WO2022257940A1 - Hydrochloride salt of calcium-sensing receptor agonist compound and pharmaceutical composition and use thereof - Google Patents

Abstract

A hydrochloride salt of a calcium-sensing receptor agonist compound and a pharmaceutical composition and use thereof. Specifically, provided are a hydrochloride salt of a polypeptide calcium-sensing receptor agonist compound and a pharmaceutical composition and application thereof. The hydrochloride salt has an agonist effect on a human calcium-sensing receptor (CaSR) so as to reduce plasma parathyroid hormone and serum calcium ion level, and can be used for the treatment of metabolic diseases such as secondary hyperparathyroidism and tumor-induced hypercalcemia.

Description

Hydrochloride salt of calcium-sensing receptor agonist compound, pharmaceutical composition and application thereof technical field

The disclosure belongs to the field of medical technology, and in particular relates to a hydrochloride salt of a compound having an agonist effect on human calcium-sensing receptors (CaSR), its composition and its application, and can be used for metabolic diseases such as primary parathyroid Treatment of hyperthyroidism, secondary hyperparathyroidism and hypercalcemia and other related metabolic diseases.

Background technique

Calcium-sensing Receptor (CaSR) refers to a family A G-protein coupled receptor (G-Protein Coupled Receptor, GPCR) distributed on the surface of human parathyroid organ cells. The secretion of parathyroid hormone is highly regulated by the calcium-sensing receptors on the surface of parathyroid cells to maintain the steady-state level of minerals in the human body. The secretion level responds accordingly.

In patients with chronic kidney disease, the need to achieve steady-state levels of calcium and phosphate ions results in a continuous secretion of parathyroid hormone from the parathyroid glands. 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 and induces secondary parathyroid hormone The formation of hyperthyroidism. Studies have shown that persistent secondary hyperparathyroidism leads to the loss of calcium-sensing receptors and vitamin D receptors on the surface of parathyroid cells. These disease-induced downstream pathological effects further contribute to the dysregulation of parathyroid regulation of mineral homeostasis in the body.

Calcimimetic generally refers to compounds whose physiological function and mechanism of action are similar to calcium ions and can directly activate calcium-sensing receptors on the surface of parathyroid cells. Cinacalcet hydrochloride is an organic small-molecule calcimimetic agent developed by Amgen, which can activate the calcium-sensing receptor on the surface of the parathyroid organ, inhibit the secretion level of parathyroid hormone and achieve the treatment of secondary parathyroid glands. Hyperfunction and other related metabolic diseases. Cinacalcet hydrochloride is clinically approved for the treatment of secondary hyperparathyroidism in dialysis patients with chronic kidney disease. Patients are administered orally, and the frequency of use is once or twice a day. The maximum dose can be 90 mg each time. Cinacalcet hydrochloride has clinically demonstrated excellent efficacy in reducing plasma parathyroid hormone levels in patients with secondary hyperparathyroidism. However, significant drug-induced side effects, such as nausea, vomiting, and diarrhea associated with gastrointestinal side effects, were observed during patient use. In addition, the oral administration of cinacalcet hydrochloride is a greater burden for chronic kidney disease patients on dialysis, and cinacalcet hydrochloride has been shown to inhibit cytochrome 450 and induce drug-drug interaction between. These side effects associated with the use of cinacalcet hydrochloride have reduced patient compliance and compliance to a certain extent.

Therefore, there is an urgent clinical need for calcium-sensing receptor agonist compounds that can be administered by intravenous injection, which can reduce the secretion of parathyroid hormone by activating the calcium-sensing receptor on the surface of parathyroid gland cells, thereby achieving the treatment of secondary parathyroid Hyperthyroidism and other related metabolic diseases. Such calcium-sensing receptor agonist compounds can significantly improve the compliance and compliance of patients with chronic kidney disease.

WO2021115272 describes a series of polypeptide calcium-sensing receptor agonist compounds, wherein the compound of formula (I) has an agonist effect on human calcium-sensing receptors to reduce plasma parathyroid hormone and serum calcium ion levels, and can be used for secondary Treatment of metabolic diseases such as hyperparathyroidism and tumor-induced hypercalcemia.

Contents of the invention

The present disclosure provides a hydrochloride salt of a calcium-sensing receptor agonist, a pharmaceutical composition and application thereof. The hydrochloride salt of the compound provided by the disclosure has high solubility and high stability, not only can effectively reduce plasma parathyroid hormone, but also has low release level of histamine and few side effects.

The hydrochloride salt of the compound provided by the disclosure, the structure of the compound is shown in formula (I):

Amino acid sequences in this disclosure are represented by the standard one-letter or three-letter codes for amino acids, namely alanine (Ala, A), cysteine (Cys, C), arginine (Arg, R), D-2 - Aminobutyric acid (D-Abu). The compound of formula (I) in the present disclosure can also be represented by the following sequence: Ac-c(C)-rr-(D-Abu)-rar-NH ₂ .

In some embodiments, the number of the compound of formula (I) combined with hydrochloric acid in the hydrochloride of the compound of formula (I) can be 1-10 (can be any value between 1-10, that is, the average value), preferably The number of hydrochloric acid is 4-8, more preferably 4-5, the optional number of hydrochloric acid includes but not limited to 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 , 8, 8.5, 9, 9.5, 10.

The present disclosure provides a use of the hydrochloride of the above-mentioned compound in the preparation of a drug for reducing the level of parathyroid hormone in a subject and treating secondary hyperparathyroidism or tumor-induced hypercalcemia.

The present disclosure also provides a hydrochloride of the above-mentioned compound, which is used for reducing the level of parathyroid hormone in a subject and treating secondary hyperparathyroidism or hypercalcemia caused by tumors.

In some embodiments, the hyperparathyroidism is secondary hyperparathyroidism in a subject suffering from chronic kidney disease.

Another aspect of the present disclosure provides a method for preparing the hydrochloride of the above compound, comprising the step of mixing the compound represented by formula (I) with hydrochloric acid.

The present disclosure provides a method for preparing the hydrochloride of the above-mentioned compound, which specifically includes: (1) using a resin to sequentially couple 7 amino acids of the main chain and acetic anhydride through solid phase synthesis to obtain a main chain resin peptide; The lysate of H ₂ O/TIS/DPDS was stirred and reacted at room temperature to obtain the crude peptide of the main chain; (3) reacted with HL-Cys-OH in aqueous solution to obtain the crude peptide; (4) purified by high pressure preparation and nanofiltration, and concentrated to obtain the finished product .

The content disclosed in WO2021115272 is incorporated in this disclosure in its entirety.

Description of drawings

Fig. 1 is the hemolytic effect of the compound of formula (I) on human red blood cells in vitro, wherein *: positive control (polyethylene glycol octylphenyl ether), #: PBS buffer solution;

Fig. 2 is the effect that formula (I) compound reduces parathyroid hormone level in normal rat body;

Fig. 3 is the efficacy of compounds of formula (I) in reducing serum calcium ion levels in normal rats.

Detailed ways

The present disclosure will be explained in more detail below in conjunction with the examples. The examples of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the essence and scope of the present disclosure. The pharmaceutical excipients used in the present disclosure can be purchased through commercial channels.

1. Experimental reagents

serial number	Reagent		source
1	Rink-amide MBHA resin	Xi'an Lanxiao Technology
2	HCTU	Suzhou Haofan Technology
3	4-Methylmorpholine	TCI Chemicals
4	Acetonitrile (chromatographic grade)	Sigma-Aldrich
5	Nitrogen, Nitrogen-Dimethylformamide	Sinopharm Reagent

6	Dichloromethane	Sinopharm Reagent
7	Trifluoroacetate	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	Jill Biochemical
13	Fmoc-D-Arg(Pbf)-OH	Jill Biochemical
14	Fmoc-D-Ala-OH	Jill Biochemical
15	Fmoc-D-Abu-OH	Jill Biochemical
16	2,2-dipyridine disulfide	Jill Biochemical

2. Experimental equipment

serial number	instrument		source
1	Prelude-X Multi-channel Peptide Synthesizer	Protein Technology
2	H-CLASS Analytical Ultra High Performance Liquid Chromatography	Waters
3	Xevo LC/MS	Waters
4	Labconco Multifunctional Freeze Dryer	Thermo-Fisher Scientific
5	Prep150 Preparative High Performance Liquid Chromatography	Waters
6	Multi-channel high-speed centrifuge	Sigma

Example 1

Solid-phase peptide synthesis was performed using the Fmoc/tBu synthesis strategy on a Prelude-X automated peptide synthesizer, starting from Rink-amide MBHA resin (0.1 mmole) using 10 equivalents of HCTU and 4-methylmorpholine activated Amino acid residues (HCTU, 4-methylmorpholine and amino acid residues in a molar ratio of 1:2:1) were coupled in nitrogen, nitrogen-dimethylformamide at room temperature for 25 minutes.

After completing the synthesis of the above-mentioned peptide-resin, in a solution containing 90:5:5 (v/v/v) trifluoroacetic acid:triisopropylsilane:water and 2,2-dipyridine disulfide (1 mmole) In the solution, at room temperature, the cleavage of the polypeptide from the solid phase resin, the removal of the side chain protecting group and the activation of the D-Cys side chain sulfhydryl group were simultaneously completed within 2 hours. After the reaction, filter and wash the resin twice with trifluoroacetic acid, combine the filtrates, add a large amount of frozen methyl tert-butyl ether to precipitate solids, and remove the supernatant after centrifugation to obtain crude polypeptides, which are dried and weighed.

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

After the mixed solution obtained above was filtered through a 0.22um membrane, it was separated with a WATERS Prep150 preparative reversed-phase high-performance liquid chromatography system, and the buffer solution was A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, 90 % acetonitrile, in water). Among them, the preparation chromatographic column is X-SELECT OBD C-18 (WATERS) reversed-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 220nm, and the flow rate is 15mL/min. After collecting product-related fractions and freeze-drying, the pure polypeptide product of Example No. 1 was obtained, with a yield of 45%. The purity and compound identity of the pure peptide were determined by analytical ultra-high performance liquid chromatography and ultra-high performance liquid chromatography/mass spectrometry. The purity of the compound was 96.78%, and the molecular weight of the compound was 1109.60Da.

Compound number		sequence
1	Ac-c(C)-(D-Phg)-rrrar-NH 2

"Ac-c(C)" means that the D-configuration cysteine (c) at the amino terminal is acetylated and linked to another cysteine in the L-configuration (C) through a disulfide bond; "r-NH ₂ "means that the D-configuration arginine (r) at the carboxy-terminus is amidated.

Example 2

The compound of formula (I) is synthesized by a synthetic scheme similar to that of Example 1, and the purity and molecular weight of the synthetic polypeptide are determined by analytical ultra-high performance liquid chromatography and ultra-high performance liquid chromatography/mass spectrometry, wherein the purity of the compound of formula (I) is 95.79%, the molecular weight of the compound is: 1062.29Da.

Example 3

The preparation of formula (I) compound hydrochloride

1. Weigh Rink Amide-AM resin (1034.4g) into a glass reactor, and then add DMF to swell the resin. Add 20% PIP/DMF (V/V) solution, respectively react to remove Fmoc, then wash with DMF, ninhydrin detection is blue. Weigh Fmoc-D-Arg(Pbf)-OH (1167.6g), Oxyma (383.6g), dissolve with DMF (5.0L) and DCM (5.0L), and then add DIC (340.5g). Stir and mix well and add to the glass reactor for reaction, use ninhydrin to detect the end of the reaction (if the resin is colorless and transparent, stop the reaction, if the resin is colored, prolong the reaction time, the same below), after the reaction, use DMF and IPA in sequence , DMF, IPA, DMF washing resin. Weigh Ac ₂ O (368.1 g), DIEA (467.4 g) and add DMF (5.0 L) and DCM (5.0 L). Stir and mix well and add it to a glass reactor for reaction. Take a small amount of resin ninhydrin and detect it until the end of the reaction. After the reaction, wash the resin with DMF, IPA, DMF, IPA, and DMF in sequence.

2. According to the same coupling method, sequentially couple subsequent amino acids to obtain N-Ac-D-Cys(Trt)-D-Arg(Pbf)-D-Arg(Pbf)-D-Abu-D-Arg( Pbf)-D-Ala-D-Arg(Pbf)-Rink Amide Resin 2.5Kg.

3. Add TFA/TIS/H2O (V:V:V=97:2.5:0.5) lysate into a 50L reactor, then add DPDS (793.2g), stir and dissolve. The product in 2 (2.5Kg) was added into the reactor, the reaction was stirred at room temperature, and filtered. The filtrate was added to IPE/ACN (V:V=7:1), and 850 g of precursor polypeptide was collected with a yield of 75.4%.

4. Add 6.73L of water into the 20L reactor, add HL-Cys-OH.HCl.H ₂ O (118.4g), stir and dissolve, then add the precursor polypeptide of 3 (850g) to react. After the end, slowly rinse into 0.01mol/L HCl/IPA solution, centrifuge after stirring, and collect the filter cake. After drying, 700 g of the target polypeptide crude product was obtained, with a yield of 79%.

5. Take 700g of the crude peptide in 4, use the Hanbang purification system, wavelength 254nm, filler C18, mobile phase 0.1% TFA/H ₂ O and 0.1% TFA/acetonitrile, purify, collect the target peak fraction, and obtain 90L of purified liquid , The process yield is 64.1%.

6. Use a nanofiltration system, add hydrochloric acid solution, and remove the trifluoroacetate group in the sample solution at the same time to obtain the compound hydrochloride solution of formula (I). After concentration and lyophilization, 240 g of compound hydrochloride of formula (I) is obtained. , the yield is 90%, the mass spectrum signal is 1061.5367Da (theoretical value is 1061.5447Da), the HPLC purity is 98.33%, and the total yield is 34.4%.

According to the 0701 potentiometric titration method of the four general rules of the Chinese Pharmacopoeia 2020 edition, the number of hydrochloric acid bound by the compound of formula (I) was measured to be 4.4.

Example 4

Investigation on the Physicochemical Properties of Formula (I) Compound Hydrochloride

Investigate the physicochemical properties and solubility of the formula (I) compound hydrochloride that embodiment 1 makes, the results are shown in the following table.

Example 5

Stability investigation of formula (I) compound hydrochloride

The formula (I) compound hydrochloride that embodiment 1 makes is respectively at 40 ℃ ± 2 ℃ and RH75% ± 5%, 25 ℃ and total illumination greater than 1.2 * 106Lux hr, near ultraviolet energy is greater than 200w hr/m ^2. Put it under the conditions of 25℃ and RH60% for a period of time to investigate its stability. The results are shown in the table below.

Conclusion: Under high temperature conditions, after being placed for 30 days, the moisture and hydrochloric acid content indicators are basically unchanged, and the total amount of impurities is increased. Under high-humidity conditions, after 10 days of storage, the water content increased significantly, and the total amount of impurities also increased. After being placed under light conditions for 30 days, the contents of water and hydrochloric acid remained basically unchanged, and the total amount of impurities increased significantly.

The hydrochloride of the compound of formula (I) prepared in Example 1 was subjected to an accelerated test at 5°C±3°C, and the results are shown in the table below.

Conclusion: After 3 months of investigation under accelerated and long-term conditions, the indicators of the three batches of samples are basically unchanged. It can be seen that the compound of formula I has good stability under the condition of 5°C±3°C.

Example 6

The following further describes and explains the present disclosure in conjunction with specific embodiments in the present disclosure, but these embodiments are not meant to limit the scope of the present disclosure.

1. Experimental reagents required for in vitro and in vivo biological test evaluation

serial number	Reagent		source
1	FBS,500ml	Thermo Fisher Scientific
2	DMEM, High Glucose, GlutaMAX, 500ml	Thermo Fisher Scientific
3	Penicillin-Streptomycin, Liquid, 100ml (100X)	Thermo Fisher Scientific
4	1X PBS pH 7.2-7.4 (500ml)	Solarbio
5	1X TrypLE Express Enzyme, no phenol red (500ml)	Thermo Fisher Scientific
6	Hygromycin B Gold solution(5g,1x 50ml,100mg/ml)	Invivogen
7	HEPES,1M	Gibco
8	MgCl 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 2 ,1M	Sigma-Aldrich
14	IP-One-Gq Kit(1,000tests)	Cisbio

2. Experimental equipment

serial number	instrument		source
1	EnVision detector	Perkin Elmer

Evaluation of Agonist Activity of Compounds of Formula (I) on the Human Calcium Sensing Receptor (CaSR)

experimental method:

The HEK293/CaSR stably transfected cell line (source: Pharmaron) was cultured in complete medium (ingredients: DMEM, high glucose+10% FBS+2mM GlutaMAX+1X Penicillin-Streptomycin+200μg/ml Hygromycin B), at 37°C , and incubate to 70%-90% confluence in a 5% CO2 environment. The cell lines were digested with TrypLE and inoculated in 384-well cell culture plates, and cultured overnight at 37°C in 5% CO2. After the cells were exchanged, the stimulation buffer (HEPES 10mM, MgCl2 0.5mM, KCl 4.2mM, NaCl 146mM, glucose 5.5mM, LiCl 50mM, CaCl2 1.2mM) and different concentrations of the compounds to be tested were added at 37°C. Incubate for 60 minutes, and detect the production of IP-One in the cells according to the steps in the instructions of the Cisbio IP-One Tb kit. After collecting the raw data of each example, software is used to calculate the EC50 value of each example to be tested on human calcium-sensing receptors, and to evaluate the agonist activity of the examples against human calcium-sensing receptors.

Experimental data processing method:

Signal readout for HTRF was performed using an EnVision detector with excitation at 320 nm and emission at 620 nm and 665 nm. Calculate the signal ratio (665nm/620nm*10,000), and use the four-parameter equation to perform nonlinear fitting between the signal ratio and the sample concentration in GraphPad Prism 6, and obtain the EC50 value of Example 1 to be tested. The specific values are shown in Table 1 below .

Table 1 Calcium-sensing receptor agonist activity in vitro

Example number	sequence	Calcium Sensing Receptor EC 50 (uM)
Compound of formula (I)	Ac-c(C)-rr-(D-Abu)-rar-NH 2	6.28
Etekatide	Ac-c(C)-arrrar-NH 2	6.78
Etekatide analogs	Ac-c(C)-rrarar-NH 2	6.74

The compound of formula (I) of the present disclosure has excellent in vitro efficacy, corresponding to an EC50 value lower than 10uM in the in vitro evaluation of human calcium-sensing receptor agonist activity, which is equivalent to the activity of the positive drug etekatide.

Evaluation of in vitro histamine-releasing activity of compounds of formula (I) on rat peritoneal mast cells

Experimental methods and data processing:

In order to evaluate the in vitro histamine release level of some of the examples to be tested, rat peritoneal mast cells were isolated by lavage of rat peritoneum with lavage buffer (cold HBSS+25mM HEPES containing 5 U/mL heparin, pH 7.4). After separation, the cells were centrifuged, the lavage buffer was removed, and the stimulation buffer (HBSS+25mM HEPES+1mM CaCl2, pH 7.4) was added to resuspend the cells and washed twice. Plate at a density of 105 cells/well (200 μl/well), add positive control Compound 48/80 (final concentration 4 μg/mL), test example compound (final concentration 10 μM) or vehicle control, and incubate at 37°C for 15 minutes. Centrifuge, take the cell supernatant, and detect the histamine concentration of the supernatant according to the instructions of the LDN Histamine ELISA kit (BAE-1000). The specific values are shown in Table 2 below.

Table 2 Level of side effects of histamine release in vitro

Example number	Relative histamine release multiple in vitro
PBS buffer	1.00
Compound of formula (I)	0.97
Etekatide	1.70

Etecartide can obviously cause the release of histamine from rat peritoneal mast cells in vitro, which is specifically reflected in the fact that the relative histamine release multiple is higher than 1.50 relative to PBS buffer. Unexpectedly, the compound of formula (I) greatly reduces the release level of histamine in rat peritoneal mast cells in vitro compared with etecartide.

Evaluation of the hemolytic effect of compounds of formula (I) on human red blood cells in vitro

Experimental methods and data processing:

In order to evaluate the hemolytic effect of the compounds of Examples on red blood cells in vitro, human whole blood (100ul) was taken and mixed with phosphate buffer, centrifuged at 4°C for 10 minutes, and the supernatant was discarded. Red blood cells were resuspended in PBS buffer (900ul) and centrifuged at 4°C for 10 minutes, then the supernatant was discarded and the above steps were repeated once. The compound of formula (I) was dissolved in 1×PBS buffer to a final concentration of 100 ug/ml. The erythrocytes were resuspended by the compound solution of the example to be tested, polyethylene glycol octylphenyl ether-100 solution and PBS buffer respectively and incubated at 37° C. for 1 hour. After incubation, centrifuge at 4°C for 10 minutes and extract the supernatant (100ul), transfer to a 96-well plate and measure the absorbance at 540nm to evaluate the hemolytic effect of the compound of formula (I) on red blood cells in vitro.

3.3.3 Experimental results

Formula (I) compound does not observe obvious erythrocyte hemolysis effect under the concentration of 100ug/ml, and corresponding polyethylene glycol octyl phenyl ether-100 solution observes obvious erythrocyte hemolysis effect under experimental conditions, see Attached Figure 1.

Evaluation of the in vivo efficacy of a compound of formula (I) after a single administration on a normal rat model

Experimental methods and data processing:

Normal adult rats (Sprague Dawley, SD) of SPF grade were used in the experiment, weighing 250-350 grams, and were restored to normal diet for 7 days in the animal room. Rats were divided into random groups, 6 in each group, half male and half male, and numbered respectively. One day before the start of the experiment, 540 μL of blood was collected from each rat, and the plasma parathyroid hormone level and serum calcium ion concentration were measured as the control values before administration. The plasma separation method is anticoagulated with K2-EDTA, blood is collected through the jugular vein, put on ice after collection, and then the whole blood is centrifuged at 6,800 rpm for 6 minutes at 2-8°C, and the upper layer is gently taken out to be the plasma, 2 Store at ~8°C. The serum separation method is to collect blood through the jugular vein, let the whole blood stand at room temperature for 1 hour, then centrifuge at room temperature at 3,500rpm for 10 minutes, gently take out the upper layer, which is the serum, and store it at room temperature. The day before the experiment, the animals were fasted overnight and had free access to water. The next day after blood collection, the compound of formula (I) and etekatide were dissolved in phosphate buffered saline (Phosphate buffered saline, PBS, Gibco), and each rat was given intravenous injection of compound of formula (I) and etekatide 3mg/ kg or an equal volume of PBS buffer solution, and then draw blood to determine the corresponding indicators according to the following method. 1 hour, 2 hours, and 4 hours after administration, 100 μL of blood was collected respectively, and the plasma was separated according to the above method. Rat Intact PTH ELISA Kit (Quidel-Immunotopics, Cat.#:60-2500) was used to detect plasma parathyroid glands according to the kit instructions. Determination of hormone levels (ELISA: Enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay). The detailed steps are: use the streptavidin pre-coated reaction strip provided by the kit, and add 25 μL of standard substance, control substance or plasma sample to each well. Mix biotinylated rat parathyroid hormone antibody and rat parathyroid hormone/HRP-conjugated antibody 1:1, and add 100 μL of this mixed solution to each well. Seal the reaction strips with a sealing film, wrap the reaction strips with aluminum foil for storage in the dark, and oscillate on a horizontal shaker at 220 rpm for 3 h at room temperature. Remove the solution in the wells, wash each well with 350 μL of cleaning working solution, and then remove the solution in the wells; wash in the same way for a total of 5 times, and finally aspirate the solution in each well. Add 150 µL of horseradish peroxidase ELISA substrate to each well. Seal the reaction strips with a sealing film, wrap the reaction strips with aluminum foil for storage in the dark, and oscillate on a horizontal shaker at a speed of 180-220 rpm for 30 min at room temperature. Add 100 μL of ELISA stop solution to each well, and shake on a horizontal shaker at 180-220 rpm for 1 minute at room temperature. Within 10 min after adding the ELISA stop solution, the absorbance of each well was read at 450 nm, and the absorbance at 620 nm was used as the background subtraction. 150 μL of horseradish peroxidase ELISA substrate plus 100 μL of ELISA stop solution was used as a blank control for absorbance measurement. Draw a standard curve according to the absorbance of the standard, and then combine the absorbance of other samples with the standard curve to calculate the actual concentration of plasma parathyroid hormone. The determination of serum calcium ion concentration is carried out according to the steps of the relevant kit.

Experimental results

The compound of formula (I) completely reduces the plasma parathyroid hormone level of normal rats within 4 hours at a dose of 3 mg/kg, and the serum calcium ion level is also correspondingly reduced, see accompanying drawings 2 and 3.

Claims (6)

1. A kind of hydrochloride of compound, the structure of described compound is shown in formula (I):

   
2. A pharmaceutical composition comprising the hydrochloride salt of the compound as claimed in claim 1.
3. Use of the hydrochloride salt of the compound as claimed in claim 1 or the pharmaceutical composition as claimed in claim 2 in the preparation of medicines for treating diseases related to abnormal parathyroid hormone levels.
4. The use according to claim 3, characterized in that the disease related to the abnormal level of parathyroid hormone is hyperparathyroidism.
5. The use according to claim 4, wherein said hyperparathyroidism is secondary hyperparathyroidism of a subject suffering from chronic kidney disease.
6. A method for preparing compound hydrochloride as claimed in claim 1, comprising the step of mixing the compound shown in formula (I) with hydrochloric acid.

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