Application of hERG channel inhibitor in preparation of medicine for prolonging heart QT interval

Abstract

The invention relates to the technical field of biological medicines, in particular to application of an hERG potassium channel inhibitor in preparation of a medicine for prolonging a heart QT interval. The compound 1, 3-(4-chlorphenyl)-N-{3-[(1-1-phenyl-1H-tetrazole-5-yl) thio]-propyl}-2-propenyl-1-amine or the pharmaceutically acceptable salt of the compound 1, 3-(4-chlorphenyl)-N-{3-[(1-1-phenyl-1H-tetrazole-5-yl) thio]-propyl}-2-propenyl-1-amine can target an hERG potassium ion channel; in a rabbit heart rate monitoring experiment, compared with a solvent control, the intraperitoneal injection of 10 mg / kg of 1, 3-(4-chlorphenyl)-N-{3-[(1-1-phenyl-1H-tetrazole-5-yl) thio]-propyl}-2-allyl-1-amine can obviously prolong the QT interval and reduce the heart rate, and has the application potential of regulating the heart function.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of a hERG potassium channel inhibitor in the preparation of a drug for prolonging the QT interval of the heart. BACKGROUND

[0002] The hERG (The human Ether-à-go-go-Related Gene) potassium channel is encoded by the KCNH2 gene, which is expressed in multiple tissues in the human body and plays different roles. The potassium channel encoded by the hERG gene mediates rapid activation of delayed rectifier potassium current (Ikr), which is closely related to QT interval prolongation and Torsades de Pointes (TdP), and is one of the key targets of many drugs that cause adverse reactions of the heart. The hERG potassium channel is a tetramer structure composed of four identical subunits, and potassium ions can selectively pass through the central pore region. IKr is one of the main K+ currents involved in the repolarization of the action potential of the human heart. Various genetic or drug-induced long QT syndrome (LQTS) in humans is related to the functional and structural changes of the IKr conduction channel. LQTS is a type of arrhythmia, and its clinical manifestations include palpitations, syncope, convulsions, and even sudden death. Many compounds can affect the function of the hERG channel and cause LQTS. These compounds can be divided into two categories according to the mechanism of action: blockers that block the function of the hERG channel on the cell membrane and inhibitors that inhibit the intracellular transport of hERG. Since the late 1990s, several clinical drugs have been withdrawn from the market due to their effects on the hERG channel. Currently, new drug development requires hERG pharmacological testing to assess the side effects of the compound on the heart. In addition, some refractory arrhythmias (such as short QT syndrome and specific ventricular tachycardia) require prolongation of the ventricular repolarization to increase the refractory period, but existing class III antiarrhythmic drugs (such as sotalol) have non-selective ion channel effects, which pose a risk of hypotension, bradycardia, or TdP. Currently, there is a lack of hERG channel-targeted drugs that can precisely regulate the extent of QT interval prolongation and have controllable safety. Traditional models (such as dogs and guinea pigs) are costly or have significant differences in cardiac electrophysiological characteristics compared to humans. Rodents have a rapid heart rate, making it difficult to accurately measure the QT interval. Existing hERG inhibitors (such as dofetilide) have unstable effects in some animal models, a narrow therapeutic window, and a lack of standardized dose-effect relationship. Rabbits have a moderate heart rate (200-300 bpm) and their cardiac ion channel distribution is more similar to that of humans, making them suitable for simulating QT interval prolongation.

[0003] By screening inhibitors targeting the hERG channel on a cell model stably expressing the hERG channel, and based on the rabbit, a high-efficiency heart rate monitoring model is established for the treatment of short QT syndrome or the establishment of a model simulating long QT syndrome in humans, the obtained hERG potassium channel inhibitor can precisely regulate the extent of QT interval prolongation. SUMMARY

[0004] The present application aims to provide a compound for prolonging QT interval and its application. The compound in the present application has obvious inhibitory effect on hERG potassium ion channel, has obvious effect of prolonging QT interval, and can be used for preparing a drug for simulating a human long QT syndrome model and treating short QT syndrome.

[0005] The present application is realized by the following technical scheme.

[0006] In the first aspect, the present application provides a use of a hERG potassium ion channel inhibitor in the preparation of a drug for prolonging QT interval of heart, wherein the inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0007]

[0008] The compound of the above formula has the effect of prolonging QT interval of heart, and the name of the compound is 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine, the molecular formula is C 19 H 20 Cl N5 S, the molecular weight is 385.92 g / mol, the CAS number is 892603-52-0, and the compound belongs to a heterocyclic compound. The compound targets hERG potassium ion channel.

[0009] Preferably, the active ingredient of the drug is administered at a dose of 50 ng / kg to 50 mg / kg.

[0010] Further, the drug is administered orally or by injection.

[0011] Preferably, the compound also includes a drug for simulating a human long QT syndrome model and a use for preparing a drug for treating short QT syndrome.

[0012] In the second aspect, the present application provides a pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof according to the first aspect, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0013] Preferably, the pharmaceutically acceptable carriers, diluents or excipients include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol.

[0014] The present application has the following beneficial effects:

[0015] The application provides application of hERG potassium ion channel inhibitor in prolonging QT interval, and compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine is an inhibitor targeting hERG channel and has obvious inhibiting effect on hERG potassium ion channel. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure shows inhibiting effect of model compound E-4031 on hERG channel.

[0017] Figure 2 The figure shows inhibiting effect of compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on hERG channel.

[0018] Figure 3 The figure shows effect of compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on QT interval of rabbit.

[0019] Figure 4 The figure shows effect of compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on HR of rabbit.

[0020] Figure 5 The figure shows effect of compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on RR interval of rabbit. DETAILED DESCRIPTION

[0021] The specific embodiment of the application is described in detail below in combination with the drawings. It should be understood that the specific embodiment described herein is only used for illustrating and explaining the application, and is not used for limiting the application.

[0022] The compound of the present application is 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H- tetrazol-5-yl)thio]propyl}-2-propenyl-1-amine, which can be prepared according to the common knowledge in the art or obtained commercially without special limitation.

[0023] The present application will be described in detail below by way of examples.

[0024] In the following examples, the various reagents and materials used are commercially available unless otherwise specified.

[0025] In the following examples, the effect of the compound on the hERG channel is detected by FLIPR-TERA instrument (Molecular Devices Corporation), which is a homogeneous, dynamic, cell level fluorescence detection method using thallium ion (Tl+) as a fluorescent sensitive substitute for potassium ion (K + ) to indirectly reflect the function of hERG potassium channel by detecting the inflow of thallium ion.

[0026] In the following examples, rabbits are used as a heart rate detection model. The heart rate of rabbits is moderate (200-300 bpm), and the distribution of cardiac ion channels is close to that of humans, which is suitable for simulating QT interval prolongation.

[0027] In the following examples, the experimental rabbits used are purchased from Beijing Jinmuyang Experimental Animal Breeding Company, male, body weight 3.5-4.0 kg.

[0028] Example 1

[0029] Effect of model compound E-4031 on hERG potassium channel

[0030] Stable hERG-transfected CHO cells (hERG-CHO) are inoculated into 384-well black-bottom transwell culture plates at 20,000 cells / well, and 20 μl of culture medium is added to each well, which is incubated at 37°C, 5% CO2 for 24 h. When the confluence of cells in the well reaches 90%, the culture medium in the well is removed, and 20 μl of FLIPR Potassium assay dye is added to each well, which is incubated at 37°C in the dark for 1 h.

[0031] E-4031 is a reported specific inhibitor of hERG channel, which was used as a positive control in this example to evaluate the pharmacological function of hERG-CHO cells. Freshly prepared gradient dilution of E-4031 was added to the compound well plate (maximum concentration 20 μM, gradient dilution by 2.5 fold, 8 concentrations in total, 5 replicates for each concentration). Different concentrations of E-4031 were added to the 384-well cell plate using FLIPR automatic sample adding system, and incubated for 20 min, then 2 mM TI + and 20 mM K + As a stimulator, fluorescence reading was performed according to the parameter setting, excitation wavelength 470-495 nm, emission wavelength 515-575 nm, exposure time 0.05 s, data collection once per second, 200 s in total, and different concentrations of E-4031 were detected by real-time sample adding and recording to determine the change of fluorescence signal over time.

[0032] According to Figure 1 , under the condition of 20 mM K + activating hERG channel, the change of fluorescence signal caused by inhibition of hERG channel by different concentrations of E-4031 showed an "inverted S" type increase-decrease curve, and there was an obvious dose-effect relationship. The IC 50 value of the inhibitory effect of E-4031 on hERG channel was 0.32 μM. The above results showed that the hERG-CHO cell model used in the present application could well reflect the effect of compounds on hERG channel, and was suitable for in vitro evaluation of the effect of compounds on hERG channel.

[0033] Example 2

[0034] Effect of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on hERG potassium ion channel

[0035] hERG-CHO in Example 1 was inoculated into a 384-well black-bottom transwell culture plate at 20000 cells / well, 20 μl of culture medium was added to each well, and the cells were incubated at 37°C, 5% CO2 for 24 h. When the confluence of cells in the wells reached 90%, the culture medium in the wells was removed, 20 μl of FLIPR calcium assay dye was added to each well, and the cells were incubated at 37°C in the dark for 1 h.

[0036] Freshly prepared 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]- propyl}-2-propenyl-1-amine was added to the compound well plate (maximum concentration 10 μM, gradient dilution according to 2-fold proportion, a total of 10 concentrations, 5 repeats for each concentration); different concentrations of 1,3-(4-chlorophenyl)-N-{3-[(1-1- phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine were added to the 384-well cell plate using the FLIPR automatic sample adding system and incubated for 20 min, then 2 mM TI + and 20 mM K + As the stimulant, fluorescence reading was performed according to the parameter settings, excitation wavelength 470-495 nm, emission wavelength 515-575 nm, exposure time 0.05 s, data collection once per second, lasting 120 s, and different concentrations of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine were detected in real time to determine the change in fluorescence signal over time.

[0037] According to Figure 1 , 20 mM K + In the case of activating the hERG channel, different concentrations of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine inhibited the change in fluorescence signal caused by the hERG channel to exhibit an "inverted S" type increase-decrease curve, and there was a clear dose-effect relationship. The IC 50 value of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2- propenyl-1-amine on the inhibitory effect of the hERG channel was 0.068 μM, which was less than that of the positive compound E-4031

[0038] The above results show that 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]- propyl}-2-propenyl-1-amine is an inhibitor of the hERG channel, and its inhibitory activity on the hERG channel is better than that of the model compound E-4031.

[0039] Example 3

[0040] Effect of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2- propenyl-1-amine on the QT interval, PR interval and HR interval of rabbits

[0041] To further determine the drug effect, a validation was performed using rabbits, and a physiological signal telemetry system was used to evaluate the effect of compound 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine on the function of the hERG channel of the rabbit heart, and the QT interval, PR interval, and HR interval were detected by electrocardiogram (ECG), and the results are shown in Table 1. Figures 3 to 5

[0042] The rabbits were fixed on a board in a supine position, and the chest and abdomen of the animals were shaved. After shaving, the small fluff was removed using depilatory cream, and the animals were wiped clean with alcohol cotton. Four kinds of color connectors, black, white, red, and green, were installed on the electrode patches, and the patches were attached to the rabbit body surface one by one. The emka PACK4G animal physiological signal acquisition system was started, and the normal ECG waveform was recorded (stabilized for 30 minutes), and the data were saved as a baseline control. After stabilizing the ECG waveform for 30 minutes, the rabbits were given a subcutaneous injection of 10 mg / kg of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine, and the ECG changes were immediately recorded for 60 minutes, and the average value of the data every 5 minutes was plotted and statistically analyzed. The control group was injected with the same amount of normal saline, and the same operation was performed.

[0043] The effect of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine compound on the QT interval of rabbits: by repeated measurement two-way ANOVA and Bonferroni test, the effect of AN-465 drug on the QT interval of rabbits was significant (F 药物 (1,6) = 14.54, P = 0.008, F 时间 (3.69, 22.13) = 12.34, P < 0.001, F 交互作用 (17, 102) = 20.31, P < 0.001), and the QT interval change was related to the administration time, and the drug effect was time-dependent. Compared with the baseline value, the QT interval of rabbits was significantly prolonged after administration of 10 mg / kg of AN-465 for 15 minutes, and the drug effect lasted for more than 60 minutes (##p < 0.01, ###p < 0.001). Compared with the normal saline control group, the QT interval of rabbits given 10 mg / kg of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine was significantly prolonged compared with the normal saline control group (*p < 0.05, **p < 0.01, ***p < 0.001) at the same time period after administration for 10 minutes.

[0044] ​1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2- propenyl-1-amine compound effects on HR in rabbits: AN-465 drug effects on HR in rabbits were significant (F 药物 (1,6) = 26.19, P = 0.0022, F 时间 (2.20, 13.22) = 5.36, P = 0.018, F 交互作用 (17, 102) = 4.185, P < 0.001) and HR changes were related to time of drug administration, with a time-dependent drug effect. The 10 mg / kg dose of 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine significantly decreased HR in rabbits 40 minutes after drug administration compared to baseline values (#p < 0.05). HR in rabbits given 10 mg / kg AN-465 was significantly lower than the solvent control group 5 minutes after drug administration compared to the saline control group.

[0045] 1,3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2- propenyl-1-amine compound effects on RR interval in rabbits: AN-465 drug effects on RR in rabbits were significant (F 药物 (1,6) = 29.26, P = 0.0016, F 时间 (2.69, 16.14) = 5.72, P = 0.0086, F 交互作用 (17, 102) = 7.00, P < 0.001) and RR interval changes were related to time of drug administration, with a time-dependent drug effect. The 10 mg / kg dose of AN-465 significantly prolonged RR interval in rabbits 25, 35, and 55 minutes after drug administration compared to baseline values (#p < 0.05). RR interval in rabbits given 10 mg / kg AN-465 was significantly prolonged compared to the saline control group from 5 minutes after drug administration compared to RR in the same time period (*p < 0.05, **p < 0.01, ***p < 0.001).

[0046] In summary, compound 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine prolonged the QT interval, decreased the HR, and prolonged the RR interval in rabbits. The RR interval is the inverse of the HR, and a decrease in heart rate (HR↓) is associated with an increase in the RR interval (RR↑), and the results of this study are consistent with this theory. In summary, 1, 3-(4-chlorophenyl)-N-{3-[(1-1-phenyl-1H-tetrazol-5-yl)thio]-propyl}-2-propenyl-1-amine can act on the heart, change the heart rate and the QT interval, and has the potential to regulate the function of the heart.

Claims

1. Use of a compound that inhibits hERG potassium channels in the preparation of a drug for prolonging cardiac QT interval, characterized in that: The inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

2. Use of the compound for inhibiting hERG potassium channels according to claim 1 in the preparation of a drug for prolonging cardiac QT interval, characterized in that: The active ingredient is administered in a dose ranging from 50 ng / kg to 50 mg / kg.

3. Use of the compound for inhibiting hERG potassium channels according to claim 2 in the preparation of a drug for prolonging cardiac QT interval, characterized in that: The administration method is oral administration or injection.

4. Use of the compound for inhibiting hERG potassium channels according to claim 1 in the preparation of a drug for prolonging cardiac QT interval, characterized in that: The compound can also be used to prepare drugs for simulating the human long QT syndrome model and drugs for treating short QT syndrome.

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable carriers, diluents or excipients include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.

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