bone surface of the peptide III treatment group is significantly increased, indicating active bone formation.

[0300] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A polypeptide compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its tautomer, or its polymorph, or its solvate, or its N-oxide, or its isotopically labeled compound, or its metabolite, or its prodrug, characterized in that, It contains the following structural formula (XX); R 1 Independently selected from H, halogens, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl; R 2 Independently selected from C1-C6 alkyl, H, halogen, C1-C6 alkoxy and C1-C6 haloalkyl; Y 1 -Y 4 Selected independently from amino acid residues; X is selected from CH3, COOH, SO3H, or OH; The subscripts m, n, q, and w are each selected individually from any value between 0 and 6; The 'o' in the subscript is selected from any value between 1 and 4; The subscript r is selected from any value between 0 and 2; The subscript p is selected from any value between 10 and 20.

2. The polypeptide compound according to claim 1, characterized in that, Where Y 4 Selected from citrulline residues, N6-(3-(pyridin-3-yl)propionyl)-lysine residues, high-citrulline residues, leucine residues, (N-methyl)leucine residues, isoleucine residues, ortholeucine residues, (N-methyl)isoleucine residues, and high-phenylalanine acid residues; Y 3 Selected from glutamic acid residues, glutamine residues, aspartic acid residues, or asparagine residues; Y 2 Selected from serine residues, threonine residues, tyrosine residues, or cysteine ​​residues; Y 1 It is selected from valine residues, isoleucine residues, leucine residues, methionine residues, or oroleucine.

3. The polypeptide compound according to claim 1, characterized in that, Where -Y 4 -YY 2 -Y 1 -Selected from -Cit-Glu-Ser-Val-.

4. The polypeptide compound according to claim 1, wherein -Y 4 -Y 3 -Y 2 -Y 1 -(AEEA)n- is selected from -Cit-Glu-Ser-Val-(AEEA)n-, m=0-2, n=0-2, o=4, r=2.

5. The polypeptide compound according to claim 1, characterized in that, Including the following structural formula (Ⅰ); In structural formula (Ⅰ), n is any value of 0, 1, 2, 3, 4, 5, 6; m is any value of 0, 1, 2, 3, 4, 5, 6; p is any value of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; X is selected from CH3, COOH, SO3H or OH.

6. A pharmaceutical composition, characterized in that, The product comprises a polypeptide compound as described in any one of claims 1-5, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a tautomer thereof, or a polymorph thereof, or a solvate thereof, or an N-oxide thereof, or an isotopically labeled compound thereof, or a metabolite thereof, or a prodrug thereof, and one or more pharmaceutically acceptable excipients or carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutically acceptable salts include any one or more of sodium, lithium, potassium, calcium, magnesium, and ammonium salts.

8. The pharmaceutical composition according to claim 6, characterized in that, This includes pharmaceutically acceptable excipients, carriers, adjuvants, solvents, or combinations thereof.

9. A pharmaceutical box product comprising: a) a container; b) at least one compound of any one of claims 1 to 5, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a tautomer thereof, or a polymorph thereof, or a solvate thereof, or an N-oxide thereof, or an isotopically labeled compound thereof, or a metabolite thereof, or a prodrug thereof, located in said container; and c) optionally present packaging and / or instructions.

10. A drug conjugate comprising the compound of any one of claims 1 to 5, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a deuterated compound thereof, or a tautomer thereof, or a polymorph thereof, or a solvate thereof, or an N-oxide thereof, or an isotopically labeled compound thereof, or a metabolite thereof, or a prodrug thereof.

11. A method for preventing, treating, or alleviating osteoporosis and promoting fracture healing, characterized in that, The pharmaceutical composition of any one of claims 1 to 5, the pharmaceutical product of claim 9, or the pharmaceutical conjugate of claim 10 may be administered to a receptor in a therapeutically effective amount.

12. A method for promoting bone formation, characterized in that, The pharmaceutical composition of any one of claims 1 to 5, the pharmaceutical product of claim 9, or the pharmaceutical conjugate of claim 10 may be administered to a receptor in a therapeutically effective amount.

13. The use of the polypeptide compound of any one of claims 1-5, or its stereoisomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its tautomer, or its polymorph, or its solvate, or its N-oxide, or its isotopically labeled compound, or its metabolite, or its prodrug, or the pharmaceutical composition of claims 6-8, the kit product of claim 9, or the drug conjugate of claim 10, in the preparation of a medicament, characterized in that, The drug is used to activate cell signaling in osteoprogenitor cells, thereby promoting bone formation.

14. The use of the polypeptide compound of any one of claims 1-6, or its stereoisomer, or its pharmaceutically acceptable salt, or its deuterated compound, or its tautomer, or its polymorph, or its solvate, or its N-oxide, or its isotopically labeled compound, or its metabolite, or its prodrug, or the pharmaceutical composition of claims 6-8, the kit product of claim 9, or the drug conjugate of claim 10, in the preparation of a medicament, characterized in that, The drug is used to prevent, treat or alleviate osteoporosis.

15. A pharmaceutical composition for the prevention, treatment, or relief of osteoporosis and for promoting fracture healing, said pharmaceutical composition comprising a compound of any one of claims 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a deuterated compound thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, a prodrug thereof, a pharmaceutical composition of claims 6-8, a kit product of claim 9, or a drug conjugate of claim 10.

16. A pharmaceutical composition for promoting bone formation, said pharmaceutical composition comprising a compound of any one of claims 1 to 5, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a deuterated compound thereof, a tautomer thereof, a polymorph thereof, a solvate thereof, an N-oxide thereof, an isotopically labeled compound thereof, a metabolite thereof, a prodrug thereof, a pharmaceutical composition of claims 6-8, a box product of claim 9, or a drug conjugate of claim 10.

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