Selective kcnq4 channel openers and their use in medicine

Abstract

The application discloses a group of KCNQ4 potassium ion channel selective regulator compounds, which have high activity and high selectivity on KCNQ4 channels, and have no or low open activity on KCNQ1, KCNQ2, KCNQ3 and KCNQ5. Since the compounds provided by the application are high-activity and high-selectivity KCNQ4 channel openers, the application also provides application of the compounds in preparation of KCNQ4 potassium ion channel openers and drugs for treating hearing impairment.

Description

Technical Field

[0001] This invention relates to KCNQ potassium ion channel openers, and more specifically to KCNQ4 potassium ion channel selective modulators and their use in pharmaceutical formulations. Background Technology

[0002] KCNQ potassium channels are a class of voltage-gated potassium ion channels, with genes encoding five subtypes (KCNQ1–KCNQ5), widely distributed in tissues such as the heart, neurons, and smooth muscle. Different subtypes of KCNQ differ in tissue distribution, physiological function, and pathological function. KCNQ1 is mainly distributed in the myocardium; KCNQ2 / 3 is mainly distributed in the nervous system and forms the molecular basis for M-type potassium ion channels in nerve cells; mutations in these genes can lead to a series of neurological diseases, such as benign familial neonatal epilepsy; KCNQ4 and KCNQ5 are highly expressed in visceral tissues and are considered drug targets for diseases such as visceral pain. KCNQ4 is also distributed in inner ear hair cells; mutations in this gene can lead to hereditary deafness.

[0003] The KCNQ4 channel plays a crucial role in maintaining ion homeostasis and regulating hair cell membrane potential. KCNQ4 encodes a gene associated with the cochlea. Reduced KCNQ4 channel activity due to gene mutations is a cause of non-syndromic hearing loss, a typical late-onset, initial high-frequency hearing loss that develops over time. Most KCNQ4 mutations associated with hearing loss cluster around the porous regions of the protein and lead to KCNQ4-mediated loss of potassium current. Furthermore, KCNQ4 mutants are also associated with noise-induced hearing loss and age-related hearing loss. The link between the KCNQ4 channel and hearing loss has been confirmed by research, making the KCNQ4 channel a well-defined drug target for the treatment of hearing impairment.

[0004] Due to their well-defined targets, KCNQ4 channel openers have been extensively studied in recent years as innovative drugs for treating hearing loss. Numerous highly active KCNQ4 channel openers have been reported. However, the vast majority of these compounds exhibit low selectivity for KCNQ channel subtypes, which severely hinders their application in pharmaceutical formulations. In summary, KCNQ channel selectivity is crucial in drug development and treatment, affecting not only the therapeutic target but also drug safety. Currently, highly selective and highly active KCNQ channel modulators, especially selective modulators of KCNQ4 channels, are lacking. Therefore, the discovery of highly selective and highly active KCNQ4 channel openers is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a KCNQ4 channel opener with higher activity and better selectivity, and to provide the application of the KCNQ4 channel opener in the preparation of pharmaceutical formulations.

[0006] The objective of this invention is achieved in the following ways:

[0007] The selective KCNQ channel opener provided by this invention has the chemical structure shown in formula (A) or formula (B).

[0008]

[0009] The compound shown in formula (A) (hereinafter referred to as compound A) has the chemical name -[3-[2-[2-[3-[(2,4-dihydroxy-3,3-dimethylbutanoyl)amino]propanoylamino]ethyldisulfo]ethylamino]-3-oxopropyl]-2,4-dihydroxy-3,3-dimethylbutanamide (N-[3-[2-[2-[3-[(2,4-dihydroxy-3,3-dimethylbutanoyl)amino]propanoylamino]ethyldisulfanyl]ethylamino]-3-oxopropyl]-2,4-dihydroxy-3,3-dimethylbutanamide), and the CAS registry number is 16816-67-4. Compound A is commercially available.

[0010]

[0011] The compound shown in Formula B (hereinafter referred to as Compound B) has the chemical name N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N'-(5-aminopentyl)-N'-hydroxybutanediamide (N-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxobutanoyl]-hydroxyamino]pentyl]-N'-(5-aminopentyl)-N'-hydroxybutanediamide), and CAS Registry Number: 70-51-9. Compound B is commercially available.

[0012] The beneficial effects of this invention are that compounds A and B provided exhibit high activity in opening KCNQ4 channels, and compound A has high selectivity, while it has no opening activity or only low activity for KCNQ1, KCNQ2, KCNQ3, and KCNQ5. Therefore, it can effectively avoid the toxic side effects of existing KCNQ4 channel openers in pharmaceutical formulation applications.

[0013] Given that compounds A and B provided by this invention are highly active and selective KCNQ4 channel openers, this invention also proposes their applications in pharmaceutical formulations as follows:

[0014] The use of compound A or its pharmaceutically acceptable salt or solvate in the preparation of KCNQ4 potassium ion channel openers.

[0015] The use of compound B or its pharmaceutically acceptable salts or solvates in the preparation of KCNQ4 potassium ion channel openers.

[0016] Use of compound A or its pharmaceutically acceptable salts or solvates in the preparation of pharmaceutical formulations for the treatment of hearing impairment.

[0017] Use of compound B or its pharmaceutically acceptable salts or solvates in the preparation of pharmaceutical formulations for the treatment of hearing impairment.

[0018] Compounds A, B and their salts can exist in non-solventized forms or in forms that are solvated with pharmaceutically acceptable solvents such as water and ethanol.

[0019] The pharmaceutically acceptable salts described in this invention may include, but are not limited to: acid addition salts, such as inorganic acid salts (hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, nitrate, etc.), organic acid salts (formate, acetate, propionate, trifluoroacetate, lactate, tartrate, oxalate, malonate, succinate, fumarate, malate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, hydroxyethanesulfonate, glucuronate, glucuronide, aspartate, glutamate, etc.). Attached Figure Description

[0020] Figure 1 It is a KCNQ4 high-throughput filter based on FLIPR.

[0021] Figure 2 It is a selective evaluation of KCNQ2 / 3 based on ICR8000.

[0022] Figure 3 The figure shows the selectivity of compound A for different subtypes of Kv7 channels. Figure (A) shows the opening activity of compound A for Kv7.1 channel (i.e., KCNQ1), Figure (B) shows the opening activity of compound A for Kv7.2 channel (i.e., KCNQ2), and Figure (C) shows the opening activity of compound A for Kv7.5 channel (i.e., KCNQ5).

[0023] Figure 4The figure shows the selectivity of compound B for different subtypes of the Kv7 channel. Figure (A) shows the opening activity of compound B for the Kv7.1 channel (i.e., KCNQ1), Figure (B) shows the opening activity of compound B for the Kv7.2 channel (i.e., KCNQ2), and Figure (C) shows the opening activity of compound B for the Kv7.5 channel (i.e., KCNQ5).

[0024] Figure 5 The figure shows the opening activity of compound A for the Kv7.4 channel. (A) shows the I / I0 values ​​corresponding to different concentrations of compound A, (B) shows the change of Kv7.4 channel current with the concentration of compound A, and (C) shows the dose-response curve of high-throughput screening for the opening activity of compound A for the Kv7.4 channel.

[0025] Figure 6 The effect of compound A on the voltage dependence of the Kv7.4 channel is shown in the figure. (A) is the classical current curve of 3 μM compound A and 10 μM RTG. (B) is the voltage activation curve of 3 μM compound A and 10 μM RTG at clamping voltage of -120 mV, step voltage of 10 mV, transition to +90 mV, and repolarization to -120 mV. (C) is the classical current curve after drug administration at -60 mV, depolarization to 0 mV, and repolarization to -60 mV.

[0026] Figure 7 The figure shows the opening activity of compound B for the Kv7.4 channel. (A) shows the I / I0 values ​​corresponding to different concentrations of compound B, (B) shows the change of Kv7.4 channel current with the concentration of compound B, and (C) shows the dose-response curve of high-throughput screening for the opening activity of compound B for the Kv7.4 channel.

[0027] Figure 8 The effect of compound B on the voltage dependence of the Kv7.4 channel is shown in the figure. (A) is the classical current curve of 3 μM compound B and 10 μM RTG. (B) is the voltage activation curve of 3 μM compound B and 10 μM RTG at clamping voltage of -120 mV, step voltage of 10 mV, transition to +90 mV, and repolarization to -120 mV. (C) is the classical current curve after drug administration at -60 mV, depolarization to 0 mV, and repolarization to -60 mV.

[0028] Figure 9 The distribution of compounds A and B in mice was detected by HPLC-MS. In the figure, (A) shows blood and brain samples taken from mice after intraperitoneal injection of compound A, and the distribution of compound A in the body shows a bimodal distribution. (B) shows blood and brain samples taken from mice after intraperitoneal injection of compound B, and compound B can enter the blood-brain barrier.

[0029] Figure 10The figure shows the activity of coenzyme A on the KCNQ4 channel. In the figure, (A) is the opening activity of 10 μM coenzyme A on Kv7.4, and (B) is the current diagram of 10 μM coenzyme A depolarizing to 0 mV and repolarizing to -60 mV after depolarization at a voltage of -60 mV.

[0030] Figure 11 Typical diagrams showing the protective effect of compound B against hearing loss in mice. In the diagram, (A) represents the ABR hearing thresholds of wild-type, model, and drug-treated mice at different frequency bands (***P<0.001), (B) represents the hearing thresholds of different groups under click stimulation (***P<0.001), and (C) represents the representative ABR waveforms of wild-type, model, and drug-treated mice.

[0031] In the above figures, F08-10 represents compound A, and F03-09 represents compound B. Detailed Implementation

[0032] The present invention will be further illustrated below through specific embodiments.

[0033] Materials and methods

[0034] 1. Materials

[0035] 1.1 Main drugs and cells

[0036] Retigabine (RTG) was purchased from Shanghai Titan Chemical Co., Ltd.

[0037] Compound QO-83 was synthesized in our laboratory. The synthesis method can be found in the master's thesis of Hebei Medical University, "Synthesis, Screening and Preliminary Establishment of Quality Standards for Novel KCNQ Potassium Channel Openers", Cui Fuchun, 2018 (Synthesis of Compound 2).

[0038] Compounds A and B are derived from commercial compound libraries and can also be purchased from Taoshu Biotechnology Co., Ltd.

[0039] The human embryonic kidney cells (HEK293 cells) with KCNQ4 potassium channels were obtained from our department's stable cell line bank.

[0040] Chinese hamster ovary cells (CHO cells) were purchased from Peking Union Medical College Cell Resource Center.

[0041] All plasmids were provided by our laboratory's plasmid library.

[0042] 1.2 Instruments and Software

[0043] FLIPR real-time fluorescence monitoring system, manufactured by Molecular Devices, USA.

[0044] ICR8000 Ion Channel Reader, WuXi AppTec (Foshan) Biotechnology Co., Ltd.;

[0045] Multiclamp 700B patch clamp amplifier, manufactured by Axon Corporation, USA;

[0046] Liquid chromatograph manufactured by Shimadzu Corporation, Japan.

[0047] Example 1: Effects of compounds A and B on KCNQ4 channels

[0048] Screening of KCNQ4 channel openers based on FLIPR high-throughput screening:

[0049] FLIPR was used to screen KCNQ4 stable cell lines. Parameters were set as follows: Excitation LED: 470-495nm; Emission Flilter: 515-575nm; Camera Gate: 40%; Camera Gain: 80%; Exposure: 0.3s; LED Intensity: 60%; Addition Volume: 50μl; Addition Height: 70μl; Addition Speed: 100μl / s; Tip-up Speed: 20mm / s. Each injection was 100μL at concentrations of 1μM and 10μM. QO-83 was used as a positive control at concentrations of 1μM and 10μM. Three replicates were set for each concentration, and three blank controls were included. Compounds A and B, initially screened, showed KCNQ4 opening activity. See details... Figure 1 .

[0050] This shows that compounds A and B have an opening effect on the KCNQ4 channel.

[0051] Example 2: Effects of compounds A and B on KCNQ2 and KCNQ3 channels

[0052] KCNQ4 channel opener screening based on ICR8000 screening:

[0053] ICR8000 was used to screen CHO stable cell lines in the KCNQ4 channel with the following parameters: Average Current: 8 mA; Entrance Slit: 0.2 nm; Peak WL; Auto gain; Integration: 10 s; Delay: 0 s; Autozero every; 100 μl injection per sample; Standard curve concentrations were set to 0 ppm, 1 ppm, 2 ppm, 4 ppm, and 5 ppm, with a correlation coefficient (R value) greater than 0.998. The compound was diluted to the highest concentration to be detected using Open Buffer, and then serially diluted to seven concentrations in a 96-well plate, with one blank control and one positive control, and three replicates for each concentration. Figure 2 The statistical results show that compounds A and B have no significant opening effect on KCNQ2 / 3 at concentrations of 1 μM and 10 μM, but exhibit good selectivity for KCNQ4.

[0054] Example 3: Selectivity of compounds A and B for different KCNQ channel isoforms

[0055] Compounds A and B were used to verify their activity against other KCNQ channels on KCNQ1, KCNQ2, and KCNQ5. KCNQ1, KCNQ2, and KCNQ5 channels were co-expressed in CHO cells at a concentration of 3 μM, and all were evaluated using whole-cell patch-clamp techniques.

[0056] Figure 3 The figure shows the selectivity of compound A (F08-10) for different Kv7 channel isoforms. In the figure, (A) shows that 3 μM of compound A has no significant opening effect on Kv7.1 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ1 channel; (B) shows that 3 μM of compound A has no significant opening effect on Kv7.2 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ2 channel; (C) shows that 3 μM of compound A has no significant opening effect on Kv7.5 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ5 channel.

[0057] Figure 4 The results show the selectivity of compound B (F03-09) for different Kv7 channel isoforms. (A) shows that 3 μM of compound B has a certain opening effect on Kv7.1 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ1 channel. (B) shows that 3 μM of compound B has a certain opening effect on Kv7.2 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ2 channel. (C) shows that 3 μM of compound B has a certain opening effect on Kv7.5 channel (n=5-7), and the cells are CHO cells derived from transiently transfected KNCQ5 channel.

[0058] from Figure 3 Figure 4 The statistical results show that compound A has no opening activity for KCNQ1, KCNQ2, and KCNQ5 channels, while compound B has some opening activity for all three channels. Therefore, compound A exhibits better selectivity for the KCNQ4 channel.

[0059] Example 4: Compounds A and B exhibit KCNQ4 channel opening activity in a concentration-dependent manner.

[0060] Electrophysiological patch-clamp validation: Patch-clamp technique is the gold standard for ion channel research. The current of KCNQ4 channels stably translocated on CHO cells was recorded by whole-cell patch-clamp. Compounds A and B were administered continuously at 3 μM, followed by 10 μM of the positive control drug RTG, to verify their activity.

[0061] The specific method is as follows:

[0062] (1) Cell culture: Spread the cell suspension on a round coverslip and incubate overnight in a small dish.

[0063] (2) Prepare extracellular solution (mM): NaCl 160, KCl 2.5, HEPES 10, Glucose 8, MgCl2 1, CaCl2 5, adjust pH to 7.4 with NaOH, store at 4℃; prepare intracellular solution (mM): KCl 160, HEPES 5, MgCl2 3, CaCl2 1, adjust pH to 7.4 with KOH, dispense into portions, store at 4℃.

[0064] (3) Whole-cell aspiration patch clamps were used. The patch clamp amplifier was AXON Multiclamp 700B. The electrodes were drawn by a horizontal electrode drawing instrument through a multi-step process. The measured liquid resistance was 2-4 MΩ. The digital-to-analog converter was Digidata 1440A. The micromanipulator was MP-225. The filter was set to 2 kHz, the sampling frequency was 5 kHz, and the series resistance compensation was 60%-80%. After the microelectrode formed a giant resistance seal with the cell membrane, the current was recorded under voltage clamp.

[0065] (4) Data processing: The raw data was exported and processed using Clampfit 10.7, and then statistical analysis was performed using Origin 8.0. The dose-response curve was fitted using the logistic formula: y=A2+(A1-A2) / [1+(x / x0)] p ].

[0066] See results Figures 5 to 8 Compounds A and B exhibit KCNQ4 channel opening activity in a concentration- and voltage-dependent manner.

[0067] The cells were derived from CHO cells transiently transfected with the KNCQ4 channel.

[0068] Figure 5(A) shows the continuous administration of compound A at concentrations of 0.01, 0.1, 0.3, 1, and 3 μM. The corresponding I / I0 values ​​for each concentration were 1.13±0.05, 1.38±1.14, 1.63±2.02, 1.88±2.14, and 2.13±2.27, respectively, which were statistically different from the normal control current (**P<0.01, ***P<0.001); (B) shows the increase in Kv7.4 channel current with increasing compound A concentration; (C) shows the maximum effect (Emax) of the outward current induced by compound A, fitted to a logistic function, EC50. 50 =0.30μM.

[0069] Figure 6 (A) shows the classical current curves of 3 μM compound A and 10 μM RTG; (B) shows the voltage activation curves of 3 μM compound A and 10 μM RTG at clamp voltage of -120 mV, step voltage of 10 mV, transition to +90 mV, and repolarization to -120 mV; (C) shows the classical current curves after drug administration at -60 mV, depolarization to 0 mV, and repolarization to -60 mV.

[0070] Figure 7 (A) shows the I / I0 values ​​of compound B administered at continuous doses of 0.1, 0.3, 1, 3, 5, and 10 μM, respectively, which were 1.12±0.05, 1.21±0.06, 1.37±0.07, 1.48±1.03, 1.63±1.43, and 1.90±2.32, respectively. These values ​​showed statistically significant differences compared to the normal control current (*P<0.05, **P<0.01, ***P<0.001). (B) shows the increase in Kv7.4 channel current with increasing compound B concentration. (C) shows the maximum effect (Emax) of the outward current induced by compound B, fitted to a logistic function, EC0. 50 =3.95μM.

[0071] Figure 8 In the middle section, (A) is the classical current diagram of 3 μM compound B and 10 μM RTG, (B) is the voltage activation curve of 3 μM compound B and 10 μM RTG at clamp voltage of -120 mV, step voltage of 10 mV, transition to +90 mV, and repolarization to -120 mV, and (C) is the classical current diagram after drug administration at -60 mV, depolarization to 0 mV, and repolarization to -60 mV.

[0072] Example 5: Detection of the in vivo distribution of compounds A and B based on HPLC-MS

[0073] In blank mice, 20 mg / kg was administered intraperitoneally. Blood was collected from the canthus of the eye and placed in heparinized EP tubes. The samples were centrifuged at 4500 rpm for 10 min at 4°C on a frozen centrifuge. The supernatant was transferred to a new EP tube and stored at -40°C. The brain was dissected and stored at -40°C. 100 μL of blood sample was mixed with 300 μL of pure methanol and centrifuged at 14000 rpm for 10 min. 100 μL of the supernatant was transferred to an autosampler vial for analysis. Brain tissue was added to 50% methanol-water at a ratio of 1 g / 4 L, ground at 1500 rpm for 10 min, centrifuged at 14000 rpm for 10 min, and the supernatant was collected. After centrifugation once more, 100 μL of the supernatant was transferred to an autosampler vial for HPLC-MS / MS analysis.

[0074] Mass spectrometry conditions: ESI source; detection mode: multiple reaction monitoring (MRM); negative ion monitoring mode: electrospray voltage (IS): -5500V; ion source temperature (TEM): 450℃; curtain gas (CUR): 20psi; nebulizer gas (GS1): 45psi; auxiliary gas (GS2): 50psi.

[0075] Liquid chromatography conditions: Column: Kinetex XB-C18 (2.6 μm, 50 × 3 mm); Detection mobile phase for compound F08: methanol (A) - pure water (B), 45% A, 55% B, isocratic elution for 3 min; flow rate: 0.4 mL / min; column temperature: 40 °C; injection volume: 5 μL. Detection mobile phase for compound F03: methanol (B) - 0.5% formic acid water (C) - 5 mM ammonium acetate (D); 40% B–90% B, 30% C–5% C, 30% D–5% D; gradient elution for 0–4 min, 40% B–90% B; flow rate: 0.4 mL / min; column temperature: 40 °C; injection volume: 5 μL.

[0076] See results Figure 9 The original drug was detected in both blood and brain samples of the drug-treated compound A at the same retention time as the standard. Figure 9 A); while two peaks were detected in blood and brain samples of the administered compound B ( Figure 9 B), which is converted in the body into another drug, coenzyme A, see details. Figure 10 According to literature review, B is a prodrug of coenzyme A.

[0077] Example 6: Protective effect of compound B against gentamicin ototoxicity

[0078] Mice were randomly divided into three groups of 10 mice each. The model group received gentamicin sulfate 100 mg / kg intraperitoneally once daily for 14 days. The DFO group received DFO (compound B, purchased from MCE Pharmaceuticals, USA) 150 mg / kg intraperitoneally, followed by gentamicin 100 mg / kg intraperitoneally 10 minutes later, once daily for 14 days. The control group received an equal volume of physiological saline intraperitoneally once daily for 14 days. Animal weight was measured daily during the experiment to adjust drug dosages.

[0079] After 14 days of continuous drug administration, the ABR hearing threshold of mice was measured. Figure 11 The results showed that the ABR threshold in the model group (GM group) was significantly increased, with significant differences compared to both the control group (WT group) and the drug-treated group (DFO+GM group). Figure 11 A)(***P<0.001), while there was no statistically significant difference between the treatment group (DFO+GM group) and the control group (WT group).

[0080] Example 7: Preparation of pharmaceutical formulation

[0081] Given existing research reports, the link between the KCNQ4 channel and hearing loss has been confirmed, and the KCNQ4 channel has become a well-defined drug target for the treatment of hearing impairment. Compound A described in this invention is a highly active and selective KCNQ4 channel opener, while exhibiting no activity against KCNQ1, KCNQ2, KCNQ3, and KCNQ5.

[0082] Therefore, compounds A, B, or their pharmaceutically acceptable salts or solvates can be used as active pharmaceutical ingredients in the preparation of KCNQ4 potassium ion channel openers and in the preparation of drug formulations for the treatment of hearing loss.

[0083] The specific preparation method can use conventional pharmaceutical preparation techniques to prepare pharmaceutical preparations such as oral liquids, tablets, capsules, injections, and sustained-release preparations.

Claims

1. Use of a compound having a chemical structure represented by Formula (A) or Formula (B) or a pharmaceutically acceptable salt thereof in the manufacture of a KCNQ4 potassium ion channel opener: ， （A）； ， （B）。 2. Use of a compound represented by Formula (A) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of hearing impairment.

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