Application of asarinol and derivatives thereof in treatment of Dravet syndrome

Abstract

The invention belongs to the technical field of medicines, and discloses application of asarinol and derivatives thereof in treatment of the Dravet syndrome. The structural general formula of asarinol and the derivatives thereof is shown in (I). Researches find that in an scn1lab / zebra fish model, alpha-asarone, beta-asarone and derivatives thereof can reduce epilepsy-like discharge by 23%-93%, and the effect is obviously superior to that of stiripentanol and cannabidiol. Related pharmaceutical preparations can be prepared into capsules, dry suspensions or gels and the like, the dry suspensions are more suitable for children to take, and the capsules and the gels are suitable for adults to take. In addition, toxicological studies show that the compound is high in safety and free of remarkable developmental toxicity. The invention provides a safe and efficient novel treatment scheme for the Dravot syndrome and intractable epilepsy, and has important clinical transformation value. (I)

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and discloses the application of asarol and its derivatives in the preparation and treatment of Dravet syndrome. Background Technology

[0002] Although many antiepileptic drugs (AEDs) possess broad-spectrum antiepileptic activity, Dravet syndrome (DS), a severe developmental epileptic encephalopathy caused by mutations in the SCN1A gene, exhibits a high degree of selectivity in treatment based on the mechanism of drug action. Many conventional AEDs are not only ineffective but may even worsen symptoms. Specifically, sodium channel blockers such as carbamazepine, oxcarbazepine, phenytoin sodium, and lamotrigine can further inhibit neuronal sodium channel function, exacerbating Dravet syndrome seizures. For example, GABAergic drugs such as gabapentin, pregabalin, tiagabine, and vigabatrin may disrupt the neuronal excitation-inhibition balance. Phenobarbital (high doses), zonisamide, and topiramate are also unsuitable for Dravet syndrome due to insufficient efficacy or potential risk of worsening.

[0003] The inventors' preliminary research revealed that α-asarone, β-asarone, and their derivatives possess GABA... A Receptor positive regulation and lactate dehydrogenase (LDH) inhibitory activity (Isolation, antiepileptic activity and mechanism of α-asarol, an active molecule in Acorus tatarinowii [D]. Northwest University, 2019; European Journal of Medicinal Chemistry 183 (2019) 111650). Clinical studies have shown that some GABA-containing... ADrugs with positive receptor modulation effects (such as diazepam, lorazepam, and phenobarbital) can exert anticonvulsant effects by enhancing GABAergic neurotransmission, but they cannot effectively control chronic myoclonic / tonic seizures of Dravet syndrome, nor are they clearly effective against the characteristic nonconvulsive seizures of Dravet syndrome. Furthermore, long-term use can easily lead to severe cognitive impairment, behavioral abnormalities, and other adverse reactions; therefore, they are not used as routine treatments for Dravet syndrome. On the other hand, lactate dehydrogenase (LDH) inhibitory activity is not a sufficient condition for antiepileptic effect or therapeutic efficacy in Dravet syndrome. For example, oxalic acid, a typical LDH inhibitor, only shows weak anticonvulsant activity in animal models and lacks evidence of effectiveness against Dravet syndrome; therefore, it cannot be used to treat Dravet syndrome. It is worth noting that while stetaminophen is an antiepileptic drug with LDH inhibitory activity and has been proven to be effective in treating Dravet syndrome, its core mechanism of action stems from enhancing GABAergic transmission and inhibiting sodium channels, rather than LDH inhibition. Summary of the Invention

[0004] Further research by the inventors revealed that α-asarol, β-asarol, and their derivatives exhibit unique therapeutic potential. They do not directly block sodium channels, but rather mitigate the risk of exacerbating SCN1A dysfunction through multi-target synergistic regulation (including regulation of the GABAergic system, regulation of lactate dehydrogenase activity, and inhibition of neuroinflammation). Furthermore, in scn1lab... − / − In EEG and behavioral experiments on zebrafish models (with the SCN1A homolog knocked out, exhibiting epileptic seizures and behavioral deficits highly similar to Dravet syndrome), it was confirmed that it could effectively suppress seizures. More importantly, its developmental toxicity was significantly lower than that of existing Dravet syndrome treatments such as stearyl alcohol and cannabidiol. This safety advantage and unique mechanism of action have opened up new directions for the treatment of Dravet syndrome.

[0005] Furthermore, the diverse symptoms of Dravet syndrome lead to significant differences in treatment needs and drug selection: some patients are primarily troubled by frequent seizures (such as febrile status seizures, focal seizures, and tonic-clonic seizures), or have concurrent sleep disorders, irritability, and hyperactivity; others are characterized by cognitive delay, language impairment, and impaired motor function. Patients with different symptom characteristics benefit significantly from antiepileptic drugs of varying sedative strengths: the former may be more suited to drugs with some sedative effect, while the latter should prioritize drugs with weak sedative effects. The drug of this invention can treat Dravet syndrome through sedation.

[0006] This invention provides antiepileptic active ingredients α-asarone, β-asarone, and their derivatives. They may exert their effects through multiple mechanisms, including: inhibiting key energy metabolism pathways and reducing the metabolic basis of abnormal discharges; enhancing GABAergic transfer and regulating GABA. A Receptor function enhances inhibitory nerve signals, has a sedative effect, etc. (In scn1lab) − / − In zebrafish models, the above-mentioned components reduced epileptiform discharges by 23-93%, demonstrating significantly better efficacy than staepentol and cannabidiol. Toxicological studies confirmed their high safety profile, with no significant developmental toxicity, and their developmental toxicity was significantly lower than that of staepentol. Formulations have been developed as dry suspensions, gels (suitable for children), and capsules (suitable for adult patients).

[0007] Therefore, the present invention provides the application of asarone and its derivatives in the preparation of sedative drugs.

[0008] Building upon previous anti-epileptic studies, the inventors further evaluated safety through developmental toxicity experiments, combining the gene-knockout scn1lab − / − Anti-epileptic experiments were conducted using a zebrafish model (including behavioral observation and electroencephalogram monitoring), comprehensively validating the potential application value of this type of compound in treating Dravet syndrome. This invention also provides the application of asarol and its derivatives in the preparation of drugs for treating Dravet syndrome.

[0009] The structural formulas of the above-mentioned asarone and its derivatives are shown in (I):

[0010] (I) In formula (I): When the olefin bond is trans-structured and R is H, formula (I) is α-asarone; When the alkene bond is in the trans configuration and R is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ether compound; The alkene bond is in the trans configuration and R is When R1 is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ester compound; When the olefin bond is in the trans configuration and R is an amino acid group, formula (I) is also an α-asarone ester compound; When the olefin bond is cis-structured and R is H, formula (I) is β-asarone.

[0011] In this invention, α-asarol, β-asarol, and their derivatives can be formulated into pharmaceutical compositions with any pharmaceutically permissible excipient, or they can be formulated into compound preparations with other therapeutic agents that do not antagonize α-asarol, β-asarol, and their derivatives. These preparations can be any pharmaceutically permissible type, including but not limited to tablets, granules, pills, gels, oral liquids, injections, films, capsules, liposomes, and nanoformulations. Preferred solid dosage forms are capsules, dry suspensions, tablets, dispersible tablets, gels, granules, and gels. Attached Figure Description

[0012] Figure 1 shows the results of α-asarone on the general developmental toxicity of zebrafish larvae in the examples; (A) embryo hatching rate at 48 hpf and 72 hpf; (B) dose-dependent malformation rate at 144 hpf; (C) median lethal concentration (LC50) at 96, 120, and 144 hpf. 50 (μM), data are expressed as mean ± standard error (n=20 tails / group, 3 independent experiments).

[0013] Figure 2 shows the results of the general developmental toxicity of β-asarone and β-asarone to zebrafish larvae in the examples; (A) morphological phenotypes of embryos treated with β-asarone and β-asarone (5~100 μM) at 24, 48, and 72 hpf; (B, C) embryo hatching rate at 48 hpf and 72 hpf; (D) phenotypic abnormalities of larvae treated with β-asarone and β-asarone at 120 hpf and 144 hpf; (E) dose-dependent malformation rate at 144 hpf; (F) median lethal concentration (LC50, μM) at 96, 120, and 144 hpf; data are expressed as mean ± standard error (n=20 fish / group, 3 independent experiments); scale bar: 1000 μm; statistical significance: ** P<0.01 *** P<0.001 (vs. control group).

[0014] Figure 3 shows the results of the neurotoxic effects of β-asarone and β-asarone on zebrafish in the examples; (A) Representative fluorescence micrographs of vmat2:GFP transgenic zebrafish embryos treated with β-asarone and β-asarone (5-100 μM) for 96 hpf (red boxes mark the distribution areas of dopaminergic (DA) neurons, and red brackets indicate the total length of labeled neurons); (B) Quantitative analysis of the number of DA neurons in different concentration (5-100 μM) treatment groups; (C) Quantitative analysis of the total length of labeled neurons in different concentration (5-100 μM) treatment groups. Data are expressed as mean ± standard error (n=10 fish / group, 3 independent experiments); scale bar: 100 μm; statistical significance: ** P<0.01 *** P<0.001 (vs. control group).

[0015] Figure 4 shows the results of the hepatotoxic effects of β-asarone and β-asarone on zebrafish in the examples; (A) Representative fluorescence micrographs of lfabp:EGFP transgenic zebrafish embryos treated with β-asarone and β-asarone (5~100 μM) for 144 hpf (the liver region is marked by a red box); (B) Quantitative analysis of liver area; (C) Quantitative analysis of relative fluorescence intensity in the liver region. Data are expressed as mean ± standard error (n=10 fish / group, 3 independent experiments); scale bar: 100 μm; statistical significance: ** P<0.01 *** P<0.001 (vs. control group).

[0016] Figure 5 shows the effects of α-asarone and TMCA-α-asarone ester on scn1lab in the examples. − / − Results of the effects of Dravet syndrome in mutant zebrafish; (A) scn1lab − / − (a) Total migration distance of mutant juvenile fish after exposure to 0.05% DMSO (solvent control, Ctl), stearyl alcohol (STP), cannabidiol (CBD), α-asarone, α-asarone, or TMCA-α-asarone ester; (b) Representative electroencephalograms of the STP, CBD, α-asarone, α-asarone, or TMCA-α-asarone ester treatment groups; (c) Frequency of epileptiform events during the recording period; (d) Mean cumulative amplitude of abnormal discharges during the recording period; Statistical significance: **** P<0.0001 (vs. solvent control group) #### P<0.0001 (vs.scn1lab) − / − (Mutant group).

[0017] Figure 6 shows the effect of β-asarone on scn1lab in the examples. − / − Results of the effects of Dravet syndrome in mutant zebrafish; (A) 4–6 dpf wild-type (WT) and scn1lab − / − Morphological comparison of mutant juveniles; (B) 3~4 dpfscn1lab − / − Total migration distance of juvenile fish after treatment with 0.05% DMSO (solvent control), 12.5 μM stearyl alcohol (STP), 1 μM cannabidiol (CBD), or β-asarone (25–100 μM); (C) Representative waveforms of local field potential (LFP) of the optic tectum in juvenile fish at 7 dpf (n=3 / group); (D) 7 dpfscn1lab − / − Frequency of abnormal neuronal firing in juvenile fish. Data are expressed as mean ± standard error (n=3); statistical significance: ### P<0.001 (vs. control group) *** P<0.001 (vs.scn1lab) − / − (Mutant group).

[0018] Figure 7 shows the results of the sedative effect study of α-asarone and β-asarone in the zebrafish model in the examples. Detailed Implementation

[0019] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be noted that the following embodiments are only some examples of the present invention. Based on the technical concept of the present invention, those skilled in the art can optimize the process by adjusting the relevant reaction conditions. In the following embodiments, unless specific conditions are specified, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise stated, the terminology used herein is understood based on the conventional understanding of those skilled in the art.

[0021] Example 1: Synthesis of α-asarone ether compounds General synthetic method: Sodium hydride (NaH, 60% mineral oil dispersion, 80 mg, 2 mmol) was slowly added to a solution of α-asarone (0.224 g, 1 mmol) in tetrahydrofuran (THF, 10 mL). The solution was stirred at room temperature for 1 hour, followed by dropwise addition of an iodinated compound (2 mmol). The reaction mixture was stirred overnight, quenched with water, extracted with ethyl acetate, and all organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 50:1, 20:1, 10:1, 5:1) to obtain α-asarone ether products.

[0022] (1) α-Asaoctanol methyl ether α-Octanol methyl ether was synthesized according to the above method, wherein the iodinated compound was iodomethane, yielding 205 mg of the target product with a yield of 86%. 1 H NMR (600 MHz, cdcl3) δ 6.99 (s, 1H), 6.87 (d,J= 16.0 Hz,1H), 6.49 (s, 1H), 6.16 (dt,J= 16.0, 6.3 Hz, 1H), 4.08 (dd,J= 6.3, 1.1 Hz,2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H), 3.37 (s, 3H). 13 C NMR (151 MHz, cdcl3) δ 151.54 (s), 149.65 (s), 143.40 (s), 127.39 (s), 124.36 (s), 117.57(s), 110.10 (s), 97.84 (s), 73.81 (s), 58.02 (s), 56.82 (s), 56.62 (s), 56.24 (s). (2) α-Asaoctanol ether α-Octyl alcohol ethyl ether was synthesized according to the above method, wherein the iodinated compound was iodoethane, yielding 215 mg of the target product with a yield of 85%. 1H NMR (600 MHz, cdcl3) δ 6.96 (s, 1H), 6.83 (d,J= 16.0 Hz,1H), 6.45 (s, 1H), 6.19 – 6.09 (m, 1H), 4.09 (d,J= 6.3 Hz, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H), 3.50 (q,J= 7.0 Hz, 2H), 1.20 (t,J= 7.0 Hz, 3H). 13 CNMR (151 MHz, cdcl3) δ 151.37 (s), 149.47 (s), 143.24 (s), 126.93 (s), 124.54(s), 117.48 (s), 109.94 (s), 97.68 (s), 71.84 (s), 65.49 (s), 56.65 (s), 56.47 (s), 56.08 (s), 15.26 (s). (3) α-Asaoctanol propyl ether α-Octanol propyl ether was synthesized according to the above method, wherein the iodinated compound was iodopropane, yielding 193 mg of the target product with a yield of 73%. 1 H NMR (600 MHz, cdcl3) δ 6.97 (s, 1H), 6.84 (d,J= 16.0 Hz,1H), 6.46 (s, 1H), 6.15 (dt,J= 16.0, 6.3 Hz, 1H), 4.10 (dd,J= 6.4, 1.1 Hz,2H), 3.85 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.40 (t,J= 6.8 Hz, 2H), 1.63 –1.57 (m, 2H), 0.91 (t,J= 7.4 Hz, 3H). 13 C NMR (151 MHz, cdcl3) δ 151.38 (s), 149.47 (s), 143.27 (s), 126.80 (s), 124.67 (s), 117.54 (s), 109.96 (s), 97.72(s), 71.99 (s), 56.67 (s), 56.49 (s), 56.09 (s), 22.99 (s), 10.61 (s). (4) α-Asaoctanol Butyl Ether α-Octanol butyl ether was synthesized according to the above method, wherein the iodinated compound was iodobutane, yielding 186 mg of the target product, with a yield of 66%. 1 H NMR (600 MHz, cdcl3) δ 6.96 (s, 1H), 6.83 (d,J= 16.0 Hz,1H), 6.45 (s, 1H), 6.15 (dt,J= 16.0, 6.3 Hz, 1H), 4.09 (d,J= 6.3 Hz, 2H),3.84 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.44 (t,J= 6.7 Hz, 2H), 1.60 – 1.52(m, 2H), 1.40 – 1.33 (m, 2H), 0.89 (t,J= 7.4 Hz, 3H). 13 C NMR (151 MHz, cdcl3)δ 151.36 (s), 149.46 (s), 143.25 (s), 126.78 (s), 124.52 (s), 117.53 (s), 109.95 (s), 97.70 (s), 72.03 (s), 70.05 (s), 56.56 (d,J= 26.8 Hz), 56.37 (s), 56.07 (s), 31.88 (s), 19.37 (s), 13.92 (s). (5) α-Asarol cyclopropyl methyl ether α-Octamethol cyclopropyl methyl ether was synthesized according to the above synthetic method, wherein the iodinated compound was (iodomethyl)cyclopropane, yielding 199 mg of the target product, with a yield of 71%. 1 H NMR (600 MHz, cdcl3) δ 6.96 (s, 1H), 6.82 (d,J=16.0 Hz, 1H), 6.45 (s, 1H), 6.15 (dt,J= 13.0, 6.3 Hz, 1H), 4.11 (d,J= 6.3 Hz,2H), 3.84 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H), 3.28 (d,J= 6.9 Hz, 2H), 1.08 –1.02 (m, 1H), 0.50 (d,J= 7.8 Hz, 2H), 0.21 – 0.14 (m, 2H), 0.22 – 0.14 (m,2H). 13C NMR (151 MHz, cdcl3) δ 151.36 (s), 149.48 (s), 143.26 (s), 126.83 (s), 124.50 (s), 117.49 (s), 109.86 (s), 97.61 (s), 74.89 (s), 71.80 (s), 56.58 (s), 56.40 (s), 56.01 (s), 10.70 (s), 3.04 (s). (6) α-Asaoctanol pentanol (7) α-Octanol ether was synthesized according to the above synthesis method, wherein the iodinated compound was iodopentane, and 156 mg of the target product was obtained, with a yield of 53%. 1 H NMR (600 MHz, cdcl3) δ 6.98 (s, 1H), 6.84 (d,J= 16.0 Hz,1H), 6.47 (s, 1H), 6.16 (dt,J= 16.0, 6.3 Hz, 1H), 4.10 (dd,J= 6.3, 1.2 Hz,2H), 3.86 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H), 3.45 (t,J= 6.8 Hz, 2H), 1.63 –1.56 (m, 2H), 1.35 – 1.29 (m, 4H), 0.88 (t,J= 7.0 Hz, 3H). 13 C NMR (151 MHz, cdcl3) δ 151.42 (s), 149.52 (s), 143.33 (s), 126.86 (s), 124.75 (s), 117.61 (s), 109.99 (s), 97.78 (s), 72.10 (s), 70.45 (s), 56.75 (s), 56.54 (s), 56.15(s), 29.57 (s), 28.44 (s), 22.62 (s), 14.11 (s). (7) α-Asaoctanol anisole α-Octanol anisole was synthesized according to the above synthetic method, wherein the iodinated compound was benzyl iodine, yielding 210 mg of the target product with a yield of 67%. 1H NMR (600 MHz, cdcl3) δ 7.41 – 7.32 (m, 4H), 7.31 – 7.25 (m,1H), 6.99 (s, 1H), 6.89 (d,J= 16.0 Hz, 1H), 6.50 (s, 1H), 6.21 (dt,J= 16.0,6.4 Hz, 1H), 4.57 (s, 2H), 4.20 (dd,J= 6.3, 1.1 Hz, 2H), 3.89 (s, 3H), 3.85(s, 3H), 3.82 (s, 3H). 13 C NMR (151 MHz, cdcl3) δ 151.55 (s), 149.68 (s), 143.44 (s), 138.55 (s), 128.51 (s), 127.98 (s), 127.69 (s), 127.39 (s), 124.42 (s), 117.61 (s), 110.02 (s), 97.78 (s), 72.17 (s), 71.62 (s), 56.79(s), 56.58 (s), 56.20 (s). Example 2: Study on the developmental toxicity of α-asarone, β-asarone and TMCA-α-asarone ester to zebrafish 1. General developmental toxicity 1.1 Experimental Methods Healthy AB strain zebrafish embryos at 4 hpf (hours post-fertilization) were selected and randomly assigned to 12-well plates (20 embryos / well). They were given 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM of the test compounds (α-asarone, β-asarone, TMCA-α-asarone ester) for 6 consecutive days. The blank control group was incubated with E3 zebrafish embryo culture medium, and the culture medium was changed every 24 h. General developmental toxicity was evaluated by morphological changes (embryo coagulation and absence of heartbeat were considered as death), including: (1) Photography method: 1~3 dpf (days post-fertilization) were photographed using stacked white light, magnification ×10, and exposure time 70 ms; 4~6 dpf were photographed using single white light, magnification ×26.6, and exposure time 130 ms; (2) Statistical indicators: 1~6 dpf Mortality rate (number of zebrafish deaths per hole / total number of zebrafish per hole × 100%), hatching rate at 2-3 dpf (number of hatched fry per hole / total number of zebrafish per hole × 100%), deformity rate at 4-6 dpf (number of deformed zebrafish per hole / total number of surviving zebrafish per hole × 100%), deformity types include spinal curvature, missing swim bladder, body curvature, smaller eyes / brain, yolk sac edema, pericardial edema, etc.

[0023] 1.2 Experimental Results (1) α-Asauropin: At concentrations of 5-100 μM, the developmental morphology and hatching rate of zebrafish at 24, 48, and 72 hpf after fertilization (Fig. 1A) showed no significant difference from the blank control group; only at a concentration of 100 μM, 26.7% of zebrafish showed swim bladder absence (Fig. 1B); in terms of mortality, the median lethal concentration (LC50) of α-asauropin at 96, 120, and 144 hpf were 257.56, 180.34, and 151.90 μM, respectively (Fig. 1C), indicating extremely low toxicity.

[0024] (2) Comparison of β-asarone and β-asarone: In the 5~100 μM β-asarone treatment groups, the developmental morphology (Fig. 2A) and hatching rate (Fig. 2B, 2C) of zebrafish 24, 48 and 72 hpf were not significantly different from those of the blank control group; only 100 μM concentration induced 30% mild swim bladder atrophy, without other deformities (Fig. 2E). β-Asarum exhibits concentration-dependent developmental toxicity: ≥10 μM induces four types of malformations, including scoliosis and swim bladder absence, with the malformation rate increasing with concentration (Figure 2E); it also significantly inhibits embryo hatching (Figures 2B and 2C); in terms of mortality, at the same concentration (25–100 μM) within 24–144 hpf, β-asarol has a 5–10 times lower lethality than β-asarrum; the LC50 of β-asarol at 96, 120, and 144 hpf (233.78, 193.83, and 160.10 μM) is approximately 3.5 times higher than that of β-asarrum (63.31, 52.61, and 47.58 μM) (Figure 2F).

[0025] (3) TMCA-α-asarone ester: Its developmental toxicity is similar to that of α-asarone and β-asarone, but its safety is significantly better.

[0026] 2. Neurodevelopmental toxicity 2.1 Experimental Methods Healthy Tg vmat2:EGFP transgenic zebrafish embryos at 4 hpf post-fertilization were selected and randomly assigned to sterile 6-well plates (30 embryos / well). β-asarol was administered at concentrations of 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM for 4 consecutive days. A blank control group was incubated in E3 medium. Fluorescence images were collected from at least 10 zebrafish in each group. Using the blank control group as a reference, the number of dopaminergic neurons and the total length of neurons were counted to assess the effect of β-asarol on zebrafish neural development.

[0027] 2.2 Experimental Results Using a Tg vmat2:EGFP transgenic zebrafish dopaminergic system model, the number of dopaminergic neurons (Figure 3A, marked with red rectangles) and the total length of marked neurons (Figure 3A, indicated by red brackets) were used for evaluation. After exposure to 5–100 μM β-asarone for 96 hpf, the above indicators showed no significant difference from the blank control group. Figure 3 (B, 3C) confirmed that β-asarol has excellent neurodevelopmental safety.

[0028] α-Asauropinol and TMCA-α-Asauropinol ester also did not show significant neurodevelopmental toxicity.

[0029] 3. Hepatotoxicity 3.1 Experimental Methods Healthy Tg lfabp:EGFP transgenic zebrafish embryos at 4 hpf post-fertilization were selected and randomly assigned to sterile 6-well plates (30 embryos / well). They were administered β-asarone at concentrations of 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM for 6 consecutive days. The control group was incubated in E3 medium. At 6 dpf, images of the liver were acquired using a fluorescence inverted microscope. Image-Pro Plus software was used to analyze liver area and relative fluorescence intensity, and the results were compared with the control group to assess liver developmental toxicity.

[0030] 3.2 Experimental Results The hepatotoxicity of the compound was assessed using a Tg lfabp:EGFP transgenic zebrafish model. After 144 hpf exposure to β-asarol, the liver morphology and tissue transparency of the treated group and the blank control group were normal (Fig. 4A), and there were no significant differences in liver area (Fig. 4B) and relative fluorescence intensity (Fig. 4C); no hepatotoxicity was observed at concentrations ≤100 μM.

[0031] α-Asauropinol and TMCA-α-Asauropinol ester also did not show significant liver developmental toxicity.

[0032] Example 3: Compounds of α-asarone, β-asarone, α-asarone esters and α-asarone ethers in scn1lab ⁻ / ⁻ Evaluation of antiepileptic activity in mutant zebrafish models 1 Experimental Methods Select scn1lab with 3 dpf ⁻ / ⁻Mutant zebrafish juveniles were treated with the following drugs for 24 h: stiripentol (STP, 12.5 μM), cannabidiol (CBD, 1 μM), α-asarol (25, 50, 100 μM), β-asarol (25, 50, 100 μM), and α-asarol ester (50 μM). At 4 dpf, the activity was assessed by the following methods: (1) Motor activity analysis: The juvenile fish were transferred to 48-well plates (1 fish / well, containing 500 μL of culture water), and after acclimatization at 28℃ for 5 min, the total swimming distance (i.e., total movement distance) was recorded for 30 min using behavioral instruments; (2) Electroencephalogram (EEG) recording: The juvenile fish were pretreated with 2 mM tubocurarine (Sigma-Aldrich, USA) and then fixed with 2% low melting point agarose (Solarbio, China); after acclimatization at 28℃ for 5 min, the glass microelectrode containing 3% (w / v) potassium acetate was used to locate the midbrain region, and the EEG signal was recorded for 30 min using the German NPI Electronic ELC-03XS system and analyzed using Lab Chart 7 software (ADInstruments, Australia).

[0033] Epileptiform events are defined as multi-peaked signals with membrane potential deviations exceeding 3 times the baseline noise and lasting ≥ 500 ms.

[0034] The percentage decrease in total swimming distance = (scn1lab) - / - Total swimming distance of mutant zebrafish (total swimming distance of the test compound) / scn1lab - / - Total swimming distance of mutant zebrafish × 100%.

[0035] Epilepsy event reduction rate = (scn1lab) - / - Mutant Zebrafish Epilepsy Event Frequency – Testing Compound Epilepsy Event Frequency / scn1lab - / - Frequency of epileptic events in mutant zebrafish × 100%.

[0036] 2. Experimental Results 2.1 Activity of α-asarone and α-asarone ester The concentrations of all compounds were selected within the range of maximum tolerable concentration (MTC) and set in multiples of 12.5 μM: (1) Motility activity: All drugs significantly inhibited the kinetic activity of 4 dpf mutant juveniles (Figure 5A). α-Asauropinol showed concentration-dependent inhibition. Compared with the blank control group, the percentage reduction in total swimming distance in each treatment group was: STP (40.3%), CBD (55.6%), α-Asauropinol (25 μM, 49.9%), α-Asauropinol (25 μM, 57.1%, 50 μM, 64.7%, 100 μM). 76.6%); (2) EEG activity: Local field potential (LFP) recordings of the optic tectum in the midbrain showed (Figure 5B) that all drugs significantly reduced the frequency (Figure 5C) and amplitude (Figure 5D) of epileptiform events: the reduction rates of STP, CBD, and α-asarone were 75%, 80%, and 75%, respectively, and the amplitudes were reduced by 70%, 80%, and 75%, respectively, while the α-asarone (100 μmol / L) treatment group almost completely eliminated such abnormal discharges. This result indicates that α-asarone can significantly inhibit scn1lab ⁻ / ⁻ The mutant zebrafish juveniles exhibited abnormal electrical discharges, showing significantly better efficacy than the positive control drugs STP and CBD, and possessed the potential to treat Dravet syndrome.

[0037] Similar to the above scheme, α-asarone esters and α-asarone ethers were tested in scn1lab. ⁻ / ⁻ The anti-epileptic activity in the mutant zebrafish model is shown in Table 1.

[0038] Table 1. α-Octanol esters and α-Octanol ethers (50 μM) in scn1lab ⁻ / ⁻ Antiepileptic activity in mutant zebrafish models

[0039] Remark: a The concentration was 12.5 μM. b The concentration is 1 μM.

[0040] 2.2 Activity of β-asarone The scn1lab⁻ / ⁻ mutant juveniles exhibited typical phenotypes: excessive melanin deposition, incomplete swim bladder development, and progressive bending of the tail axis at 4-6 dpf (Fig. 6A). Motility and convulsive-like behavior (generalized tonic-clonic seizures, abnormal movement patterns) peaked at 3-5 dpf. Therefore, 4 dpf was chosen as the detection time window: (1) Motility activity: β-asarone (25, 50, 100 μM) inhibited motility activity in a concentration-dependent manner, and both β-asarone (12.5 μM) and CBD (1 μM) significantly reduced the total distance of movement. Figure 6 B). Compared with the blank control group, the total distance of movement decreased by 55.9%, 68.7%, and 73.8% for β-asarone (25, 50, 100 μM), respectively; (2) EEG activity: mutant juvenile fish showed spontaneous bursts of movement and epileptiform discharges (monospin waves, polyspin waves, ... Figure 6 (C) β-Asauropinol reduced the discharge frequency in a dose-dependent manner (Figure 6D), with an inhibitory efficacy 3.9 times higher than STP, 3.2 times higher than CBD, and 14.3 times higher than the untreated mutant group. β-Asauropinol (25, 50, and 100 μM) reduced epileptiform events by 75%, 83%, and 93%, respectively. These results indicate that β-Asauropinol can significantly inhibit scn1lab ⁻ / ⁻ The mutant zebrafish juveniles exhibited abnormal electrical discharges, showing significantly better efficacy than the positive control drugs STP and CBD, and possessed the potential to treat Dravet syndrome.

[0041] Example 4: Study on the sedative effect of β-asarone in a zebrafish model 1 Experimental Methods Juvenile AB strain zebrafish with a dpf of 6 were selected, and sedation activity was quantitatively assessed by total movement distance. Each juvenile was placed in a 48-well plate (containing 500 μL of culture water) and allowed to acclimatize in the dark at 28°C for 10 min before being replaced with a vehicle control (VHC) solution. The total movement distance over 30 min was immediately recorded using Zeblab software from Viewpoint (France). Six juveniles were selected from each group, and the experiment was repeated six times (n=8 / repeat). The compound was dissolved in DMSO and then diluted with culture water until the final DMSO concentration was ≤0.5%. Data are expressed as mean ± standard error (using GraphPad Prism software), and differences between groups were compared using one-way ANOVA.

[0042] 2. Experimental Results Treatment with 100–150 μM β-asarone significantly reduced the basal kinetic activity of zebrafish (compared to the control group). Figure 7This inhibitory effect reflects the sedative properties of β-asarone, confirming its sedative activity.

Claims

1. The application of asarol and its derivatives in the preparation of sedative drugs, characterized in that, The structural formulas of the asarol and its derivatives are shown in formula (I): （I） In formula (I): When the olefin bond is trans-structured and R is H, formula (I) is α-asarone; When the alkene bond is in the trans configuration and R is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ether compound; The alkene bond is in the trans configuration and R is When R1 is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ester compound; When the olefin bond is in the trans configuration and R is an amino acid group, formula (I) is also an α-asarone ester compound; When the olefin bond is cis-structured and R is H, formula (I) is β-asarone.

2. The application of asarol and its derivatives in the preparation of drugs for treating Dravet syndrome, characterized in that, The structural formula of the asarol and its derivatives is the same as that of formula (I) in claim 1: （I） In formula (I): When the olefin bond is trans-structured and R is H, formula (I) is α-asarone; When the alkene bond is in the trans configuration and R is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ether compound; The alkene bond is in the trans configuration and R is When R1 is a branched alkyl group with 1-12 carbon atoms, a straight-chain alkyl group with 1-12 carbon atoms, a cycloalkyl group with 3-9 carbon atoms, a substituted cycloalkyl group with 3-9 carbon atoms, a cycloalkenyl group with 3-9 carbon atoms, a substituted cycloalkenyl group with 3-9 carbon atoms, an aryl group, a monosubstituted aryl group, a polysubstituted aryl group, a heterocyclic aryl group, a monosubstituted heterocyclic aryl group, or a polysubstituted heterocyclic aryl group, formula (I) is an α-ascorbic acid ester compound; When the olefin bond is in the trans configuration and R is an amino acid group, formula (I) is also an α-asarone ester compound; When the olefin bond is cis-structured and R is H, formula (I) is β-asarone.

3. The application according to claim 1 or 2, characterized in that, The drug formulation is prepared by adding pharmaceutically acceptable excipients, and the formulation type includes solid dosage forms.

4. The application according to claim 1 or 2, characterized in that, The formulation types of the drug include tablets, granules, gels, pills, oral liquids, injections, films, capsules, liposomes, or nanoparticles.

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