Calcium-sensing receptor agonist compounds and uses thereof

Abstract

The present disclosure provides calcium-sensing receptor agonist compounds and uses thereof. Specifically, the present disclosure provides a series of polypeptide calcium-sensing receptor agonist compounds and pharmaceutical compositions of pharmaceutically acceptable salts thereof that have agonistic activity against the human calcium-sensing receptor (CaSR), thereby lowering plasma parathyroid hormone and serum calcium ion levels, and can be used to treat metabolic diseases such as primary hyperparathyroidism, secondary hyperparathyroidism, and tumor-induced hypercalcemia.

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 201911250088.1, filed on December 9, 2019, the entire text of which is incorporated herein by reference.

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to compounds having agonistic activity against the human calcium-sensing receptor (CaSR) and pharmaceutically acceptable salts thereof, compositions containing the same, and uses thereof in the treatment of related metabolic diseases such as primary hyperparathyroidism, secondary hyperparathyroidism, and hypercalcemia. [Background technology]

[0003] Secondary hyperparathyroidism is a chronic compensatory clinical manifestation of parathyroid glands that secrete excess parathyroid hormone due to prolonged hypocalcemia, hypomagnesemia, or hyperphosphatemia in patients with chronic renal failure, intestinal malabsorption syndrome, Fanconi syndrome, renal tubular acidosis, vitamin D deficiency or resistance, and pregnancy and lactation. This condition is accompanied by parathyroid hyperplasia, which ultimately leads to the formation of functionally autonomous adenomas.

[0004] The calcium-sensing receptor (CaSR) is a family A G-protein coupled receptor (GPCR) distributed on the cell surface of the human parathyroid gland. Parathyroid hormone secretion is highly regulated by the calcium-sensing receptor on the surface of parathyroid cells to maintain steady-state mineral levels in the body. The calcium-sensing receptor continuously monitors subtle changes in calcium ion concentration in the body and responds accordingly by altering the secretion level of parathyroid hormone.

[0005] In patients with chronic kidney disease, parathyroid hormone is continuously secreted from the parathyroid gland due to the need to achieve steady-state levels of calcium and phosphorus ions in the body. This continuous secretion of parathyroid hormone is initially adaptive, but as chronic kidney disease progresses, it eventually leads to parathyroid hyperplasia and excessive parathyroid hormone levels in the body, as well as the formation of secondary hyperparathyroidism. Research has shown that persistent secondary hyperparathyroidism results in the loss of calcium-sensing receptors and vitamin D receptors on the surface of parathyroid cells. These disease-induced downstream pathological effects further lead to parathyroid dysregulation of mineral homeostasis in the body.

[0006] Calcimimetics generally refer to compounds whose physiological functions and mechanisms of action are similar to calcium ions and can directly activate calcium-sensing receptors on the surface of parathyroid cells. Cinacalcet hydrochloride, a small-molecule organic calcimimetic developed by Amgen, activates calcium-sensing receptors on the surface of parathyroid glands and inhibits parathyroid hormone secretion levels, thereby achieving the goal of treating related metabolic disorders such as secondary hyperparathyroidism. Cinacalcet hydrochloride has been clinically approved for the treatment of secondary hyperparathyroidism in dialysis patients with chronic kidney disease. It is administered orally once or twice daily, with a maximum dose of 90 mg per administration. Cinacalcet hydrochloride has demonstrated excellent therapeutic efficacy in reducing plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. However, significant adverse drug reactions, such as nausea, vomiting, and diarrhea associated with gastrointestinal side effects, have been observed during its use in patients. Furthermore, the oral administration of cinacalcet hydrochloride is a significant burden for dialysis patients with chronic kidney disease, and cinacalcet hydrochloride has been proven to inhibit cytochrome 450 and induce related drug-drug interactions. These side effects associated with the use of cinacalcet hydrochloride reduce patient compliance and adaptability to a certain extent.

[0007] Therefore, it is expected to develop calcium-sensing receptor agonist compounds that can be administered intravenously and that can reduce parathyroid hormone secretion by activating calcium-sensing receptors on the surface of parathyroid cells, thereby achieving therapeutic effects in treating related metabolic diseases such as secondary hyperparathyroidism. Such calcium-sensing receptor agonist compounds can significantly improve compliance and adaptability in treating patients with chronic kidney disease. Summary of the Invention

[0008] The present disclosure provides a compound comprising a peptide and a conjugate group, wherein the peptide comprises an amino acid sequence represented by formula (I): X1-X2-X3-X4-X5-X6-X7(I)(SEQ ID NO:39) Among them, X1 is D-Cys, X2 is selected from D-Phg, D-Phe(4-CH3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, D-Arg, D-Trp or D-His; X3 is D-Arg, X4 is D-Arg, D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2-NaI, D-hPhe, D-Abu, D - selected from Tle, D-hLeu, D-Cha, D-Ser, D-Gln, D-Tyr, D-Ile, D-Ser, D-His, D-Val or D-Chg, X5 is D-Arg, X6 is selected from D-Ala, D-Abu, D-Ser or Gly; X7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond, the conjugate group is L-Cys, and the X1 residue of the peptide is covalently bonded to the conjugate group by a disulfide bond; The present invention aims to provide a compound or a pharmaceutically acceptable salt thereof, wherein the N-terminal X1 of the peptide is modified by acetylation and the C-terminal X7 of the peptide is modified by amidation.

[0009] In one embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof is a peptide of the general formula (I): X1 is D-Cys, X2 is selected from D-Phg, D-Phe(4-CH3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, or D-His; X3 is D-Arg, X4 is D-Arg, X5 is D-Arg, X6 is selected from D-Ala, D-Abu, D-Ser or Gly; X7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; the conjugate group is L-Cys, and the residue X1 of the peptide is covalently bonded to the conjugate group by a disulfide bond; The N-terminus X1 of the peptide is modified by acetylation and the C-terminus X7 of the peptide is modified by amidation.

[0010] In another embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof is a peptide of the general formula (I): X1 is D-Cys, X2 is D-Arg, X3 is D-Arg, X4 is selected from D-Arg, D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2-NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, D-Ser, D-Cha, D-Gln, D-Tyr, D-His or D-Val; X5 is D-Arg, X6 is selected from D-Ala, D-Abu, D-Ser or Gly; X7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; the conjugate group is L-Cys, and the residue X1 of the peptide is linked to the conjugate group by a disulfide bond; The N-terminus X1 of the peptide is modified by acetylation and the C-terminus X7 of the peptide is modified by amidation.

[0011] In another embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof is a peptide of the general formula (I): X1 is D-Cys, X2 is D-Arg, X3 is D-Arg, X4 is selected from D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, D-Ser, D-Cha, D-Gln, D-Tyr, D-Ile, D-His or D-Val; X5 is D-Arg, X6 is selected from D-Ala, D-Abu, D-Ser or Gly; X7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; the conjugate group is L-Cys, and the residue X1 of the peptide is linked to the conjugate group by a disulfide bond; The N-terminus X1 of the peptide is modified by acetylation and the C-terminus X7 of the peptide is modified by amidation.

[0012] In another embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof is a peptide of the general formula (I): X1 is D-Cys, X2 is selected from D-Arg; X3 is D-Arg, X4 is selected from D-Phe(4-CH3), D-2-Thi, D-Abu, D-hLeu or D-Val; X5 is D-Arg, X6 is selected from D-Ala or D-Ser; X7 is D-Arg, the conjugate group is L-Cys, and the residue X1 of the peptide is covalently bonded to the conjugate group by a disulfide bond; The N-terminus X1 of the peptide is modified by acetylation and the C-terminus X7 of the peptide is modified by amidation.

[0013] In some embodiments, X4 is selected from D-Abu or D-Val, while in some other embodiments, X4 is selected from D-Abu.

[0014] The present disclosure provides a method for manufacturing a semiconductor device having a structure in which both ends are joined as follows: R1-X1-X2-X3-X4-X5-X6-X7-R2 (II) Among them, R1 is H, an alkyl group, an acetyl group, a formyl group, a benzoyl group, a trifluoroacetyl group, D-pGlu, or L-pGlu; R2 is —NH2 or —OH; X1, X2, X3, X4, X5, X6, and X7 are defined as in the general formula (I) above, and further relate to a compound having the general formula (II) or a pharmaceutically acceptable salt thereof.

[0015] In one embodiment, R1 is selected from an acetyl group, X1 is selected from the amino acid residue of D-Cys, X2 is selected from the amino acid residues of D-Phg, D-Phe(4-CH3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, D-Arg, or D-His, X3 is selected from the amino acid residue of D-Arg, and X4 is selected from the amino acid residues of D-Arg, D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2-NaI, D-hPh X5 is selected from the amino acid residues of D-Ala, D-Abu, D-Tle, D-hLeu, D-Cha, D-Ser, D-Gln, D-Tyr, D-Ile, D-Ser, D-His, D-Val or D-Chg; X5 is selected from the amino acid residue of D-Arg; X6 is selected from the amino acid residues of D-Ala, D-Abu, D-Ser or Gly; X7 is selected from the amino acid residue of D-Arg; and R2 is selected from -NH2.

[0016] In one embodiment, the compound has the general formula (II) or a pharmaceutically acceptable salt thereof, wherein R1 is selected from an acetyl group, X1 is selected from a D-Cys amino acid residue, X2 is selected from a D-Phg, D-Phe(4-CH3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, or D-His amino acid residue, X3 is selected from a D-Arg amino acid residue, X4 is selected from a D-Arg amino acid residue, X5 is selected from a D-Arg amino acid residue, X6 is selected from a D-Ala, D-Abu, D-Ser, or Gly amino acid residue, X7 is selected from a D-Arg amino acid residue, and R2 is selected from -NH2.

[0017] In one embodiment, R1 is selected from an acetyl group, X1 is selected from the amino acid residues of D-Cys, X2 is selected from the amino acid residues of D-Arg, X3 is selected from the amino acid residues of D-Arg, and X4 is selected from D-Arg, D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, and D-Chg. X5 is selected from the amino acid residues of D-Ala, D-Abu, D-Ser or Gly, X7 is selected from the amino acid residues of D-Arg, and R2 is selected from -NH2, or a pharmaceutically acceptable salt thereof.

[0018] In one embodiment, R1 is selected from an acetyl group, X1 is selected from an amino acid residue of D-Cys, X2 is selected from an amino acid residue of D-Arg, X3 is selected from an amino acid residue of D-Arg, and X4 is selected from D-Phg, D-Phe(4-CH3), D-2-Thi, D-Phe(4-NO2), D-2NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, or D-Ser. X5 is selected from the amino acid residues of D-Ala, D-Abu, D-Ser or Gly, X7 is selected from the amino acid residues of D-Arg, and R2 is selected from -NH2, or a pharmaceutically acceptable salt thereof.

[0019] In one embodiment, the compound has the general formula (I) or (II) or a pharmaceutically acceptable salt thereof, wherein when X1 is selected from the amino acid residues of D-Cys, the X1 residue is linked to the second sulfhydryl group by a side chain disulfide bond.

[0020] In one embodiment, the compound or a pharmaceutically acceptable salt thereof is selected from the following compounds: [Table 1]

[0021] In the structural formulas in the table above, "Ac-c(C)" indicates that the amino-terminal D-cysteine ​​(c) is acetylated and linked to another L-cysteine ​​(C) via a disulfide bond, and "r-NH2" indicates that the carboxyl-terminal D-arginine (r) is amidated.

[0022] The present disclosure further relates to a pharmaceutical composition comprising any of the above compounds or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable carrier.

[0023] The present disclosure further relates to the use of any of the foregoing compounds or pharmaceutically acceptable salts thereof, and compositions thereof, in the preparation of a medicament for reducing parathyroid hormone levels in a subject and treating secondary hyperparathyroidism or tumor-induced hypercalcemia.

[0024] The polypeptide compounds provided by the present disclosure belong to the amphoteric class of compounds, and those skilled in the art can react acidic or basic compounds therewith to form salts using known techniques.

[0025] Pharmaceutical compositions containing the polypeptide compounds of the present disclosure can be used to treat patients in need of such treatment by parenteral administration. The parenteral administration route can be selected from subcutaneous injection, intramuscular injection, or intravenous injection. The polypeptide compounds of the present disclosure can also be administered via a transdermal route, such as via a scalp patch, an iontophoretic patch, or a transmucosal route. Such pharmaceutical compositions and their preparation methods are known in the art, and the preferred administration route is intravenous injection.

[0026] The polypeptide compounds provided by the present disclosure are prepared by solid-phase synthesis. The synthetic support is Rink-amide-MBHA resin (Xi'an Lanxiao Technology). The α-amino group of the amino acid derivatives used in the synthesis is protected with an Fmoc group. The amino acid side chains are protected by functional groups selected from the group consisting of the mercapto group of the cysteine ​​side chain and the amino group of the glutamine side chain. The imidazolyl group of the histidine side chain is protected with a trityl group (Trt). The guanidine group of the arginine side chain is protected with a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (Pbf). The indolyl group of the tryptophan side chain is protected with a tert-butoxycarbonyl group (Boc). The phenol group of the tyrosine side chain and the hydroxyl group of the serine side chain are protected with a tert-butyl group (t-Bu). During synthesis, the carboxyl group of the C-terminal amino acid residue of the polypeptide is first condensed to the polymeric, insoluble Rink-amide MBHA resin via an amide bond. The Fmoc protecting group on the α-amino group is then removed using a solution of N,N-dimethylformamide (DMF) containing 25% 4-methylpiperidine. The solid support is then condensed with the next amino acid derivative in the sequence in excess to form an amide bond, thereby extending the peptide chain. The synthetic polypeptide chain length is then obtained by repeating the condensation, washing, deprotection, washing, and condensation of the next amino acid. Finally, the polypeptide is cleaved from the solid support by reacting the resin with a mixture of trifluoroacetic acid:water:triisopropylsilane (90:5:5, v:v:v). After further precipitation with frozen methyl tert-butyl ether, the solid crude polypeptide compound is obtained. The crude polypeptide solid product is then dissolved in a mixture of acetonitrile / water containing 0.1% trifluoroacetic acid and purified on a C-18 reverse-phase preparative chromatography column to obtain the purified polypeptide compound.

[0027] Detailed Description of the Invention Unless otherwise specified, terms used in the claims and specification have the following meanings.

[0028] The amino acid sequences of the present disclosure are represented by the standard one-letter or three-letter codes of the 20 amino acids, and unless otherwise specified, in this disclosure, D-amino acids are represented by the addition of the prefix "D-" before the standard three letters, such as D-Ser, or the corresponding lowercase letter, such as s, and L-amino acids are represented by the addition of the prefix "L-" before the standard three letters, such as L-Cys, or the corresponding uppercase letter, such as C. As one particular example, glycine is achiral and is represented as "Gly" or the corresponding uppercase letter "G".

[0029] The term agonist is defined as a substance that activates the receptor type under consideration.

[0030] The term calcium-sensing receptor agonist as used in the context of this disclosure refers to a substance or ligand capable of activating the calcium-sensing receptor. In this disclosure, the term treatment includes inhibiting, alleviating, halting, or reversing the progression or severity of an existing symptom or disease.

[0031] Parathyroid hormone, as used in this disclosure, is an 84-amino acid peptide produced by the parathyroid gland and its degradation products. In addition to full-length parathyroid hormone, various parathyroid hormone fragments exist in the blood, produced by proteolysis and other metabolic pathways. The amino-terminal 1-34 region of the complete parathyroid hormone molecule is biologically active. Various methods for measuring parathyroid hormone levels have been developed in the industry and are known in the art.

[0032] "Naturally occurring amino acid" refers to the 20 naturally occurring conventional amino acids (i.e., alanine (Ala, A), cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), phenylalanine (Phe, F), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), lysine (Lys, K), leucine (Leu, L), methionine (Met, M), asparagine (Asn, N), proline (Pro, P), glutamine (Gln, Q), arginine (Arg, R), serine (Ser, S), threonine (Thr, T), valine (Val, V), tryptophan (Trp, W), and tyrosine (Tyr, Y)).

[0033] "Non-naturally occurring amino acids" refer to amino acids that are not naturally encoded or expressed in the genetic code of any organism. They may, for example, be purely synthetic compounds. Illustrative examples include, but are not limited to, D-2-aminobutyric acid (D-Abu), 3-cyclohexyl-D-alanine (D-Cha), 3-(2-thienyl)-D-alanine (D-2-Thi), 2-naphthyl-D-alanine (D-2-NaI), D-phenylglycine (D-Phg), D-2-chlorophenylalanine (D-Phe(2-Cl)), D-4-nitrophenylalanine (D-Phe(4-NO2)), D-4-methylphenylalanine (D-Phe(4-Me)), D-homophenylalanine (D-hPhe), D-tert-leucine (D-Tle), D-homoleucine (D-hLeu), and D-cyclohexylglycine (D-Chg).

[0034] Additionally, natural or unnatural amino acids in which the C-terminal carboxyl group, the N-terminal amino group and / or their side chain functional groups have been chemically modified are also included.

[0035] Different terms such as "X is selected from A, B, or C," "X is selected from A, B, and C," "X is A, B, or C," and "X is A, B, and C" have the same meaning, i.e., X may be any one or more of A, B, and C.

[0036] Any hydrogen atom described in the present disclosure may be substituted with its isotope, deuterium, and any hydrogen atom in the compounds of the examples according to the present disclosure may be substituted with a deuterium atom.

[0037] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and the phrase includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may, but need not, be present, and the phrase includes instances where the heterocyclyl group is substituted with an alkyl group and instances where the heterocyclyl group is not substituted with an alkyl group.

[0038] "Substituted" means that one or more hydrogen atoms, preferably up to five, more preferably one to three hydrogen atoms of the group are independently replaced with a corresponding number of substituents. Needless to say, substituents are located only at chemically feasible positions, and those skilled in the art can determine possible or impossible substitutions (by experiment or theory) without undue effort. For example, an amino group or hydroxy group having free hydrogen may be unstable when bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

[0039] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs, with other chemical components, and other components, such as physiologically / pharmaceutically acceptable carriers and excipients, to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0040] A "pharmaceutically acceptable salt" refers to a salt of a compound according to the present disclosure, which is safe and effective when used in a mammalian body and possesses the desired biological activity.

[0041] As used herein, a subject refers to a human subject or an animal subject.

[0042] As used herein, a mercapto group-containing group or mercapto group-containing moiety refers to a functional group that contains a sulfur-hydrogen bond and can form a disulfide bond with another mercapto group under physiological conditions. [Brief explanation of the drawings]

[0043] [Figure 1] The hemolytic effect of Example Compounds 12, 13, 17, 19, 29, and 31 on human red blood cells in vitro is shown. *: positive control (polyethylene glycol octylphenyl ether), #: PBS buffer.

[0044] [Figure 2] 1 shows the efficacy of example compounds 13, 17, 31 and etelcalcetide (AMG-416) at 3 mg / kg in reducing parathyroid hormone levels in normal rats.

[0045] [Figure 3] 1 shows the efficacy of 3 mg / kg of example compounds 13, 17, 31 and etelcalcetide (AMG-416) in reducing serum calcium ion levels in normal rats.

[0046] [Figure 4] 1 shows the efficacy of example compound 17 and etelcalcetide (AMG-416) in reducing parathyroid hormone levels in 5 / 6 nephrectomized rats.

[0047] [Figure 5] 1 shows the efficacy of example compound 17 and etelcalcetide (AMG-416) in reducing serum calcium ion levels in 5 / 6 nephrectomized rats. DETAILED DESCRIPTION OF THE INVENTION

[0048] To more fully describe the present disclosure, the present specification provides the following detailed description of the invention, but the present disclosure is not limited to these detailed description. 1. Experimental reagents [Table 2]

[0049] 2. Experimental equipment [Table 3]

[0050] 3. Example 3.1 Chemical synthesis of compound 1 Solid-phase peptide synthesis was performed on a Prelude-X automated polypeptide synthesizer using the Fmoc / tBu synthesis protocol, starting with Rink-amide MBHA resin (0.1 mmole). Coupling was carried out for 25 min at room temperature using 10 equivalents of amino acid residues activated with HCTU and 4-methylmorpholine in N,N-dimethylformamide (1:2:1 molar ratio of HCTU, 4-methylmorpholine, and amino acid residue).

[0051] After completing the peptide-resin synthesis, the polypeptide was cleaved from the solid-phase resin, side-chain protecting groups were removed, and the sulfhydryl group of the D-Cys side chain was activated simultaneously in a solution containing 90:5:5 (v / v / v) trifluoroacetic acid:triisopropylsilane:water and 2,2-dipyridyl disulfide (1 mmole) at room temperature for 2 hours. After the reaction was completed, the resin was filtered and washed twice with trifluoroacetic acid. The filtrates were combined and a large amount of frozen methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the supernatant was removed to obtain the crude polypeptide product, which was then dried and weighed.

[0052] The crude polypeptide product obtained above and L-Cys (0.1 mmole) were dissolved in PBS buffer (pH = 7.4), and the mixture was shaken at room temperature to react, and the production of Example No. 1 was monitored by ultra-high performance liquid chromatography. After the reaction was completed, trifluoroacetic acid (300 μL) was added to the mixture to quench the reaction, and the mixture was used for subsequent purification.

[0053] The resulting mixture was filtered through a 0.22 μm membrane and then separated using a WATERS Prep150 preparative reverse-phase high-performance liquid chromatography system. Buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution) were used. The preparative chromatography column was an X-SELECT OBD C-18 (WATERS) reverse-phase chromatography column. During purification, the chromatographic detection wavelength was set to 220 nm and the flow rate was 15 mL / min. The product-related distillate was collected and lyophilized to obtain the purified polypeptide of Example No. 1 with a yield of 45%. The purity and compound identity of the purified polypeptide were determined by analytical ultra-high-performance liquid chromatography and ultra-high-performance liquid chromatography / mass spectrometry. The compound purity was 96.78% and the molecular weight of the compound was 1109.60.

[0054] 3.2 Chemical synthesis of compound 2-38 Compounds 2-38 of the present disclosure were synthesized by a synthesis method similar to that for compound 1, and the purity and molecular weight of the synthesized polypeptides were determined by analytical ultra-high performance liquid chromatography and ultra-high performance liquid chromatography / mass spectrometry, as shown in Table 1. [Table 4]

[0055] Biological Testing and Evaluation Hereinafter, the present disclosure will be further explained and interpreted in conjunction with specific examples of the present disclosure, but these examples are not intended to limit the scope of the present disclosure.

[0056] 1. Experimental reagents necessary for in vitro and in vivo biological testing and evaluation [Table 5]

[0057] 2. Experimental equipment [Table 6]

[0058] 3. Test example 3.1. Agonist activity of compounds 1-38 on the human calcium-sensing receptor (CaSR) 3.1.1 Experimental Objective: The objective of this experiment is to measure the agonist activity of Compound 1-38 on the human calcium-sensing receptor (CaSR).

[0059] 3.1.2 Experimental Method: The HEK293 / CaSR stably transformed cell line (supplied by Koryu Chemical) was cultured in complete medium (components: DMEM, high glucose + 10% FBS + 2 mM GlutaMAX + 1X Penicillin-Streptomycin + 200 μg / mL Hygromycin B) and incubated at 37°C, 5% CO2 until 70%-90% confluence. After digestion with TrypLE, the cell line was seeded into a 384-well cell culture plate and cultured overnight at 37°C, 5% CO2. After replacing the cell fluid, stimulation buffer (HEPES 10 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, CaCl2 1.2 mM) and different concentrations of the example compounds to be detected were added and incubated at 37°C for 60 minutes. The production of IP-One in the cells was detected according to the instructions in the Cisbio IP-One Tb Reagent Kit. After collecting the raw data of each example compound, the software calculated the EC of each example compound on the human calcium-sensing receptor. 50The values ​​are calculated and the agonist activity of the examples against the human calcium-sensing receptor is evaluated based on these values.

[0060] 3.1.3 Experimental data processing method: The HTRF signal was read using an EnVision detector, with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio value (665 nm / 620 nm*10,000) was calculated, and the EC values ​​of Examples 1-38 were determined by nonlinear fitting of the signal ratio and sample concentration using a four-parameter equation in GraphPad Prism 6. 50 The values ​​were obtained and the specific values ​​are shown in Table 2 below.

[0061] [Table 7] [Table 8]

[0062] In the above table, etelcalcetide and etelcalcetide analogs are prepared by the method disclosed in patent document WO2011014707 as positive controls.

[0063] A significant proportion of the examples of the present disclosure have excellent in vitro potency, corresponding to EC50 values ​​of less than 10 uM in an assessment of in vitro agonist activity at the human calcium-sensing receptor.

[0064] 3.2. Evaluation of in vitro histamine-releasing activity of compounds 1-38 in rat peritoneal mast cells 3.2.1 Experimental Objective: To evaluate the in vitro histamine release levels of Compounds 1-38 detected in rat peritoneal mast cells.

[0065] 3.2.2 Experimental methods and data processing: To assess the level of in vitro histamine release detected in some of the examples, rat peritoneal mast cells were isolated by washing the rat peritoneum with washing buffer (cold HBSS containing 5 U / mL heparin + 25 mM HEPES, pH 7.4). After isolation, the cells were centrifuged, the washing buffer was removed, and the cells were resuspended and washed twice with stimulation buffer (HBSS + 25 mM HEPES + 1 mM CaCl2, pH 7.4). 5 The cells were plated at a density of 100 cells / well (200 μL / well), and the positive control Compound 48 / 80 (final concentration 4 μg / mL), the compound to be detected (final concentration 10 μM), or the vehicle control was added and incubated at 37°C for 15 minutes. After centrifugation, the cell supernatant was collected and the histamine concentration in the supernatant was determined according to the instructions of the LDN Histamine ELISA Reagent Kit (BAE-1000). Specific values ​​are shown in Table 3 below.

[0066] [Table 9]

[0067] A significant portion of the compounds disclosed herein induce in vitro stamina release in rat peritoneal mast cells to an insignificant extent, as reflected specifically in a relative stamina release fold ratio of less than 1.50 relative to PBS buffer. Surprisingly, amino acid substitutions in some compounds reduce the in vitro stamina release levels in rat peritoneal mast cells relative to etelcalcetide, e.g., Examples 17, 29, 31, and 32.

[0068] 3.3. Evaluation of the Hemolytic Effect of Some Compounds of the Present Disclosure on Human Erythrocytes In Vitro 3.3.1 Experimental Objective: To evaluate the hemolytic effect of some compounds of the present disclosure on human red blood cells in vitro.

[0069] 3.3.2 Experimental methods and data processing: To evaluate the hemolytic effect of the compounds of the present disclosure on red blood cells in vitro, human whole blood (100 μL) was homogenously mixed with phosphate buffer and centrifuged at 4°C for 10 minutes, after which the supernatant was discarded. The red blood cells were resuspended in PBS buffer (900 μL), centrifuged at 4°C for 10 minutes, and the supernatant was discarded. The above steps were repeated once. The compounds of the examples to be detected were dissolved in 1×PBS buffer to a final concentration of 100 μg / ml. The red blood cells were resuspended in the solution of the compounds of the examples to be detected, polyethylene glycol octylphenyl ether-100 solution, and PBS buffer, and incubated at 37°C for 1 hour. After incubation, the mixture was centrifuged at 4°C for 10 minutes, and the supernatant (100 μL) was removed and transferred to a 96-well plate. The absorbance was measured at 540 nm, and the hemolytic effect of the compounds of the examples to be detected on red blood cells in vitro was evaluated based on this data.

[0070] 3.3.3 Experimental results While no obvious hemolytic effect on red blood cells was observed for some compounds 12, 13, 17, 19, 29 and 31 of the present disclosure at a concentration of 100 μg / ml, a clear hemolytic effect on red blood cells was observed for polyethylene glycol octylphenyl ether-100 solution under the experimental conditions, as shown in FIG. 1.

[0071] 3.4. Evaluation of in vivo efficacy following single-dose administration of some compounds of the present disclosure in a normal rat model 3.4.1 Experimental Objective: To evaluate the efficacy of test compounds in lowering plasma parathyroid hormone levels following a single dose in a normal rat model.

[0072] 3.4.2 Experimental methods and data processing: Normal adult SPF rats (Sprague-Dawley, SD) weighing 250-350 g were selected for the study and returned to a normal diet for 7 days in the animal room. The rats were randomly assigned to groups of six, half male and half female, and each group was individually numbered. The day before the experiment, 540 μL of blood was collected from each rat, and plasma parathyroid hormone and serum calcium ion concentrations were measured as pre-administration controls. Plasma was isolated by anticoagulation with K2-EDTA and collected from the jugular vein. After placing the blood on ice, the whole blood was centrifuged at 6,800 rpm for 6 minutes at 2-8°C. The upper layer, which contained plasma, was carefully removed and stored at 2-8°C. Serum was isolated by collecting blood from the jugular vein and allowing it to stand at room temperature for 1 hour. It was then centrifuged at 3,500 rpm for 10 minutes at room temperature. The upper layer, which contained serum, was carefully removed and stored at room temperature. The animals were fasted overnight and allowed free access to water from the day before the experiment. On the day after blood collection, Example Compounds 13, 17, and 31 and etelcalcetide (AMG-416) were dissolved in phosphate buffered saline (PBS, Gibco). Each rat was intravenously administered Example Compounds 13, 17, and 31, 3 mg / kg etelcalcetide, or an equal volume of PBS buffer solution. Blood samples were then collected according to the following method, and the corresponding indicators were measured. 100 μL of blood was collected 1 hour, 2 hours, and 4 hours after administration. Plasma was separated according to the above method, and plasma parathyroid hormone levels were measured (ELISA: enzyme-linked immunosorbent assay) using a Rat Intact PTH ELISA Kit (Quidel-Immunotopics, Cat. #: 60-2500) according to the kit's instructions. The detailed steps are as follows: Using the reaction strips provided in the reagent kit, pre-plated with streptavidin, 25 μL of the standard, control, or plasma sample was added to each well. Biotinylated rat parathyroid hormone antibody and rat parathyroid hormone / HRP-conjugated antibody were mixed in a 1:1 ratio, and 100 μL of this mixture was added to each well.The reaction strips were sealed with sealing film, wrapped in aluminum foil, and stored in the dark. They were then shaken at 220 rpm on a horizontal shaker at room temperature for 3 h. The solution in the wells was removed, and each well was washed with 350 μL of washing solution. The solution was removed again, and the wash was repeated five times in the same manner. Finally, the solution in each well was aspirated. 150 μL of horseradish peroxidase ELISA substrate was added to each well. The reaction strips were sealed with sealing film, wrapped in aluminum foil, and stored in the dark. They were then shaken at 180–220 rpm on a horizontal shaker at room temperature for 30 min. 100 μL of ELISA stop solution was added to each well, and the strips were shaken at 180–220 rpm on a horizontal shaker at room temperature for 1 min. The absorbance of each well was read at 450 nm within 10 min after the addition of the ELISA stop solution, while the absorbance at 620 nm was simultaneously subtracted as background. 150 μL of horseradish peroxidase ELISA substrate and 100 μL of ELISA stop solution were used as a blank control for measuring absorbance. A standard curve was constructed based on the absorbance of the standard, and the actual plasma parathyroid hormone concentration was calculated by combining the absorbance of other samples with the standard curve. Serum calcium ion concentration was measured according to the steps in the relevant reagent kit.

[0073] 3.4.3 Experimental results As shown in Figures 2 and 3, test compounds 13, 17 and 31 completely reduced plasma parathyroid hormone levels in normal rats within 4 hours at a dose of 3 mg / kg, with a corresponding reduction in serum calcium ion levels.

[0074] 3.5. Evaluation of In Vivo Efficacy Following Continuous Administration of Some Compounds of the Present Disclosure in a 5 / 6 Nephrectomized Rat Model 3.5.1 Experimental Objective: To evaluate the efficacy of certain compounds of the present disclosure in reducing plasma parathyroid hormone and serum calcium ion levels following sequential administration in a 5 / 6 nephrectomized rat model.

[0075] 3.5.2 Experimental Methods and Data Processing: After rats were fed an adapted diet, under anesthesia, 2 / 3 of the left kidney was surgically removed. After one week of recovery, the right kidney was surgically removed to establish a 5 / 6 nephrectomy rat model. After the second surgery, the animals were maintained under normal conditions for two weeks, and creatinine (CREA) and plasma parathyroid hormone levels were measured. Based on parathyroid hormone levels, the animals were randomly divided into four groups, including a saline group, a low-dose compound 17 group, a high-dose compound 17 group, and an etelcalcetide group, with 10 rats per group. After randomization, the saline group, the low-dose compound 17 group, the high-dose compound 17 group, and the etelcalcetide group received saline, 1 mg / kg compound 17, 2 mg / kg compound 17, and 1 mg / kg etelcalcetide via the tail vein, respectively, once daily for 28 consecutive days. During the treatment period, animal weight, plasma parathyroid hormone levels, serum calcium ions, and other indicators were measured. The first day of administration after the animals were grouped is marked as day 1 of the experiment.

[0076] 3.5.3 Experimental results and conclusions: Compared with the saline group, 1 mg / kg and 2 mg / kg of Compound 17 dose-dependently reduced plasma parathyroid hormone levels in rats. After 6 h of administration on days 1, 14, and 28, all treatment groups reduced parathyroid hormone to very low levels, with the reduction in parathyroid hormone levels exceeding 90% from day 14. During the treatment period, the magnitude of inhibition of plasma parathyroid hormone levels after 6 and 16 h of administration of 1 mg / kg of Compound 17 was slightly superior to or comparable to that of the same dose of etelcalcetide (Figure 4). Reduction of blood calcium is a mechanism-related effect of such drugs. In this study, both Compound 17 and etelcalcetide induced a reversible reduction in blood calcium after administration on days 1, 14, and 28. There were no significant differences in minimum blood calcium levels between treatment groups on days 14 and 28 compared to day 1, suggesting that the degree of blood calcium reduction induced by compound 17 and etelcalcetide did not increase with increasing administration time. On days 1, 14, and 28, the maximum reduction in blood calcium induced by compound 17 was comparable to that of the same dose of etelcalcetide, suggesting that compound 17 and etelcalcetide have comparable activity in reducing blood calcium (Figure 5). It should be noted that the durability of the reduction in serum calcium ion levels by compound 17 on day 28 was superior and significantly different from that of the same dose of etelcalcetide. Furthermore, the present invention includes the following aspects. [Aspect 1] A compound or a pharmaceutically acceptable salt thereof comprising a peptide and a conjugate group, wherein the peptide comprises an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) Among them, X 1 is D-Cys, X 2 D-Phg, D-Phe(4-CH 3 ), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, D-Arg or D-His; X 3 is D-Arg, X 4 are D-Arg, D-Phg, D-Phe(4-CH 3 ), D-2-Thi, D-Phe(4-NO 2 ), D-2-NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Cha, D-Ser, D-Gln, D-Tyr, D-Ile, D-Ser, D-His, D-Val or D-Chg, X 5 is D-Arg, X 6 is selected from D-Ala, D-Abu, D-Ser or Gly, X 7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; The conjugate group is L-Cys, and X of the peptide 1 is covalently linked to said conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, The compound or a pharmaceutically acceptable salt thereof. [Aspect 2] The peptide is composed of an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) Among them, X 1 is D-Cys, X 2 D-Phg, D-Phe(4-CH 3 ), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser or D-His; X 3 is D-Arg, X 4 is D-Arg, X 5 is D-Arg, X 6 is selected from D-Ala, D-Abu, D-Ser or Gly, X 7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; The conjugate group is L-Cys, and X of the peptide 1 is covalently linked to said conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, A compound according to embodiment 1 or a pharmaceutically acceptable salt thereof. [Aspect 3] A peptide comprising a polypeptide and a conjugate group, the peptide comprising an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) Among them, X 1 is D-Cys, X 2 is D-Arg, X 3 is D-Arg, X 4 are D-Arg, D-Phg, D-Phe(4-CH 3 ), D-2-Thi, D-Phe(4-NO 2 ), D-2-NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, D-Ser, D-Cha, D-Gln, D-Tyr, D-His or D-Val, X 5 is D-Arg, X 6 is selected from D-Ala, D-Abu, D-Ser or Gly, X 7 is D-Arg, The peptide and the conjugate group are covalently bonded by a disulfide bond, the conjugate group is L-Cys, and X of the peptide 1 is linked to the conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, A compound according to embodiment 1 or a pharmaceutically acceptable salt thereof. [Aspect 4] A peptide comprising a polypeptide and a conjugate group, the peptide comprising an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) Among them, X 1 is D-Cys, X 2 is D-Arg, X 3 is D-Arg, X 4 D-Phg, D-Phe(4-CH3 ), D-2-Thi, D-Phe(4-NO 2 ), D-2NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, D-Ser, D-Cha, D-Gln, D-Tyr, D-Ile, D-His or D-Val, X 5 is D-Arg, X 6 is selected from D-Ala, D-Abu, D-Ser or Gly, X 7 is D-Arg, the peptide and the conjugate group are covalently bonded by a disulfide bond; The conjugate group is L-Cys, and X of the peptide 1 is linked to the conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, A compound according to embodiment 1 or a pharmaceutically acceptable salt thereof. [Aspect 5] A peptide comprising a polypeptide and a conjugate group, the peptide comprising an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) Among them, X 1 is D-Cys, X 2 is selected from D-Arg, X 3 is D-Arg, X 4 is D-Phe(4-CH 3 ), D-2-Thi, D-Abu, D-hLeu or D-Val; X 5 is D-Arg, X 6 is selected from D-Ala or D-Ser, X 7 is D-Arg, The conjugate group is L-Cys, and X of the peptide 1 is covalently linked to said conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, A compound according to embodiment 1 or a pharmaceutically acceptable salt thereof. [Aspect 6] X 4 is selected from D-Abu or D-Val, preferably D-Abu; A compound according to aspect 5, or a pharmaceutically acceptable salt thereof. [Aspect 7] the conjugate group is acetylated; A compound according to any one of Aspects 1 to 6, or a pharmaceutically acceptable salt thereof. [Aspect 8] The formula is 1 is covalently linked to another sulfhydryl-containing amino acid sequence by a disulfide bond via a sulfhydryl-containing group in the residue of A compound according to any one of Aspects 1 to 7, or a pharmaceutically acceptable salt thereof. [Aspect 9] The compound is selected from the compounds shown in the table below: A compound according to any one of aspects 1 to 8, or a pharmaceutically acceptable salt thereof: Table 10 Table 11 [Aspect 10] A pharmaceutical composition comprising a compound according to any one of Aspects 1 to 9, or a pharmaceutically acceptable salt thereof. [Aspect 11] Use of a compound according to any one of aspects 1 to 9 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to aspect 10, in the preparation of a medicament for treating a disease associated with abnormal parathyroid hormone levels. [Aspect 12] The disease associated with abnormal parathyroid hormone levels is hyperparathyroidism. 12. The use according to embodiment 11. [Aspect 13] The hyperparathyroidism is secondary hyperparathyroidism in a subject with chronic kidney disease. 13. The use according to embodiment 12. [Aspect 14] A method of treating a disease associated with abnormal parathyroid hormone levels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of aspects 1 to 9 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to aspect 10. method.

Claims

1. A compound or a pharmaceutically acceptable salt thereof comprising a peptide and a conjugate group, wherein the peptide comprises an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) where: X 1 is D-Cys, X 2 is D-Arg, X 3 is D-Arg, X 4 is D-Abu, X 5 is D-Arg, X 6 is selected from D-Ala, D-Abu, D-Ser and Gly; X 7 is D-Arg, The peptide and the conjugate group are covalently bonded by a disulfide bond, the conjugate group is L-Cys, and X of the peptide 1 is linked to the conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, The compound or a pharmaceutically acceptable salt thereof.

2. The peptide is composed of an amino acid sequence represented by the following formula (I): X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (I) where: X 1 is D-Cys, X 2 is D-Arg, X 3 is D-Arg, X 4 is D-Abu, X 5 is D-Arg, X 6 is selected from D-Ala and D-Ser, X 7 is D-Arg, The conjugate group is L-Cys, and X of the peptide 1 is covalently linked to said conjugate group by a disulfide bond, The N-terminal X of the peptide 1 is modified by acetylation to the C-terminal X 7 is modified by amidation, 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. the conjugate group is acetylated; 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. The formula is 1 is covalently linked to another sulfhydryl-containing amino acid sequence by a disulfide bond via a sulfhydryl-containing group in the residue of 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

5. The compound is selected from the compounds shown in the table below: A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof: Table 1

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

7. 7. The pharmaceutical composition of claim 6 for treating a disease associated with abnormal parathyroid hormone levels.

8. The disease associated with abnormal parathyroid hormone levels is hyperparathyroidism.

8. The pharmaceutical composition of claim 7.

9. The hyperparathyroidism is secondary hyperparathyroidism in a subject with chronic kidney disease.

9. The pharmaceutical composition of claim 8.

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