CALCIUM-SENSING RECEPTOR AGONIST COMPOUND AND APPLICATION THEREOF

Abstract

Provided are a calcium-sensing receptor (CaSR) agonist compound and application thereof. Specifically, provided are a series of polypeptide CaSR agonist compounds and pharmaceutically acceptable salts thereof, which have agonist effects on human CaSRs to reduce plasma parathyroid hormone and serum calcium ion levels, and can be used for treatment of metabolic diseases such as primary hyperparathyroidism, secondary hyperparathyroidism, and tumor-induced hypercalcemia.

Claims

1. A compound consisting of a peptide and a conjugated group, or a pharmaceutically acceptable salt thereof, wherein the peptide consists of an amino acid sequence of the following formula (I):
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7  (I) wherein: X.sub.1 is D-Cys; X.sub.2 is selected from the group consisting of D-Phg, D-Phe(4-CH.sub.3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser, D-Arg and D-His; X.sub.3 is D-Arg; X.sub.4 is selected from the group consisting of D-Arg, D-Phg, D-Phe(4-CH.sub.3), D-2-Thi, D-Phe(4-NO.sub.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 and D-Chg; X.sub.5 is D-Arg; X.sub.6 is selected from the group consisting of D-Ala, D-Abu, D-Ser and Gly; X.sub.7 is D-Arg; wherein the peptide and the conjugated group are covalently linked by a disulfide bond; wherein the conjugated group is L-Cys and the X.sub.1 residue of the peptide is covalently linked to the conjugated group by a disulfide bond; and the N-terminal X.sub.1 of the peptide is acetylated and the C-terminal X.sub.7 of the peptide is amidated.

2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the peptide consists of an amino acid sequence of the following formula (I):
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7  (I) wherein: X.sub.1 is D-Cys; X.sub.2 is selected from the group consisting of D-Phg, D-Phe(4-CH.sub.3), D-Phe(2-Cl), D-Tyr, D-Trp, D-Ser and D-His; X.sub.3 is D-Arg; X.sub.4 is D-Arg; X.sub.5 is D-Arg; X.sub.6 is selected from the group consisting of D-Ala, D-Abu, D-Ser and Gly; X.sub.7 is D-Arg; wherein the peptide and the conjugated group are covalently linked by a disulfide bond; wherein the conjugated group is L-Cys and the X.sub.1 residue of the peptide is covalently linked to the conjugated group by a disulfide bond; and the N-terminal X.sub.1 of the peptide is acetylated and the C-terminal X.sub.7 of the peptide is amidated.

3. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the peptide consists of an amino acid sequence of the following formula (I):
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7  (I) wherein: X.sub.1 is D-Cys; X.sub.2 is D-Arg; X.sub.3 is D-Arg; X.sub.4 is selected from the group consisting of D-Arg, D-Phg, D-Phe(4-CH.sub.3), D-2-Thi, D-Phe(4-NO.sub.2), D-2-NaI, D-hPhe, D-Abu, D-Tle, D-hLeu, D-Chg, D-Ser, D-Cha, D-Gln, D-Tyr, D-His and D-Val; X.sub.5 is D-Arg; X.sub.6 is selected from the group consisting of D-Ala, D-Abu, D-Ser and Gly; X.sub.7 is D-Arg; wherein the peptide and the conjugated group are covalently linked by a disulfide bond; wherein the conjugated group is L-Cys and the X.sub.1 residue of the peptide is linked to the conjugated group by a disulfide bond; and the N-terminal X.sub.1 of the peptide is acetylated and the C-terminal X.sub.7 of the peptide is amidated.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the peptide consists of an amino acid sequence of the following formula (I):
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7  (I) wherein: X.sub.1 is D-Cys; X.sub.2 is D-Arg; X.sub.3 is D-Arg; X.sub.4 is selected from the group consisting of D-Phg, D-Phe(4-CH.sub.3), D-2-Thi, D-Phe(4-NO.sub.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 and D-Val; X.sub.5 is D-Arg; X.sub.6 is selected from the group consisting of D-Ala, D-Abu, D-Ser and Gly; X.sub.7 is D-Arg; wherein the peptide and the conjugated group are covalently linked by a disulfide bond; wherein the conjugated group is L-Cys and the X.sub.1 residue of the peptide is linked to the conjugated group by a disulfide bond; and the N-terminal X.sub.1 of the peptide is acetylated and the C-terminal X.sub.7 of the peptide is amidated.

5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the peptide consists of an amino acid sequence of the following formula (I):
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7  (I) wherein: X.sub.1 is D-Cys; X.sub.2 is D-Arg; X.sub.3 is D-Arg; X.sub.4 is selected from the group consisting of D-Phe(4-CH.sub.3), D-2-Thi, D-Abu, D-hLeu and D-Val; X.sub.5 is D-Arg; X.sub.6 is selected from the group consisting of D-Ala and D-Ser; X.sub.7 is D-Arg; wherein the conjugated group is L-Cys and the X.sub.1 residue of the peptide is covalently linked to the conjugated group by a disulfide bond; and the N-terminal X.sub.1 of the peptide is acetylated and the C-terminal X.sub.7 of the peptide is amidated.

6. The compound or the pharmaceutically acceptable salt thereof according to claim 5, wherein X.sub.4 is selected from the group consisting of D-Abu and D Val.

7. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the conjugated group is acetylated.

8. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of the formula is covalently linked by disulfide bonds to other amino acid sequences containing a thiol via a group containing a thiol in the X.sub.1 residue.

9. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of the compounds listed below: TABLE-US-00010 SEQ Compound Sequence ID NO:  1 Ac-c©-(D-Phg)-r-r-r-a-r-NH.sub.2  1  2 Ac-c©-[D-Phe(2-Cl)]-r-r-r-a-r-NH.sub.2  2  3 Ac-c©-[D-Phe(4-CH.sub.3)]-r-r-r-a-r-NH.sub.2  3  4 Ac-c©-(D-Tyr)-r-r-r-a-r-NH.sub.2  4  5 Ac-c©-(D-Trp)-r-r-r-a-r-NH.sub.2  5  6 Ac-c©-s-r-r-r-s-r-NH.sub.2  6  7 Ac-c©-h-r-r-r-G-r-NH.sub.2  7  8 Ac-c©-s-r-r-r-G-r-NH.sub.2  8  9 Ac-c©-w-r-r-r-G-r-NH.sub.2  9 10 Ac-c©-h-r-r-r-s-r-NH.sub.2 10 11 Ac-c©-r-r-(D-Phg)-r-a-r-NH.sub.2 11 12 Ac-c©-r-r-[D-Phe(4-CH.sub.3)]-r-a-r-NH.sub.2 12 13 Ac-c©-r-r-(D-2-Thi)-r-a-r-NH.sub.2 13 14 Ac-c©-r-r-[D-Phe(4-NO.sub.2)]-r-a-r-NH.sub.2 14 15 Ac-c©-r-r-(D-2-NaI)-r-a-r-NH.sub.2 15 16 Ac-c©-r-r-(D-hPhe)-r-a-r-NH.sub.2 16 17 Ac-c©-r-r-(D-Abu)-r-a-r-NH.sub.2 17 18 Ac-c©-r-r-(D-Tle)-r-a-r-NH.sub.2 18 19 Ac-c©-r-r-(D-hLeu)-r-a-r-NH.sub.2 19 20 Ac-c©-r-r-(D-Chg)-r-a-r-NH.sub.2 20 21 Ac-c©-r-r-(D-Cha)-r-a-r-NH.sub.2 21 22 Ac-c©-r-r-s-r-G-r-NH.sub.2 22 23 Ac-c©-r-r-q-r-G-r-NH.sub.2 23 24 Ac-c©-r-r-y-r-a-r-NH.sub.2 24 25 Ac-c©-r-r-s-r-s-r-NH.sub.2 25 26 Ac-c©-r-r-q-r-s-r-NH.sub.2 26 27 Ac-c©-r-r-h-r-s-r-NH.sub.2 27 28 Ac-c©-r-r-h-r-G-r-NH.sub.2 28 29 Ac-c©-r-r-v-r-s-r-NH.sub.2 29 30 Ac-c©-r-r-v-r-G-r-NH.sub.2 30 31 Ac-c©-r-r-(D-Abu)-r-s-r-NH.sub.2 31 32 Ac-c©-r-r-(D-Abu)-r-G-r-NH.sub.2 32 33 Ac-c©-r-r-(D-hLeu)-r-s-r-NH.sub.2 33 34 Ac-c©-r-r-(D-hLeu)-r-G-r-NH.sub.2 34 35 Ac-c©-r-r-(D-Chg)-r-s-r-NH.sub.2 35 36 Ac-c©-r-r-(D-Chg)-r-G-r-NH.sub.2 36 37 Ac-c©-r-r-(D-Tle)-r-s-r-NH.sub.2 37 38 Ac-c©-r-r-(D-Tle)-r-G-r-NH.sub.2 38

10. A pharmaceutical composition, comprising the compound or the pharmaceutically acceptable salt thereof according to claim 1.

11. 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, the compound or the pharmaceutically acceptable salt thereof according to claim 1, or a pharmaceutical composition according to claim 10.

12. The method according to claim 11, wherein the disease associated with abnormal parathyroid hormone levels is hyperparathyroidism.

13. The method according to claim 12, wherein the hyperparathyroidism is a secondary hyperparathyroidism in a subject with chronic kidney disease.

14. A method for treating a disease associated with abnormal parathyroid hormone levels in a subject in need, comprising administering to the subject a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1 shows the hemolytic effect of example compounds 12, 13, 17, 19, 29 and 31 against human red blood cells in vitro, wherein * denotes positive control (polyethylene glycol octyl phenyl ether) and #denotes PBS buffer.

[0098] FIG. 2 shows the efficacy of 3 mg/kg of example compounds 13, 17, 31 and etelcalcetide (AMG-416) in reducing parathyroid hormone level in normal rats.

[0099] FIG. 3 shows the efficacy of 3 mg/kg of example compounds 13, 17, 31 and etelcalcetide (AMG-416) in reducing serum calcium level in normal rats.

[0100] FIG. 4 shows the efficacy of example compound 17 and etelcalcetide (AMG-416) in reducing parathyroid hormone level in 5/6 nephrectomized rats.

[0101] FIG. 5 shows the efficacy of example compound 17 and etelcalcetide (AMG-416) in reducing serum calcium level in 5/6 nephrectomized rats.

DETAILED DESCRIPTION

[0102] The following specific embodiments are provided herein only for illustrating the present disclosure in more detail, rather than limiting the present disclosure.

TABLE-US-00003 1. Reagents No. Reagent Source  1 Rink-amide MBHA resin Sunresin, Xi'an  2 O-(1H-6-chlorobenzotriazole- Highfine Biotech, 1-yl)-1,1,3,3- Suzhou tetramethyluronium hexafluorophosphate (HCTU)  3 4-Methylmorpholine TCI Chemicals  4 Acetonitrile  Sigma-Aldrich (chromatographic grade)  5 N,N-dimethyl formamide Sinopharm Chemical Reagent  6 Dichloromethane Sinopharm Chemical Reagent  7 Trifluoroacetic acid TCI Chemicals  8 Triisopropylsilane TCI Chemicals  9 Methyl tert-butyl ether TCI Chemicals 10 4-Methylpiperidine TCI Chemicals 11 L-cysteine Sigma-Aldrich 12 Fmoc-D-Cys(Trt)-OH GL Biochem 13 Fmoc-D-Arg(Pbf)-OH GL Biochem 14 Fmoc-D-Ala-OH GL Biochem 15 Fmoc-D-Abu-OH GL Biochem 16 Fmoc-D-Phg-OH GL Biochem 17 Fmoc-D-Phe(4-CH.sub.3)-OH GL Biochem 18 Fmoc-D-2-Thi-OH GL Biochem 19 Fmoc-D-Phe(4-NO.sub.2)-OH GL Biochem 20 Fmoc-D-2NaI-OH GL Biochem 21 Fmoc-D-hPhe-OH GL Biochem 22 Fmoc-D-Tle-OH GL Biochem 23 Fmoc-D-hLeu-OH GL Biochem 24 Fmoc-D-Chg-OH GL Biochem 25 Fmoc-D-Ser(tBu)-OH GL Biochem 26 Fmoc-D-Cha-OH GL Biochem 27 Fmoc-D-Gln(Trt)-OH GL Biochem 28 Fmoc-Gly-OH GL Biochem 29 Fmoc-D-Tyr(tBu)-OH GL Biochem 30 Fmoc-D-His(Boc)-OH GL Biochem 31 Fmoc-D-Val-OH GL Biochem 32 Fmoc-D-Phe(2-Cl)-OH GL Biochem 33 Fmoc-D-Trp(Boc)-OH GL Biochem 34 2,2′-Dipyridyldisulfide GL Biochem

2. Instruments

[0103]

TABLE-US-00004 No. Instruments Source 1 Prelude-X multichannel polypeptide Protein synthesizer Technology 2 H-CLASS analytical ultra- Waters performance liquid chromatography 3 Xevo liquid chromatography/mass Waters spectrometry 4 Labconco multifunctional freeze Thermo-Fisher dryer Scientific 5 Prep 150 preparative high Waters performance liquid chromatography 6 Multichannel high-speed centrifuge Sigma

3. Examples

3.1 Chemical Synthesis of Compound 1

[0104] Solid phase peptide synthesis was performed on a Prelude-X automatic polypeptide synthesizer using the Fmoc/tBu synthesis strategy starting from Rink-amide MBHA resin (0.1 mmol). Coupling was performed using 10 equivalents of amino acid residues activated with HCTU and 4-methylmorpholine (the molar ratio of HCTU:4-methylmorpholine:amino acid residues was 1:2:1) in N,N-dimethylformamide at room temperature for 25 min.

[0105] After completion of the above peptide-resin synthesis, in a solution of 90:5:5 (v/v/v) trifluoroacetic acid:triisopropylsilane:water and 2,2′-dipyridyldisulfide (1 mmol) at room temperature, cleavage of polypeptide from solid phase resin, removal of side chain protecting group and activation of D-Cys side chain thiol group were synchronously accomplished in 2 h. After the reaction was completed, the mixture was filtered and the resin was washed for 2 times by using trifluoroacetic acid. The filtrates were combined before a large amount of frozen methyl tert-butyl ether was added to precipitate a solid. The mixture was centrifuged and the supernatant was discarded to obtain a crude product of the polypeptide, which was then dried and weighed.

[0106] The crude polypeptide obtained above and L-Cys (0.1 mmol) were dissolved in PBS buffer (pH=7.4) and reacted with shaking at room temperature. The production of compound 1 was monitored by ultra-performance liquid chromatography. After completion of the reaction, trifluoroacetic acid (300 μL) was added to the mixture to quench the reaction and for subsequent purification.

[0107] The mixture obtained above was filtered through a 0.22 μm membrane and separated by a Waters Prep150 preparative reverse-phase high performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution). The preparative chromatographic column was an X-SELECT OBD C-18 (Waters) reversed-phase chromatographic column, the detection wavelength of a chromatograph was set as 220 nm in the purification process, and the flow rate was 15 mL/min. The purified polypeptide product of compound 1 was obtained after the relevant fractions were collected and lyophilized (45% yield). The purity and the compound identity of the pure polypeptide product were determined by analytical ultra-performance liquid chromatography and ultra-performance liquid chromatography/mass spectrometry, wherein the purity of the compound was 96.78%, and the molecular weight of the compound was 1109.60.

3.2 Chemical Synthesis of Compounds 2-38

[0108] Compounds 2-38 of the present disclosure were synthesized using synthetic protocols similar to that of compound 1 and the purity and molecular weight of the synthesized polypeptides were determined by analytical ultra-performance liquid chromatography and ultra-performance liquid chromatography/mass spectrometry, as detailed in Table 1 below:

TABLE-US-00005 TABLE 1 Purity and measured molecular weight of the synthesized compounds Molecular Compound Purity weight  2 96.00% 1157.80  3 97.80% 1137.80  4 95.25% 1140.00  5 97.72% 1162.80  6 96.58% 1079.80  7 98.62% 1100.00  8 95.20% 1050.00  9 98.20% 1149.00 10 95.56% 1130.00 11 95.69% 1110.00 12 95.88% 1138.20 13 99.04% 1129.60 14 97.64% 1169.00 15 95.48% 1174.00 16 97.07% 1138.20 17 95.79% 1062.00 18 96.65% 1090.80 19 96.33% 1104.40 20 96.82% 1116.80 21 98.39% 1130.00 22 96.97% 1050.00 23 95.47% 1091.00 24 96.35% 1140.00 25 95.83% 1080.00 26 96.92% 1120.90 27 95.80% 1130.00 28 96.74% 1100.20 29 96.56% 1092.20 30 95.98% 1062.00 31 95.81% 1077.80 32 99.68% 1047.80 33 96.03% 1119.80 34 96.73% 1090.00 35 98.53% 1132.00 36 95.25% 1102.00 37 96.83% 1106.00 38 96.76% 1075.80

Biological Evaluation

[0109] The present disclosure is further illustrated in conjunction with the specific examples, which, however, are not intended to limit the scope of the present disclosure.

1. Reagent for in vitro and in vivo biological evaluation

TABLE-US-00006 No. Reagent Source  1 FBS, 500 mL ThermoFisher Scientific  2 DMEM, High Glucose, GlutaMAX, ThermoFisher 500 mL Scientific  3 Penicilin-Streptomyces, Liquid, ThermoFisher 100 mL (100×) Scientific  4 1× PBS pH 7.2-7.4 (500 mL) Solarbio  5 1× TrypLE Express Enzyme, ThermoFisher no phenol red (500 mL) Scientific  6 Hygromycin B Gold solution Invivogen (5 g, 1 × 50 mL,1 00 mg/mL)  7 HEPES, 1M Gibco  8 MgCl.sub.2, 1M Sigma-Aldrich  9 KCl, 1M Sigma-Aldrich 10 NaCl, 5M Sigma-Aldrich 11 Glucose Sigma-Aldrich 12 LiCl, 8M Sigma-Aldrich 13 CaCl.sub.2, 1M Sigma-Aldrich 14 IP-One-Gq Kit (1,000 tests) Cisbio

2. Instruments

[0110]

TABLE-US-00007 No. Instruments Source 1 EnVision detector Perkin Elmer

3. Test Examples

3.1 Evaluation of Agonist Activity of Compounds 1-38 on Human Calcium-Sensing Receptor (CaSR)

[0111] 3.1.1 Objective: The test example is intended to measure the agonist activity of compounds 1-38 on the human calcium-sensing receptor (CaSR).

3.1.2 Procedures:

[0112] Stably transfected HEK293/CaSR cells (source: Pharmaron) were cultured in a complete medium (composition: DMEM, high glucose+10% FBS+2 mM GlutaMAX+1× Penicillin-Streptomycin+200 μg/mL Hygromycin B) and incubated at 37° C./5% CO.sub.2 till 70%-90% cell confluence. The cells were digested with TrypLE, inoculated into 384-well cell culture plates, and cultured overnight at 37° C./5% CO.sub.2. After buffer exchange, stimulation buffer (HEPES 10 mM, MgCl.sub.2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, CaCl.sub.2 1.2 mM) and various concentrations of the test example compounds were added and incubated at 37° C. for 60 min. Production of IP-One in cells was detected according to the procedures in the Cisbio IP-One Tb kit instructions. The EC.sub.50 values of various test example compounds in influencing human calcium-sensing receptor was calculated by software after the raw data of the example compounds were collected, so as to evaluate the agonist activity of the example compounds on human calcium-sensing receptor.

3.1.3 Data Processing:

[0113] HTRF signal was read by an EnVision detector with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm/620 nm×10,000) was calculated and fitted non-linearly to the sample concentration in GraphPad Prism 6 using a four-parameter equation to give EC.sub.50 values of the test example compounds 1-38. The specific values are shown in Table 2 below.

TABLE-US-00008 TABLE 2 In vitro agonist activity of compounds 1-38 on calcium-sensing receptor EC.sub.50 for calcium- sensing Com- SEQ receptor pound Sequence ID NO: (μM)  1 Ac-c(C)-(D-Phg)-r-r-r-a-r-NH.sub.2  1 15.71  2 Ac-c(C)-[D-Phe(2-Cl)]-r-r-r-a-r-NH.sub.2  2  6.76  3 Ac-c(C)-[D-Phe(4-CH.sub.3)]-r-r-r-a-r-NH.sub.2  3 15.28  4 Ac-c(C)-(D-Tyr)-r-r-r-a-r-NH.sub.2  4  6.92  5 Ac-c(C)-(D-Trp)-r-r-r-a-r-NH.sub.2  5  1.14  6 Ac-c(C)-s-r-r-r-s-r-NH.sub.2  6 11.56  7 Ac-c(C)-h-r-r-r-G-r-NH.sub.2  7 18.60  8 Ac-c(C)-s-r-r-r-G-r-NH.sub.2  8 14.32  9 Ac-c(C)-w-r-r-r-G-r-NH.sub.2  9  3.16 10 Ac-c(C)-h-r-r-r-s-r-NH.sub.2 10  3.52 11 Ac-c(C)-r-r-(D-Phg)-r-a-r-NH.sub.2 11 15.71 12 Ac-c(C)-r-r-[D-Phe(4-CH.sub.3)]-r-a-r-NH.sub.2 12  1.28 13 Ac-c(C)-r-r-(D-2-Thi)-r-a-r-NH.sub.2 13  1.21 14 Ac-c(C)-r-r-[D-Phe(4-NO.sub.2)]-r-a-r-NH.sub.2 14  4.33 15 Ac-c(C)-r-r-(D-2-NaI)-r-a-r-NH.sub.2 15  5.59 16 Ac-c(C)-r-r-(D-hPhe)-r-a-r-NH.sub.2 16  2.37 17 Ac-c(C)-r-r-(D-Abu)-r-a-r-NH.sub.2 17  6.28 18 Ac-c(C)-r-r-(D-Tle)-r-a-r-NH.sub.2 18 13.37 19 Ac-c(C)-r-r-(D-hLeu)-r-a-r-NH.sub.2 19  2.05 20 Ac-c(C)-r-r-(D-Chg)-r-a-r-NH.sub.2 20  7.53 21 Ac-c(C)-r-r-(D-Cha)-r-a-r-NH.sub.2 21  5.59 22 Ac-c(C)-r-r-s-r-G-r-NH.sub.2 22 15.41 23 Ac-c(C)-r-r-q-r-G-r-NH.sub.2 23 30.40 24 Ac-c(C)-r-r-y-r-a-r-NH.sub.2 24  2.03 25 Ac-c(C)-r-r-s-r-s-r-NH.sub.2 25 10.08 26 Ac-c(C)-r-r-q-r-s-r-NH.sub.2 26  9.24 27 Ac-c(C)-r-r-h-r-s-r-NH.sub.2 27 10.81 28 Ac-c(C)-r-r-h-r-G-r-NH.sub.2 28 21.50 29 Ac-c(C)-r-r-v-r-s-r-NH.sub.2 29  9.80 30 Ac-c(C)-r-r-v-r-G-r-NH.sub.2 30 19.57 31 Ac-c(C)-r-r-(D-Abu)-r-s-r-NH.sub.2 31  5.93 32 Ac-c(C)-r-r-(D-Abu)-r-G-r-NH.sub.2 32 13.09 33 Ac-c(C)-r-r-(D-hLeu)-r-s-r-NH.sub.2 33  5.03 34 Ac-c(C)-r-r-(D-hLeu)-r-G-r-NH.sub.2 34 12.19 35 Ac-c(C)-r-r-(D-Chg)-r-s-r-NH.sub.2 35 12.94 36 Ac-c(C)-r-r-(D-Chg)-r-G-r-NH.sub.2 36 17.95 37 Ac-c(C)-r-r-(D-Tle)-r-s-r-NH.sub.2 37 15.45 38 Ac-c(C)-r-r-(D-Tle)-r-G-r-NH.sub.2 38 18.73 Etelcalcetide Ac-c(C)-a-r-r-r-a-r-NH.sub.2 40  6.78 Etelcalcetide Ac-c(C)-r-r-a-r-a-r-NH.sub.2 41  6.74 analogue

[0114] Positive controls etelcalcetide and the etelcalcetide analogue in the above table were prepared according to the method disclosed in Patent No. WO2011014707.

[0115] A considerable portion of the example compounds disclosed herein demonstrated excellent in vitro efficacy, corresponding to EC.sub.50 values less than 10 μM in the in vitro agonist activity evaluation on human calcium-sensing receptor.

3.2 Evaluation of In Vitro Activity of Compounds 1-38 to Induce Histamine Release in Rat Peritoneal Mast Cells

3.2.1 Objective: To Evaluate the In Vitro Activity of the Test Compounds 1-38 to Induce Histamine Release in Rat Peritoneal Mast Cells

3.2.2 Procedures and Data Processing:

[0116] To evaluate the in vitro histamine release levels induced by some of the test example compounds, rat peritoneal mast cells were collected by lavaging rat peritoneum with a lavage buffer (cold HBSS+25 mM HEPES containing heparin 5 U/mL, pH 7.4). After collection, the cells were centrifuged, and the lavage buffer was discarded. The cells were resuspended and washed twice with a stimulation buffer (HBSS+25 mM HEPES+1 mM CaCl.sub.2), pH 7.4). The cells were plated at a density of 10.sup.5 cell/well (200 μL/well) and incubated at 37° C. for 15 min with positive control compound 48/80 (final concentration: 4 μg/mL), test example compounds (final concentration: 10 μM) or vehicle control. The cells were centrifuged, and cell supernatant was collected and tested for histamine concentration according to LDN Histamine ELISA kit (BAE-1000) instructions. Specific data are shown in Table 3 below.

TABLE-US-00009 TABLE 3 Histamine release levels in vitro induced by some of the compounds disclosed herein Relative fold of histamine Example release in vitro PBS buffer 1.00 Compound 8.78 48/80  1 1.50  2 4.96  4 3.94  5 4.13  6 1.65  7 2.24  8 1.50 10 3.66 11 1.11 12 2.81 13 2.38 16 2.94 17 0.97 18 1.24 19 2.07 20 2.33 21 3.42 22 1.63 24 3.18 25 1.65 26 1.73 27 2.23 29 0.99 30 1.51 31 1.29 32 1.41 33 2.50 34 2.28 35 2.06 37 2.23 Etelcalcetide 1.70

[0117] A considerable portion of the compounds disclosed herein did not significantly induce histamine release in rat peritoneal mast cells in vitro, in particular at a relative histamine release fold less than 1.50 relative to PBS buffer. Surprisingly, amino acid substitutions in some compounds resulted in a reduction in histamine release levels in rat peritoneal mast cells in vitro relative to the etelcalcetide, e.g., examples 17, 29, 31 and 32.

3.3 Evaluation of Hemolytic Effect of Some of the Compounds Disclosed Herein on Human Red Blood Cells In Vitro

3.3.1 Objective: To Evaluate the Hemolytic Effect of Some of the Compounds Disclosed Herein on Human Red Blood Cells In Vitro.

3.3.2 Procedures and Data Processing:

[0118] To evaluate the hemolytic effect of the compounds disclosed herein on red blood cells in vitro, human whole blood (100 μL) was taken and mixed with a phosphate buffer. The mixture was centrifuged at 4° C. for 10 min and the supernatant was discarded. The red blood cells were resuspended in PBS buffer (900 μL) and centrifuged at 4° C. for 10 min with the supernatant discarded, and the procedures above were repeated once. The test example compounds were dissolved in 1×PBS buffer to a final concentration of 100 μg/mL. The red blood cells were resuspended in solutions of various test example compounds, an octylphenoxy poly(ethyleneoxy)ethanol-100 solution or PBS buffer, and incubated at 37° C. for 1 h. After incubation, the cells were centrifuged at 4° C. for 10 min and the supernatant (100 μL) was pipetted and transferred to a 96-well plate. The absorbance at 540 nm was detected for evaluating the hemolytic effect of the test example compounds on red blood cells in vitro.

3.3.3 Results

[0119] At a concentration of 100 μg/mL, no significant hemolytic effect on red blood cells was observed for compounds 12, 13, 17, 19, 29 and 31 of the present disclosure, while the octylphenoxy poly(ethyleneoxy)ethanol-100 solution demonstrated a significant hemolytic effect on red blood cells under the experimental conditions, as shown in FIG. 1.

3.4 Evaluation of In Vivo Efficacy of Some of the Compounds Disclosed Herein in a Normal Rat Model after a Single Dose
3.4.1 Objective: To Evaluate the Efficacy of the Test Compounds in Reducing Plasma Parathyroid Hormone Levels after a Single Dose in a Normal Rat Model.

3.4.2 Procedures and Data Processing:

[0120] SPF normal adult rats (Sprague Dawley, or SD) with weight of 250-350 g were fed with normal diet in an animal room for 7 days. Rats were randomized into groups of 6, half female and half male, and numbered. One day before the start of treatment, 540 μL of blood was collected from each rat, and the plasma parathyroid hormone level and the serum calcium concentration were measured as baseline. The plasma was separated by K2-EDTA anticoagulation. Blood was collected through jugular vein and preserved on ice after collection. The whole blood was centrifuged at 6,800 rpm for 6 minutes at 2-8° C. The supernatant, i.e., the plasma, was collected and preserved at 2-8° C. For serum separation, blood was collected through jugular vein, let stand at room temperature for 1 h, and centrifuged at room temperature at 3,500 rpm for 10 min. The supernatant, i.e., the serum, was collected and preserved at room temperature. The animals were fasted overnight with free access to water the day before treatment. The day after blood sampling, example compounds 13, 17, 31 and etelcalcetide (AMG-416) were dissolved in a phosphate buffered saline (PBS, Gibco). The rats were intravenously administered with example compounds 13, 17, 31 or etelcalcetide 3 mg/kg or an equal volume of PBS buffer. Subsequently, blood samples were collected as per the following procedures for measuring the parameters. 100 μL of blood was collected at 1 h, 2 h and 4 h post-dose, and the plasma was separated according to the procedures above. The plasma parathyroid hormone levels were measured using the Rat Intact PTH ELISA Kit (Quidel—Immunotopics, Cat. #: 60-2500; ELISA: Enzyme-linked immunosorbent assay) according to the kit instructions. The detailed procedures are as follows: using the streptavidin-preplated reaction strips provided in the kit, 25 μL of reference standard, control or plasma samples were added to the wells. Biotinylated rat parathyroid hormone antibody and rat parathyroid hormone/HRP binding antibody were mixed at a ratio of 1:1, and 100 μL of the mixed solution was added to each well. The reaction strip was sealed with a sealing film, wrapped with an aluminum foil for storage in dark, and shaken on a horizontal shaker at room temperature for 3 h at a rotation speed of 220 rpm. The solutions in the wells were discarded. 350 μL of cleaning working solution was added to the wells for washing and then discarded; 5 washes were performed with the same procedures. Finally the wells were dried. To each well was added 150 μL of horseradish peroxidase ELISA substrate. The reaction strip was sealed with a sealing film, wrapped with an aluminum foil for storage in dark, and shaken on a horizontal shaker at room temperature for 30 min at a rotation speed of 180-220 rpm. 100 μL of ELISA terminating solution was added to each well, and the strip was shaken at 180-220 rpm for 1 min on a horizontal shaker at room temperature. The absorbance at 450 nm in each well was detected within 10 min after the addition of the ELISA terminating solution, while the absorbance at 620 nm was subtracted as background. A mixture of horseradish peroxidase ELISA substrate 150 μL and ELISA terminating solution 100 μL was used as the blank control in the absorbance detection. A standard curve according to the absorbance of the reference standard was plotted, and the actual plasma parathyroid hormone concentration was calculated according to the absorbance of other samples and the standard curve. The determination of the serum calcium concentration was conducted according to the procedures of the relevant kit.

3.4.3 Results

[0121] Test compounds 13, 17 and 31 completely reduced the plasma parathyroid hormone level in normal rats within 4 h at a dose of 3 mg/kg, and a corresponding reduction in serum calcium level was also observed, as shown in FIGS. 2 and 3.

3.5 Evaluation of In Vivo Efficacy of Some of the Compounds Disclosed Herein in a 5/6 Nephrectomized Rat Model after Continuous Administration

[0122] 3.5.1 Objective: to evaluate the efficacy of some of the compounds disclosed herein in reducing plasma parathyroid hormone level and serum calcium level after continuous administration in a 5/6 nephrectomized rat model.

3.5.2 Procedures and Data Processing:

[0123] The rats were adapted. After anesthesia, ⅔ of the left kidney was surgically resected, and after 1 week of recovery, the right kidney was resected to establish the 5/6 nephrectomized rat model. After the second resection, the animals were normally fed for 2 weeks, tested for creatinine (CREA) and plasma parathyroid hormone level, and randomized as per the parathyroid hormone level into 4 groups of 10, including normal saline group, compound 17—low dose group, compound 17—high dose group and etelcalcetide group. After randomization, the normal saline group, compound 17—low dose group, compound 17—high dose group and etelcalcetide group were respectively administered with 1 dose of normal saline, 1 mg/kg of compound 17, 2 mg/kg of compound 17 and 1 mg/kg of etelcalcetide through the tail vein daily for 28 days. During the treatment period, parameters such as animal weight, plasma parathyroid hormone level and serum calcium were detected. The first day of treatment was taken as day 1.

3.5.3 Results and Conclusions:

[0124] Compared with the normal saline group, the example compound 17 1 mg/kg and 2 mg/kg reduced the plasma parathyroid hormone level in rats in a dose dependent manner. The parathyroid hormone level was reduced to an extremely low level in various treatment groups at 6 h post-dose on days 1, 14 and 28, and the parathyroid hormone reduction was greater than 90% since day 14. During the treatment period, compound 17 1 mg/kg suppressed the plasma parathyroid hormone level at 6 h and 16 h post-dose by a slightly superior or comparable magnitude to that of etelcalcetide at the same dose (FIG. 4). Serum calcium reduction is a mechanism-related effect for drugs of the type. In this study, after administration on days 1, 14 and 28, both compound 17 and etelcalcetide induced reversible serum calcium reduction. There was no significant difference in the minimum of serum calcium on days 14 and 28 compared to day 1 in various treatment group, suggesting that the extent of serum calcium reduction induced by compound 17 and etelcalcetide did not increase with the period of treatment. The maximal reduction in serum calcium induced by compound 17 was comparable to etelcalcetide at the same dose on days 1, 14 and 28, suggesting that compound 17 has comparable serum calcium-reducing activity to etelcalcetide (FIG. 5). Notably, the persistence of compound 17 to reduce serum calcium levels at day 28 was superior and significantly different as compared to etelcalcetide at the same dose.