Small molecules for treating amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) by targeting C9ORF72 hexanucleotide G-quadruplexes

Abstract

The present invention relates to a pharmaceutical composition comprising a series of compounds selectively targeting G-quadruplexes (G4) formed by GGGGCC (G4C2) hexanucleotide repeat sequences (HRE) (G4C2) C9orf72 n (G4C2) n The composition comprises: compounds G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. The present invention also relates to a method for treating amyotrophic lateral sclerosis (ASL) and / or frontotemporal dementia (FTD) comprising administering to a subject an effective amount of the composition. These compounds can improve the pathological features of ALS models caused by C9orf72 (G4C2) n the sequence.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 744,866, filed January 14, 2025, the entire contents of which are incorporated herein by reference, including any tables, figures, or drawings. Technical Field

[0003] This application relates to the biomedical field. More specifically, this application relates to a pharmaceutical composition comprising selectively targeted components... C9orf72 The GGGGCC (G4C2) hexanucleotide repeat sequence (G4C2) n The compound formed by the G-quadruplex, and the method of using the pharmaceutical composition to rescue amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD). Background Technology

[0004] Amyotrophic lateral sclerosis (ALS), also known as Lugris' disease, is a devastating neurodegenerative disease characterized by the rapid, progressive loss of motor neurons, ultimately leading to respiratory failure within 3 to 5 years of symptom onset (see P. Masrori, P. Van Damme, Amyotrophic lateral sclerosis: a clinical review). Eur J Neurol , 27 (2020) 1918-1929). In particular, approximately 50% of ALS patients have cognitive impairment, and 13% develop frontotemporal dementia (FTD) (see references O. Hardiman, A. Al-Chalabi, A. Chio, EMCorr, G. Logroscino, W. Robberecht, PJ Shaw, Z. Simmons, LH van den Berg, Amyotrophic lateral sclerosis, Nat Rev Dis Primers, 3 (2017) 17071). Approximately two out of every 100,000 people suffer from ALS, characterized by the degeneration of motor neurons, leading to muscle weakness and atrophy (see M. DeJesus-Hernandez, IR Mackenzie, BF Boeve, AL Boxer, M. Baker, NJ Rutherford, AM Nicholson, NA Finch, H. Flynn, J. Adamson, N. Kouri, A. Wojtas, P. Sengdy, GY Hsiung, A. Karydas, WW Seeley, KA Josephs, G. Coppola, DH Geschwind, ZK Wszolek, H. Feldman, DS Knopman, RC Petersen, BL Miller, DW Dickson, KB Boylan, NR Graff-Radford, R. Rademers, Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS). Neuron , 72 (2011) 245-256).

[0005] Regarding the etiology of ALS, it has been reported that sporadic ALS (sALS) accounts for approximately 90% of all ALS cases, while the remaining 10% of ALS patients have familial ALS (fALS), which is caused by gene mutations (see reference X. Liu, X. Zhao, J. He, S. Wang, X. Shen, Q. Liu, S. Wang, Advances in the Structure of GGGGCCRepeat RNA Sequence and Its Interaction with Small Molecules and Protein Partners). Molecules, 28 (2023)). Abnormalities in the function of SOD1, FUS / TLS, and TDP-43 caused by mutations lead to neurotoxicity, resulting in fALS (see EL Feldman, SA Goutman, S. Petri, L. Mazzini, MG Savelieff, PJ Shaw, G. Sobue, Amyotrophic lateral sclerosis, 28 (2023)). Lancet , 400 (2022) 1363-1380). Furthermore, it has been shown that, C9orf72 Amplification of the GGGGCC (G4C2) hexanucleotide repeat sequence in the non-coding region of the gene is a pathogenic factor for ALS and FTD (see CM Rodriguez, PK Todd, New pathologic mechanisms in nucleotide repeat expansion disorders, Neurobiol Dis, 130 (2019) 104515; Q. Zhu, J. Jiang, TF Gendron, M. McAlonis-Downes, L. Jiang, A. Taylor, S. Diaz Garcia, S. Ghosh Dastidar, MJ Rodriguez, P. King, Y. Zhang, AR La Spada, H. Xu, L. Petrucelli, J. Ravits, S. Da Cruz, C. Lagier-Tourenne, DW Cleveland, Reduced C9ORF72 function exacerbates gain of toxicity from ALS / FTD-causing repeat expansion in C9orf72, Nat Neurosci, 23 (2020) 615-624). Aberrant amplification of the G4C2 repeat sequence was observed in 8% of sporadic ALS (sALS) patients and over 40% of familial ALS (fALS) cases (see references: AE Renton, E. Majounie, A. Waite, J. Simón-Sánchez, S. Rollinson, JR Gibbs, JCSchymick, H. Laaksovirta, JC van Swieten, L. Myllykangas, H. Kalimo, A. Paetau, Y. Abramzon, AM Remes, A. Kaganovich, SW Scholz, J. Duckworth, JH Ding, DW Harmer, DG Hernandez, JO Johnson, K. Mok, M. Ryten, D. Trabzuni, RJ Guerreiro, RW Orrell, J. Neal, A. Murray, J. Pearson, IE Jansen, D. Sondervan, H. Seelaar, D. Blake, K. Young, N. Halliwell, JBCallister, G. Toulson, A. Richardson, A. Gerhard, J. Snowden, D. Mann, D.Neary, MA Nalls, T. Peuralinna, L. Jansson, VM Isoviita, AL Kaivorinne, M. Hölttä-Vuori, E. Ikonen, R. Sulkava, M. Benatar, J. Wuu, A. Chiò, G.Restagno, G. Borghero, M. Sabatelli, D. Heckerman, E. Rogaeva, L. Zinman,JD Rothstein, M. Sendtner, C. Drepper, EE Eichler, C. Alkan, Z.Abdullaev, SD Pack, A. Dutra, E. Pak, J. Hardy, A. Singleton, NMWilliams, P. Heutink, S.Pickering-Brown, HR Morris, PJ Tienari, BJTraynor, I. Consortium, A Hexanucleotide Repeat Expansion in Is the Cause ofChromosome 9p21-Linked ALS-FTD,. Neuron , 72 (2011) 257-268). The average number of repeat sequences in individuals with ALS typically ranges from 700 to 1600, while healthy individuals have fewer than 25 repeat sequences (see A. Nordin, C. Akimoto, A. Wuolikainen, H. Alstermark, P. Jonsson, A. Birve, SL. Marklund, KS. Graffmo, K. Forsberg, T. Brannstrom, PM. Andersen, Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD, Hum Mol Genet , 24 (2015) 3133-3142). In particular, aberrant amplifications of short nucleotide repeat sequences, such as (CAG), have been identified in many neurodegenerative diseases. n The aberrant expansion of these abnormal nucleotide repeat sequences leads to spinocerebellar ataxia 3 (SCA3) (see I. Malik, CP Kelley, ET Wang, PK Todd, Molecular mechanisms underlying nucleotide repeat expansion disorders, Nat Rev Mol Cell Biol, 22(2021) 589-607). Therefore, elucidating the pathological mechanisms by which these abnormal nucleotide repeat sequences cause fatal diseases is a core research focus, with the aim of discovering effective treatments.

[0006] Currently, three proposed mechanisms elucidate the hexanucleotide repeat sequence (G4C2). nThe pathological basis of aberrant amplification (see R. Balendra, AM Isaacs, C9orf72-mediated ALS and FTD: multiplepathways to disease), Nat Rev Neurol , 14 (2018) 544-558). First, abnormal G4C2 amplification can lead to C9orf72 First, the gain or loss of gene function. Second, transcribed RNA G4C2 amplification can recruit RNA-binding proteins (RBPs) to form toxic RNA aggregates (RNA foci), leading to impaired protein function. Third, non-AUG translation of RNA G4C2 amplification can produce neurotoxic dipeptide repeats (DPRs) in the central nervous system, leading to neuronal death. Notably, the formation mechanism of toxic RNA aggregates has received the most attention. In particular, C9orf72 G4C2 repeat sequences (including both DNA and RNA sequences) can fold into secondary structures, such as G4 and hairpin structures (see AR Haeusler, CJ Donnelly, G. Periz, EAJ Simko, PG Shaw, MS Kim, NJ Maragakis, JC Troncoso, A. Pandey, R. Sattler, JD Rothstein, J. Wang, nucleotide repeat structures initiate molecular cascades of disease, Nature, 507 (2014)195-+; YY Geng, CD Liu, QX Cai, ZP Luo, HT Miao, X. Shi, NN Xu, CP Fung, TT Choy, B. Yan, N. Li, PY Qian, B. Zhou, G. Zhu, Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence, Nucleic Acids Res, 49 (2021) 5881-5890; YY Geng, CD Liu, NN Xu, MC Suen, HT Miao, YY Xie, BC Zhang, XQ Chen, YJ Song, ZX Wang, QX Cai, G. Zhu, Crystal structure of a tetrameric RNAG-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS / FTD, Nucleic Acids Res , 52 (2024) 7961-7970; ZF Wang, A. Ursu, JL Childs-Disney, R. Guertler, WY Yang, V. Bernat, SG Rzuczek, R. Fuerst, YJZhang, TF Gendron, I. Yildirim, BG Dwyer, JE Rice, L. Petrucelli, MDDisney, The Hairpin Form of r(G4C2)exp in c9ALS / FTD Is Repeat-Associated Non-ATG Translated and a Target for Bioactive Small Molecules, Cell Chem Biol , 26(2019) 179-190e12). Most importantly, the formation of G4 is C9orf72 G4C2 repeat sequences have an important aspect of toxicity in ALS / FTD, and have been reported to... C9orf72 RNA aggregates formed by G4C2 repeat sequences are mainly composed of RNA G4 (see X. Liu, X. Zhao, J. He, S. Wang, X. Shen, Q. Liu, S. Wang, Advances in the Structure of GGGGCC Repeat RNA Sequence and Its Interaction with SmallMolecules and Protein Partners). Molecules, 28 (2023); EG Conlon, L. Lu, A.Sharma, T. Yamazaki, T. Tang, NA Shneider, JL Manley, The C9ORF72 GGGGCCexpansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disruptsplicing in ALS brains, Elife , 5 (2016)). Therefore, C9orf72 Structural studies of G4C2 repeat sequences are currently a very important research area because the corresponding structures may become potential drug targets for treatment.

[0007] Currently, due to insufficient understanding of the disease mechanisms of ALS / FTD, it remains an incurable disease. The U.S. Food and Drug Administration (FDA) has approved seven drugs for the treatment of ALS: Qalsody. ® Relyvrio ® ,Radicava ® Rilutek ® Tiglutik ® Exservan ® and Nuedexta ® (See RJ Mead, N. Shan, HJ Reiser, F. Marshall, PJ Shaw, Amyotrophic lateral sclerosis: aneurodegenerative disorder poised for successful therapeutic translation, Nat Rev Drug Discov, 22 (2023) 185-212; U. Ansari, M. Alam, D. Nadora, Z.Muttalib, V. Chen, I. Taguinod, M. FitzPatrick, J. Wen, Z. Ansari, F. Lui, Assessing the efficacy of amyotrophic lateral sclerosis drugs in slowingdisease progression: A literature review, AIMS Neurosci, 11 (2024) 166-177;SA Johnson, T. Fang, F. De Marchi, D. Neel, D. Van Weehaeghe, JD Berry,S. Paganoni, Pharmacotherapy for Amyotrophic Lateral Sclerosis: A Review ofApproved and Upcoming Agents, Drugs , 82 (2022) 1367-1388). Qalsody ® Alternatively, Tofersen, which was approved in 2023, targets mutant superoxide dismutase 1 (SOD). SOD1 Antisense oligonucleotides of the mRNA of the gene (see HA Blair, Tofersen: First Approval, Drugs , 83 (2023) 1039-1043). Relyvrio ® It is a fixed composition of sodium phenylbutyrate and taurine glycol, which prevents nerve cell death by inhibiting stress signals in intracellular mitochondria and endoplasmic reticulum. (Relyvrio) ® It was approved in 2022 (see the literature DB Tiz, L. Bagnoli, O. Rosati, F. Marini, C. Santi, L. Sancineto, FDA-Approved SmallMolecules in 2022: Clinical Uses and Their Synthesis). Pharmaceutics Radicava (14(2022)) was discontinued and withdrawn from the market in the United States and Canada in 2024 based on the results of Phase 3 trials. ®Edaravone was first manufactured and marketed in Japan in 2015 and received FDA approval for ALS treatment in 2017. (Radicava) ® It is a potent free radical scavenger that can combat reactive oxygen species-driven motor neuron death and inflammation (see P. Neupane, PK Thada, P. Singh, AR Faisal, N. Rai, P. Poudel, MS Waleed, J. Quinonez, S. Ruxmohan, E. Jain, Investigating Edaravone Use for Management of Amyotrophic Lateral Sclerosis (ALS): A Narrative Review, Cureus, 15 (2023)e33746). Rilutek ® Riluzole, the first ALS drug approved by the FDA in 1995, is an inhibitor of the sodium channel alpha subunit and has been shown to extend the lifespan of ALS patients by approximately three months (see MC Bellingham, A Review of the Neural Mechanisms of Action and Clinical Efficiency of Riluzolein Treating Amyotrophic Lateral Sclerosis: What have we Learned in the Last Decade?). Cns Neurosci Ther , 17 (2011) 4-31). Tiglutik ® It was reapproved by the FDA in 2018. It is an oral suspension variant of riluzole designed specifically for ALS patients who have difficulty swallowing tablets (see B. Brooks, P. Bettica, S. Cazzaniga, Riluzole Oral Suspension: Bioavailability Following Percutaneous Gastrostomy Tube-modeled Administration Versus Direct Oral Administration). Clin Ther , 41 (2019) 2490-2499). Exservan ®This is an oral film formulation of riluzole, specifically designed for ALS patients with severe dysphagia. It was approved in 2019 (see literature J. Wymer, S. Apple, A. Harrison, BA Hill, Pharmacokinetics, Bioavailability, and Swallowing Safety With Riluzole Oral Film). Clin Pharmacol Drug Dev , 12 (2023)57-64). Nuedexta ® Approved in 2010, it is an oral combination of dextromethorphan hydrobromide and quinidine sulfate for the treatment of excessive emotional instability known as pseudobulbar mood (PBA) (see reference Y. Sun, M. Benatar, J. Mascias Cadavid, D. Ennist, P. Wicks, K. Staats, M. Beauchamp, S. Jhooty, G. Pattee, A. Brown, T. Bertorini, P. Barkhaus, M. Bromberg, G. Carter, R. Bedlack, X. Li, ALSUntangled #71: Nuedexta, Amyotroph Lateral Scler Frontotemporal Degener (25 (2024) 218-222). However, the detailed mechanisms by which these drugs treat ALS remain unclear. In particular, more than 5,000 new cases of ALS are diagnosed globally each year. Therefore, there is an urgent need to discover and identify new targets and drugs that are effective in treating ALS. Summary of the Invention

[0008] In one aspect, the present invention relates to a novel composition for treating ALS comprising 7H-pyridocarbazole and a derivative of 7H-pyrazinocarbazole, selectively targeting [the affected component]. C9orf72 The hexanucleotide repeat sequence (HRE) of GGGGCC (G4C2) (G4C2) n The resulting G-quadruplex (G4). In some embodiments, the composition comprises: one or more compounds including G4008, G4009, G4010, G4012 and G4013, or pharmaceutically acceptable salts, solvates or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0009] On the other hand, the present invention relates to a novel method for treating ALS, comprising administering an effective amount of the composition of the present invention to a subject. In some embodiments, the novel compound of the present invention targets... C9orf72G4C2 G4. In a preferred embodiment, the composition comprises: one or more compounds selected from the group consisting of G4008, G4010, G4012, and G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In a more preferred embodiment, the composition comprises: one or more compounds selected from the group consisting of G4010 and G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In the most preferred embodiment, the composition comprises: compound G4013 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0010] In some implementations, administration of a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012 and G4013 significantly reduced the number of RNA aggregates and reactive oxygen species (ROS) levels in subjects.

[0011] In some embodiments, application of the composition can significantly improve the condition caused by… C9orf72 (G4C2) n The sequence evokes the pathological features of an ALS model. In other embodiments, administration of the composition can significantly improve one or more symptoms and / or the progression of ALS and / or FTD in a subject. Attached Figure Description

[0012] Figure 1 The structures of six compounds designed based on chrexanthomycin A are shown. Figure 1 Small molecules G4008 to G4013 are designed based on the chemical structural skeleton of chrexanthomycin A, 7H-pyridocarbazole and derivatives of 7H-pyrazinocarbazole.

[0013] Figure 2 The compound and C9orf72 NMR titration of DNA (G4C2)4G4. For small molecules G4008, G4009, G4010, G4011, G4012, and G4013, titrations with compounds (without G4: black, 1:1 red, 1:5 green, 1:10 purple) are shown. C9orf72 DNA (G4C2)4 1D 1 The imino region of the H spectrum.

[0014] Figure 3 The compound and C9orf72NMR titration of RNA (G4C2G4) G4. For small molecules G4008, G4009, G4010, G4011, G4012, and G4013, titrations with compounds (without G4: black, 1:1 red, 1:5 green, 1:10 purple) are shown. C9orf72 RNA (G4C2G4) G4's 1D 1 The imino region of the H spectrum.

[0015] Figures 4A to 4B Cellular assays of compounds in HT22 cells using MTT and ROS assays are shown. Figure 4A The MTT assays for six compounds are shown (the logarithmic concentrations corresponding to the six data points are 0.064 µM, 0.32 µM, 1.6 µM, 8 µM, 40 µM and 200 µM). Figure 4B ROS detection for 6 compounds is shown (x-axis: 6 compounds and related control group, y-axis: normalized ROS fluorescence intensity).

[0016] Figures 5A to 5B The compound was shown to treat [the disease] in a cell model. C9orf72 (G4C2) 29 Amplification-related symptoms. Figure 5A The results were shown in DMSO control or new compound (8 µM treatment) using C9orf72 DNA (G4C2) 29 Representative RNA FISH images of transfected Neuro2a cells. Detected using Cy3-labeled probes. C9orf72 RNA aggregates of G4C2 repeat sequences, with DAPI used as a counterstain (blue). Figure 5B The expression (G4C2) treated with the new compound is shown. 29 Each cell in the cells C9orf72 Quantification of the number of RNA G4C2 aggregates. n = 15-20 cells, p-value determined by one-way ANOVA.

[0017] Figures 6A to 6B The compound was shown in fruit flies (Drosophila melanogaster) Drosophila Treatment in the model C9orf72 (G4C2) 49 Amplification-related symptoms. Figure 6A Seven-day-old wild-type (WT) fruit flies fed with DMSO (100 μM), compounds G4010 (100 μM) and G4013 (100 μM) are shown, along with GMR-GAL4-(G4C2) in the larval stage. 49 Representative external eye images and scanning electron microscope (SEM) images of fruit flies. Figure 6BThe results show WT and GMR-GAL4-(G4C2) animals fed with DMSO (100 μM), compound G4010 (100 μM), and G4013 (100 μM). 49 Quantitative analysis of the percentage of eye degeneration in fruit flies 。 n = 7-18 animals; p-value was determined by one-way ANOVA (Tukey multiple comparison test).

[0018] Figure 7 The G4010 and G4013 are shown with C9orf72 ITC assays for DNA (G4C2)4 and RNA (G4C2G4) G4 were performed. ITC assays were conducted in a buffer containing 20 mM potassium phosphate (pH 7.0) and 70 mM KCl for G4010+ DNA (G4C2)4, G4010+ RNA (G4C2G4), G4013+ DNA (G4C2)4, and G4013+ RNA (G4C2G4). The compounds were dissolved in DMSO to a stock solution concentration of 10 mM.

[0019] Brief description of the sequence

[0020] SEQ ID NO: 1 corresponds to C9orf72 The DNA sequence d[GGGGCCGGGGCCGGGGCCGGGCC] of DNA (G4C2)4, consisting of 24 nucleotides, was purchased from Accurate Biotechnology (Hunan) Co., Ltd.

[0021] SEQ ID NO: 2 corresponds to C9orf72 The RNA sequence r[GGGGCCGGGG] of RNA (G4C2G4) consists of 10 nucleotides and was synthesized by Hunan Aikerui Biotechnology Co., Ltd.

[0022] SEQ ID NO: 3 corresponds to C9orf72 DNA (G4C2) 29The DNA sequence d[GGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCG GGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCC], consisting of 174 nucleotides, was inserted into the DNA plasmid pHR-Tre3G-29xGGGGCC-12xMS2 (Addgene #99149) purchased from Addgegne. Detailed Implementation

[0023] The chosen definition

[0024] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Furthermore, with respect to the use of the terms “comprising,” “including,” “containing,” “having,” “with,” or variations thereof in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional terms / phrases (and any grammatical variations thereof) “comprising,” “including,” “substantially consisting of,” and “consisting of” are used interchangeably.

[0025] The phrase “consistent essentially of” indicates that the claim covers embodiments that include the specified materials or steps as well as materials and steps that do not substantially affect the essential and novel features of the claim.

[0026] The term "about" refers to an acceptable range of error for a specific value, as determined by a person skilled in the art, where the error depends in part on how the value was measured, i.e., limitations of the measurement system. In the case of compositions containing a certain amount of component for which the term "about" is used, these compositions contain the amount of component whose variation (error range) around the value is 0% to 10% (X ± 10%). In other cases, the term "about" provides for a variation (error range) of 0% to 10% around a given value (X ± 10%). Clearly, this variation represents a range above or below the given value by up to 10%, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0027] In this disclosure, ranges are stated in a simplified form to avoid having to state and describe every and all values ​​within the range in detail. Where appropriate, any suitable value within the range may be chosen as the upper limit, lower limit, or endpoint of the range. For example, a range of 0.1 to 1.0 represents the endpoints 0.1 and 1.0, and the intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges included within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, 0.7 to 1.0, and so on. It is conceivable that a range has at least two significant figures; for example, a range of 5 to 10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including the endpoints. When ranges are used herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments thereof are explicitly included.

[0028] As used herein, the term "subject" refers to an animal, particularly a human, that needs or expects to receive the benefits provided by a therapeutic compound. The term "subject," as used herein, encompasses both humans and non-human animals. The term "non-human animal" includes all vertebrates, such as mammals, like non-human primates (especially higher primates), sheep, dogs, rodents (like mice or rats), guinea pigs, goats, pigs, cats, rabbits, and cows; and non-mammals, such as chickens, amphibians, reptiles, etc. These benefits may include, but are not limited to, treating a health condition, disease, or symptom; preventing a health condition, disease, or symptom; improving immune health; or enhancing the function of organs, tissues, or systems in the body. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute used as a disease model. In some embodiments, the term "subject" refers to mammals, including but not limited to rats, apes, humans, felines, canines, horses, cattle, farm mammals, racing mammals, and pet mammals. In the context of this invention, the subject is preferably a human. This term does not indicate a specific sex. Therefore, it is intended to cover both male and female subjects. Subjects can be at any age or developmental stage, including infancy, early childhood, adolescence, youth, adulthood, or old age.

[0029] As used herein, the terms “therapeutic effective amount,” “therapeutic effective dose,” “effective amount,” and “effective dose” refer to the amount or dose of a compound or composition that, when administered to a subject, is capable of treating, preventing, inhibiting, or improving the subject’s condition, disease, or symptom. In other words, the amount is “therapeutic effective” when administered to a subject. The actual amount will vary depending on many factors, including but not limited to the specific condition, disease, or symptom being treated or improved; the severity of the condition; the specific organ, tissue, or body system for which health or function is to be enhanced; the patient’s weight, height, age, and health status; and the route of administration.

[0030] As used herein, the term "treatment" means eradication; reduction; inhibition; improvement; elimination; relief; symptom reduction or delay of symptom onset; slowing of the rate of degeneration or worsening; lessening of the degree of weakness at the endpoint of degeneration; and / or improvement of the physical or mental health of a subject, or reversal to any extent of the signs or symptoms of a health condition, disease, or symptom, and includes, but does not require, a complete cure of the condition, disease, or symptom. Treatment can cure, improve, or partially improve a condition. "Treatment" can also include improving or enhancing a condition or characteristic, for example, bringing the function of a specific system in the body to a higher state of health or homeostasis.

[0031] As used herein, the terms “prevent,” “reduce,” “inhibit,” “block,” “prevent,” “relieve,” “delay,” “preemptively block,” “minimize,” or “mitigate” refer to the onset of a specific sign or symptom of a condition, disease, or symptom. Inhibition may be, but is not required to be, absolute or complete; this means that the sign or symptom may still progress later. Inhibition may include reducing the severity of the onset of the condition, disease, or symptom, and / or inhibiting the progression of the condition, disease, or symptom to a more severe condition, disease, or symptom.

[0032] When referring to a compound, “inhibition” means that the compound reduces the incidence, severity, lesion size, volume, or associated symptoms of ALS by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% compared to the normal presentation of pathological features in an ALS / FTD model without the application of the compound or with a composition containing the compound.

[0033] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to the mentioned nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implies coverage of variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologous sequences, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted with a mixture of bases and / or deoxyinosine residues at the third position. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0034] As used herein, the terms “low nucleotide” and “oligonucleotide” are used interchangeably to describe short single strands of synthetic DNA or RNA, such as sequences of about 5 to about 500 nucleic acid bases. “Low nucleotide delivery” and “oligonucleotide delivery” are also used interchangeably to describe methods of delivering oligonucleotides into cells or targeting specific cell types, tissues, or organs.

[0035] The term "G-tetrachain" refers to its common and general meaning. A G-tetrachain is a square arrangement of four guanine atoms, stabilized by Husstan hydrogen bonds. The G-tetrachain is helical and formed by stacking interconnected guanine tetrads (or "G-tetrads"), each a flattened ring structure of four guanine atoms, which can also be stabilized by the presence of a monovalent cation (such as potassium) at the center of the tetrad. G-tetrachains can be formed from DNA, RNA, or any combination thereof.

[0036] As used herein, the “isolated” or “purified” compound is substantially free of other compounds. In some embodiments, the purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the formulation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the target compound. For example, the purified compound is one in which the desired compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% by weight. Purity is determined by any suitable standard method, such as column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC).

[0037] In some implementations, treatment involves administering medication to a subject to significantly reduce or eliminate the formation and number of RNA aggregates in neurons. Compared to untreated subjects, this refers to a reduction in the number of aggregates of at least (or at least about) 30%. Therefore, treated subjects exhibit a significantly reduced or eliminated number of RNA aggregates, ranging from about 30% to about 99.99%, about 35% to about 99.99%, about 40% to about 99.99%, about 45% to about 99.99%, about 50% to about 99.99%, about 60% to about 99.99%, and about 70% to about 99.99%.

[0038] The term "gene" refers to a segment of DNA that is involved in the production of polypeptide chains; a gene includes regions before and after the coding region (leader and tail) that are involved in the transcription / translation and transcription / translation regulation of the gene product, as well as insertion sequences (introns) between individual coding segments (exons).

[0039] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. In particular, such non-toxic salt can be an inorganic or organic acid addition salt and a base addition salt.

[0040] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or carrier used for administering the compounds of the present invention. "Pharmaceutically acceptable carrier" refers to a substance that is non-toxic, biologically tolerable, and biologically suitable for administration to a subject, such as an inert substance, added to or otherwise used to facilitate the administration of a pharmaceutical composition, and is compatible with it. Examples of carriers include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0041] "Reduction" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0042] "Increase" refers to a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0043] As used herein, the terms “determine,” “measure,” “evaluate,” and “test” are used interchangeably and include both quantitative and qualitative determinations.

[0044] In some embodiments of the invention, the method includes administering multiple doses of the composition of the invention. The method may include administering a therapeutically effective dose of a composition comprising the compound or composition thereof described herein at frequencies of twice daily, once daily, every other day, three times weekly, once weekly, once monthly, or lower. In some embodiments, a dose is administered over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years. Furthermore, treating a subject with a therapeutically effective amount of the composition of the invention may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound or composition thereof used for treatment may be increased or decreased during a particular treatment. As is known in the art, dose variations can be determined by and are readily apparent from the results of diagnostic tests. In some embodiments of the invention, the method includes administering the composition once daily to several times daily, including but not limited to twice daily, three times daily, and four times daily.

[0045] In this document, the description of any chemical group listed in the definition of a variant includes the definition of a variant as any single group or combination of the listed groups. In this document, the description of embodiments of a variant or situation includes embodiments as any single embodiment or in combination with any other embodiment or part thereof.

[0046] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0047] The method of the present invention

[0048] In one aspect, the present invention relates to a novel composition for treating amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD), comprising: one or more compounds including, but not limited to, G4008, G4009, G4010, G4012 and G4013, or pharmaceutically acceptable salts, solvates or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0049] On another front, the present invention relates to a method for treating ALS and / or FTD, comprising administering to a subject an effective amount of a composition comprising, but not limited to, one or more compounds of G4008, G4009, G4010, G4012, and G4013. In some embodiments, one or more compounds of the composition may selectively recognize [a specific type of compound] in cells. C9orf72G4C2 G4. In a preferred embodiment, administration of the composition to a subject significantly reduces, inhibits, or improves one or more symptoms of ASL and / or FTD by about 70%. In a preferred embodiment, it significantly reduces, inhibits, or improves the progression of ASL and / or FTD by about 70%.

[0050] In preferred embodiments, the composition includes, but is not limited to: compounds G4008, G4010, G4012, and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In more preferred embodiments, the composition includes, but is not limited to: compounds G4010 and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In the most preferred embodiment, the composition comprises: compound G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition comprises: compound G4013 as the sole active agent, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition comprises, but is not limited to: compound G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0051] In some embodiments, by administering a composition comprising one or more of compounds G4008, G4010, G4012, and G4013, the number of RNA aggregates in the cells of subjects suffering from ALS and / or FTD is significantly reduced. In some embodiments, the number of aggregates is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In a preferred embodiment, the number of aggregates is reduced by an average of about 50% or more.

[0052] In some implementations, the application of the composition significantly reduces the level of reactive oxygen species (ROS) in the subject's cells.

[0053] In a preferred embodiment, G4013 is applied with high binding affinity targeting. C9orf72 G4C2 G4, where G4013 and C9orf72 DNA (G4C2)4 G4 binding affinity ( K d The value is approximately 114 μM, and it is similar to... C9orf72 The binding affinity of RNA (G4C2G4) G4 is approximately 68 μM.

[0054] In some embodiments, the subject is a mammal. In a preferred embodiment, the mammal is a primate. In a more preferred embodiment, the primate is a human. In some embodiments, the subject suffers from ALS and / or FTD.

[0055] In some embodiments, the therapeutically effective amount of the compositions of the present invention can be formulated for application by injection (e.g., bolus), parenteral administration, intravenous administration, intraperitoneal administration, or continuous infusion; or by oral, rectal, bronchial, nasal, topical, oral cavity, sublingual, percutaneous, vaginal, intramuscular, intraarterial, intracerebral, or intraocular administration; or by administration in forms suitable for inhalation or blowing, including powders and liquid aerosols; or by administration via a sustained-release system such as a semi-permeable membrane matrix of a solid hydrophobic polymer. Administration can also be carried out via other carriers or delivery systems, such as patches, micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc.

[0056] Depending on the method of application, the composition can be administered in various unit dosage forms. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained-release formulations, lipid complexes, etc.

[0057] In various embodiments, the active agents described herein (e.g., G4008, G4009, G4010, G4012, and G4013) can be combined with pharmaceutically acceptable carriers (excipients) to form pharmacological compositions. In some embodiments, pharmaceutically acceptable carriers include those approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals, and more particularly for humans. "Carrier" refers to, for example, a diluent, adjuvant, excipient, auxiliary agent, or transporter used for administering the active agents of the present invention.

[0058] Furthermore, with or without preservatives, the composition may be present in unit dose form in ampoules, pre-filled syringes, and small-volume infusions, or in multi-dose containers. The composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier. The composition may also contain formulation reagents such as suspending agents, stabilizers, and / or dispersants. In other embodiments, prior to use, the active ingredient of the composition according to the invention may be in powder form, obtained by aseptic separation of a sterile solid, or by lyophilization of a solution with a suitable carrier such as sterile pyrogen-free water.

[0059] The compositions of the present invention may also comprise one or more pharmaceutically acceptable carriers and / or excipients, and may be formulated into liquid forms, such as solutions or suspensions for injection. Solutions or suspensions may comprise suitable non-toxic, parenteral-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution; or suitable dispersants or wetting agents and suspending agents, such as sterile, non-irritating fixed oils, including synthetic monoglycerides or diglycerides, and fatty acids including oleic acid.

[0060] The carriers and / or excipients according to the invention may include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions suitable for, for example, intravenous use, with or without organic cosolvents, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners (e.g., carbomer, gelatin, or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, compatibilizers (e.g., lactose, mannitol), etc. The use of carriers and / or excipients in the pharmaceutical and supplement fields is well known. The use of carriers or excipients in the compositions of this invention may be considered, except for any conventional media or reagents incompatible with the target health-promoting substance or composition.

[0061] Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that function, for example, to stabilize the composition or to increase or decrease the absorption of the active agent. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; protective and uptake enhancers such as lipids; compositions that reduce the scavenging or hydrolysis of the active agent; or excipients or other stabilizers and / or buffers.

[0062] Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives specifically designed to inhibit the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Those skilled in the art will understand that the choice of pharmaceutically acceptable carriers, including physiologically acceptable compounds, depends, for example, on the route of administration of the active agent and the specific physicochemical properties of the active agent.

[0063] In one embodiment, the excipient is sterile and generally free of unwanted substances. These compositions can be sterilized using conventional and well-known sterilization techniques.

[0064] In some therapeutic applications, a compound or composition is therapeutically administered to a subject scheduled for chemotherapy in an amount sufficient to reduce and / or inhibit the symptoms, morbidity, severity, and / or progression of the disease. The amount sufficient to achieve this goal is defined as the “therapeutic effective dose.” The effective dose for therapeutic use will depend on the subject’s expected response to the active agent. Single or multiple administrations of the active agent may be used, depending on the dose and frequency required and tolerated by the subject. In any case, treatment should provide an adequate amount of the compound or composition to effectively reduce, inhibit, or improve one or more symptoms and the progression of ASL and / or FTD.

[0065] The concentration / amount of the compound or composition can vary widely and is selected primarily based on the activity of the active ingredient, body weight, and the chosen specific route of administration and patient needs. However, commonly chosen concentrations provide doses ranging from about 0.0005 mg / kg / day to about 1000 mg / kg / day, and sometimes higher doses. Typical dose ranges are from about 1.0 mg / kg / day to about 250 mg / kg / day, preferably from about 1.0 mg / kg / day to about 200 mg / kg / day, more preferably from about 1.0 mg / kg / day to about 150 mg / kg / day, and most preferably from about 1.0 mg / kg / day to about 100 mg / kg / day. Other dose ranges are preferably from about 600 mg / kg / day to about 1000 mg / kg / day, more preferably from 600 mg / kg / day to about 750 mg / kg / day; and most preferably from 600 mg / kg / day to about 650 mg / kg / day. It should be understood that such doses can be varied to optimize the treatment regimen for a particular subject.

[0066] In some instances, the composition is administered at a dose of about 10 mg / kg to about 250 mg / kg of the subject's body weight. In preferred instances, the composition is administered at a dose of about 15 mg / kg to about 200 mg / kg of the subject's body weight. In more preferred instances, the composition is administered at a dose of about 25 mg / kg to about 200 mg / kg of the subject's body weight.

[0067] In some embodiments of the invention, the method includes administering multiple doses of the compound of the invention. The method may include administering a composition comprising the compound of the invention described herein in therapeutically effective doses once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, thirty times, thirty-five times, forty times, or more. In some embodiments, the dose is administered over a period of 1 day, two days, three days, four days, five days, six days, seven days, ten days, fourteen days, twenty days, thirty days, or more than thirty days, or at a frequency of once weekly, once monthly, once quarterly, twice a year, once a year, or less. Furthermore, treating a subject with a therapeutically effective amount of the compound of the invention may include a single treatment or may include a series of treatments. It will also be understood that the effective dose of the compound used for treatment may be increased or decreased during a particular treatment course. Variations in dose may be determined by and are readily apparent from the results of diagnostic tests or imaging techniques known in the art for detecting the progression of ALS or FTD. In some embodiments of the invention, the method includes administering the compound multiple times daily, including but not limited to twice daily, three times daily, and four times daily. In some embodiments, the compound or composition is administered to the patient for approximately three to five days.

[0068] In some implementations, at least one dose of the composition is applied at least once daily, three times a week, twice a week, once a week, twice a month, once a year, or at least every 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 52 weeks, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, or longer. In some embodiments, repeated application of at least one dose of the composition occurs for a duration of at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 52 weeks, or longer. In a preferred embodiment, at least one dose of the composition is repeated every about 2 to about 5 days for a duration of about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. In a preferred embodiment, the composition is applied once daily for a duration of about 8 weeks to about 52 weeks. In a more preferred embodiment, at least one dose of the composition is repeatedly administered every about 2 to about 5 days for about 1 to about 5 years. Other embodiments of the invention include, but are not limited to, the following experimental methods and results: Materials and Methods

[0069] G4 DNA / RNA Sample Preparation

[0070] DNA / RNA oligonucleotides were purchased from Hunan Aikerui Biotechnology Co., Ltd. 100 µM DNA / RNA samples were placed in annealing buffer containing 20 mM potassium phosphate (pH 7.0) and 70 mM KCl, annealed at 97°C, and then slowly cooled to room temperature overnight. For 1D NMR screening, samples were prepared at a concentration of 50 µM using 5% D₂O.

[0071] NMR spectrum

[0072] Experiments were conducted on a 500 MHz and 800 MHz Varian spectrometer. All samples were diluted from the initial stock solution to 50 µM, with a final volume of 400 µL for NMR detection. 11H NMR spectroscopy identified the imino protons of guanine, and the data were analyzed using MestReNova. NMR titration was performed by adding a compound pre-dissolved in DMSO-d6 at a stock concentration of 100 mM.

[0073] One-dimensional 1 H-NMR titration experiment

[0074] To verify whether the compound interacts with G4 DNA / RNA, we ran a one-dimensional (1D) test at 25°C. 1 NMR titration experiments were performed using 1H NMR spectroscopy. All compounds were dissolved in isotopically labeled d6-DMSO at a concentration of approximately 100 mM as stock solutions. To avoid chemical shift changes in G4 DNA / RNA due to the addition of d6-DMSO, 10 μL or 15 μL of d6-DMSO were added to 400 μL of 0.1 mM G4 DNA / RNA NMR buffer (20 mM potassium phosphate buffer and 70 mM KCl, pH 7.0, 10% D2O), respectively, followed by 1D NMR titration. 1 ¹H-NMR spectra. In NMR titration experiments where each compound was titrated into G4 DNA / RNA solution, the maximum volume of d6-DMSO solution for each compound, 10 μL or 15 μL, was considered the final data point.

[0075] Isothermal titration calorimetry (ITC)

[0076] Intracellular titration (ITC) is commonly used for the quantitative analysis of interactions between two molecules. It provides valuable information through association and dissociation constants, such as the binding stoichiometry, kinetics, and affinity of the complex. The Malvern Panalytical MicroCal PEAQ-ITC is used to determine the affinity between ligands and DNA / RNA. Since the cell volume is 280 µL, sample preparation requires at least 400 µL, while the ligand used as the titrant requires 40 µL. The molar ratio depends on the specific case and typically begins at 10–20:1 (titer:cell). All samples must be kept under the same buffer conditions (including additives). A default titration procedure is typically 18 injections, with each injection titrating 2 µL (the first injection being 0.4 µL).

[0077] Cell culture

[0078] Neuro2a cells were initially seeded onto glass coverslips in 12-well plates. Then, the DNA plasmids pHR-Tre3G-29xGGGGCC-12xMS2 (Addgene #99149) and pCAG-TetON-3G (Addgene #96963) were co-transfected into the cells using a Lipofectamine 3000 (Thermo Fisher Scientific). 1 µg of plasmid was transfected per well according to the manufacturer's protocol. The plasmids were incubated for over 10 hours for expression and induced overnight at 37°C with doxycycline (1000 ng / mL).

[0079] transfection

[0080] Transfect the DNA construct at 7 DIV using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) or Lipofectamine LTX with Plus reagent (Thermo Fisher Scientific, 15338100). 4 to 6 h post-transfection, replace the transfection medium with fresh medium and incubate the cells for another 48 h (HT22) or longer (primary neurons; 5 to 8 days) to allow for recovery and construct expression.

[0081] MTT testing

[0082] The cytotoxicity levels of different concentrations of the compound were determined using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to determine cell growth inhibition (IC50) (see van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay). Methods Mol Biol , 2011;731:237-45. doi: 10.1007 / 978-1-61779-080-5_20. PMID: 21516412). Mitochondrial activity in cells can be reflected by converting tetrazolium salt to formazan crystals, which are then further dissolved in DMSO for measurement. First, 5 × 10⁻⁶... 3HT22 cells were seeded in 96-well plates and cultured overnight. Cells were then treated with different concentrations of the added compound and diluted in culture medium overnight. 10 µL of MTT (5 mg / ml) diluted in PBS buffer was added to each well, and the plates were incubated at 37°C for 4 hours. Then, 100 µL of DMSO was added to each well to dissolve the thoroughly pre-washed crystals. Absorbance was then recorded at 570 nm using a CLARIOstar PLUS microplate reader (BMG LABTECH). IC50 was then analyzed using a GraphPad Prism.

[0083] Reactive oxygen species (ROS) detection

[0084] HT22 cells were prepared in 96-well plates in DMEM medium supplemented with 5 mM L-glutamate and incubated overnight at 37°C. Then, following the manufacturer's (Beyotime) protocol, the ROS detection kit was used to test reactive oxygen species levels. 10 µM of the DCFH-DA fluorescent probe was added to the cells, followed by pre-washing with DMEM-only medium at 37°C for 30 minutes, gently agitated every 5 minutes. The cells were then washed three times with DMEM-only medium to remove excess probe. Subsequently, the appropriate compound (2 µM) and control (Rosup: 50 µg / mL) were added, and incubation was performed for one hour. The fluorescence intensity of the oxidation product, dichlorofluorescein (DCF), was measured using a CLARIOstar PLUS microplate reader (BMG LABTECH) with a 488 / 525 nm filter.

[0085] fruit fly strains

[0086] (G4C2) n The repetitive sequence-related Drosophila strain and eye development expression promoters were obtained from Bloomington. Drosophila Stock Centre (BDSC), while wild-type strains such as w (CS10) were provided by Professor Yukinori's research group. All fruit flies were reared at 25°C and humidity controlled between 56% and 60%. Fruit fly food was prepared the day before the addition of DMSO control and 100 µM of various promising compounds. Then, the next day, parent fruit flies were crossbred on solidified food containing the test compounds, and the larvae were also fed food containing the compounds until emergence.

[0087] Eye imaging using bright-field microscopy

[0088] All adult fruit flies that emerged after molting were anesthetized with CO2 and photographed on day 5 using a camera (Canon EOS Kiss X7 Dual Zoom Kit) on a microscope (Nikon SMZ 745T) to capture the condition of the entire eye, followed by scanning electron microscopy examination. The level of eye degeneration was then examined accordingly and the flies were collected individually.

[0089] Eye imaging using scanning electron microscopy (SEM)

[0090] The fruit flies isolated in the previous step were further treated with 2.5% glutaraldehyde and fixed overnight at 4°C. Then, the fruit flies were dehydrated sequentially in a gradient of acetone for 12 hours daily (30%, 50%, 70%, 90%, 95%, 100% twice). The dehydrated fruit flies were then air-dried, and the compound eyes were dissected using an optical microscope. Finally, the compound eyes were gold-plated before SEM (JSM-6390).

[0091] All patents, patent applications, provisional applications and publications referenced or cited herein are incorporated herein by reference in their entirety, including all figures and tables, unless they contradict the express teachings of this specification.

[0092] The following are examples illustrating the process of carrying out the present invention. These examples should not be construed as limiting. Unless otherwise stated, all percentages are by weight and all solvent mixture proportions are by volume.

[0093] Example 1—Design of a new compound based on chrexanthomycin A

[0094] ALS is a fatal degenerative neurological disease that is currently incurable. C9orf72 The six nucleotides (G4C2) in the first non-coding region of the gene. n Abnormal amplification (n ranging from 30 to thousands) is the most common genetic cause of ALS / FTD. C9orf72 DNA / RNA G4C2 repeat sequences can fold into the secondary structure G4. Several studies have shown that... C9orf72G4C2 G4On-Off-Current-Current-Also-AlS-Current-Current-Current-Current-CurveZ. Su, Y, Zhang, TF Gendron, PBauer, J Chew, WY Yang, E Fostvedt, K Jansen-West, VV Belzil, P Desaro, A Johnston, K Overstreet, SY Oh, PK Todd, JD Berry, MECudkowicz, BF Boeve, D Dickson, MK Floeter, BJ Traynor, C Morelli, A.Ratti, V. Silani, R. Rademakers, RH Brown, JD Rothstein, KB Boylan, L.Petrucelli, MD Disney, Discovery of a Biomarker and Lead Small Molecules toTarget r(GGGGCC)-Associated Defects in c9FTD / ALS, 1999; Neuron , 84 (2014) 239;R.Simone, P. Fratta, S. Neidle, GN Parkinson, AM Isaacs, G-quadruplexes:Emerging roles in neurodegenerative diseases and the non-codingtranscriptome, FEBS Lett , 589 (2015) 1653–1668A. Cheng, C. Liu, W. Ye, D.Huang, W. She, X. Liu, CP Fung, N. Xu, MC Suen, W. Ye, HHY Sung, IDWilliams, G. Zhu, PY Qian, Selective C9orf72 G-Quadruplex-Binding SmallMolecules Ameliorate Pathological Signatures of ALS / FTD Models J Med Chem,65 (2022) 12825-12837). In our previous study, we identified the marine-derived natural product chrexanthomycin A, which can specifically recognize C9orf72 DNA / RNA G4C2 G4. However, chrexanthomycin A has a weaker binding affinity, among which... K d ~2.2 ± 0.1 mM, 3.0 ± 0.1 mM, and 2.8 ± 0.1 mM. To develop highly selective, specific, and affinity targeting... C9orf72 Based on the DNA / RNA G4C2 G4 lead compound, we designed and synthesized six small molecules (G4008 to G4013) based on the structure of chrexanthomycin A. These are derivatives of 7H-pyridocarbazole and 7H-pyrazinocarbazole and contain four fused rings. Figure 1 ).

[0095] Example 2—Using C9orf72 NMR titration of these designed compounds by DNA / RNA G4C2 G4

[0096] As we all know, C9orf72 DNA G4C2 repeat sequences can fold into G4, including both antiparallel and obliquity forms (see YY Geng, CD Liu, QX Cai, ZP Luo, HT Miao, X.Shi, NN Xu, CP Fung, TT Choy, B. Yan, N. Li, PY Qian, B. Zhou, G.Zhu, Crystal structure of parallel G-quadruplex formed by the two-repeat ALS-and FTD-related GGGGCC sequence). Nucleic Acids Res , 49 (2021) 5881-5890; B. Zhou, C. Liu, Y. Geng, G. Zhu, Topology of a G-quadruplex DNA formed byC9orf72 hexanucleotide repeats associated with ALS and FTD, Sci Rep , 5 (2015)16673). We use C9orf72 DNA / RNA G4C2 G4 were used to titrate these compounds using NMR. For C9orf72 The DNA G4C2 sequence was determined by NMR titration using d(G4C2)4. Figure 2 As shown, compounds G4008, G4009, G4010, G4012, and G4013 exhibit significant chemical shift changes in the imino proton region, indicating that these compounds are related to... C9orf72 d(G4C2)4 (SEQ ID NO: 1) binding.

[0097] for C9orf72 The RNA G4C2 sequence was analyzed by NMR titration using r(G4C2G4) (SEQ ID NO: 2). r(G4C2G4) folded into a forward-parallel G4 form (see reference YY Geng, CD Liu, NN Xu, MCSuen, HT Miao, YY Xie, BC Zhang, XQ Chen, YJ Song, ZX Wang, QXCai, G. Zhu, Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS / FTD). Nucleic Acids Res , 52(2024) 7961-7970). For example Figure 3 As shown, five of the six compounds, namely G4008, G4009, G4010, G4012, and G4013, exhibited significant chemical shift changes in the imino proton region, indicating that these compounds are related to... C9orf72 r(G4C2G4) binding. Further MTT assays for cytotoxicity showed that G4011 did not exhibit any associated cytotoxicity.

[0098] Example 3—The compound is able to rescue cells at the cellular level C9orf72 Symptoms related to G4C2 repeat sequences

[0099] Cytotoxicity is crucial for determining whether cells can withstand and tolerate high concentrations of drug treatment without significant cell death. To evaluate the cytotoxicity of these six novel compounds, the MTT assay was applied to HT22 cells, an immortalized mouse hippocampal neuronal cell line, to determine cell viability. Figure 4AFirst, a high concentration of 200 µM was applied, followed by five consecutive dilutions to obtain a total of six data points (0.064 µM, 0.32 µM, 1.6 µM, 8 µM, 40 µM, 200 µM). The calculated IC50 values, from highest to lowest toxicity, were as follows: G4008 (2.5 µM) > G4013 (10 µM) > G4009 (63 µM) > G4010 (79.4 µM) > G4012 (199.5 µM) > G4011 (no value). Even at concentrations up to 200 µM, compound G4011 showed no detectable cytotoxicity, therefore no inhibitory concentration was expected.

[0100] According to reports, in ALS / FTD, C9 Mutated astrocytes produce insufficient antioxidants, leading to loss of motor neuron defense capabilities and increased oxidative stress, ultimately resulting in neurodegeneration (see A. Birger, I. Ben-Dor, M. Ottolenghi, T. Turetsky, Y. Gil, S. Sweetat, L. Perez, V. Belzer, N. Casden, D. Steiner, M. Izrael, E. Galun, E. Feldman, O. Behar, B. Reubinoff, Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity). EBioMedicine , 50(2019) 274-289). Therefore, it is crucial to test whether these compounds have the ability to protect neurons from oxidative stress. Comparing the positive control (Rosup) with increased oxidative stress levels to the untreated negative control, all six compounds showed a significant reduction in glutamate-induced cellular ROS levels ( Figure 4B Overall, we selected G4008, G4009, G4010, G4012, and G4013 for expression. C9orf72 (G4C2) 29 Further in vivo testing will be conducted in cell models of the sequence. 。

[0101] Because NMR titration studies revealed C9orf72 The interaction between DNA / RNA G4C2 G4 and these compounds suggests that these compounds have similar biological activities. Therefore, the encoding... C9orf72 (G4C2) 29 The plasmid containing the DNA sequence (SEQ ID NO:3) was transfected into Neuro2a cells used as an ALS / FTD cell model. The amplified G4C2 repeat sequence can lead to ALS / FTD by isolating RNA-binding proteins (RBPs) and forming nuclear RNA G4C2 aggregates, which recruit specific RBPs (such as hnRNP H). Based on NMR titration results combined with cell viability assays (MTT assay), compounds G4008, G4009, G4010, G4012, and G4013 were selected for RNA FISH experiments to determine their bioactivity against RNA G4C2 aggregates via immunostaining. Figure 5A ).

[0102] like Figure 5B As shown, the number of RNA aggregates was significantly reduced in cells treated overnight with G4008, G4010, G4012, and G4013. Considering the IC50 values ​​in the MTT assay, we ultimately used G4010 and G4013 to target the expression of RNA aggregates. C9orf72 (G4C2) 49 Bioactivity tests were performed on the Drosophila sequence model.

[0103] Example 4—A compound can rescue fruit flies in... C9orf72 Symptoms related to G4C2 repeat sequences

[0104] In addition, we tested the G4010, G4012, and G4013 against... C9orf72 The bioactivity of ALS in Drosophila. Therefore, the effects on GMR-GAL4-(G4C2) are investigated. 49 The fruit flies were engineered to express (G4C2) only in their eyes. 49 RNA. Expresses a pathogenic repeat sequence of 49 characters (G4C2). 49 of C9 A Drosophila model of ALS confirmed that amplified G4C2 repeat sequences induce ocular degeneration. Figure 6B During the larval stage, mutant and wild-type (WT) Drosophila were fed solid food containing compounds G4010 and G4013 at an effective concentration of 100 μM. In external eye experiments, we observed GMR-GAL4-(G4C2) in 7-day-old larvae. 49 Adult fruit flies have black spots around their eyes, which are much rougher than those of age-matched WT fruit flies, indicating eye degeneration. Interestingly, the G4013 treatment makes the eyes smoother and more reflective. Figure 6A ), significantly inhibited (G4C2) 49The ocular degeneration in Drosophila significantly treated the structurally disordered ocular phenotype. In summary, these data demonstrate that G4013 rescued the ocular defects in Drosophila caused by the G4C2 repeat sequence. （ Figure 6A and Figure 6B However, G4010 cannot treat ocular degeneration. It is noteworthy that ITC trials showed that G4010 and G4013... C9orf72 DNA (G4C2)4 G4 and C9orf72 RNA (G4C2G4) G4 has a binding affinity approximately 100 times stronger than chrexanthomycin A, with G4010 and G4013 being particularly effective. C9orf72 DNA (G4C2)4 G4 K d They are approximately 46 μM and 114 μM, respectively; and G4010 and G4013 are... C9orf72 RNA (G4C2G4) G4 K d They are approximately 35 μM and 68 μM, respectively. Figure 7 ).

[0105] Therefore, this invention confirms the binding of these compounds. C9orf72 The ability of G4C2 to amplify specific targets and rescue pathological features in ALS / FTD cell and Drosophila models. C9orf72 G4C2 amplification is the most common genetic cause of ALS. All these data indicate that G4013 can recognize [the virus] with high binding affinity. C9orf72 G4C2 G4, and rescued the eye defects in fruit flies caused by G4C2 repetitive sequences.

[0106] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes will be apparent to those skilled in the art, and such modifications or changes will be included within the spirit and scope of this application and the appended claims. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are within the scope of this invention but are not limited thereto.

[0107] Exemplary Implementation

[0108] Implementation Scheme 1. A pharmaceutical composition comprising: one or more compounds selected from the group consisting of G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0109] Implementation Scheme 2. A method for treating a subject with amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD), the method comprising administering to the subject an effective amount of the composition according to Implementation Scheme 1.

[0110] Implementation Scheme 3. The method according to Implementation Scheme 2, wherein the composition comprises G4008, G4010, G4012 or G4013.

[0111] Implementation Scheme 4. The method according to Implementation Scheme 2, wherein the composition comprises G4010 or G4013.

[0112] Implementation Scheme 5. The method according to Implementation Scheme 2, wherein the composition comprises compound G4013.

[0113] Implementation Scheme 6. The method according to Implementation Schemes 2 to 5, wherein the composition is applied once, twice, three times, four times, or five times per week.

[0114] Implementation Scheme 7. The method according to Implementation Schemes 2 to 6, wherein the composition is administered at a dose of about 0.1 mg / kg to about 200 mg / kg.

[0115] Implementation Scheme 8. The method according to Implementation Schemes 2 to 7, wherein the composition is applied topically via intravitreal, intracranial, or intradiscal application, or systemically via intramuscular, intravascular (e.g., intravenous), intraoral, intradermal, intranasal, intrathecal, or subcutaneous application.

[0116] Implementation Scheme 9. The method described according to Implementation Schemes 2 to 8, wherein the subject is a human being.

[0117] Implementation Scheme 10. The method according to Implementation Schemes 2 to 9, wherein one or more compounds of the composition selectively target and bind to... C9orf72 The hexanucleotide repeat sequence (HRE) of GGGGCC (G4C2) (G4C2) n The resulting G-quadruplex (G4) is formed.

[0118] Implementation Scheme 11. The method according to Implementation Schemes 2 to 10, wherein the application of the composition improves one or more symptoms of ASL and / or FTD by about 70%.

[0119] Implementation Scheme 12. The method according to Implementation Schemes 2 to 10, wherein the application of the composition reduces the progress of ASL and / or FTD by about 70%.

[0120] Implementation Scheme 13. The method according to Implementation Scheme 3, wherein the number of RNA aggregates is significantly reduced by applying a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012 and G4013.

[0121] Implementation Scheme 14. The method according to Implementation Scheme 13, wherein the number of RNA aggregates is reduced by approximately 50%.

[0122] Implementation Scheme 15. The method according to Implementation Schemes 2 to 14, wherein the administration of the composition reduces the reactive oxygen species (ROS) level of the subject by approximately 40%.

[0123] Implementation Scheme 16. The method according to Implementation Scheme 5, wherein G4013 is applied with high binding affinity targeting C9orf72 G4C2 G4.

[0124] Implementation Scheme 17. The method according to Implementation Scheme 16, wherein G4013 and C9orf72 The binding affinity of DNA (G4C2)4G4 ( K d The value is approximately 114 μM.

[0125] Implementation Scheme 18. The method according to Implementation Scheme 16, wherein G4013 and C9orf72 The binding affinity of RNA (G4C2G4)G4 ( K d The value is approximately 68 μM.

[0126] Implementation Scheme 19. The method according to Implementation Scheme 2, wherein the composition comprises G4013 as the sole active agent.

Claims

1. A pharmaceutical composition comprising: one or more compounds selected from the group consisting of G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

2. A method for treating a subject with amyotrophic lateral sclerosis and / or frontotemporal dementia, the method comprising administering to the subject an effective amount of the composition according to claim 1.

3. The method of claim 2, wherein the composition comprises G4008, G4010, G4012 or G4013.

4. The method of claim 2, wherein the composition comprises G4010 or G4013.

5. The method of claim 2, wherein the composition comprises compound G4013.

6. The method of claim 2, wherein the composition is applied once, twice, three times, four times, or five times per week.

7. The method of claim 2, wherein the composition is administered at a dose of about 0.1 mg / kg to about 200 mg / kg.

8. The method of claim 2, wherein the composition is administered locally via intravitreal, intracranial, or intradiscal application, or systemically via intramuscular, intravascular (e.g., intravenous), intraoral, intradermal, intranasal, intrathecal, or subcutaneous application.

9. The method of claim 2, wherein the subject is a human.

10. The method of claim 2, wherein one or more compounds of the composition selectively target and bind to... C9orf72 The G-quadruplex (G4) is formed by the hexanucleotide repeat sequence (G4C2)n of GGGGGCC (G4C2).

11. The method of claim 2, wherein the application of said composition improves one or more symptoms of ASL and / or FTD by about 70%.

12. The method of claim 2, wherein the application of the composition reduces the progress of ASL and / or FTD by about 70%.

13. The method of claim 3, wherein the number of RNA aggregates is significantly reduced by applying a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012 and G4013.

14. The method of claim 13, wherein the number of RNA aggregates is reduced by about 50%.

15. The method of claim 2, wherein the administration of the composition reduces the reactive oxygen species (ROS) level of the subject by about 40%.

16. The method of claim 2, wherein G4013 is applied with high binding affinity to target C9orf72 G4C2G4.

17. The method of claim 16, wherein G4013 and C9orf72 DNA (G4C2)4 G4 binding affinity ( K d The value is approximately 114 μM.

18. The method of claim 16, wherein G4013 and C9orf72 RNA (G4C2G4) G4 binding affinity ( K d The value is approximately 68 μM.

19. The method of claim 2, wherein the composition comprises G4013 as the sole active agent.

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