Improved ambroxol for medical use

Abstract

The present invention relates to analogs of ambroxol and related compounds, compositions comprising the analogs of ambroxol and / or related compounds, and methods of preventing and / or treating various diseases and medical conditions by administering the analogs of ambroxol and related compounds.

Description

Technical Field

[0001] This invention relates to ambroxol, ambroxol hydrochloride and / or bromhexine derivatives or analogs, which are applicable to a variety of medical scenarios. Background Technology

[0002] Ambroxol, ambroxol hydrochloride, and their structurally related parent compound bromhexine are all mucolytics. Since the late 1970s, these drugs have been approved in several countries for the treatment of acute and chronic respiratory diseases and conditions associated with the production of excessive and / or highly viscous mucus.

[0003] Existing research suggests that ambroxol triggers lysosomal exocytosis through pH-dependent calcium release from acidic calcium stores (Fois G et al., Cell Calcium, 56(6): 628-637, 2015), thereby promoting mucus clearance, such as breaking down sputum. Simultaneously, it can stimulate type II alveolar epithelial cells to produce surfactant (Seiffert C et al., Toxicol Appl Pharmacol, 203(1): 27-35, 2005), reducing the adhesion of mucus to the respiratory tract wall. Furthermore, studies have found that ambroxol can effectively inhibit neuronal sodium channels, especially when administered in lozenge form, rapidly relieving pain associated with acute pharyngitis (de Mey C et al., Arzneimittel-Forschung, 28(5a): 889-898, 1978).

[0004] The reuse of ambroxol in many other medical applications has attracted considerable interest. For example, studies have reported that ambroxol can act as a “molecular chaperone” for the lysosomal enzyme β-glucocerebroside lipase (GCase, UniProt accession number P04062), thereby increasing the enzyme’s content and activity. This property suggests that ambroxol may be suitable for the treatment of Gaucher disease (Maegawa GHB et al., J Biol Chem, 284(35): 23502-23516, 2009), the most common lysosomal storage disease caused by GCase deficiency. Similarly, the ability of ambroxol to increase GCase activity may have therapeutic benefits for patients with Parkinson’s disease (PD) with loss-of-function mutations in the glucocerebroside lipase gene (GBA1) (McNeill A et al., Brain, 137(5): 1481-1495, 2014). A recent study showed that daily administration of ambroxol increased GCase activity in the brains of healthy non-human primates (Migdalska-Richards A et al., *Symcpse*, 71(7): e21967, 2017). The study also found that ambroxol enhanced transcription factor EB (TFEB)—a key regulator of autophagosome / lysosome genes—thereby increasing the levels of other lysosomal proteins, including cathepsin D. (For details, see, for example: Magalhaes, J., Gegg, ME, Migdalska-Richards, A., Schapir, AH, 2018. "Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons." *Sci Rep-uk*, 8, 1385).

[0005] Therefore, ambroxol remains a research hotspot, and related research investment continues to increase. In the process of researching and developing novel treatment methods and compositions based on ambroxol, the inventors designed modified ambroxol (i.e., ambroxol analogues). Research suggests that these modified compounds may possess one or more advantages over ambroxol, ambroxol hydrochloride, and bromhexine, such as higher stability, thereby prolonging their half-life and duration of action in vivo. Summary of the Invention

[0006] This invention is intended to introduce some concepts in a simplified form, which will be further elaborated in detail in the following specific embodiments. This invention is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. Other features, details, applications, and advantages of the claimed subject matter will become apparent from the following written specific embodiments, including the aspects shown in the drawings and the scope defined in the appended claims.

[0007] This invention relates to ambroxol analogues and related compounds, compositions comprising these compounds, and methods for preventing and / or treating various diseases and medical conditions by administering ambroxol analogues and related compounds.

[0008] More specifically, the present invention provides compounds of Formula I:

[0009]

[0010] in:

[0011] The R or X groups in the above diagram can be attached to any available carbon atom on the benzene ring;

[0012] R a Selected from hydrogen (H), hydroxyl (OH), and lower alkyl groups (e.g., C). 1-3 Alkyl groups, such as CH3 and CH2CH3, and lower alcohols (e.g., C45, CH32, CH2CH3) 1-3 Alcohols, such as CH2OH;

[0013] R b Selected from hydrogen (H) and lower alkyl groups (e.g., C1-3 alkyl groups, such as CH3 and CH2CH3);

[0014] R c With R d Each is independently selected from hydrogen (H), lower alkyl groups (e.g., C), and C2. 1-3 Alkyl groups, such as CH3 and CH2CH3; or R c With R d These are parts of a 4-membered, 5-membered, 6-membered, or 7-membered ring structure connecting the two (e.g., -R). c -NR d -(CH2) n - (where n is an integer selected from 1, 2, 3, and 4);

[0015] R 1 To R 14 They are independently selected from hydrogen (H) and deuterium (D);

[0016] X 1 With X 2Each is independently selected from fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and lower alkyl groups (e.g., C). 1-3 Alkyl groups, such as CH3, CH2CH3), and lower alkoxy groups (e.g., C10, CH2CH3). 1-3 Alkoxy groups, such as OCH3 and OCH2CH3), and lower alkylamines (e.g., C42-C ... 1-3 Alkylamines, such as NR b CH3 and NR b CH2CH3), lower acyl groups (e.g., C2) -4 Acyl groups, such as C(O)CH3, C(O)CH2CH3), nitro (NO2), nitrile (CN), sulfoxide (SO-R), sulfonate (SO2-R), and sulfate (O-SO2-OR), under the condition X 1 With X 2 It is not simultaneously bromine (Br); or its pharmaceutically acceptable salt, solvate, or prodrug.

[0017] In some embodiments, the present invention provides compounds of formula Ia:

[0018]

[0019] in:

[0020] Among them, R a R b R c and R d R 1 To R 14 X 1 With X 2 Consistent with the compound of formula I, under condition X 1 With X 2 It is not simultaneously bromine (Br); or its pharmaceutically acceptable salt, solvate, or prodrug.

[0021] Optionally, the present invention envisions that any one or all of the hydrogen atoms in the compound of Formula I may be replaced by deuterium atoms.

[0022] The analogues presented in this article unexpectedly exhibited superior metabolic stability, which may help improve the overall cost of the analogues. Furthermore, the improved stability of the analogues may also lead to greater exposure during administration. Simultaneously, the improved stability of the analogues compared to ambroxol alone may also reduce the need for dosing regimens. An additional potential advantage of the Formula I compounds is their antimetabolite activity (compared to ambroxol), for example, their ability to resist metabolism through the cleavage (e.g., oxidation) of covalent carbon-nitrogen bonds connecting the ring structures of compounds according to Formula I.

[0023] The present invention also provides a pharmaceutical composition comprising a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), and provides a method for preventing and / or treating a subject with a variety of diseases and medical conditions by administering a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof).

[0024] More specifically, the present invention provides methods for preventing and / or treating diseases and medical conditions selected from respiratory diseases and conditions (e.g., bronchopulmonary diseases, especially those associated with the production of excessive and / or highly viscous mucus), including pain associated with acute sore throat, lysosomal storage diseases (LSDs) (e.g., Gaucher disease), neurological diseases and conditions (e.g., Parkinson's disease), and other age-related diseases involving autophagy dysfunction (Dockrill P., ScienceAlert, February 2020).

[0025] In addition, the present invention provides methods for treating, alleviating and / or stabilizing symptoms associated with neurological diseases and conditions such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, frontotemporal dementia, Pick's disease, Gaucher disease and amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease)).

[0026] Furthermore, the present invention provides a method for extending the life expectancy of a subject. Specifically, the compound represented by Formula I can be used in the following methods: (a) treating, inhibiting, or slowing down aging in a subject; (b) treating, inhibiting, or slowing down age-related symptoms or age-related diseases in a subject; and / or (c) improving the subject's healthy lifespan, life expectancy, and / or mental acuity.

[0027] In a preferred embodiment, the subject is a mammal; in a more preferred embodiment, the mammal is a human, a domesticated animal (e.g., a dog, cat, horse), or a farm animal (e.g., a cow, pig). Attached Figure Description

[0028] The purpose and features of the present invention can be more clearly understood by referring to the following specific embodiments and accompanying drawings.

[0029] Figure 1 The structures of representative compounds (compounds 1-6) of this invention are provided;

[0030] Figure 2 A-2B demonstrates a single oral administration of ambroxol hydrochloride to female C57BL / 6 mice. Figure 2 A) with ZW010 hydrochloride (compound 1, also referred to herein as "ZW-010"; dosage 10 mg / kg, based on free base) Figure 2Following B), the mean curves (including standard deviation) of drug concentrations in plasma and brain (ng / mL and ng / g, respectively) over time (h) were analyzed. The results indicate that the exposure of ambroxol halogen analogues in brain tissue is significantly increased, making them ideal compounds for treating the neurological diseases described in this article.

[0031] Figure 3 This study demonstrated that both difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010") and dimethoxysubstituted ambroxol (compound 5, also referred to herein as "ZW-011") increased lysosomal volume. To obtain data, neurons derived from artificial pluripotent stem cells (iPSCs) were cultured in medium for 14 days; lysosomes were stained with a lysosomal tracker, fixed, and imaged using confocal microscopy; lysosomal volume was measured using IMARIS software (Bitplane).

[0032] Figure 4 This study demonstrated that both difluorosubstituted ambroxol (compound 1, also referred to as "ZW-010" in this paper) and dimethoxysubstituted ambroxol (compound 5, also referred to as "ZW-011" in this paper) could drive the expression of lysosomes, autophagosomes, and TFEB genes. To obtain data, N2A neuroblastoma cells were treated for 3 days with either 10 μmol DMSO, ZW-010 (difluoro substituted), or ZW-011 (dimethoxy substituted); cell samples were extracted, and mRNA was quantified using real-time quantitative polymerase chain reaction (qPCR).

[0033] Figure 5 This study demonstrates that in iPSC-derived human neurons, difluorosubstituted ambroxol (compound 1, also referred to as "ZW-010" in this paper) and dimethoxysubstituted ambroxol (compound 5, also referred to as "ZW-011" in this paper) upregulated lysosomal and autophagosome gene expression to a greater extent than ambroxol. To obtain data, iPSC-derived human neurons were treated for 3 days with DMSO, ZW-010 (difluorosubstituted), or ZW-011 (dimethoxysubstituted) (all at a concentration of 10 μmol); cells were extracted and mRNA was quantified by qPCR.

[0034] Figure 6 A and Figure 6 B demonstrated that difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010") can induce TFEB translocation to the nucleus. Figure 6A). To obtain data, iPSC-derived human neurons were co-cultured with difluorosubstituted ambroxol for 14 days; after cell fixation, endogenous TFEB was immunostained, and the nucleus was identified by 4',6-diamidinyl-2-phenylindole (DAPI) staining; imaging with a Leica SP8 confocal microscope showed that TFEB signals were distributed in a punctate pattern in the cytoplasm and nucleus. Figure 6 B) Quantitative analysis of TFEB punctate signals in the cell nucleus was performed using IMARIS software (Bitplane).

[0035] Figure 7 A and Figure 7 B demonstrated that both difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010") and dimethoxysubstituted ambroxol (compound 5, also referred to herein as "ZW-011") reduced β-amyloid (Aβ) 40 in iPSC-derived human neurons carrying Swedish amyloid precursor protein mutants. Figure 7 A) and Aβ42 ( Figure 7 B) Secretion levels. To obtain data, human neurons derived from iPSCs carrying the Swedish mutant were treated for 5 days with solvent (DMSO), ambroxol, ZW-010 (difluoro-substituted), or ZW-011 (dimethoxy-substituted) (all at a concentration of 10 μmol); the culture medium was collected and analyzed by enzyme-linked immunosorbent assay (ELISA);

[0036] Figure 8 Animal data were provided showing that ambroxol could prolong the lifespan of the B6D2F1 mouse model. Group 1 represented the control group animals that were not given ambroxol; Group 2 represented the animals that were given 50 mg / kg (by body weight) of ambroxol daily via feed starting from 2 months of age.

[0037] Figure 9 This study presents the results of a novel location recognition test performed on mice given ambroxol from 2 months of age and at 9 months of age. This cognitive test assesses short-term working memory, including the recognition of familiar objects placed in unfamiliar locations (see Magen et al., *Eur J Neurosc*, 35:870-880, 2012; Magen and Chesselle, *Journal of Parkinson's Disease*, 1:217-227, 2011). The discrimination index (DI) was used, calculated as: (time spent exploring an object in a novel location (t...)). novel - Time spent exploring familiar objects in their locations (t) familiar )) / (Time to explore objects in new locations (t) novel+ Time spent exploring familiar locations and objects (t) familiar This is used to assess the time (t) mice spend exploring the vicinity of objects in a new location. novel The ratio of the discrimination index to the total exploration time. A discrimination index greater than 0 indicates that the mouse has good location recognition memory;

[0038] Figure 10 A- Figure 10 D illustrates the effect of ambroxol on basal macroautophagy in mouse cells. Cultured mouse fibroblasts (NIH3T3 cells) expressing the tandem reporter gene mCherry-GFP-LC3 were exposed to a specified concentration of ambroxol in complete culture medium for 24 hours. Figure 10 A is a schematic diagram of the autophagy compartment being analyzed; Figure 10 B- Figure 10 D represents autophagy (AV) ( Figure 10 B) Autophagosomes (APG) Figure 10 C) and autolysostomy bodies (AUT) Figure 10 D) Quantity. All values ​​are mean ± standard error (sem); at least 2500 cells were quantitatively analyzed under each condition in three different experiments using high content microscopy. Significant differences were observed compared to the untreated group (0 μM ambroxol): *p<0.05, **p<0.01, ****p<0.001;

[0039] Figure 11 A and Figure 11 B shows the treatment results of ambroxol in a mouse model of Alzheimer's disease (AD). Detailed Implementation

[0040] I. Definition

[0041] The following definitions are provided for the specific terms used in the written description below.

[0042] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0043] This invention may include (open-ended) or "consistent with" the components mentioned herein, and may also include other ingredients or elements described herein. As used herein, "comprising" means including the listed elements or their structural or functional equivalents, as well as any other elements not listed. Unless the context otherwise suggests, "having" and "comprising" should also be understood as open-ended expressions. As used herein, "consistent with" means that the invention may include, in addition to the components listed in the claims, other components that do not fundamentally alter the essential and novel features of the claimed invention.

[0044] As used herein, "subject" refers to a vertebrate, preferably a mammal, more preferably a human, and even more preferably a domesticated animal, such as a pet; or a farm animal. Mammals include, but are not limited to, rodents, primates, humans, farm animals, sports animals, and pets. In other preferred embodiments, "subject" is a rodent (e.g., guinea pig, hamster, rat, mouse), a canine (e.g., dog), a feline (e.g., cat), an equine (e.g., horse), a primate (e.g., monkey or ape), a monkey (e.g., marmoset, baboon), or an ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In other embodiments, non-human mammals may be used, especially mammals commonly used as models of human therapeutic effects (e.g., rodents, primates, pigs, dogs, or rabbits). In preferred embodiments, "individual" or "patient" (i.e., treatment subject) refers to a mammal, particularly a non-human primate (e.g., ape, monkey), and most preferably a human.

[0045] As understood herein, an “effective amount” of the pharmaceutical composition of the present invention refers to an amount in which the composition can elicit a therapeutically beneficial response in a subject, such as promoting mucus clearance associated with respiratory diseases and symptoms, relieving pain associated with acute pharyngitis, improving symptoms of lysosomal storage diseases (LSDs) and neurological diseases and symptoms, or prolonging and / or increasing healthy lifespan, life expectancy and / or cognitive acuity (e.g., improving subject survival and / or healthy aging, reducing morbidity or the incidence of age-related diseases).

[0046] As used herein, “dosage” or “prescription ratio” refers to a physical discrete unit suitable for administration to a subject, each dose containing a predetermined amount of active pharmaceutical ingredient that is calculated to produce the expected therapeutic response.

[0047] The term "about" or "approximately" refers to a specific numerical value that is acceptable to those skilled in the art, a range that depends in part on the method of measurement or determination of the value (e.g., limitations of the measurement system). For example, "about" may refer to a range of ±20% of a given numerical value, preferably ±10%, more preferably ±5%, and even more preferably ±1%. Alternatively, particularly for biological systems or processes, the term may refer to a value within an order of magnitude, preferably within 5 times, more preferably within 2 times. Unless otherwise stated, "about" refers to an acceptable error range for a specific numerical value, such as ±1-20%, preferably ±1-10%, more preferably ±1-5%. In further embodiments, "about" should be understood as ±5%.

[0048] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other value or intermediate value within that range, is included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are also included within the scope of this invention, except for any explicitly excluded limits within those ranges. If the range contains one or two limits, then the range excluding either one or both of those limits is also included within the scope of this invention.

[0049] All percentages and proportions used herein are based on the total weight of the composition unless otherwise stated. All temperatures are in degrees Celsius unless otherwise specified. All measurements were performed at 25°C and standard atmospheric pressure unless otherwise specified.

[0050] All ranges listed herein include endpoints, including ranges expressed as "between two values." Terms such as "about," "usually," "substantially," and "approximately" should be interpreted as modifying a term or value to make it not absolute, but without affecting the understanding of the prior art. The definitions of these terms will be determined in conjunction with the specific circumstances and the term they modify, and based on the understanding of someone skilled in the art, and for the purpose of measuring a given value, include at least the degree of expected experimental error, technical error, and instrumental error.

[0051] As used herein, when “and / or” is used in a list of two or more items, it means that any one item in the list may be present, or any combination of two or more items in the list may be present. For example, if a composition describing the invention comprises features A, B and / or C, then the composition may comprise only feature A; only feature B; only feature C; a combination of features A and B; a combination of features A and C; a combination of features B and C; or a combination of features A, B and C.

[0052] As used herein, “lower alkyl” includes straight-chain alkyl, branched alkyl and cycloalkyl (e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, etc.) having 1 to 8 carbon atoms.

[0053] As used herein, “lower alcohols” include straight-chain, branched, or cycloalkyl alcohols (e.g., methanol, ethanol, propanol, etc.) having 1 to 8 carbon atoms and containing one or more hydroxyl groups (OH).

[0054] As used herein, “pharmaceutically acceptable salt” means a salt that retains the intended biological activity of a Formula I compound, including pharmaceutically acceptable acidic addition salts and basic addition salts. Suitable pharmaceutically acceptable acidic addition salts of Formula I compounds can be prepared from inorganic or organic acids. Examples of such inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, alicyclic, aromatic, heterocyclic, carboxylic acid, and sulfonic acid organic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkyl sulfonic acids, and aryl sulfonic acids. For more information on pharmaceutically acceptable salts, see Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, PA, 1995.

[0055] "Solvate" refers to any form formed by the solvation of a compound of formula I with a suitable solvent, such as a crystalline solvate or a complex formed by a solvent and a dissolved compound.

[0056] "Prodrugs" refer to compounds that are typically converted into Formula I compounds within a biological system via metabolic pathways (e.g., hydrolysis, reduction, or oxidation). For example, ester prodrugs of Formula I compounds containing a hydroxyl group can be converted into Formula I compounds in vivo via hydrolysis. Suitable esters of Formula I compounds containing a hydroxyl group include, but are not limited to, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene bis-β-hydroxynaphthyl esters, oxalates, ethanesulfonates, ditolyl tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylaminosulfonates, and quinates. As another example, ester prodrugs of Formula I compounds containing a carboxyl group can be converted into Formula I compounds in vivo via hydrolysis. Examples of ester prodrugs can be found in Leinweber FJ, *Drug Metabolism Review*, 1987, Vol. 18, pp. 379-439. Similarly, acyl prodrugs containing amino groups of formula I can be converted into formula I compounds in vivo via hydrolysis. For examples of prodrugs with these and other functional groups (including amino groups), see Prodrugs: Challenges and Rewards, edited by Valentino J. Stella, Springer, 2007.

[0057] As used herein, a "derivative" compound refers to a second compound derived from a first compound, such as a brominated derivative of a non-brominated parent compound. For example, ambroxol is a derivative of the parent compound bromhexine.

[0058] As used in this article, "lifespan" refers to the time until death; "healthy lifespan" or "healthy aging" refers to the time of life without serious illness (or in optimal health); "mental acuity" refers to a measure of a subject's mental abilities, such as the ability to concentrate, the duration of attention, and the speed of thought.

[0059] As used in this article, “nutrient sensing” refers to the ability of cells to sense and respond to fluctuations in nutrient levels. For a detailed description, see Efeyan et al., “Nutrient Sensing Mechanisms and Pathways”, Nature, 517:302-310 (2015) (in full by reference).

[0060] As used in this article, expressions such as "treat, suppress and / or alleviate aging, aging-related symptoms and / or aging-related diseases" refer to reducing the risk of developing symptoms of aging and / or degenerative diseases, delaying their onset, slowing their progression and / or reducing their severity and manifestations, including but not limited to preventing the occurrence, development or progression of symptoms of aging and degenerative diseases.

[0061] As used herein, “pharmaceuticalally acceptable carrier” means any carrier, diluent, or excipient that is compatible with the other components of the composition and has no harmful effect on the intended subject receiving the composition.

[0062] Compounds of the present invention

[0063] The compounds of this invention are analogs of ambroxol and related compounds (including bromhexine and ambroxol hydrochloride), and their structures are defined by Formula I. Table 1 shows the structures of ambroxol (chemical name: trans-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol), bromhexine (chemical name: 2-amino-3,5-dibromo-N-cyclohexyl-N-methylbenzylamine), and ambroxol hydrochloride.

[0064] Table 1. Structures of Ambroxol, Bromhexine, and Ambroxol Hydrochloride

[0065]

[0066] In some embodiments, the compound is selected from Figure 1The examples shown are halogenated or alkoxylated analogs of ambroxol and bromhexine. Studies suggest that these compounds may possess antimetabolites (compared to their counterparts), for example, resistance to metabolism mediated by the breaking of covalent carbon-nitrogen bonds in the intercyclic linkers of Formula I compounds (e.g., via oxidation) or the oxidation of aromatic ring carbon-hydrogen bonds to form phenolic metabolites. In other words, these compounds may exhibit greater stability.

[0067] The compounds of this invention can be used for the prevention and / or treatment of a variety of diseases and medical conditions, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, and can also be used in methods to prolong the life expectancy of subjects. In these methods, the more stable compounds of this invention may exhibit one or more superior pharmacokinetic properties (e.g., compared to corresponding compounds without deuterium substitution).

[0068] Preparation of the compounds of the present invention

[0069] The compounds of this invention can be prepared by methods known to those skilled in the art of organic synthesis. For example, U.S. Patent Application Publication No. US2004 / 0242700 (in whole, incorporated herein by reference) provides a synthetic scheme for ambroxol that can be easily adapted to synthesize analogs of ambroxol and related compounds. Furthermore, Lati B et al. disclosed a synthetic scheme for ambroxol and related compounds (including deuterium-containing analogs) in the *Journal of Labeled Compounds and Radiopharm*, 53:15-23, 2010 (in whole, incorporated herein by reference), which can also be easily adapted to prepare compounds of formula I. For example, compounds of formula I can be prepared by replacing 2-amino-3,5-dibromobenzaldehyde in Scheme 4 of Lati et al.'s 2010 document with a corresponding analog. In addition, the compounds of this invention can also be prepared by methods such as those illustrated herein.

[0070] Salts of the compounds of this invention

[0071] For compounds that typically contain acidic or basic groups (such as carboxyl or amino groups), these groups are not necessarily in the form of free acids or free bases. When referring to compounds of the present invention, it should be understood that this includes the salt form of the compound. Therefore, the scope of protection of the present invention includes salts of compounds of Formula I. Preferred salts are pharmaceutically acceptable salts.

[0072] The term "salt" includes addition salts of free acids or free bases. The term "pharmaceutically acceptable salt" refers to a salt whose toxicity spectrum is within an acceptable range and is suitable for pharmaceutical use. Pharmaceutically acceptable salts may have specific properties, such as high crystallinity, which are valuable in the practice of this invention, for example, in the synthesis, purification, or formulation processes of therapeutic compounds.

[0073] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic acids. Examples include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, dihydroxynaphthyl acid (purine), methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, trifluoromethanesulfonate, 2-hydroxyethanesulfonate, p-toluenesulfonate, sulfanilates, cyclohexylaminosulfonate, stearate, alginate, β-hydroxybutyrate, salicylate, galactobionic acid, oxalate, malonate, and galacturonic acid. Pharmaceutically unacceptable examples of acid addition salts include perchlorates and tetrafluoroborates. All of these acid addition salts can be prepared by reacting a compound of formula I with the corresponding acid.

[0074] Suitable pharmaceutically acceptable base addition salts of Formula I compounds include, for example, metal salts, including alkali metal salts, alkaline earth metal salts, and transition metal salts, such as calcium, magnesium, potassium, sodium, and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts prepared from basic amines, such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine), and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanates. All of these base addition salts can be prepared by reacting a Formula I compound with a corresponding base.

[0075] Application method of the compound of the present invention

[0076] As described above, the compounds of the present invention can be used to prevent and / or treat a variety of diseases and medical conditions, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, and can also be used in methods to extend the life expectancy of subjects. For example, the compounds of Formula I can be used in the following methods: (1) treating, inhibiting, or alleviating aging in subjects; (2) treating, inhibiting, or alleviating aging-related symptoms or aging-related diseases in subjects; and / or (3) improving the healthy lifespan, lifespan, and / or mental acuity of subjects.

[0077] In some other embodiments, the invention more specifically relates to methods for preventing and / or treating a disease or medical condition in a subject, wherein the disease or condition is selected from respiratory diseases and conditions (e.g., bronchopulmonary diseases, particularly those associated with excessive and / or high-viscosity mucus production), including acute pharyngitis-related pain, lysosomal storage diseases (LSDs), such as Gaucher disease, and neurological diseases and conditions (e.g., Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease, frontotemporal dementia, Pick's disease, Gaucher disease, and amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease), as well as other age-related diseases associated with autophagy dysfunction); wherein the method comprises administering an effective amount of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) to the subject. The invention also relates to the use of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) as a medicament in the prevention and / or treatment of a disease or medical condition selected from respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions. Furthermore, the present invention relates to a pharmaceutical composition for treating diseases or medical conditions selected from respiratory diseases and conditions, lysosomal storage diseases (LSDs) and nervous system diseases and conditions, the composition comprising a therapeutically effective amount of a compound of formula I (or a pharmaceutically acceptable salt, solvate or prodrug thereof) and a pharmaceutically acceptable carrier.

[0078] In some other embodiments, the present invention relates to a method for improving the healthy lifespan, life expectancy, and / or mental acuity of a subject, the method comprising administering to the subject an effective amount of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof). The invention also relates to the use of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) as a medicament in prolonging life expectancy and / or alleviating aging or age-related diseases or symptoms. Furthermore, the present invention relates to a pharmaceutical composition for prolonging life expectancy, the composition comprising a therapeutically effective amount of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) and a pharmaceutically acceptable carrier.

[0079] Examples of age-related diseases and symptoms include, but are not limited to, cardiovascular disease, metabolic syndrome, bone loss disorders, neurodegenerative diseases, prediabetes, diabetes, obesity, osteoporosis, coronary artery disease, cerebrovascular disease, heart attack, stroke, peripheral artery disease, aortic valve disease, mild cognitive impairment, predementia, dementia, macular degeneration, cataracts, thinning hair, graying hair, loss of mobility, decreased endurance, fatigue, increased susceptibility to infection, metabolic changes, biochemical changes, cardiac hypertrophy, heart failure, myocardial infarction, ischemia-reperfusion injury, inflammatory diseases, pro-inflammatory states, arthropathy, autoimmune diseases, and / or Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, frontotemporal dementia, Pick's disease, Gaucher disease, and amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease).

[0080] In the method of this invention, the daily dose of the compound administered to the subject may be approximately 20-500 mg / day, 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, or 250 mg-1500 mg / day. In some cases, doses higher or lower than the above ranges may also be used. The daily dose may be administered in multiple divided doses, for example, divided into 2-4 administrations per day. Furthermore, for long-term administration (such as in methods used to increase and / or improve healthy lifespan, life expectancy, and / or cognitive acuity in subjects), the preferred dosage of the compound is about 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 1200 mg / day, 1250 mg / day. g / day, 1300 mg / day, 1350 mg / day, or between 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, 250-1000 mg / day, 1000-1500 mg / day, 1500-2000 mg / day or between 1000-2000 mg / day, or less than 1000 mg / day, or about 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, and / or between 4-12 mg / kg / day.

[0081] In some preferred embodiments, the method of the present invention can be particularly applied to subjects who: suffer from acute pharyngitis-related pain, suffer from bronchopulmonary diseases associated with excessive and / or high-viscosity mucus production, suffer from lysosomal storage diseases (LSDs, such as Gaucher disease), and suffer from neurological diseases and conditions (such as Parkinson's syndromes (including Parkinson's disease and Lewy body dementia), Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Pick's disease, Gaucher disease, and / or frontotemporal dementia (FTD)).

[0082] Parkinson's disease and other diseases associated with autophagy dysfunction

[0083] Ambroxol (and ambroxol hydrochloride, bromhexine) and the ambroxol analogues of the present invention are particularly suitable for the treatment of age-related diseases associated with proteolytic / lysosomal, autophagy and chronic inflammatory dysfunction, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, frontotemporal dementia, Pick's disease, Gaucher disease and amyotrophic lateral sclerosis (ALS, i.e. Lou Gehrig's disease).

[0084] As mentioned above, ambroxol's ability to enhance GCase activity may offer therapeutic benefits to Parkinson's disease patients with loss-of-function mutations in the glucocerebrosidase gene (GBA1) (McNeill A et al., 2014, ibid.), and GBA1 mutation is a major genetic risk factor for Parkinson's disease (Do J et al., Neurodegenerative Diseases, 2019, 14:36). The anticipated enhancement of GCase activity from ambroxol treatment is expected to improve lysosomal degradation capacity, which could help clear intracellular accumulations of α-synuclein (aSyn)—a protein associated with the pathogenesis of Parkinson's disease (Migdalska-Richards A et al., 2016, ibid.). In addition, GCase deficiency is also associated with lysosomal and mitochondrial dysfunction observed in Parkinson's disease (Brooker and Kranc, 2021, Essays in Biochemistry, 65(7), pp. 873-883), which may explain, to some extent, the ameliorative effect of ambroxol on lysosomal and mitochondrial function (Magalhaes et al., 2018, Scientific Reports, 8(1), p. 1385). Based on the above research and promising preclinical data, the application of ambroxol as a disease-modifying compound for Parkinson's disease (see, for example, Silvera CRA et al., BMC Neurol, 19:20, 2019; Mullin S et al., JAMA Neurol, 77:427-434, 2020) and for lipid storage disease Gaucher disease (Zimran, Altarescu and Elstein, Blood Cells, Molecules & Diseases, 55(2), pp.134-137; Narita et al., Annals of Clinical and Translational Neurology, 3(3), pp.200-215) is being investigated in clinical trials.

[0085] However, the potential beneficial biological activities of ambroxol extend far beyond its role as a GCase molecular chaperone. Specifically, ambroxol has effects on the autophagy-endolysosomal network (AELN) (McNeill et al., 2014, Brain: A Journal of Neurology, 137(Pt 5), pp. 1481-1495; Fois et al., 2015, Cell Calcium, 58(6), pp. 628-637; Magalhaes et al., 2018, ibid.) and induced immune responses (Beeh et al., 2008, European Journal of Medical Research, 13(12), pp. 557-562; Kern and Schwickert, 2017, Journal of Pain Research). Ambroxol has broad implications for the treatment of a variety of diseases characterized by defects in the autophagy-endolysosomal network, immune system, or nociceptive perception. This includes the blocking of chronic pain-related pathways (Russo et al., 2022, *Pain* [preprint], doi.org / 10.1097 / j.pain.0000000000002693). Therefore, ambroxol may be helpful in treating a range of diseases. For example, many neurodegenerative diseases, including Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Alzheimer's disease, all exhibit defects in the production or degradation of neurotoxic proteins within the autophagy-endolysosomal network, which in turn leads to a chronic inflammatory environment in brain tissue.

[0086] The mechanisms by which ambroxol affects cellular processes have been explained. Ambroxol is an amphiphilic amine compound that can easily cross the cell membrane via passive diffusion, acting directly on the cytoplasm and organelles. Intracellularly, ambroxol is protonated as a weak base and is trapped in acidic subcellular compartments, including lysosomes and other structures in the late endosomal pathway (Magalhaes J et al., 2018, ibid.; Fois G et al., 2015, ibid.). These properties explain the high bioavailability of ambroxol and its tendency to accumulate in lipid-rich organ systems such as the brain, lungs, and skin (Mullin S et al., 2020, ibid.). In the cytoplasm, ambroxol also acts as a potent scavenger and stabilizer, scavenging free radicals generated during cellular metabolism and innate immune responses (Stetinova, Herout, and Kvetina, 2004, Clinical and Experimental Medicine, 4(3), pp. 152-158). Finally, studies have shown that ambroxol can exert its analgesic effect by directly blocking the Nav1.7 / 8 channels associated with neuropathic pain (Kern & Schwickert, 2017, ibid.).

[0087] Ambroxol's amphiphilic capture in acidic compartments affects the autophagy-endolysosomal network system in multiple ways. A key consequence of ambroxol protonation in lysosomes is the deacidification of the lysosomal cavity (Mullin S et al., 2020, ibid.; Lu S et al., PLOS One, 12:e0173771, 2017). This process mobilizes intracellular calcium stores, thereby activating lysosomal-associated transcription factor EB (TFEB). Activated TFEB repositions itself in the nucleus to initialize gene network transcription through key regulation of lysosomal organisms (Medina DL et al., Nature Cell Biology, 17:288-199, 2015). Upregulation of this gene network can enhance the cell’s ability to degrade longevity proteins (Sardiello et al., 2009, Science, 325(5939), pp.473-477), particularly proteins associated with neurodegenerative diseases, such as hyperphosphorylated tau protein (Martini-Stoica et al., 2018, The Journal of Experimental Medicine, 215(5), pp.2355-2377). Ambroxol can effectively activate TFEB in cell culture and in vivo experiments. Application of ambroxol derived from the brain (Magalhaes et al., 2018, ibid.), skin (McNeill et al., 2014, ibid.), or immune-related tissues (Choi et al., 2018, Antimicrobial Agents and Chemotherapy, 62(9)) results in upregulation of TFEB at the transcriptional, protein, and / or activity levels.Enhanced TFEB activity can drive the expression of various lysosomal enzymes, including cathepsin D, a known protease that degrades α-synuclein, β-amyloid, and tau proteins (see, for example, Suire CN et al., “Cathepsin D: A Candidate Link between Amyloid β-Protein and Tauopathy in Alzheimer Disease,” *J Exp Neurology*, 2, 10-15 (2021); and Sevlever D et al., “Cathepsin D Is the Main Lysosomal Enzyme Involved in the Degradation of α-Synuclein and Generation of Its Carboxy-Terminally Terminated Fragment”). Truncated Species, Biochemistry-US, 47, 9678-9687 (2008).

[0088] Relatedly, high doses of ambroxol can drive cellular autophagy into a secretory autophagy mode (see, for example, McNeill et al., 2014, ibid.; Silverira CRA et al., 2019, ibid.). Secretory autophagy involves the packaging of normal and defective proteins into membrane-bound organelles called autophagosomes and their secretion outside the cell. It is an alternative to degradative autophagy (where autophagosomes fuse with lysosomes to degrade and recycle their contents) (Rabouille C et al., *Journal of Cell Science*, 125:5251-5255, 2012). Upregulation of autophagy-dependent secretion is considered a potential pathway for disease modification in neurodegenerative diseases (Ponpuak M et al., *Curr Opin Cell Biol*, 35:106-116, 2015).

[0089] In Parkinson's disease and Alzheimer's disease, for example, the aggregateable proteins α-synuclein (in Parkinson's disease, see, for example, Dehay B et al., *Journal of Neuroscience*, 30:12535-1544, 2010) and β-amyloid (in Alzheimer's disease, see, for example, Vickers JC et al., *Exp Neurol*, 141:1-11, 1996) form degradation-resistant aggregates and accumulate in autophagic vesicles. In both diseases, these protein aggregates cause dysfunction at multiple points in the autophagy processing pathway, which may be alleviated by secretory unloading of the aggregates.

[0090] Ambroxol can act directly on the secretory autophagy system. In primary neuronal cultures derived from cells expressing Parkinson's disease pathological features and unmodified control cells, ambroxol promotes the secretion of α-synuclein while clearing intracellular aggregated forms of this protein (Magalhaes J et al., 2018, ibid.). This result can be further extended to Alzheimer's disease pathology research, where bromhexine, when applied to brain cell cultures, drives the clearance of intracellular aggregated tau protein (Chauhan S et al., *Nature Communications*, 6:8620, 2015). In summary, these data suggest that ambroxol can play a beneficial role in the autophagy-lysosomal network by enhancing the inherent degradation capacity of cells and unloading toxic protein aggregates from cells with lower inherent degradation capacity, such as neurons.

[0091] Another important human health system affected by ambroxol is the immune response. Epidemiological and preclinical studies have shown that promoting the immune response from an inherent inflammatory response to an anti-inflammatory / adaptive immune state may be beneficial for a variety of chronic disease states. For example, in a variety of neurodegenerative diseases, longitudinal sampling of patients' serum and cerebrospinal fluid has shown a gradual increase in inflammatory markers, such as cytokines like interleukin-1β, 6, 8, and tumor necrosis factor-α, as well as the level of NOD-like receptor heat protein domain-associated protein 3 (NLRP3)-associated inflammasomes, which reflects a decline in cognitive function (Heneka et al., 2015, The Lancet Neurology, 14(4), pp. 388-405; Wang, Liu and Zhou, 2015, Translational Neurodegeneration, 4, p. 19). Correspondingly, many gene variations with innate immune regulatory functions are associated with an increased risk of Alzheimer's disease and Parkinson's disease (Hollingworth et al., 2011, Nature Genetics, 43(5), pp.429-435; Griciuc and Tanzi, 2021, Current Opinion in Neurology, 34(2), pp.228-236). Conversely, upregulation of anti-inflammatory and adaptive immune markers, such as interferon-γ and interleukin-12, is associated with a decreased incidence of Alzheimer's disease in the elderly (Yang et al., 2022, Alzheimer's & Dementia, 18(4), pp.645-653).

[0092] Neurodegenerative diseases are a classic example of chronic inflammation, which occurs when the immune system is unable to alleviate immunostimuli over a prolonged period. In neurodegenerative diseases, these immunostimuli may originate in part from toxic aggregates produced by cells within the brain. Chronic inflammation can also occur in cases of persistent infection, such as mycoplasma pneumonia and inappropriate immune activation caused by autoantigens.

[0093] Regarding inflammation, studies have shown that ambroxol can significantly remodel the immune system’s response to pathogens and stimuli of its own origin. Studies across multiple organ systems, particularly the brain (Jiang et al., 2020, BioMedResearch International, 2020, p.e8131286), the lungs (Takeda et al., 2016, Immune Network, 16(3), pp.165-175; Zhang et al., 2016; Kókai et al., 2021, Microorganisms, 9(4), p.880), and the gut (Schneider et al., 2021, EMBOMolecular Medicine, 13(1), p.e12724; Cavalu et al., 2022, The FASEB Journal, 36(9), p.e22496), have demonstrated that ambroxol can reduce the pro-inflammatory response of the immune system to these stimuli while preserving the adaptive characteristics of the immune response. Specifically, studies have shown that ambroxol can reduce the expression of pro-inflammatory cytokines, such as interleukin-1β, 6, 8, 10 and tumor necrosis factor-α (Bianchi et al., 1990, Agents and Actions, 31(3-4), pp. 275-279; Jang et al., 2003, Pharmacology & Toxicology, 92(4), pp. 173-179; Wang et al., 2011; Zhongguo YingYong et al., Chinese Journal of Applied Physiology, 27(2), pp. 231-235), and can also reduce the activation of upstream pathways of inflammasomes, such as nuclear factor κB (NFκB) (Cavalu et al., 2022, ibid.). This inhibitory effect is at least partly attributable to the ability of ambroxol to scavenge free radicals (Peroni et al., 2013, International Journal of Immunopathology and Pharmacology, 26(4), pp. 868-876), and may also involve direct blockade of specific inflammation-related pathways (Schneider et al., 2021, ibid.).

[0094] Unlike nonspecific immunosuppressants, such as NSAIDs, ambroxol preserves and, in some cases, enhances the function of the anti-inflammatory and adaptive immune systems. This includes upregulation of the anti-inflammatory cytokines interleukin-10 and 12, as well as adaptive immune-associated interferon-γ in lung tissue in response to pathogen and ovalbumin attack (Takeda et al., 2016, ibid.; Kókai et al., 2021, ibid.). These immune effector factors may explain the known clinical benefits of ambroxol in chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) (Plomer and de Zeeuw, 2017, *MMW Fortschritte der Medizin*, 159(Supplement 5), pp. 22-33); furthermore, they may explain preclinical observations of reduced inflammation in a model of ulcerative colitis (Schneider et al., 2021, ibid.) and decreased microglial activation in a model of cerebral hemorrhage (Jiang et al., 2020, ibid.).

[0095] The aforementioned anti-inflammatory effects may be related to ambroxol's influence on autophagy. Autophagy has been noted to play a crucial role in regulating immune responses. Specifically, activation of TFEB function can drive the degradation of key inflammatory mediators, including inflammasome components such as NLRP3 and apoptosis-associated speckle-like protein (ASC) containing a caspase recruitment domain (Shi et al., *Nature Immunology*, 13(3), pp. 255-263 (2012); *Deretic*, 54(3), pp. 437-453 (2021)). Inflammasome inactivation achieved through degradation can suppress inflammatory responses, including reducing the production and release of pro-inflammatory cytokines such as interleukin-1β. Therefore, the immunomodulatory function of ambroxol may depend on its influence on autophagy.

[0096] Ambroxol also has a potent analgesic effect. From a mechanistic perspective, this effect is attributed to ambroxol's ability to block voltage-dependent sodium channels, including Nav1.7 (Lefler, Reckzeh and Nau, 2010, European Journal of Pharmacology, 630(1-3), pp.19-28) and 1.8 (Weiser and Wilson, 2002, Molecular Pharmacology, 62(3), pp.433-438), which are preferentially expressed in neurons involved in nociception (Bennett et al., 2019, Physiological Reviews, 99(2), pp.1079-1151). This mechanism explains, at least to some extent, the traditional use of ambroxol as a treatment for moderate to severe pain in acute pharyngitis (Fischer et al., 2002, Research on Drugs (Arzneimitte-Forschung), 52(4), pp.256-263). In addition, there has been increased research interest in using ambroxol to treat neuropathic pain (Russo et al., 2022, ibid.).

[0097] Furthermore, a novel mechanism for regulating TFEB nuclear transport and transcriptional function has recently been discovered—poly(ADP-ribosyl) ribosylation (PARsylation) (Kim et al., 2021; Chen et al., 2024). PARsylation is a reversible post-translational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) to transfer ADP-ribose units to target proteins. This modification recruits ubiquitin ligases to ubiquitinate target proteins, ultimately increasing target protein turnover through the ubiquitin-proteasome system (Vivelo et al., 2019).

[0098] For TFEB, poly(ADP-ribosylation) can drive its nuclear localization in a non-phosphorylation-dependent manner. As expected, nuclear localization enhances TFEB-initiated transcription, but there is an important limitation: in the poly(ADP-ribosylation) state, TFEB complexes with the Wnt / β-catenin protein, leading to transcriptional initiation of a gene set completely different from that of the CLEAR network (Kim et al., 2021). Essentially, poly(ADP-ribosylation) allows TFEB to flexibly adjust the proteins it activates according to the cell's real-time needs.

[0099] If ambroxol regulates the Wnt / β-catenin pathway via TFEB, it could explain several unexplained phenomena observed in ambroxol-treated brain cells. For example, in a mouse stroke model (dMCAO model), ambroxol preferentially induced cells from the brain's proliferative zone (called the subventricular zone) to differentiate into new neurons, rather than the more common reactive astrocytes. In a parallel in vitro model (hypoxic-glucose hypoxia in human induced pluripotent stem cells (iPSCs), researchers found that ambroxol enhanced β-catenin signaling and GCase (Ge et al., 2021). This is consistent with the expected effect that Wnt / β-catenin transcriptional regulation tends to result in neural progenitor cells having a neuronal fate rather than a glial cell fate (Gao et al., 2021; Kriska et al., 2021).

[0100] Other studies support a link between ambroxol and the TFEB-mediated Wnt / β-catenin pathway. One study confirmed the link between GCase and Wnt / β-catenin transcriptional regulation: in human induced pluripotent stem cell lines induced to differentiate into dopaminergic cells, Gaucher disease mutations lead to weakened Wnt / β-catenin signaling; while the addition of recombinant GCase restores Wnt / β-catenin signaling to wild-type levels (Awad et al., 2017).

[0101] In summary, while not wishing to be bound by theory, the above observations suggest that the analogues described herein, similar to ambroxol, may enhance the degradation and / or secretion of toxic intracellular proteins, protein fragments, misfolded proteins, protein aggregates, or debris associated with autophagy-lysosomal network impairment. These analogues may also modulate the immune response to a low-inflammatory state by scavenging free radicals and interacting with inflammation-associated pathways. Finally, these analogues may block pathways upregulated in disease states characterized by chronic pain.

[0102] Application significance—The ambroxol analogues described in this article offer great potential for the treatment of neurological diseases and conditions such as Parkinson's disease, Alzheimer's disease, and other age-related diseases associated with autophagy dysfunction. Even in the case of autophagy dysfunction, they can still induce affected cells to secrete toxic aggregates (such as aggregated α-synuclein, β-amyloid, and tau proteins) and maintain organelle health.

[0103] In some cases, co-administration of the ambroxol analogues described herein with drugs used to prevent and / or eliminate toxic aggregates may be advantageous.

[0104] Therefore, another aspect of the present invention provides a method for preventing, alleviating symptoms, and / or stabilizing the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD)), said method comprising administering to a subject an effective amount of ambroxol (or a related compound, such as ambroxol hydrochloride, bromhexine) or an analogue of the present invention, and one or more suitable anti-β-amyloid antibodies or fragments thereof. Such combination therapy may produce a synergistic effect, such as more effectively clearing disease-associated aggregated β-amyloid protein, as in Alzheimer's disease.

[0105] The ambroxol analogue and an anti-β-amyloid antibody or a fragment thereof may be administered as part of the same treatment regimen, even if they are administered as separate compositions. In this case, the ambroxol analogue and the anti-β-amyloid antibody or a fragment thereof may be administered simultaneously or sequentially in any order (e.g., at intervals of seconds, minutes, or even hours (e.g., 2-48 hours)).

[0106] The anti-β-amyloid antibody or a fragment thereof may be selected from antibodies known to those skilled in the art. Suitable antibodies may include human or humanized anti-Aβ monoclonal antibodies, such as bapineuzumab (Pfizer Inc. / Janssen Pharmaceutieals, Inc.), solanezumab (Eli Lilly and Company), gantenerumab (Hoffman-La Roche), crenezumab (Genentech, Inc.), ponezumab (Pfizer Inc.), donanemab (Eli Lilly and Company), and BAN2401 / lecanamab (BioArctic Neuroscience, AB / Eisai). Co., Ltd / Biogen, Inc.) and Aducanumab (Aduhelm™), (Biogen, Inc.) (see also van Dyck CH, Biol Psychiatry, 83(4): 311-319, 2018). Suitable antibody fragments may include fragments such as Fab fragments and single-chain antibodies targeting Aβ, including scFv molecules described by Sebollela A in J Neurochem, 142(6): 934-937, 2017, and by Zha J et al. in Scientific Reports, 6: 36631, 2016.

[0107] In some embodiments, the method is used to prevent, alleviate symptoms, and / or slow the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD). Therefore, the method can prevent the occurrence, development, or progression of the disease or condition, or prevent the occurrence, development, or progression of one or more symptoms or harmful features of the disease or condition (such as toxic aggregation of Aβ protein). It is known that the aggregation of β-amyloid and tau proteins occurs early in the onset of Alzheimer's disease; therefore, treatment methods that prevent and / or clear toxic aggregations of Aβ or tau by administering ambroxol (or related compounds) or analogues of the present invention have significant application potential.

[0108] In one variation, the present invention provides a method for preventing, alleviating symptoms, and / or slowing the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD)), the method comprising administering to the subject an effective amount of ambroxol (or a related compound, such as ambroxol hydrochloride, bromhexine) or an analogue of the present invention. That is, ambroxol or a related compound or an analogue of the present invention may be used as the sole active pharmaceutical ingredient.

[0109] In some embodiments of methods for preventing, alleviating symptoms and / or slowing the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher's disease and frontotemporal degeneration (FTD)), subjects may be selected based on appropriate biomarkers that indicate patients at risk for or in the early stages of Alzheimer's disease or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e. Lou Gehrig's disease), Pick's disease, Gaucher's disease and frontotemporal degeneration (FTD)). For Alzheimer's disease, changes in biomarker levels can be detected in cerebrospinal fluid (CSF) and / or blood in the early stages of the disease or preclinical period (see, for example, Janelidze S et al., "Plasma Ptau181 in Alzheimer's Disease: Relationship to Other Biomarkers, Differential Diagnosis, Neuropathology and Longitudinal Progression to Alzheimer's Dementia," *Nature Medicine*, 26: 379-386; also see Hansson et al., "The Alzheimer's Association Appropriate Use Recommendations for Blood Biomarkers in Alzheimer's Disease"). Disease), Alzheimer's & Dementia, 2669-2686 (2022).Therefore, in one instance, the method may further include detecting phosphorylated tau protein (p-tau) p-tau217 (i.e., tau phosphorylated at Thr-217 residues) (Palmqvist et al., "Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders", JAMA, 324:772-781 (2020)) or p-tau181 (i.e., the phosphorylated form of tau protein at Thr-181 site) (Janelidze et al., "Plasma P-tau181 In Alzheimer's Disease: Relationship To Others"). Biomarkers, Differential Diagnosis, Neuropathology and Longitudinal Progression To Alzheimer's Dementia, Nature Medicine, 26:379-386 (2020) or β-amyloid oligomers (Shea et al., SOBA: Development and Testing Of A Soluble Oligomer Binding Assay For Detection Of Amyloidogenic Toxic Oligomers, Proceedings of the National Academy of Sciences, 119:e2213157119 (2022)) to select subjects with elevated levels.These biomarkers can accurately distinguish between patients with normal cognitive function, mild cognitive impairment, or memory impairment in Alzheimer's disease; furthermore, studies have shown that these biomarkers can accurately predict which patients will progress to Alzheimer's disease (Janelidze et al., 2020; Palmqvist et al., "Prediction of Future Alzheimer's Disease Dementia Using Plasma Phospho-Tau Combined With Other Accessible Measures", *Nature Medicine*, 27:1034-1042 (2021); Shea et al., 2022; Jia J et al., "Biomarker Changes During 20 Years Preceding Alzheimer's Disease", *New England Journal of Medicine*). Journal of Medicine, 2024, Vol. 390, No. 8, pp. 712-722 (2024)). These same substances can be longitudinally monitored in treated subjects to assess treatment efficacy. Other p-tau subtypes can also be detected, such as p-tau231 (Ashton N.J. et al., Acta Neuropathol, 1-16 (2021)) or p-tau235 (Lantero-Rodriguez J. et al., Embo Molecular Medicine, 13, e15098 (2021)). Other potential biomarkers include glucose metabolism, or aggregates such as β-amyloid or tau protein detected by positron emission tomography (PET) (Therriault J et al., Nature Aging, 1-10 (2022), doi:10.1038 / s43587-022-00204-0; Iaccarino L et al., Journal of Alzheimer's Disease, 59, pp.603-614 (2017)).Biomarkers based on retinal imaging, including amyloid deposition and thinning of the neurofibrillary layer, can also be used for the early diagnosis of Alzheimer's disease (Snyder PJ et al., Alzheimer's Disease and Dementia: Diagnosis, Assessment and Disease Monitoring, 4, 169-178 (2016); Koronyo Y et al., JCI Insight, 2 (2017)).

[0110] Other appropriate biomarkers indicating patients at risk of Alzheimer's disease or other amyloidosis, or those in the early stages of disease progression, include, for example, the ε4 allele of the apolipoprotein E (ApoE) gene, an apolipoprotein E assay kit (C2N Diagnostics), pyroglutamate-modified β-amyloid (N3pG), pTau217, and / or the β-amyloid 42 / 40 ratio (Aβ42 / 40, C2N Diagnostics). Plasma neurofilament light chains (NFL) are elevated in a variety of neurodegenerative diseases; they are not specific to any one neurodegenerative disease but rather reflect the activity and severity of the disease. For example, in patients with multiple sclerosis, plasma neurofilament light chain levels are elevated if active demyelinating lesions are present, but decrease after immunosuppressant treatment (Barro C. and Zetterberg H., Acta Neurol Scand, (2021), doi:10.1111 / ane.13415). Plasma exosomes carrying neuronal proteins also hold promise as biomarkers for diagnosing various neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and brain injury (Rastogi S. et al., International Journal of Molecular Sciences, 22, 440 (2021)).

[0111] Other biomarkers used to identify at-risk patients include, for example, Roche's ElecsysAD (Aβ42, Aβ40, p-tau181, and total tau), Fujirebio's β-amyloid ratio assay (e.g., Aβ42 and Aβ40), Eli Lilly's Tauvid (detects tau protein on PET via fiourtaucipir) or Amyvid (detects amyloid protein on PET via florbetapir), Neuraeq (via fluorbetapen), or Vizamyl (flutemetamol).

[0112] In some embodiments of methods for preventing, alleviating symptoms and / or slowing the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher's disease and frontotemporal degeneration (FTD)), subjects may be selected based on a genotype of at least one gene or locus indicating a patient at risk for Alzheimer's disease or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e. Lou Gehrig's disease), Pick's disease, Gaucher's disease and frontotemporal degeneration (FTD)). For Alzheimer's disease, genotyping can be performed on the gene encoding apolipoprotein E (ApoE): subjects carrying the ApoE ε4 allele have a higher risk of developing Alzheimer's disease compared to subjects carrying the more common ε3 allele, while the ε2 allele may reduce the risk (Liu CC et al., *Nature Review Neurol*, 9(2): 106-118, 2013). Suitable ApoE genotyping methods are known to those skilled in the art, including, for example, suitable reverse transcription polymerase chain reaction (RT-PCR) protocols (see, for example, Zhong L et al., *Molecular Neurodegenerative Diseases*, 11:2, 2016). However, in other embodiments, subjects may be selected based on the detection results of p-tau217, p-tau181, and / or N3pG.

[0113] In some embodiments of methods for the prevention and / or treatment of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD)), it is preferred to administer to the subject a higher daily dose of ambroxol (or a related compound) or an analogue of the present invention, the daily dose being selectable from any of the following:

[0114] (i) A dose in which the peak concentration of the drug in the subject's serum is greater than 1 μM (e.g., 2-50 μM, 2-25 μM or 10-20 μM);

[0115] (ii) A dose that produces a peak drug concentration in the subject's brain tissue greater than 3 μM (e.g., 5-50 μM, 5-25 μM, or 10-20 μM); or

[0116] (iii) A dose of approximately 250 mg-1000 mg / day or 750-1000 mg / day;

[0117] This is because such high daily doses may be necessary to upregulate the secretion of toxic aggregates (e.g., aggregated Aβ) in affected cells by ambroxol (or related compounds) or analogues of the present invention.

[0118] Pharmaceutical Composition

[0119] On one hand, the present invention includes a composition comprising a therapeutically effective amount of a compound of formula I (e.g., Figure 1 The compound (as shown) or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier, are intended for the prevention and / or treatment of a variety of diseases and medical conditions in a subject, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, or for the purpose of prolonging the life expectancy of a subject. For example, a composition comprising a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) may be used for: (a) treating, inhibiting, or alleviating aging in a subject; (b) treating, inhibiting, or alleviating age-related symptoms or age-related diseases in a subject; and / or (c) improving the healthy lifespan, life expectancy, and / or mental acuity of a subject.

[0120] Compounds of Formula I (or pharmaceutically acceptable salts, solvates, or prodrugs thereof) may be administered as pharmaceutical compositions comprising a compound of Formula I and a pharmaceutically acceptable carrier. In such compositions, the weight percentage of the compound of Formula I may range from 0.1% to 99.9%.

[0121] Compounds of Formula I are preferably administered with a pharmaceutically acceptable carrier chosen based on the selected route of administration and standard pharmaceutical practice. Compounds of Formula I may be formulated into dosage forms according to standard methods in the field of pharmaceutical formulation. See Alfonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th edition (1990), Mack Publishing Co., Easton, Pennsylvania, USA. Suitable dosage forms may include, for example, tablets, capsules, solutions, injections, lozenges, suppositories, or suspensions. Suitable examples of the preparation of oral, topical, suppository, and injectable formulations of ambroxol, bromhexine, or other ambroxol derivatives (which can be easily adapted for use with the compounds of this invention) are disclosed, for example, in Examples 1-8 of WO 2005 / 007146 or their corresponding U.S. Patent Application Publication No. US2005 / 00148747, the entire contents of which are incorporated herein by reference.

[0122] On the other hand, the present invention provides the use of the compound of formula I in the preparation of a medicament for the prevention and / or treatment of a variety of diseases and medical conditions (including respiratory diseases and conditions, lysosomal storage diseases (LSDs) and neurological diseases and conditions) or for the prolongation of the life expectancy of the subject.

[0123] For injection, the Formula I compound can be mixed with a suitable carrier or diluent, such as water, oil (especially vegetable oil), ethanol, physiological saline, aqueous glucose solution and related sugar solutions, glycerol or ethylene glycol (such as propylene glycol or polyethylene glycol), or mixed with plant extracts as a supplement. The injection solution preferably contains a water-soluble salt of the active pharmaceutical ingredient. Stabilizers, antioxidants, and preservatives may also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and its salts, and sodium ethylenediaminetetraacetate (EDTA sodium). Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. The injection composition can be in the form of an aqueous solution, a non-aqueous solution, a dispersion, a suspension, or an emulsion.

[0124] For oral administration, a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) may be mixed with one or more solid inert components to prepare tablets, capsules, pills, powders, granules, or other suitable oral dosage forms. For example, a compound of formula I may be mixed with at least one excipient selected from fillers, binders, humectants, disintegrants, dissolution inhibitors, absorption enhancers, wetting agents, absorbents, or lubricants. In one tablet embodiment, a compound of formula I may be mixed with calcium carboxymethyl cellulose, magnesium stearate, mannitol, and starch, and then formulated into tablets using conventional compression methods.

[0125] For oral administration, Formula I compounds (or their pharmaceutically acceptable salts, solvates, or prodrugs) can also be formulated into liquid oral pharmaceutical compositions. Liquid oral dosage forms offer unique advantages over solid dosage forms such as tablets and capsules. For example, in many cases, the dose of the active ingredient required to treat a specific disease or condition requires multiple daily doses of tablets or capsules. Taking multiple tablets or capsules not only results in the ingestion of the active pharmaceutical ingredient but also multiple doses of the excipients used in the preparation of the tablets and capsules. These excipients (such as oils and alcohols) are often poorly tolerated by many patients and frequently cause stomach upset. Furthermore, liquid oral dosage forms have higher patient compliance than solid dosage forms because the administration of the active pharmaceutical ingredient only needs to be completed 1-2 times daily. Moreover, liquid oral dosage forms allow for rapid absorption of the active pharmaceutical ingredient from the gastrointestinal tract. Additionally, flavoring agents and / or taste enhancers can be added to liquid oral dosage forms to further improve patient acceptance and compliance.

[0126] Therefore, in some preferred embodiments, the compositions of the present invention are liquid oral pharmaceutical compositions. Such compositions may be particularly suitable for the prevention and / or treatment of lysosomal storage diseases (LSDs, such as Gaucher disease) or neurological disorders and conditions (such as Parkinson's disease). In some embodiments, the compositions comprise a "high-drug-loading" compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) to deliver an effective dose in a small volume (e.g., once, twice, or more daily doses of 5 mL). The high-drug-loading liquid oral pharmaceutical compositions of the present invention offer at least one of the following advantages: (1) better absorption in the gastrointestinal tract; (2) maintenance of effective blood drug concentrations over 24 hours; (3) fewer excipient-related adverse side effects compared to solid dosage forms; (4) fewer frequency of administration; and (5) better taste and oral comfort.

[0127] In one specific embodiment of the high-drug-load liquid oral pharmaceutical composition of the present invention, the composition comprises: (i) a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); and (ii) at least one pharmaceutically acceptable excipient; wherein the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in particulate form, the particulate having a core, the core comprising about 60 to about 97 weight percent of the active pharmaceutical ingredient and about 3 to 40 weight percent of the excipient, the weight percent being based on the total weight of the core.

[0128] In another specific embodiment of the high-drug-load liquid oral pharmaceutical composition of the present invention, the composition comprises: (i) a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); (ii) at least one pharmaceutically acceptable excipient; and (iii) a diluent; wherein the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in particulate form, the particulate having a core containing about 60 to about 97% by weight of the active pharmaceutical ingredient and about 3 to about 40% by weight of the excipient, the weight percentages being based on the total weight of the core; the core is coated with (iv) a water-soluble release coating, the amount of which increases the weight of the particulate by about 0.5 to about 5%; and (v) an enteric coating, the amount of which increases the weight of the particulate by about 0.5 to about 50%.

[0129] Such high-drug-load liquid oral pharmaceutical compositions can be prepared, for example, by the following methods: (a) preparing granules having a core containing about 60 to 97 weight percent of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) and about 3 to 40 weight percent of at least one pharmaceutically acceptable excipient, the weight percentages being based on the total weight of the core; (b) coating the granules with a water-soluble separating coating, the amount of which increases the granule weight by about 0.5 to about 5 percent; (c) coating the granules prepared in step (b) with an enteric coating, the amount of which increases the granule weight by about 0.5 to about 50 percent; and (d) preparing a liquid suspension comprising the enteric-coated granules prepared in step (c) and a liquid suspension, wherein the liquid suspension comprises a suspending agent, a carrier for improving the stability of the high-drug-load liquid oral pharmaceutical composition, and a diluent.

[0130] The compound of Formula I (or its pharmaceutically acceptable salt, solvate, or prodrug) is preferably in particulate form, said particulates having a particle size of about 0.00 micrometers to about 500 micrometers after micronization, more preferably about 100 to about 300 micrometers, about 150 to about 350 micrometers, or about 200 to about 350 micrometers; further preferably about 100 to about 200 micrometers, about 150 to about 250 micrometers, about 200 to about 300 micrometers, about 200 to about 400 micrometers, about 250 to about 350 micrometers, about 250 to about 450 micrometers, about 300 to about 400 micrometers, about 300 to about 500 micrometers, about 350 to about 450 micrometers, and / or about 400 to about 500 micrometers. Particle sizes within this range have been found to facilitate coating and eliminate the need for chewing during swallowing. For example, in a preferred embodiment, a particle size of about 250 to about 350 micrometers strikes a balance between coating feasibility and avoiding a harsh "texture." Particle size can be determined by laser light scattering, for example using a Malvern laser particle size analyzer (Malvern Mastersizer Apparatus MS2000) equipped with a Hydro S dispersion unit. For example, the micronization of compound I can be carried out in a dry state using a dry mill, such as a claw mill, pin mill, air jet mill, fluidized bed air jet mill, and ball mill.

[0131] A preferred excipient for preparing the granular core is a binder. The binder can be any water-soluble, pharmaceutically acceptable polymer. In one embodiment, the compound of Formula I (or its pharmaceutically acceptable salt, solvate, or prodrug) is in powder form. Since most powders have poor cohesiveness, a binder is needed to bind them together. Preferably, the binder is selected from povidone (polyvinylpyrrolidone), copovidone (vinylpyrrolidone-vinyl acetate copolymer), microcrystalline cellulose, powdered cellulose, crystalline cellulose, silicified microcrystalline cellulose, cellulose derivatives (such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose), starch, pregelatinized starch, polymethacrylates, compressible sugars, sucrose and sugar alcohols (such as mannitol, sorbitol, maltitol, and xylitol), and mixtures thereof. More preferably, the binder is hydroxypropyl cellulose (Klucel LF).

[0132] The granules used in high-drug-loading liquid oral pharmaceutical compositions are preferably prepared by direct spheroidization. The granule core comprises about 60 to about 97 weight percent of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) (preferably about 75 to about 97 weight percent, more preferably greater than 90 to 97 weight percent) and about 3 to about 40 weight percent of a binder (preferably about 3 to about 25 weight percent, more preferably about 3 to about 10 weight percent), the weight percentages being based on the total weight of the granule core. Granulation can be carried out under high-shear conditions (mixing granulation) or in a fluidized bed (fluidized bed granulation).

[0133] The granule core may optionally be coated with a water-soluble release coating, the amount of which may increase the granule weight by about 0.5% to about 5%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 2% to about 3%, about 2% to about 4%, or increase the granule weight by 1%, 2%, 3%, or 4%. It has been found that applying a water-soluble release coating can form smooth and uniform granules that are more readily accepted by enteric coatings. Preferred water-soluble polymers include hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (sodium and calcium salts), ethyl cellulose, methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), low-substituted hydroxypropyl cellulose (L-HPC), povidone, polyvinyl alcohol, polymers of acrylic acid and its salts, vinylpyrrolidone-vinyl acetate copolymers (e.g., Kolidon VA64, BASF), gelatin, guar gum, partially hydrolyzed starch, alginate, and xanthan gum. Most preferably, the water-soluble polymer is hydroxypropyl methylcellulose. The isolation coating is preferably applied by a bottom-spray fluidized bed coating machine equipped with a Wurster column.

[0134] In addition to the isolation coating, or in the absence of an isolation coating, the enteric coating may optionally be applied directly to the core particles or to the isolation coating previously applied to the core particles. Preferably, the amount of enteric coating may increase the particle weight by about 0.5% to about 50%, about 1% to about 40%, about 2% to about 30%, about 5% to about 20%, or about 4% to about 10% (based on the total weight of the core particles). More preferably, the amount of enteric coating may increase the particle weight by about 0.5% to about 5%, about 1% to about 4%, or about 2% to about 3% (based on the total weight of the core particles). The enteric coating is preferably applied by a bottom-spray fluidized bed coating machine equipped with a Wolster column.

[0135] The enteric coating comprises a polymer selected from acrylate polymers or aqueous cellulose dispersions. A mixture of acrylate polymers and / or aqueous cellulose dispersions may also be used. Preferably, the acrylate polymer is selected from polymethyl methacrylate copolymer (PMMA-Methyl methacrylate copolymer). L-100), polyethyl acrylate-methyl methacrylate-trimethylammonium ethyl methacrylate chloride copolymer ( RL-100, RS-100), polymethacrylic acid-ethyl acrylate copolymer ( L30D-55), Ethyl acrylate-methyl methacrylate-trimethylammonium ethyl methacrylate chloride copolymer ( RL30D), ethyl acrylate-methyl methacrylate-trimethylammonium ethyl methacrylate chloride copolymer ( RS30D) and polyethyl acrylate-methyl methacrylate copolymer ( NE30D). More preferably, the acrylate polymer is a polymethacrylic acid-ethyl acrylate copolymer (NE30D).

[0136] L30D-55). Enteric coating is preferably applied via a bottom-spray fluidized bed coating machine equipped with a Wolster column. Enteric coating can improve the delivery efficiency of the active pharmaceutical ingredient to a region of the gastrointestinal tract of the subject with a pH between about 4-5 and about 6.5; it can also improve the delivery efficiency of Formula I compound (or its pharmaceutically acceptable salt, solvate or prodrug) to the proximal or mid-small intestine (or both sites simultaneously); furthermore, enteric coating can improve the delivery efficiency of Formula I compound (or its pharmaceutically acceptable salt, solvate or prodrug) to one or more sites in the duodenum, jejunum and / or mid-ileum. Preferably, the enteric coating begins to dissolve in an aqueous solution with a pH between about 4-5 and about 5-5.

[0137] The carrier used to improve the stability of high-drug-load liquid oral pharmaceutical compositions may preferably be a protective colloid.

[0138] The high-drug-loading liquid oral pharmaceutical composition of the present invention may further comprise a plasticizer. Preferred plasticizers are diethyl phthalate, dibutyl phthalate, triethyl citrate, and glycerin; mixtures of plasticizers may also be used. More preferably, the plasticizer is triethyl citrate.

[0139] In one embodiment, the high-drug-load liquid oral pharmaceutical composition is in solution form; in another embodiment, it is in suspension form. To prepare the suspension, enteric-coated particles can be mixed with the liquid suspension. The liquid suspension may include a suspending agent, a carrier for improving the stability of the high-drug-load liquid oral pharmaceutical composition, and a diluent. Since many suspending agents can also act as stability enhancers, the suspending agent and the carrier for improving the stability of the high-drug-load liquid oral pharmaceutical composition may be the same or different substances.

[0140] Examples of suspending agents include, but are not limited to, methylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, astragalus gum, and glyceryl monostearate. More preferably, the carrier used to improve the stability of the liquid oral pharmaceutical composition is a protective colloid. Examples of protective colloids include, but are not limited to, hydroxymethylcellulose, sodium carboxymethylcellulose, polyvinyl alcohol, gelatin, and povidone; mixtures of protective colloids may also be used. Preferred protective colloids are mixtures of cellulose gum, xanthan gum, and carrageenan. Examples of diluents include, but are not limited to, water, alcohols (such as methanol, ethanol, propanol, isopropanol, etc.), acetone, glycerin, oils (such as castor oil), and other pharmaceutically acceptable diluents or mixtures thereof. Most preferably, water is used as a diluent for the suspension or solution. In addition, pH adjusters and / or antioxidants may be used.

[0141] In some specific embodiments of the liquid suspension of the present invention, the suspension comprises: microcrystalline cellulose and sodium carboxymethyl cellulose (Avicel RC-591), gum (CP Kelco), and water; or microcrystalline cellulose and sodium carboxymethyl cellulose (Avicel RC-591), cellulose gum, xanthan gum, carrageenan, and water.

[0142] According to a preferred embodiment, the liquid suspension comprises about 0.5% to about 3% of at least one suspending agent, about 0.5% to about 1% of at least one protective colloid, and about 98% of a diluent, the weight percentages being based on the weight of the liquid suspension. More preferably, the amount of the protective colloid is about 0.1% to 0.5% by weight. In some specific embodiments, the liquid suspension comprises about 1.5% of at least one suspending agent, about 0.2% of at least one protective colloid, and about 98% of a diluent.

[0143] Preferably, the viscosity of the high-drug-load liquid oral pharmaceutical composition in suspension form is less than about 5 Pa·s; more preferably, its viscosity is less than about 3 Pa·s; and most preferably, it is less than about 1 Pa·s.

[0144] In some embodiments, the high-load liquid oral pharmaceutical composition is used for the prevention and / or treatment of lysosomal storage diseases selected from Gaucher disease (including type 1, 2 and 3 Gaucher disease), Pompe disease (including infantile and late-onset types) and Fabry disease, or for the prevention and / or treatment (e.g., symptom relief) of Parkinson's disease (see, for example, Lukas J. et al., Enzyme Enhancers for the Treatment of Fabry and Pompe Disease, Molecular Therapy, March 2015, 23(3): 456-44).

[0145] In some more specific embodiments, the high-drug-load liquid oral pharmaceutical composition is used for the prevention and / or treatment of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD)). In some further specific embodiments, the high-drug-load liquid oral pharmaceutical composition is used for the prevention and / or treatment of subjects carrying a glucocerebrosidase (GCase) mutation (e.g., a β-glucocerebrosidase mutation), who may also have Gaucher disease, Alzheimer's disease, and / or Parkinson's disease. The β-glucocerebrosidase mutation may be selected from:

[0146] 1. Point mutations at any of the following locations: D140H, V15L, G46E, K79N, R119Q, P122S, R131L, K157Q, N188S, Y212H, F213I, F216V, F216Y, H225Q, F251L, R257E, P289L, A309V, H311R, W312C, Y323I, G32 5R, E326K, C342G, R353G, R359X (stop codon), S364T, N370S, L371V, G377S, V394L, V398F, P401L, D409H, D409V, P415R, L444P, A456P, V460V, R463C, G478S or R496H, and / or any combination thereof;

[0147] 2. I444P point mutation;

[0148] 3. N370S point mutation;

[0149] 4. E326K point mutation;

[0150] 5. L444P, A456P, and V460V point mutations;

[0151] 6. D140H and E326K point mutations;

[0152] 7. H255Q and D409H point mutations;

[0153] A guanine insertion mutation at position 8.84GG;

[0154] 9. A splicing site mutation in intron 2 (IVS2DS+1G-A) leads to exon 2 skipping;

[0155] 10. A single base deletion in the GCase gene (1023delC in the genome sequence);

[0156] 11. A 55-base deletion in the GCase gene (nucleotides 5879-5933 in genomic DNA);

[0157] 12. Homozygous 259C-T transition (position 1763 in genomic DNA);

[0158] 13. A homozygous deletion of one base in the GCase gene leads to a frameshift and premature termination of the protein in exon 6; and

[0159] 14. The GA substitution at position 1 of the splice site of intron 10 of the GCase gene results in the insertion of the first 11 bases of IVS10 and the deletion of the first 11 bases of exon 11.

[0160] In some embodiments, the high-drug-load oral liquid pharmaceutical composition may be administered to a subject concurrently receiving enzyme replacement therapy (e.g., combination therapy). Examples of such enzyme replacement therapies include, but are not limited to, recombinant glucocerebrosidases, such as imiglucerase, velaglucerase, and taliglucerase alfa. ), and / or Eliglustat (Eliglustat, The high-drug-load oral liquid drug composition can be administered simultaneously with, sequentially with, or at different times as enzyme replacement therapy.

[0161] In some embodiments, the high-load oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof of Formula I, for treating subjects carrying misfolded and / or mistransported glucocerebrosidase. In a further embodiment, the high-load oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof of Formula I, for treating or preventing lysosomal storage diseases in subjects. In a further embodiment, the high-load oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof of Formula I, for treating subjects carrying glucocerebrosidase mutations. In yet another embodiment, the high-load oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof of Formula I, for treating subjects carrying β-glucocerebrosidase mutations. Furthermore, in some preferred embodiments, the β-glucocerebrosidase mutation is selected from N370S, L444P, and / or E326K. In a further embodiment, the high-drug-loading oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof as shown in Formula I, for the treatment of a subject with Gaucher disease. In a further embodiment, the high-drug-loading oral liquid pharmaceutical composition comprises an ambroxol analog or bromhexine analog or a pharmaceutically acceptable salt thereof as shown in Formula I, for the treatment of a subject with Parkinson's disease.

[0162] The pharmaceutical compositions of the present invention can also be formulated into unit dosage forms, each containing about 50 to about 1000 mg, more typically about 250 to about 500 mg of a compound of formula I. A “unit dosage form” refers to a physically discrete unit suitable as a unit dose for human subjects and other mammals, each unit containing a predetermined number of a compound of formula I calculated to produce the desired therapeutic effect, and a suitable pharmaceutically acceptable carrier.

[0163] In a further preferred embodiment, the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) may be administered multiple times over a specific period of time, such as daily, for a week or longer. For example, doses of approximately 20-500 mg, 50-150 mg, 50-200 mg, 50-250 mg, 250-500 mg, 250-1000 mg, 1000-1500 mg, 1500-2000 mg, or 1000-2000 mg daily may be used. In some cases, doses higher or lower than the above ranges may also be used. The daily dose may be divided into multiple administrations, for example, divided into 2-4 doses daily.

[0164] The pharmaceutical compositions of the present invention can also be formulated to allow for the slow or controlled release of the active ingredient, for example, by using different proportions of hydroxypropyl methylcellulose (to provide the desired release profile), other polymer matrices, gels, permeable membranes, permeation systems, multilayer coatings, microparticles, liposomes and / or microspheres.

[0165] Typically, controlled-release formulations are pharmaceutical compositions that release the active ingredient at a desired rate, thereby maintaining constant pharmacological activity over a desired period of time. These dosage forms continuously deliver the drug to the body for a predetermined period, thus maintaining drug concentrations within the therapeutic range for a longer time compared to conventional non-controlled-release formulations.

[0166] U.S. Patent No. 5,674,533 discloses a liquid dosage form controlled-release pharmaceutical composition for administering the potent peripheral antitussive mogistran; U.S. Patent No. 5,059,595 describes an enteric-coated tablet controlled-release active pharmaceutical ingredient for treating organic mental disorders; U.S. Patent No. 5,591,767 describes a liquid reservoir-type transdermal patch for controlled-release ketorolac (a nonsteroidal anti-inflammatory drug with potent analgesic effects); U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprising a swellable polymer; U.S. Patent No. 5,073,543 describes a controlled-release formulation comprising a nutrient factor encapsulated by a ganglioside-liposome carrier; and U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation whose coating is derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known to be used in controlled-release formulations; U.S. Patent No. 5,354,566 discloses a controlled-release powder containing an active ingredient; U.S. Patent No. 5,733,566 describes polymeric microparticles for releasing antiparasitic compositions. Any of the above techniques can be adapted for the controlled release of compounds of Formula I (or pharmaceutically acceptable salts, solvates, or prodrugs thereof).

[0167] Controlled release of a Formula I compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) can also be triggered by a variety of inducing factors, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. Drug release occurs through various mechanisms. For example, in one embodiment, the controlled-release component may swell upon administration to a patient, forming porous openings sufficient to release the active ingredient. In the context of this invention, a "controlled-release component" is defined as one or more compounds (such as polymers, polymer matrices, gels, permeable membranes, liposomes, and / or microspheres) that facilitate the controlled release of a Formula I compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) from a pharmaceutical composition. In another embodiment, the controlled-release component is biodegradable, with degradation triggered by exposure to an in vivo aqueous environment, pH, temperature, or enzymes. In yet another embodiment, a sol-gel may be used, wherein the active ingredient is encapsulated in a sol-gel matrix that is solid at room temperature. Upon implantation of this matrix into a subject, preferably a human or other mammal, the subject's body temperature is sufficient to induce the sol-gel matrix to form a gel, thereby releasing the active ingredient into the subject.

[0168] Compositions containing the compounds of the present invention and suitable for intranasal or inhalation administration are of particular significance. Thus, compounds of Formula I can be formulated into dosage forms for intranasal or inhalation administration, typically in dry powder form (which can be used alone, as a mixture, e.g., with anhydrous or monohydrated lactose, preferably monohydrated lactose, mannitol, dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, or trehalose), or as mixed component granules (e.g., mixed with phospholipids), administered via a dry powder inhaler; or as an aerosol spray, administered via a pressurized container, pump, nebulizer, nebulizer (preferably using an electrodynamically generated fine mist nebulizer), with or without a suitable propellant, such as dichlorofluoromethane.

[0169] The pressurized container, pump, spray bottle, nebulizer, or sprayer containing a solution or suspension of a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) includes, for example, ethanol (optionally aqueous ethanol) or other suitable dispersants, solubilizers or agents that prolong the release of the compound, a propellant as a solvent, and optional surfactants such as sorbitan trioleate or oligolactic acid.

[0170] Before being used in dry powder or suspension formulations, the Formula I compound (or its pharmaceutically acceptable salt, solvate, or prodrug) needs to be micronized to a particle size suitable for inhalation administration (typically less than 5 micrometers). This can be achieved by any suitable pulverization method, such as spiral air milling, fluidized bed air milling, supercritical fluid processing (to form nanoparticles), high-pressure homogenization, or spray drying.

[0171] A suitable solution formulation of a Formula I compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) for use in an nebulizer that generates a fine mist using electrodynamics may contain 1 microgram to 20 milligrams of the compound per initiation, with an initiation volume varying from 1 microliter to 100 microliters. Typical formulations may contain a Formula I compound, propylene glycol, sterile water, ethanol, and sodium chloride. Alternative solvents that can be used instead of propylene glycol include glycerol and polyethylene glycol. Capsules, blister packs, and cartridges (e.g., made of gelatin or hydroxypropyl methylcellulose) for use in inhalers or blow-through devices may be formulated as a powder mixture containing a Formula I compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), a suitable powder matrix (such as lactose or starch), and a performance modifier (such as L-leucine, mannitol, or magnesium stearate).

[0172] Inhaled / nasal formulations of compounds of formula I (or their pharmaceutically acceptable salts, solvates, or prodrugs) can be formulated as immediate-release and / or modulated-release formulations. Modulated-release formulations include delayed-release, sustained-release, pulsatile-release, controlled-release, dual-release, targeted-release, and programmed-release formulations. Sustained-release or controlled-release can be achieved by using, for example, poly(D,L-lactic-co-glycolic acid).

[0173] Administration method of the compound of the present invention

[0174] In some preferred embodiments, the compounds of the present invention are administered to the patient orally. However, the compounds can also be administered via any route, including rectal, pulmonary, sublingual, and injectable administration. Injectable administration includes, for example, intravenous, intramuscular, arterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., injected into the bladder), intradermal, transdermal, local, or subcutaneous administration.

[0175] Dosing intervals can be once a week, twice a week, every two days, or once daily, typically once, twice, three times, or four times daily, with the dosing time evenly distributed throughout the day and night to maintain a constant presence of the drug in the body. However, those skilled in the art will appreciate that treatment regimens can be optimized for any specific subject, and the dosing frequency of the Formula I compound (or its pharmaceutically acceptable salt, solvate, or prodrug) can be less than once daily. Treatment can be continued for any length of time as needed.

[0176] The specific dose required to elicit a beneficial therapeutic response in a subject by a compound of Formula I (or its pharmaceutically acceptable salt, solvate, or prodrug) will, of course, be determined by the subject’s specific circumstances, including the subject’s body type, weight, age, sex, and route of administration.

[0177] For example, doses of approximately 20-500 mg, 50-150 mg, 50-200 mg, 50-250 mg, 250-500 mg, 250-1000 mg, 1000-1500 mg, or 1000-2000 mg daily may be used. However, in cases where doses outside these ranges may be necessary, doses higher or lower than these ranges may be used. The daily dose may be divided into multiple administrations, for example, 2-4 times daily. Furthermore, for long-term administration, such as methods that may require increasing and / or improving the subject's healthy lifespan, life expectancy, and / or cognitive acuity, the preferred dosage of the compound is approximately 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 12... 00 mg / day, 1250 mg / day, 1300 mg / day, 1350 mg / day, or between 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, 250-1000 mg / day or 1000-1500 mg / day, or less than 1000 mg / day, or about 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, and / or between 4-12 mg / kg / day.

[0178] The present invention also provides a pharmaceutical package or box comprising one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Optionally, such containers are associated with a notification in the form prescribed by a government agency responsible for regulating the production, use, or sale of pharmaceuticals or biological products, indicating that the agency approves the production, use, or sale of the pharmaceutical composition for human administration.

[0179] Without further explanation, it is believed that those skilled in the art can readily prepare and use compounds of formula I (e.g., based on the foregoing description and the following illustrative examples). Figure 1The method of the invention is carried out by using the compound shown (or its pharmaceutically acceptable salt, solvate, or prodrug) or the same compound. The following examples are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, although the invention has been described herein with reference to examples, it should be understood that these examples and examples are merely illustrative of the principles and applications of the invention. Therefore, it should be understood that various modifications can be made to the illustrative examples and particles, and other components can be designed without departing from the nature and scope of the invention. All patent applications, patents, documents, and references cited herein are incorporated herein by reference in their entirety.

[0180] Example

[0181] The following embodiments will further illustrate the present invention. It should be understood that the following are merely illustrative examples, and modifications may be made to the details without departing from the scope of the present invention.

[0182] Example 1: Synthesis of ZW-010

[0183] Figure 1 The compounds shown can be synthesized by modifying one or more synthetic schemes described by Lati B et al. in the Journal of Labeled Compounds and Radiopharm, 53:15-23, 2010, and / or by the synthetic methods of the difluoro-substituted ambroxol analogue (ZW-010) and the dimethoxy-substituted analogue (ZW-011) shown below, and / or by other methods known to those skilled in the art.

[0184] Synthesis of ZW-010 compound

[0185]

[0186] 1. Synthesis of (2-amino-3,5-difluorophenyl)methanol

[0187] Under nitrogen atmosphere, a 2.4 M lithium aluminum hydride solution in tetrahydrofuran (2.54 mL, 6.11 mmol) was added dropwise to a cooled (0 °C) solution of 2-amino-3,5-difluorobenzoic acid (500 mg, 2.8 mmol) in anhydrous tetrahydrofuran (THF, 15 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour, then cooled to 0 °C, and the reaction was quenched with a 2.0 N aqueous sodium hydroxide (NaOH) solution. The aqueous solution was filtered through diatomaceous earth, and the filtrate was extracted with dichloromethane (2 × 50 mL). The organic extracts were combined, dried over sodium sulfate (Na₂SO₄), filtered, and concentrated to give an orange crystalline solid (302 mg, 66% yield) that could be used directly without further purification. Mass spectrometry (APCI): m / z 160.

[0188] Synthesis of 2,2-amino-3,5-difluorobenzaldehyde

[0189] A solution of (2-amino-3,5-difluorophenyl)methanol (302 mg, 1.90 mmol) in toluene (15 mL) was treated with manganese dioxide (1.50 g, 1.20 mmol, 88%). The reaction mixture was stirred at 80 °C for 30 min. After cooling to room temperature, excess reagent was removed by filtration through a diatomaceous earth filter, and the filtrate was concentrated. Elution was performed by a small, rapid silica gel column (silica gel 60, 230-400 mesh, n-hexane:ethyl acetate (EtOAc) v / v = 7:3) to give a yellow oil, which crystallized upon standing (143 mg, 48% yield). Mass spectrometry (APCI): m / z 158.

[0190] 3. Synthesis of trans-4-{[(2-amino-3,5-difluorophenyl)methyl]amino}cyclohexanol, ZW-010, as a hydrochloride salt.

[0191] A mixture of 2-amino-3,5-difluorobenzaldehyde (140 mg, 0.89 mmol) and trans-4-aminocyclohexanol (103 mg, 0.891 mmol) in ethanol (15 mL) was heated at 80 °C for 5 hours, then cooled to room temperature. Sodium borohydride (81 mg, 2.14 mmol) was added, and the mixture was stirred for another 2 hours at room temperature. The reaction was then quenched with a saturated aqueous solution of ammonium chloride (NH4Cl). The mixture was adjusted to alkalinity with a saturated sodium bicarbonate solution, and the product was extracted with ethyl acetate. The organic extract was dried over sodium sulfate (Na2SO4), filtered, and concentrated. Elution was performed using a rapid chromatography column (silica gel 60, 230-400 mesh, methanol:ethyl acetate = 1:9, v / v) to give a clear oily product whose mass spectra matched the target product (APCI: m / z 257). The free base was dissolved in ethyl acetate, and a solution of 5-6 N hydrochloric acid in isopropanol was added dropwise to the solution, precipitating a hydrochloride salt as a white solid. The precipitate was filtered, washed with ethyl acetate, and dried under vacuum at 35 °C to give pure ZW-010 hydrochloride (180 mg, yield 69%). Mass spectrometry (ESD): m / z 257.1 (M+H) + ; Hydrogen nuclear magnetic resonance spectrum (MeOD-d4) δ (ppm): 1.35 (m, 2H), 1.60 (m, 2H), 2.1 (m, 2H), 2.25 (m, 2H), 3.25 (m, 1H), 3.6 (m, 1H), 4.3 (s, 2H), 7.1 (m, 2H).

[0192] Example 2: Synthesis of compound ZW-011

[0193] 1. Synthesis of (2-amino-3,5-dimethoxy)methanol

[0194] Under nitrogen atmosphere, a 2.4 M lithium aluminum hydride solution in tetrahydrofuran (4.44 mL, 10.65 mmol) was added dropwise to a cooled (0 °C) solution of 2-amino-3,5-dimethoxybenzoic acid (1.0 g, 5.07 mmol) in anhydrous tetrahydrofuran (THF, 30 mL). After the addition was complete, the reaction mixture was stirred at room temperature for 1 hour, cooled again to 0 °C, and the reaction was quenched with a 2.0 N aqueous sodium hydroxide (NaOH) solution. The aqueous solution was filtered through diatomaceous earth, and the filtrate was extracted with dichloromethane (2 × 50 mL). The organic extracts were combined, dried over sodium sulfate (Na₂SO₄), filtered, and concentrated. Elution was performed by a rapid column (silica gel 60, 230-400 mesh, ethyl acetate containing 7% methanol) to give an orange crystalline solid (870 mg, 94% yield). Mass spectrometry (APCI): m / z 184.

[0195] Synthesis of 2,2-amino-3,5-dimethoxybenzaldehyde

[0196] A solution of (2-amino-3,5-dimethoxy)methanol (870 mg, 4.75 mmol) in dichloromethane (40 mL) was treated with manganese dioxide (3.75 g, 38.00 mmol, 88%), and the reaction mixture was stirred at room temperature for 1.5 h. Excess reagent was removed by filtration through a diatomaceous earth mat, and the filtrate was concentrated. The solution was eluted by a rapid column (silica gel 60, 230-400 mesh, n-hexane:ethyl acetate v / v = 1:1) to give 2-amino-3,5-dimethoxybenzaldehyde as a yellow oil (270 mg, yield 31%). Mass spectrometry (APCI): m / z 182.

[0197] 3. Synthesis of trans-4-[(2-amino-3,5-dimethoxyphenyl)methyl]amino]cyclohexanol ZW-011 as its hydrochloride salt.

[0198] A mixture of 2-amino-3,5-dimethoxybenzaldehyde (270 mg, 1.49 mmol) and trans-4-aminocyclohexanol (172 mg, 1.49 mmol) in ethanol (25 mL) was heated at 80 °C for 4 hours, then cooled to room temperature. Sodium borohydride (135 mg, 3.58 mmol) was added, and the mixture was stirred for another hour at room temperature. The reaction was then quenched with a saturated aqueous solution of ammonium chloride (NH4Cl). The mixture was adjusted to alkaline with a saturated aqueous solution of sodium bicarbonate (NaHCO3) and extracted with ethyl acetate. The organic extract was dried over sodium sulfate (Na2SO4), filtered, and concentrated. Elution was performed by a rapid column chromatography (silica gel 60, 230-400 mesh, ethyl acetate containing 45% methanol) to give an orange oil (APCI: m / z 281). The free base was dissolved in ethyl acetate. A solution of 5-6N hydrochloric acid in isopropanol was added dropwise to the solution, precipitating ZW-011 hydrochloride as a light green amorphous solid. The precipitate was filtered, washed, and dried under vacuum at 35°C (207 mg, yield 44%). Mass spectrometry (ESD): m / z 281.0 (M+H)+; 1H NMR (MeOD-d4) δ (ppm): 1.35 (m, 2H), 1.60 (m, 2H), 2.1 (m, 2H), 2.25 (m, 2H), 3.3 (m, 1H), 3.6 (m, 1H), 3.9 (s, 3H), 4.0 (s, 3H), 4.3 (s, 2H), 6.8 (s, 2H).

[0199] Example 3: Metabolic stability of ambroxol analogues

[0200] The metabolic stability of ambroxol and its analogues was investigated in the presence of human liver microsomes.

[0201] Study Design: A 1 mM stock solution of the test compound (ambroxol and its analogues) was prepared by dissolving it in dimethyl sulfoxide (DMSO). The compound was added to a human liver microsomal formulation (200 μL per incubation, protein concentration 1 mg / mL) to a final concentration of 1 μM. The sample was incubated at 37°C in the presence of a reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system (NADP, 1 mM, pH 7.4; glucose-6-phosphate, 5 mM, pH 7.4; glucose-6-phosphate dehydrogenase, 1 unit / mL). A positive control (midazolam, 1 μM, protein concentration 0.1 mg / mL) was incubated simultaneously to ensure the microsomal formulation performed as expected. Samples were taken at 0, 30, 60, and 120 minutes of incubation, and the reaction was quenched with acetonitrile.

[0202] Quenched samples were analyzed by liquid chromatography-tandem mass spectrometry (LC / MS-MS). The concentrations of the tested compounds at each time point were converted to the percentage remaining relative to the compound concentration at time 0 (set as 100%). All data points were included in the data processing. A single exponential decay formula (A0) was fitted to the experimental data. t =A0e -k el t A t (where A0 is the percentage remaining at time t), A0 is 100%, k el Determine the elimination rate constant (ke) for each test compound (t is time). el ,minute -1 Based on k el According to formula t 1 / 2 =ln(2) / k el Calculate the in vitro half-life (t) 1 / 2 The elimination rate constant is also used to calculate the in vitro intrinsic clearance rate (CL). 肠微粒体 (μL / min / mg protein).

[0203] Results: The in vitro elimination half-life (t) of compounds ZW-010 and ZW-011 was determined. 1 / 2 ) and inherent clearance rate (CL) 肠微粒体 As shown in Table 2, the values ​​are compared with the corresponding values ​​of the parent compound ambroxol.

[0204] Table 2 shows the in vitro elimination half-life (t) of the compounds studied. 1 / 2 ) and inherent clearance rate (CL) 肠微粒体 )

[0205] <![CDATA[t 1 / 2 (minutes) <![CDATA[CL 肠微粒体 (μL / min / mg protein) ZW-010 >120 <5.775 ZW-011 >120 <5.775 Ambroxol 60.8 11.4

[0206] In the presence of cofactors, after 120 minutes of incubation, the remaining amounts of ZW-010 and ZW-011 were 62.2% and 66.8%, respectively.

[0207] As shown in Table 2, compared to ambroxol, the substitution of the bromine substituent in ZW-010 (difluoro-substituted) and ZW-011 (dimethoxy-substituted) significantly improved the metabolic stability of ambroxol, with both compounds exhibiting elimination half-lives more than twice that of ambroxol. This improvement in stability is unexpected. The increased stability of the analogues may lead to higher post-dose exposure; furthermore, the improved stability compared to ambroxol alone may allow for a reduction in dosing frequency. In summary, these improvements could reduce the overall manufacturing cost of the analogues by lowering the dose levels and dosing frequency required to achieve sustained efficacy (compared to ambroxol).

[0208] Example 4: Comparative pharmacokinetics (PK) of ambroxol and ZW-010

[0209] After acclimatizing in the laboratory for at least 3 days, female C57BL / 6 mice (6-8 weeks old, weighing 16-30g, 21 mice per group) were administered ambroxol hydrochloride or ZW-010 hydrochloride via a single oral gavage. The dosage of both compounds was 10 mg / kg (based on free base), prepared as a 1 mg / mL clear solution (DMSO: saline = 5:95). The volume of administration was calculated based on the animal's most recent body weight. Mice were fasted overnight before administration and resumed eating and drinking 4 hours later. Three mice from each group were sacrificed at 0.15, 0.25, 1, 2, 4, 8, and 24 hours post-administration. Blood (using K2EDTA blood collection tubes) and brain samples were collected from each animal. Plasma was separated from blood samples by centrifugation. Brain tissue was homogenized with 50% methanol aqueous solution at a ratio of 1:2 (w / v) to prepare brain homogenate samples. Quantitative analysis (internal standard: verapamil) was performed on the samples using high-performance liquid chromatography-tandem mass spectrometry (HPLC / MS-MS) (C18 column, gradient elution; standard curve range 1 to 3000 ng / mL; electrospray ionization, positive ion mode, multiple reaction monitoring (MRM) mode). Drug concentrations (ng / mL in plasma, ng / g in brain) were calculated, and curves showing the change in mean drug concentration (including standard deviation) in plasma and brain over time after administration were plotted, as shown below. Figure 2 A (ambroxol) and Figure 2 As shown in B(ZW-010).

[0210] like Figure 2 A and Figure 2 As shown in B, both compounds can cross the blood-brain barrier, with brain / plasma content ratios >1 at all time points. Furthermore, at the same dose (10 mg / kg), ZW-010 showed significantly higher plasma and brain exposures than ambroxol. These results indicate that the halogenated analogue of ambroxol has greater stability in vivo, thereby increasing exposure in both the brain and peripheral systems. These properties make this novel compound an ideal candidate for treating the diseases described herein that involve both the nervous system and peripheral systems.

[0211] Table 3. Pharmacokinetic parameters of the brain and plasma in female C57BL / 6 mice after a single oral administration of 10 mg / kg (based on free base) of ambroxol or ZW-010 (hydrochloride form).

[0212]

[0213]

[0214] *Plasma is measured in ng / mL, while brain plasma is measured in ng / g.

[0215] **Plasma units are measured in ng·h / mL, while brain units are measured in ng·h / g.**

[0216] As shown in Table 3, the exposure levels of ZW-010 in both the brain and plasma were higher than those of ambroxol. Both compounds could cross the blood-brain barrier, with brain / plasma ratios >1.0, although the ratio for ambroxol (11.0) was higher than that for ZW-010 (3.6). However, the absolute exposure level of ZW-010 in the brain (ng·h / g) was higher than that of ambroxol. It should be noted that ZW-010 had a higher molar exposure (AUClasts of ZW-010 and ambroxol were 42.0 μM·h and 12.3 μM·h, respectively). Furthermore, the elimination half-life (t) of ZW-010 was also higher. 1 / 2 It is approximately twice that of ambroxol.

[0217] Therefore, ZW-010 exhibits greater metabolic stability compared to ambroxol (see Example 3), resulting in superior pharmacokinetic properties: at the same dose, ZW-010 shows significantly higher exposure in brain tissue and a significantly longer elimination half-life. These results suggest that the compounds of this invention hold promise for achieving therapeutic efficacy at doses far lower than those required for ambroxol, with a more optimized dosing regimen (once or twice daily), thereby reducing the risk of off-target side effects and potentially lowering the cost of compound production.

[0218] In summary, these data suggest that the dosage of this novel ambroxol analogue may be lower than that of the parent compound, and the dosing frequency may be lower. This is significant because dropouts are common in clinical trials of high-dose ambroxol due to low subject compliance (see, for example, Stirnemann J. et al., "Ambroxol Therapy in Type 1 Gaucher Disease: Focusing on Patients with Poor Response to Enzyme Replacement Therapy or Substrate Reduction Therapy," *International Journal of Molecular Sciences*, 2023, 24, 6732; and clinical trial number NCT02941822).

[0219] Example 5: Testing the bioactivity of the compound

[0220] Figure 3Human neuronal induced pluripotent stem cells (iPSCs) were obtained from the New York StemCell Foundation. The cultured cells were differentiated and cultured in proprietary medium from Stem Cell Technology. Compounds including ambroxol, difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010"), and dimethoxysubstituted ambroxol (compound 5, also referred to herein as "ZW-011") were dissolved in dimethyl sulfoxide (DMSO). After 14 days of treatment, lysosomes were stained with Lysotracker Green (Invitrogen) according to the manufacturer's instructions. After cell fixation, 3D image stacks were acquired using a Leica SP8 Laser Scanning Confocal Microscope. Lysosome volume was quantitatively analyzed using IMARIS 7.7 software (Bitplane). For the origin of human neurons, see (Kwart D, Gregg A, Scheckel C, Murphy EA, Paquet D, Duffield M, Fak J, Olsen O, Darnell RB, Tessier-Lavigne M: A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP beta-CTFs (Not Abeta), Neuron, 2019, 104: 256-270 e255.31416668).

[0221] Figure 4N2a neuroblastoma cells were obtained from the American Type Culture Collection (ATCC). Cells were seeded at a density of 100,000 cells / well in 6-well plates. After culturing in MEM medium containing 10% fetal bovine serum (FBS) for 24 hours, cells were differentiated by serum starvation in MEM medium for 24 hours. Cells were then treated with solutions of dimethyl sulfoxide (DMSO), ambroxol or difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010"), and dimethoxysubstituted ambroxol (compound 5, also referred herein as "ZW-011") in MEM medium containing 10% FBS. After culturing in the treatment medium for 24 hours, RNA was extracted with TRIzol-chloroform. cDNA was synthesized using the Script cDNA Synthesis Kit (Bio-Rad), and reverse transcription polymerase chain reaction (RT-PCR) was performed using the SsoAdvanced Universal SYBR Green Supermix Kit (Bio-Rad). The results showed that, compared with ambroxol, difluoro-substituted and dimethoxy-substituted compounds more significantly upregulated the transcriptional levels of lysosome-related genes LAMP1, cathepsin B, GBA1, and autophagy-related genes LC3 and TFEB.

[0222] Figure 5 In this process, neurons derived from human iPSCs (same as...) Figure 3 Cells were seeded at a density of 150,000 cells / well in two 6-well plates. For in vitro diagnostics, one 6-well plate was treated with an equal volume of dimethyl sulfoxide (solvent control), while the other 6-well plate was treated with 10 μmol of either ambroxol or difluorosubstituted ambroxol (compound 1, also referred to herein as “ZW-010”) and dimethoxysubstituted ambroxol (compound 5, also referred to herein as “ZW-011”) at the first 50% / 50% medium change. Cells were extracted with TRIzol-chloroform 3 days after treatment. cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad), and reverse transcription-polymerase chain reaction (RT-PCR) was performed using the SsoAdvanced Universal SYBR Green Supermix Kit (Bio-Rad). The results showed that, compared with ambroxol, difluoro-substituted and dimethoxy-substituted compounds more significantly upregulated the transcriptional levels of lysosome-related genes LAMP1, cathepsin D, cathepsin B, GBA1, as well as autophagy-related genes LC3 and TFEB.

[0223] Figure 6A-6B shows that difluorosubstituted ambroxol (compound 1, also referred to herein as "ZW-010") induces TFEB translocation to the nucleus. Human iPSC-derived neurons were cultured with difluorosubstituted ambroxol for 14 days. After cell fixation, endogenous TFEB was immunostained, and the nucleus was identified using DAPI staining. A 3D image stack was acquired using a Leica SP8 confocal microscope, revealing TFEB signals distributed in a punctate pattern in the cytoplasm and nucleus. Figure 6 A). Quantitative analysis of TFEB punctate signals in the cell nucleus was performed using IMARIS 7.7 software (Bitplane). Figure 6 B).

[0224] Figure 7 A-7B shows that human iPSC-derived neurons carrying the Swedish mutation were differentiated and then cultured using culture medium from Stem Cell Technologies. Cells were treated for 5 days with dimethyl sulfoxide (solvent), ambroxol, or difluoro-substituted ambroxol (compound 1, also referred to herein as "ZW-010"). The culture medium was collected, and Aβ40 was detected by enzyme-linked immunosorbent assay (ELISA, Invitrogen) according to the manufacturer's instructions. Figure 7 A) and Aβ42 ( Figure 7 The results showed that ambroxol and its derivatives could effectively reduce the secretion of β-amyloid protein.

[0225] Example 6: Experimental verification of the compound's ability to extend lifespan

[0226] Male BDF1 mice, raised from weaning to pup stage, were housed in cages of 3-4, with free access to Teklad 7013NIH-31 rodent feed and water. At 2 months of age, the mice were divided into two groups of approximately 22-25 mice each. One group continued to be fed the Teklad feed as before (control group); the other group was fed a Teklad feed supplemented with 300 mg / kg of any one of compounds 1-6. Based on the average free-flow intake of adult male BDF1 mice, the formulation was designed to deliver 50 mg / kg / day (by body weight) of the compound to each mouse in the treatment group. Mice were fed this feed until natural death or reaching 16 months of age, at which point the experiment ended. During the experiment, all mice were periodically removed from their cages and manipulated for various sensorimotor, cognitive, and / or behavioral tests.

[0227] Figure 8Animal data from the above experiments using ambroxol are provided, showing that ambroxol can prolong lifespan in mouse models. Group 1 was a control group of animals that did not receive ambroxol; Group 2 was a group of animals that received daily supplemental ambroxol at 50 mg / kg via feed starting from 2 months of age. The data also indicate that ambroxol not only prolongs lifespan, but also that at sacrifice, the surviving animals in Group 2 had a mean health status at least equivalent to those in Group 1, suggesting that healthy lifespan and lifespan are extended simultaneously. Similar results are expected for compounds 1 through 6.

[0228] Example 7: Activity study of the compound of the present invention in improving tremor in a mouse model of Parkinson's disease

[0229] The activity of the compounds of this invention in improving tremor in a mouse model of Parkinson's disease can be studied using the following methods.

[0230] 6-hydroxydopamine (6-OHDA) mice exhibiting resting tremor (i.e., mice injected with 6-hydroxydopamine into the striatum) were selected. Mice were divided into three groups of at least 10 each, receiving either a "high dose" (Group 1: 150 mg / kg of the compound of the present invention daily), a "low dose" (Group 2: 50 mg / kg of the compound of the present invention daily), or no administration of the compound of the present invention (control group). Tremor was monitored daily using electromyography or a force plate-based measurement method (Bekar L et al., *Nature Medicine*, 1:75-80, 2008) to assess the effect of the compound of the present invention on resting tremor.

[0231] Example 8: Study on the effect of the compound of the present invention on aggregated Aβ in a mouse model of Alzheimer's disease

[0232] The effects of the compounds of this invention on the aggregation and deposition of β-amyloid protein (Aβ) in a mouse model of Alzheimer's disease can be studied using the following methods. Figure 11 The use of ambroxol was shown. Figure 11 B) compared with the control group ( Figure 11 A) Results from tests conducted in the following mouse models. The analogues described herein can also be tested using the same models, and due to their superior pharmacokinetic properties, significantly higher exposure in brain tissue at the same dose, and significantly longer elimination half-life in other models, their efficacy is expected to be comparable to or better than ambroxol.

[0233] Alzheimer's disease mouse models with significantly elevated β-amyloid (Aβ) production (e.g., mice carrying the Swedish mutation of human amyloid precursor protein (APP)) were selected. Mice were divided into two groups: a "treatment group" (with 2400 mg / kg of the compound of the present invention added to their feed) and a "control group" (without the compound of the present invention added). After treatment for an appropriate period (e.g., 2 months for age-appropriate animals exhibiting obvious Alzheimer's disease-related pathological features), the relative development of diffuse and fibrillary aggregated Aβ deposition was analyzed, for example, by comparing the results of silver staining or Aβ immunohistochemistry with Congo red or thiamine S histological staining (Jankowsky J et al., *Molecular Neurodegenerative Diseases*, 12:89, 2017).

[0234] Example 9: Activity study of the compound of the present invention in improving cognitive function

[0235] The activity of the compounds of this invention in improving the mental function of mouse models can be studied using the following methods.

[0236] Mice were divided into three groups of at least 13 mice each, and their mental acuity was tested at 9 months of age. The three groups of mice were a "high-dose" group (administered 150 mg / kg of the compound of the present invention daily), a "low-dose" group (administered 50 mg / kg of the compound of the present invention daily), and a control group (not administered the compound of the present invention).

[0237] Figure 9 The results of the above experiments using ambroxol (not the compounds of this invention) are shown, revealing a dose-dependent improvement in mental acuity: the "high-dose" group showed the best results, the "low-dose" group's results were between those of the control and "high-dose" groups, and the control group's cognitive acuity results were at baseline. The dosage used in this experiment was significantly lower than the dosage previously observed to effectively promote GCase molecular chaperone activity in mice (see, for example, Migdalska-Richards et al., *Annals of Neurology*, 80: 766-775, 2016).

[0238] Example 10: Study on macroautophagy induced by the compound of the present invention

[0239] The ability of the compounds of this invention to induce macroautophagy in in vitro cultured mouse cells can be studied using the following methods.

[0240] Mouse fibroblasts (NIH3T3 cells) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Duchenne Modified Eagle Medium (DMEM, Sigma, St. Louis, Missouri, USA) containing 10% newborn calf serum (NCS), 50 μg / mL penicillin, and 50 μg / mL streptomycin at 37°C and 5% CO2. Cells were seeded in 96-well glass-bottomed plates, fixed after specified time intervals, and images were acquired using a high-content microscope (Operetta, PerkinElmer). For example, nine different fields of view were captured per well, yielding an average of 2500–3000 cells. Nuclei and punctate structures were identified using the manufacturer's software. In unsaturated images, after thresholding, the number of granules / punctate structures per cell was quantified in the cytoplasmic region using the "Particle Recognizer" function. In all cases, the focal plane thickness was set to 0.17 μm, and slices with the largest nuclei diameter were selected for quantitative analysis. The numerical value can be expressed as the number of puncta per cell slice, which, under the acquisition conditions, represents 10%–20% of the total number of puncta per cell. Autophagy activity in intact cells was measured by transducing cells carrying the mCherry-GFP-LC3 tandem reporter gene via lentiviral transduction (Kimura S et al., Autophagy, 3(5): 452–460, 2007). Cells were seeded in 96-well glass-bottomed plates, and fluorescence signals were read in two channels. Punctate structures containing both fluorophores corresponded to autophagosomes, while punctate structures containing only red fluorophores corresponded to autolysosomes. Autophagic flux was determined by the conversion of autophagosomes (yellow punctate structures) to autolysosomes (red punctate structures only).

[0241] Figure 10 Results of equivalent experiments using ambroxol are presented. The results indicate that, in addition to its pharmacological activity as a GCase molecular chaperone, ambroxol may also inhibit nutrient sensing. By interfering with nutrient sensing, ambroxol may trigger animal cells to respond to nutrient restriction or fasting, thereby inducing a "catabolism signaling" mode characterized by lysosomal biogenesis and autophagy induction, which could extend lifespan, improve healthy lifespan, and / or mental alertness (see, for example, Efeyan et al., *Nature*, 517: 302-310, 2015) (incorporated hereby by citation). Therefore, ambroxol, its analogues, and related compounds may induce a catabolism signaling mode in the entire organism by systematically inhibiting nutrient sensing.

[0242] Based on the "low-dose" and "high-dose" mouse dosages in this study, the human equivalent doses (HEDs) were calculated to be approximately 4 mg / kg / day and 12 mg / kg / day, respectively. For a human with an average weight of 62.5 kg, this would be approximately equivalent to 250 mg / day or 750 mg / day. Therefore, in a preferred embodiment, a compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is administered over a long period at doses of approximately 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 1200 mg / day, or between 50 and 150 mg / day. / day, 50-200mg / day, 50-250mg / day, 250-500mg / day, 250-1000mg / day, 1500-2000mg / day, 1000-1500mg / day or between 1000-2000mg / day, or less than 1000mg / day, or about 1mg / kg / day, 2mg / kg / day, 3mg / kg / day, 4mg / kg / day, 5mg / kg / day, 6mg / kg / day, 7mg / kg / day, 8mg / kg / day, 9mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, and / or between 4-12mg / kg / day, are expected to effectively improve healthy lifespan, life expectancy and / or mental acuity.

[0243] Example 11: Preparation and properties of a high-drug-loading liquid oral pharmaceutical composition of Formula I

[0244] Particle Preparation – Suitable particles of any one of compounds 1 to 6 (as representative examples of compounds of the present invention) can be prepared by the following method: Micronized compound powder is placed in a rotary granulator (GXR-35 rotary granulator, Freund-Vector), and a hydroxypropyl cellulose (HPC-Klucel LF) binder solution is sprayed onto the powder to form a particle core. Then, additional compound powder is sprayed together with the binder solution to allow particle growth. As compound layers are added, the particles become closer to spherical shapes. The final spherical particles may contain 97% by weight of the compound of formula I and 3% by weight of HPC, with a particle size (x50) of approximately 350 micrometers and a density of approximately 0.7 g / mL.

[0245] Applying a water-soluble release coating to the granules—Using a bottom-spray fluidized bed coating machine equipped with a Wurster column, the compound A granules are coated to form a smooth, uniform substrate. The batch size is approximately 750 grams. A suitable release coating contains 9.1% by weight hydroxypropyl methylcellulose (HPMC), 0.9% by weight triethyl citrate (TEC), and 90% by weight water. Coating the granules with the release coating increases their weight by 2%.

[0246] Enteric coating is applied to the particles—using a bottom-spray fluidized bed coating machine equipped with a Wolster column, enteric coating is applied to the particles coated with the isolation coating. The batch size is approximately 750 grams. A suitable enteric coating comprises 58.0 wt% Eudragit L30D55, 0.9 wt% triethyl citrate (TEC), 8.7 wt% Plasacryl T20, and 32.5 wt% water. Applying enteric coating to the particles coated with the isolation coating increases the particle weight by 35%.

[0247] Dissolution properties—Enteric-coated particles were tested for dissolution properties. Dissolution parameters of equivalent particles containing ambroxol hydrochloride are shown in Table 4.

[0248] Table 4 Dissolution parameters

[0249]

[0250] Every patent, patent application, and publication cited in this article is incorporated herein in its entirety through citation.

[0251] Although the present invention has been disclosed in conjunction with specific embodiments, those skilled in the art can devise other embodiments and variations of the invention without departing from its true spirit and scope. The appended claims are intended to cover all such embodiments and equivalent variations.

Claims

1. A compound represented by Formula I: Its features are: In the above diagram, the R or X group can be attached to any available carbon atom on the benzene ring; R a Selected from hydrogen (H), hydroxyl (OH), and lower alkyl groups (e.g., C). 1-3 Alkyl groups, such as CH3, CH2CH3) and lower alcohols (e.g., C45, CH2CH3) 1-3 Alcohols, such as CH2OH; R b Selected from hydrogen (H) and lower alkyl groups (e.g., C1-3 alkyl groups, such as CH3 and CH2CH3); R c With R d Each is independently selected from hydrogen (H), lower alkyl groups (e.g., C), and C2. 1-3 Alkyl groups, such as CH3 and CH2CH3; or R c With R d These are parts of a 4-membered, 5-membered, 6-membered, or 7-membered ring structure connecting the two (e.g., -R). c -N-Rd-(CH2) n - (where n is an integer selected from 1, 2, 3, and 4); R 1 To R 14 They are independently selected from hydrogen (H) and deuterium (D); X 1 With X 2 Each is independently selected from fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and lower alkyl groups (e.g., C). 1-3 Alkyl groups, such as CH3, CH2CH3), and lower alkoxy groups (e.g., C10, CH2CH3). 1-3 Alkoxy groups, such as OCH3 and OCH2CH3), and lower alkylamines (e.g., C42-C ... 1-3 Alkylamines, such as NR b CH3 and NR b CH2CH3), lower acyl groups (e.g., C) 2-4 Acyl groups, such as C(O)CH3, C(O)CH2CH3), nitro (NO2), nitrile (CN), sulfoxide (SO-R), sulfonate (SO2-R), and sulfate (O-SO2-OR), under the condition X 1 With X 2 It is not simultaneously bromine (Br); or its pharmaceutically acceptable salt, solvate, or prodrug.

2. The compound according to claim 1, characterized in that... It has the following formula Ia:

3. The compound according to any one of claims 1 to 2, characterized in that... X 1 With X 2 All are fluorine (F).

4. The compound according to any one of claims 1 to 2, characterized in that... X 1 With X 2 All are OCH3.

5. The compound according to any one of claims 1 to 2, characterized in that... X 1 With X 2 Both are chlorine (Cl).

6. The compound according to any one of claims 1 to 5, characterized in that... R a For hydroxyl group (OH), R b It is hydrogen (H).

7. The compound according to any one of claims 1 to 5, characterized in that... R a For hydrogen (H), R b It is CH3.

8. The compound according to any one of claims 1 to 2, characterized in that... The compound is selected from:

9. The compound according to any one of claims 1 to 2, characterized in that... The compound is selected from:

10. A pharmaceutical composition comprising a compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) according to any one of claims 1 to 9, optionally combined with a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 9, characterized in that... The composition is a liquid oral pharmaceutical composition.

12. The pharmaceutical composition according to claim 10, comprising a high drug loading of the compound of formula I according to any one of claims 1 to 9 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), characterized in that, The composition comprises: (1) a compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); and (2) at least one pharmaceutically acceptable excipient; wherein the compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in particulate form, the particulate having a core, the core comprising about 60 to about 97% by weight of the active pharmaceutical ingredient and about 3 to about 40% by weight of the excipient, the weight percentages being based on the total weight of the core.

13. The pharmaceutical composition according to claim 10, comprising a high drug loading of the compound of formula I according to any one of claims 1 to 9 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), characterized in that, The composition comprises: (1) a compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); (2) at least one pharmaceutically acceptable excipient; and (3) a diluent; wherein the compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in particulate form, the particulate having a core containing about 60 to about 97% by weight of the active pharmaceutical ingredient and about 3 to about 40% by weight of the excipient, the weight percentages being based on the total weight of the core; wherein the core is coated with (4) a water-soluble protective coating, the amount of which increases the weight of the particulate by about 0.5 to about 5%; and (5) an enteric coating, the amount of which increases the weight of the particulate by about 0.5 to about 50%.

14. A method for preventing and / or treating a disease or medical condition in a subject, said disease or medical condition being selected from respiratory diseases and conditions, lysosomal storage diseases (LSDs), and nervous system diseases and conditions, said method comprising administering to the subject an effective amount of a compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) according to any one of claims 1 to 9, or a pharmaceutical composition according to any one of claims 10 to 13.

15. The method according to claim 14, characterized in that, The disease or medical condition to be prevented and / or treated is bronchopulmonary disease, or Gaucher disease, Pompe disease or Fabry disease, or Parkinson's disease, Lewy body dementia, Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e. Lou Gehrig's disease), frontotemporal dementia (FTD), Pick's disease or Gaucher disease.

16. A method comprising administering to a subject an effective amount of a compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) according to any one of claims 1 to 9, or a pharmaceutical composition according to any one of claims 10 to 13; wherein the method is intended to prolong the life expectancy of the subject, or to treat, inhibit, or alleviate aging of the subject, or to treat, inhibit, or alleviate aging-related symptoms or aging-related diseases of the subject, or to improve the subject's healthy lifespan, lifespan, and / or mental acuity.

17. A method for preventing and / or treating, alleviating symptoms and / or delaying the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease and frontotemporal degeneration (FTD)), said method comprising administering to a subject an effective amount of ambroxol (or a related compound, such as ambroxol hydrochloride, bromhexine) or a compound according to any one of claims 1 to 9 (or a pharmaceutically acceptable salt, solvate or prodrug thereof), and one or more suitable anti-β-amyloid antibodies or fragments thereof.

18. A method for preventing, alleviating symptoms and / or delaying the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease and frontotemporal degeneration (FTD)), said method comprising administering to a subject an effective amount of ambroxol (or a related compound, such as ambroxol hydrochloride, bromhexine) or a compound according to any one of claims 1 to 9 (or a pharmaceutically acceptable salt, solvate or prodrug thereof).

19. The method according to claim 17 or 18, characterized in that, The subjects were selected by detecting biomarkers indicating that they were at risk for Alzheimer's disease or in the early stages of disease development in patients with other diseases associated with the misfolding, aggregation, and deposition of pathological proteins, including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher's disease, and frontotemporal degeneration (FTD).

20. The method according to claim 19, characterized in that, The subjects were selected by detecting phosphorylated tau protein (p-tau) in patients who were at risk of Alzheimer's disease or in the early stages of Alzheimer's disease.

21. The method according to claim 17 or 18, characterized in that, The subjects were selected by genotyping at least one gene or locus indicating a risk of Alzheimer's disease or other diseases associated with misfolding, aggregation, and deposition of pathological proteins, including Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, i.e., Lou Gehrig's disease), Pick's disease, Gaucher disease, and frontotemporal degeneration (FTD).

22. The method according to claim 21, characterized in that, The subjects were selected by genotyping the apolipoprotein E (ApoE) gene or by detecting p-tau217, p-tau181, p-tau231, p-tau235 and / or N3pG.

23. The method according to claim 22, characterized in that, The subjects were selected by genotyping the ε4 allele of the apolipoprotein E (ApoE) gene, or by detecting p-tau217, p-tau181, p-tau231, p-tau235 and / or N3pG.

24. The method according to any one of claims 17 to 23, characterized in that, The daily dose of ambroxol (or a related compound) or a compound according to any one of claims 1 to 9 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) administered to the subject is selected from any of the following doses: (1) A dose that produces a peak drug concentration in the serum of the subject greater than 1 μM (e.g., 2-50 μM, 2-25 μM, or 10-20 μM); or (2) A dose that produces a peak drug concentration in the brain tissue of the subject greater than 3 μM (e.g., 5-50 μM, 5-25 μM, or 10-20 μM); or (3) A dose of approximately 250-1000 mg / day, 750-1000 mg / day, 1000-2000 mg / day, 1000-1500 mg / day or 1500-2000 mg / day.

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