Pharmaceutically active compounds, compositions and methods for modulating petrine

Abstract

The present invention relates to a novel compound represented by the following Chemical Formula 1 as a pentralin correcting agent, and a composition for preventing or treating deafness-goiter syndrome and diseases related thereto, comprising the compound as an active ingredient. [Chemical Formula 1] # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a novel compound as a pendrin corrector, and a composition for preventing or treating deafness - goiter syndrome and related diseases, which comprises the compound as an active ingredient. Background Art

[0002] Pendrin is an anion exchange channel protein encoded by SLC26A4 (PDS) and is a member of the SLC26A family. Pendrin is expressed on the apical cell membrane and mediates the transport of Cl - , HCO3 - , OH - and I - ions as well as formate, nitrate and thiocyanate. Pendrin is significantly detected in the inner ear, thyroid and kidney, although other tissues also show induced expression of pendrin under certain conditions. In the inner ear, pendrin is expressed in the endolymphatic sac and hair cells. Defects in pendrin caused by gene mutations lead to endolymph acidification and reduced Ca 2+ reabsorption, resulting in auditory sensory conduction defects, including deafness - goiter syndrome (Pendred Syndrome) (Bassot, C. et al., 2017; Biochimie. 132: 109 - 120)).

[0003] Some types of pharmacological modulators have been reported to rescue the ion channel function of ion transporters by directly interacting with the targeted malfunctioning proteins (such as cystic fibrosis transmembrane conductance regulator (CFTR)). Among these modulators, correctors (also known as pharmacological chaperones) and potentiators are the main types of such modulators that rescue the function of specific membrane transporters. Correctors or pharmacological chaperones help mutant polypeptides fold correctly into functional transporters. Thus, correctors enable mutant proteins to form a functionally complete structure, and thus, through appropriate post - translational modifications, cross the Golgi trans - membrane network and target to the plasma membrane. On the other hand, potentiators are modulators that enhance channel gating function by stabilizing the protein and / or by increasing the transduction function of cell - surface transporters (Collawn, J.F., et al., 2014; Am. J. Physiol. Lung Cell. Mol. Physiol. 307: L431 - L434)). A variety of compounds with the above strategies have been developed and marketed for certain diseases, such as cystic fibrosis caused by CFTR gene defects (Lopes - Pacheco, M., 2020; Front. Pharmacol.). Summary of the Invention

[0004] Technical Problem

[0005] The present invention is based on the following discovery: certain compounds can act as pantethine correctors with the potential for preventing, ameliorating or treating deafness-goiter syndrome and related diseases.

[0006] Technical Solution

[0007] On the one hand, the present invention provides a compound represented by the following Chemical Formula 1, its pharmaceutically acceptable salts, its optical isomers, mixtures of its two isomers, its precursors, its pharmaceutically acceptable salts or its solvates:

[0008] [Chemical Formula 1]

[0009]

[0010] X 1 Each occurrence is independently selected from CH, C-Z and N;

[0011] X 2 Each occurrence is independently selected from CH, C-Z and N;

[0012] X 3 Each occurrence is independently selected from CH, C-Z and N;

[0013] X 4 Each occurrence is independently selected from CH, C-Z and N;

[0014] X 5 Each occurrence is independently selected from CH, C-Z and N;

[0015] n is independently selected from 0, 1 and 2 each occurrence;

[0016] R 1 Each occurrence is independently selected from the group consisting of hydrogen, C1-C6 alkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C1-C6 alkyls; C3-C10 cycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C3-C10 cycloalkyls; C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R7 one or more substituted C3-C10 heterocycloalkyls; C6-C12 aryls, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 one or more substituted C6-C12 aryls; C3-C12 heteroaryls, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 the group consisting of one or more substituted C3-C12 heteroaryls;

[0017] Z is any structure of group A below;

[0018]

[0019] Y 1 、Y 2 and Y 3 each occurrence is independently selected from CH and N;

[0020] R 2 and R 3 each occurrence is independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 wherein any one is optionally substituted;

[0021] R 4 and R 5 each occurrence is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, wherein any one is optionally substituted;

[0022] R 6 and R 7 each occurrence is independently selected from the group consisting of hydrogen, C1-C4 haloalkyl, C1-C6 alkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 8 and NR 8 R 9 one or more substituted C1-C6 alkyls; C3-C10 cycloalkyl, halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and NR 8 R 9one or more of the following substituted C3-C10 cycloalkyls; C3-C10 heterocycloalkyls, halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and NR 8 R 9 and one or more of the following substituted C3-C10 heterocycloalkyls;

[0023] R 8 and R 9 each occurrence of which is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, and C1-C4 haloalkyl, any of which is optionally substituted.

[0024] On the other hand, the present invention provides a pharmaceutical composition for preventing, ameliorating or treating deafness-goiter syndrome or related diseases thereof, which comprises at least one of the said compounds, at least one of its optical isomers, at least one of a mixture of its two isomers, at least one of its precursors, at least one of its pharmaceutically acceptable salts or at least one of its solvates as an active ingredient.

[0025] In another aspect, the present invention provides a composition comprising a compound represented by Chemical Formula 1 or a mixture thereof, and a composition comprising a compound represented by Chemical Formula 1 or a mixture thereof and a pharmaceutically acceptable carrier.

[0026] In another aspect, the present invention provides the use of a compound represented by Chemical Formula 1 and its pharmaceutical composition as a pantothenate corrector.

[0027] In another aspect, the present invention provides the use of a compound represented by Chemical Formula 1, a mixture thereof and its pharmaceutical composition as an active ingredient in a health functional food for preventing or ameliorating deafness-goiter syndrome or related diseases thereof.

[0028] In another aspect, the present invention provides the use of the said compound and its pharmaceutical composition as a pantothenate corrector, as an active ingredient in a health functional food for preventing or ameliorating deafness-goiter syndrome or related diseases thereof.

[0029] Beneficial effects

[0030] According to the present invention, the novel compound can be used as a pantothenate corrector, and thus can be effectively used as a composition for preventing, treating or ameliorating deafness-goiter syndrome or related diseases thereof. Description of the drawings

[0031] Figure 1Shows the results of Western blot analysis of patient-derived epithelial cells (derived from patient nasal epithelial cells) treated with compound 9. (A, B) The B-form, which represents the specific non-glycosylated precursor form of pantethine, shows little significant change in response to treatment with compound 9; however, in the C-form, which represents the fully glycosylated functional pantethine, a significant dose-dependent increase was observed, particularly at the high concentration of 10 μM. Extrapolating these observations to patient-derived nasal epithelial cells indicates a concentration-dependent effect of compound 9 treatment on C-type expression in cells carrying the H724R mutation.

[0032] Figure 2 Shows the immunofluorescence staining results of compounds 9, 18, and 105. (A) Illustrates the immunofluorescence staining results of epithelial cells under different conditions. In the vehicle and IL-4 groups, pantethine expression was absent, as indicated by the absence of fluorescence. Conversely, treatment with compound 9, compound 18, and compound 105 at 10 μM showed clear red fluorescence, indicating enhanced expression of pantethine localized on the epithelial cell membrane. Scale bar = 20 μm. (B) Is a quantitative representation comparing the mean fluorescence intensity levels between the vehicle, IL-4, and compound 9, compound 18, and compound 105 (10 μM) groups. Statistical significance indicates a significant difference in pantethine expression intensity between experimental conditions. *p < 0.05. Detailed Description

[0033] 1. Definitions

[0034] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, unless otherwise specified, the following terms have the following meanings.

[0035] The term "or" as used herein is understood to be inclusive unless specifically stated or clear from the context.

[0036] The term "about" as used herein is understood to be within the generally accepted range in the art, e.g., within two standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context otherwise indicates, all numerical values provided herein are modified by the term "about".

[0037] The terms "activator", "drug", and "pharmaceutical formulation" are used interchangeably herein and refer to a chemical material or compound that, when administered to a subject (e.g., any animal including a human or non-human animal) by any means described herein, induces the desired pharmacological effect (e.g., such as a reduction in inflammation).

[0038] As used herein, "additive" can refer to any additional ingredient that can be added to the compositions and chemical formulas described herein. For example, assuming the additional ingredient is pharmaceutically acceptable for the particular condition being treated, additives can include excipients (e.g., one or more excipients), antioxidants (e.g., one or more antioxidants), stabilizers (e.g., one or more stabilizers), preservatives (e.g., one or more preservatives), pH adjusters and / or buffers (e.g., one or more pH adjusters and / or buffers), isotonicity regulators (e.g., one or more isotonicity regulators), thickeners (e.g., one or more thickeners), suspending agents (e.g., one or more suspending agents), binders (e.g., one or more binders), tackifiers (e.g., one or more tackifiers), etc. In addition, additives can include therapeutic agents and drug delivery regulators, as well as enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, and ion exchange resins, and combinations of any two or more thereof.

[0039] As used herein, the term "administering" refers to oral administration, rectal administration, topical administration, intravenous administration, parenteral administration, transmucosal administration, intratympanic administration, intra-tympanic administration, intracochlear administration, intraperitoneal administration, intramuscular administration, intralesional administration, intracapsular administration, intranasal administration, intravitreal administration, or subcutaneous administration, or implanting a sustained release device, such as a mini-osmotic pump, into a subject. Administration is by any route including parenteral and transmucosal (e.g., oral, intranasal, pulmonary, rectal, buccal, vaginal, intraocular, and dermal) routes.

[0040] "Analogue" and "derivative" are used interchangeably herein and refer to a compound that has the same nucleus as the parent compound in the absence or presence of one or more atoms and / or groups of atoms and combinations thereof, but differs from the parent compound in the order of bonding. A derivative can differ from the parent compound in terms of one or more substituents present on the core, which can include, for example, one or more atoms, functional groups, or substructures. In addition, a derivative can differ from the parent compound in the order of bonding between the atoms in the core. Generally, a derivative can be at least theoretically predicted to be formed from the parent compound by chemical and / or physical processes.

[0041] As used herein, "antioxidant" can refer to a synthetic or natural substance that can prevent or delay a certain type of damage and / or oxidation. Antioxidants are present in many foods, including fruits and vegetables. In addition, they can be used as dietary supplements. Exemplary antioxidants can include beta-carotene, lutein, lycopene, selenium, vitamin A, vitamin C, and vitamin E. In addition, other antioxidants known to those skilled in the art can be used. The antioxidants described herein can be used in any suitable amount.

[0042] "Co-administration" means that the compounds or compositions described herein are administered simultaneously immediately before or after administering the additional therapeutic agent or activator or additive described herein. The compounds or compositions of the present disclosure can be administered alone or in co-administration to a patient. Co-administration is construed to include administering the compounds (one or more compounds or agents) alone or in combination simultaneously or sequentially. If desired, the formulation can also be combined with other active substances.

[0043] In the present disclosure, terms such as "comprise", "comprising", "containing", and "having" can have the meanings ascribed to them and can mean "include", "including", etc.; and "consisting essentially of" or "consisting essentially of" can similarly have the meanings ascribed to them, and the terms are open-ended, allowing for more to be present than those enumerated, unless the basic or novel features enumerated are changed due to the presence of more features than those enumerated, but excluding examples of the prior art.

[0044] "Simultaneous administration" as used herein includes at least partial overlap in duration. For example, when two agents (e.g., any agent or class of agents having biological activity as described herein) are administered simultaneously, their administrations occur within a certain desired time period. The administration of the formulation can start and end on the same day. In addition, the administration of one formulation can occur before the administration of the second formulation, as long as the two formulations are taken at least once on the same day. Similarly, the administration of one formulation can extend beyond the administration of the second formulation, as long as the two formulations are taken at least once on the same day. It is not necessary for the bioactive agent / formulation to be taken at the same time every day in order to include simultaneous administration.

[0045] As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to effect a desired biological effect (e.g., a beneficial outcome including a clinical result). Thus, an "effective amount" depends on the circumstances of its application. The effective amount can vary according to factors known in the art, such as the disease state, age, sex, and weight of the subject being treated. It can be administered in several doses per day, or the dose can be proportionally reduced as indicated by the exigencies of the treatment situation. In addition, the compositions / formulations of the present disclosure can be administered as often as needed to achieve a therapeutically effective amount.

[0046] As used herein, the term "intermittent dosing" includes a period of time during which a formulation is administered (which can be considered the "first dosing time"), followed by a period of time during which the formulation is not ingested or is ingested at a lower dose (which can be considered the "intermittent time"), and then followed by a period of time during which the formulation is administered again (which can be considered the "second dosing time"). Generally, during the second dosing time, the dose level of the formulation is consistent with the dosing level during the first dosing time, but it can be increased or decreased according to medical need.

[0047] As used herein, a "liquid" is a dosage form of a pharmaceutical composition that consists of a liquid composition. A liquid is spillable; and at room temperature, it will flow and exhibit various behaviors in a container. A liquid exhibits Newtonian or pseudoplastic flow behavior.

[0048] In an embodiment, a "semi-liquid" as used herein can have the characteristics of a liquid and other formulations (i.e., suspensions, emulsions, solutions, creams, gels, jellies, etc.).

[0049] As used herein, the term "ointment" can refer to a highly viscous liquid or semi-liquid formulation that can be used for the therapeutic treatment of a disease, syndrome, or disorder.

[0050] As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically suitable. The type of carrier can be selected based on the intended route of administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile topical solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. As long as any conventional media or agents are incompatible with the composition (e.g., the chemical formula 1, derivatives or analogs of chemical formula 1, or pharmaceutically acceptable salts, solvents, hydrates, or polymorphs thereof described herein), their use in the composition is contemplated by the present disclosure.

[0051] As used herein, "pharmaceutical carrier" or "carrier" may also include pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals at the employed dosages and concentrations. Physiologically acceptable carriers are usually aqueous pH-buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 polypeptide residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions that form salts such as sodium; and / or nonionic surfactants such as Tween TM ), polyethylene glycol (PEG) and Pluronics TM ). Additionally, "pharmaceutically acceptable" means that it has been or can be approved by federal or state government regulatory agencies or corresponding agencies in countries other than the United States for use in animals, more specifically in humans, or is listed in the United States Pharmacopeia or other generally recognized pharmacopeias.

[0052] The term "pharmaceutically acceptable salt or complex" refers to salts or complexes represented by the following specific Chemical Formula 1. Examples of such salts include base addition salts formed by reacting a compound represented by Chemical Formula 1 with a metal cation selected from the group consisting of, for example, alkali metals (such as sodium, potassium or lithium) and alkaline earth metals (such as calcium or magnesium) in an organic or inorganic base (such as hydroxide, carbonate or bicarbonate), or base addition salts formed by reacting with a primary, secondary or tertiary alkylamine, but not limited thereto. Amine salts induced by methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, morpholine, N-methyl-D-glutamine, N,N'-bis(benzyl)-1,2-ethanediamine, tromethamine, ethanolamine, diethanolamine, ethylenediamine, N-methylmorpholine, procaine, piperidine, piperazine, etc. are considered to be within the scope of the present invention.

[0053] In addition, the "salt" or "salt form" or "pharmaceutically acceptable salt" used herein may include base addition salts (formed from free carboxyl or other anionic groups) derived from inorganic bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organic bases (such as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, etc.). The salt is formed as an acid addition salt with any free cationic group, and for example, it is usually formed from inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, or from organic acids such as formic acid, acetic acid, citric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts of the present disclosure may include amine salts formed by protonation of an amino group with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc. In addition, the salts of the present disclosure include amine salts formed by protonation of an amino group with a suitable organic acid such as p-toluenesulfonic acid, acetic acid, etc.

[0054] The term "pH agent" or "buffer" used herein may refer to a compound or buffer used as a pH regulator. This may include, but is not limited to, glycerol buffer, citrate buffer, borate buffer, acetate buffer, gluconate buffer, phosphate buffer, or citrate-phosphate buffer. The pH agent or buffer may be used in any suitable amount.

[0055] The term "preservative" used herein may refer to a substance or chemical that prevents undesired changes in the compounds or compositions or chemical formulas described herein. Suitable preservatives may include, for example, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenethyl alcohol, disodium edetate, sorbic acid, polyquaternium, cetyl bromide, cetylpyridinium chloride, benzyl bromide, EDTA, phenylmercuric nitrate, phenylmercuric acetate, thimerosal, acetate and phenylboric acid mercury, polymyxin B sulfate, methyl and propyl parabens, quaternary ammonium chloride, sodium benzoate, sodium propionate, and sodium perborate, as well as other reagents known to those skilled in the art or combinations thereof. The preservative may be used in any suitable amount.

[0056] The terms "prevent", "preventing", or "prevention" and other grammatical equivalents used herein include those for reducing the incidence of a syndrome, as well as for preventing the development, occurrence, interference, or avoidance of a syndrome of a disease or disorder. Prevention may be complete (i.e., without detectable symptoms) or partial, such that fewer symptoms may be observed compared to no treatment. The term also includes prophylactic benefits. To prevent a disease or disorder, a composition may be administered to a patient at risk of developing a particular disease, or a patient reporting one or more physiological syndromes of the disease, although the disease may not necessarily be diagnosed.

[0057] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 10 is understood to include not only all intermediate decimal values between the above integers (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9), but also any numbers, combinations of numbers, that form sub-ranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. With respect to sub-ranges, “nested sub-ranges” that extend from one of the endpoints of the range are specifically contemplated. For example, the nested sub-ranges of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or can include 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. The range can be expressed herein as “about” a particular value and / or “about” other particular values. When expressing such a range, other aspects include a particular value and / or other particular values. Similarly, when a value is expressed as an approximation using the antecedent “about”, it is to be understood that the particular value forms another aspect. It should also be understood that each endpoint of a range is significant relative to the other endpoint and independent of the other endpoint. In addition, it should be understood that throughout the application, data is provided in a variety of different formats, and these data represent endpoints and starting points and ranges of any combination of data points. For example, when a particular data point “10” and a particular data point “15” are disclosed, it is considered that what is disclosed is between 10 and 15, as well as greater than, greater than or equal to, less than, less than or equal to, and equal to. In addition, it should be understood that each unit between two particular units is disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0058] Other excipients contemplated for use in the practice of the present disclosure are those available to those skilled in the art.

[0059] A “semi-solid gel” according to the present disclosure is semi-solid. The apparent viscosity of a semi-solid preparation can increase with increasing concentration.

[0060] “Sequential administration” as used herein includes administration of two formulations (e.g., the compounds or compositions described herein) occurring separately on the same day or not on the same day (e.g., occurring on consecutive days).

[0061] A “solution” according to the present disclosure can be a clear, homogeneous liquid dosage form that contains one or more chemical substances dissolved in a solvent or a mixture of solvents that are miscible with each other. Since the molecules of the drug substance in a solution are uniformly dispersed, using a solution as a dosage form generally provides good accuracy in ensuring uniform dosing upon administration and upon dilution or otherwise mixing the solution.

[0062] As used herein, the term "solvent" refers to an aqueous or non-aqueous liquid solvent. The choice of solvent depends particularly on the solubility of the composition and the mode of administration. An aqueous solvent may consist solely of water or may consist of water and one or more miscible solvents, and may contain dissolved solutes such as sugars, buffers, salts or other excipients. More commonly used non-aqueous solvents are short-chain organic alcohols such as methanol, ethanol and propanol, short-chain ketones such as acetone, and polyols such as glycerol.

[0063] "Subject" or "patient" refers to a human or non-human animal, such as a mammal. "Subject" can include any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cows, fish and birds. A human subject can refer to a patient.

[0064] As used herein, a "suspension" is a liquid dosage form containing solid particles dispersed in a liquid carrier.

[0065] As used herein, "viscosity" refers to the resistance to flow of a fluid. Viscosity agents can be used herein, and for example, they include polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, other reagents known to those skilled in the art or combinations thereof.

[0066] The term "weight percentage" or "%(w / w)" refers to the percentage of a component in a solution calculated based on the weight of the component and the solvent. For example, a 1%(w / w) solution of a component may have 1 g of the component dissolved in 100 g of the solvent. The term "volume percentage" or "%(v / v)" refers to the percentage of a component in a solution calculated based on the volume of the component and the solvent. For example, a 1%(v / v) solution of a component may have 1 ml of the component dissolved in 100 ml of the solvent. The term "weight / volume percentage" or "%(w / v)" refers to the percentage of a component in a solution calculated based on the weight of the component and the volume of the solvent. For example, a 1.0%(w / v) solution of a component may have 1 g of the component dissolved in 100 ml of the solvent.

[0067] As used herein, the term "syndrome" refers to a disorder characterized by a group of symptoms or a series of related symptoms that occur together continuously. A syndrome (e.g., acute respiratory distress syndrome) can be a group of medical signs and symptoms that are interrelated and are usually associated with a specific disease. On the other hand, a disease can be a health condition with a well-defined cause behind it. However, a syndrome (from the Greek meaning "to run together") can result in many symptoms without a determinable cause. They can imply the possibility of an underlying disease or the likelihood that a disease will develop.

[0068] As used herein, the terms "treat", "treating", or "treatment" and other grammatical equivalents include alleviating, reducing, ameliorating, or preventing a disease, disorder (e.g., acute respiratory distress syndrome), or symptom, preventing additional symptoms, ameliorating or preventing the underlying metabolic cause of a symptom, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder, alleviating a disease or disorder, resolving a disease or disorder, alleviating the conditions caused by a disease or disorder, or stopping the symptoms of a disease or disorder, and are intended to include prevention. The term also includes achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit means eradicating or ameliorating the underlying disorder being treated. Additionally, since a therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder, an improvement is observed in the patient even if the patient may still be afflicted with the underlying disorder.

[0069] The term "health functional food" refers to a food or food supplement prepared or processed with raw materials, functional ingredients, active pharmaceutical ingredients, or additives for improving and / or nourishing and / or maintaining the physiological functions of the human body.

[0070] The term 'PDS' can also be used as pendrin protein encoded by the gene SLC26A4 (PDS).

[0071] Unless the definition of a single substituent otherwise limits, all substituents are understood to be optionally substituted in their entirety.

[0072] 2. Compound

[0073] One aspect of the present invention provides a compound represented by the following Chemical Formula 1, its optical isomers, a mixture of its two isomers, its precursors, its pharmaceutically acceptable salts or solvates, and pharmaceutically acceptable salts thereof:

[0074] [Chemical Formula 1]

[0075]

[0076] X 1 Each occurrence is independently selected from CH, C-Z, and N;

[0077] X 2 Each occurrence is independently selected from CH, C-Z, and N;

[0078] X 3 Each occurrence is independently selected from CH, C-Z, and N;

[0079] X 4 Each occurrence is independently selected from CH, C-Z, and N;

[0080] X 5independently selected from CH, C-Z and N each time it appears;

[0081] n is independently selected from 0, 1 and 2 each time it appears;

[0082] R 1 independently selected from the group consisting of hydrogen, C1-C6 alkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C1-C6 alkyl; C3-C10 cycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C3-C10 cycloalkyl; C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C3-C10 heterocycloalkyl; C6-C12 aryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C6-C12 aryl; C3-C12 heteroaryl, halogen, C1-C six alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and one or more of the substituted C3-C12 heteroaryl;

[0083] Z is any structure of the following group A;

[0084]

[0085] Y 1 、Y 2 and Y 3 independently selected from CH and N each time it appears;

[0086] R 2 and R 3 independently selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR6 and NR 6 R 7 A group consisting of any one of which is optionally substituted;

[0087] R 4 and R 5 Each occurrence is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, any one of which is optionally substituted;

[0088] R 6 and R 7 Each occurrence is independently selected from the group consisting of hydrogen, C1-C4 haloalkyl, C1-C6 alkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 8 and NR 8 R 9 And one or more of NR 8 and NR 8 R 9 And one or more of NR 8 and NR 8 R 9 A group consisting of C3-C10 heterocycloalkyl substituted with one or more of;

[0089] R 8 and R 9 Each occurrence is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl and C1-C4 haloalkyl, any one of which is optionally substituted.

[0090] In a preferred embodiment, Z in Chemical Formula 1 is any structure of the following Group B;

[0091]

[0092] Wherein, R 2 and R 3 As defined in claim 1; and

[0093] Y 1 , Y 2 and Y 3 As defined in claim 1.

[0094] In a preferred embodiment, in Chemical Formula 1, Z is

[0095] wherein R 2 and R 3 are as defined above; and

[0096] Y 1 、Y 2 and Y 3 are as defined above.

[0097] In a preferred embodiment, the compound of Formula 1 has the general formula II,

[0098]

[0099] wherein R 1 、R 2 and R 3 are as defined above; and

[0100] X 1 、X 2 、X 3 and X 4 are as defined above; and

[0101] n is as defined above.

[0102] In a preferred embodiment, in Formula 1, R 1 is independently selected, each time it appears, from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 cycloalkyl, C3-C10 cycloalkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 heterocycloalkyl, C3-C10 heterocycloalkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 .

[0103] In a preferred embodiment, in Formula 1, R 1 is independently selected, each time it appears, from the group consisting of C6-C12 aryl, C6-C12 aryl substituted with one or more of halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR6 and NR 6 R 7 one or more of the following substituted C6-C12 aryl groups; C3-C12 heteroaryl groups, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 selected from the group consisting of one or more of the following substituted C3-C12 heteroaryl groups.

[0104] In a preferred embodiment, in Formula 1, R 2 and R 3 are each independently selected from the group consisting of OR 6 and NR 6 R 7 each time they appear.

[0105] In a preferred embodiment, the compound of Formula 1 has one of Formulas 1)-120), as follows:

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] 3. Composition / Preparation

[0124] In another embodiment, the present invention provides a composition of Formula 1 as an active ingredient, and at least one pharmaceutically acceptable carrier, excipient and / or diluent.

[0125] In a preferred embodiment, the composition can be used as a pharmaceutical active agent, preferably for methods of treating diseases.

[0126] The present invention includes the compounds described herein and pharmaceutical compositions comprising formulations suitable for administering the compounds described herein. Formulations including pharmaceutical compositions suitable for administration by any medically acceptable means. The pharmaceutical formulations may comprise pharmaceutically acceptable additives or carriers suitable for the mode of administration, and pharmaceutically acceptable compounds (compositions).

[0127] The compounds described herein can be formulations (including pharmaceutical compositions) with additives such as excipients (e.g., one or more excipients), antioxidants (e.g., one or more antioxidants), stabilizers (e.g., one or more stabilizers), preservatives (e.g., one or more preservatives), pH regulators and / or buffers (e.g., one or more pH regulators and / or buffers), isotonicity regulators (e.g., one or more isotonicity regulators), thickeners (e.g., one or more thickeners), suspending agents (e.g., one or more suspending agents), binders (e.g., one or more binders), tackifiers (e.g., one or more tackifiers), etc., and are provided as pharmaceutically acceptable additional components for the specific conditions to be treated. In some embodiments, the formulation may comprise a combination of the additional components described herein (e.g., 2, 3, 4, 5, 6, 7, 8 or more additional components). In some embodiments, the additives may comprise, for example, therapeutic agents and drug delivery modifiers, as well as enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starches, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, glucose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low melting point waxes and ion exchange resins, and combinations of any two or more thereof.

[0128] Other suitable pharmaceutically acceptable excipients are described in "Remington's Pharmaceutical Sciences", Mack Pub Co., New Jersey (1991) and "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005), which are incorporated herein by reference.

[0129] The formulations of the compositions described herein may be suitable for oral administration and may consist of inhalants, nasal sprays, intravenous, intramuscular, intravitreal injections, ointments or solutions, suspensions, semi - liquids, semi - solids, gels, semi - solid gels, jellies, emulsions, ointments, oils, tablets, liquids, and creams. The tablet dosage form may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, humectants, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically suitable carriers. Capsules may contain appropriate excipients along with the compound, or the compound may be used alone in the shell. All of these formulated compounds may be administered alone or in combination, or intermittently, or sequentially, or simultaneously.

[0130] 4. Administration

[0131] The compositions of the present invention include compositions that can be administered by any method including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, transtympanic, intratympanic, intracochlear, by inhalation, buccal, or intranasal administration, or by a combination thereof. Parenteral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intracapsular, and intra - arterial administration. Additionally, the compositions of the present invention can be administered as implants, and this allows for slow release of the composition, as well as slow - controlled intravenous administration.

[0132] The dose administered to an individual in a single dose or multiple doses will vary depending on various factors, including pharmacokinetic characteristics, patient condition and characteristics (gender, age, weight, health, body size), symptom severity, concomitant therapy, treatment frequency, and the desired effect.

[0133] According to one embodiment of the present invention, the compounds according to the present invention and their pharmaceutical formulations may be administered alone or in combination with useful adjuvants for the treatment of respiratory disorders or diseases. According to another embodiment of the present invention, the compounds according to the present invention and their pharmaceutical formulations may be administered in combination with radiotherapy.

[0134] The present invention includes the administration of the compounds according to the present invention or their pharmaceutical formulations, and the compounds according to the present invention and their pharmaceutical formulations are administered to a subject simultaneously or sequentially in a therapeutically effective amount prior to other therapies or adjuvants (e.g., multi - drugs) for the treatment of deaf - goiter syndrome. The compounds or pharmaceutical formulations according to the present invention may be administered simultaneously with the adjuvant in the same or different compositions by the same or different routes of administration.

[0135] In one embodiment, a patient according to the present invention can be a patient suffering from Pendred syndrome or its related diseases, such as hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, volume depletion, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or sinusitis.

[0136] 5. Use according to the present invention

[0137] In another embodiment, the present invention provides the use of a compound represented by Chemical Formula 1 for preventing, ameliorating, or treating Pendred syndrome or its related diseases, a mixture of such compounds, or a pharmaceutical composition thereof.

[0138] In another embodiment, the present invention provides the use of a compound represented by Chemical Formula 1 and its pharmaceutical composition as a pendrin corrector.

[0139] In another embodiment, the present invention provides a use for treating diseases related to Pendred syndrome, the diseases related to Pendred syndrome being selected from one or more of the group consisting of hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, volume depletion, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or sinusitis.

[0140] In another embodiment, the present invention provides the use of a compound represented by Chemical Formula 1 for preventing or ameliorating Pendred syndrome or its related diseases, a mixture of such compounds, or a pharmaceutical composition thereof as an active ingredient in a health functional food.

[0141] In another embodiment, the present invention provides the use of a compound represented by Chemical Formula 1 and its pharmaceutical composition as a pendrin inhibitor for preventing or ameliorating Pendred syndrome or its related diseases, and as an active ingredient in a health functional food.

[0142] Other aspects and advantages of the present invention will become apparent to those skilled in the art upon reference to the detailed description and the drawings.

[0143] Embodiments of the present invention

[0144] [Embodiment]

[0145] This document describes non - restrictive embodiments of detailed experiments without limiting the entire experiment. The description of the present invention herein is by way of example, and those skilled in the art should understand that it can be easily changed into other specific fields or forms without changing the technical spirit or essential characteristics of the present invention. The description of the present invention is illustrated only by the following embodiments, but is not limited thereto.

[0146] References cited herein are incorporated herein by reference in their entirety. The scope of the present invention is not limited to the specific embodiments described herein, which are intended to be single embodiments of a single aspect of the present invention, and functionally equivalent methods and components are within the scope of the present invention. In practice, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0147] Scheme 1 - General Synthesis Route I

[0148]

[0149] General synthesis steps of A2

[0150] A mixture of A1, A1 - 1, Na2CO3, cyclopentyl(diphenyl)phosphine, dichloropalladium, iron / di alkane and H2O was degassed and purged with N2 three times, and then the mixture was stirred at 100 °C for 2 h under a N2 atmosphere. The reaction mixture was poured into water and extracted with ethyl acetate, washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel to give A2.

[0151] General synthesis steps of A3

[0152] K2CO3 and tert - butyl 2 - cyanoacetate were added to a DMF solution of A2. The mixture was stirred at 120 °C for 16 h. The reaction mixture was poured into water and extracted with ethyl acetate, washed with brine, dried over Na2SO4 and concentrated under reduced pressure to give A3.

[0153] General synthesis steps of A4

[0154] Concentrated HCl was added to a mixture of A3 in EtOH and the mixture was stirred at 80 °C for 40 minutes. The mixture was quenched with saturated NaHCO3 to pH = 7 and then extracted with ethyl acetate. The combined organic layers were dried over Na2SO4. The filtrate was concentrated to give a crude product, which was purified by flash column chromatography on silica gel to give A4.

[0155] General synthesis steps of A5

[0156] At 20 °C, pyridine was added to a solution of A4 in dichloroethane. At 40 - 50 °C, 2-chloroacetyl chloride was added dropwise to the reaction mixture. The mixture was stirred at 100 °C for 20 minutes. The reaction mixture was used in the next step without further purification. At 20 °C, pyridine was added to a solution of A4 in dichloroethane. At 40 - 50 °C, 2-chloroacetyl chloride was added dropwise to the reaction mixture. The mixture was stirred at 100 °C for 20 minutes. The reaction mixture was used in the next step without further purification.

[0157] General synthesis steps of A6

[0158] DMF and B1 were added to a dichloroethane solution of A5 from the previous step. The mixture was stirred at 100 °C for 10 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative-HPLC and lyophilized to obtain A6. DMF and B1 were added to a dichloroethane solution of A5 from the previous step. The mixture was stirred at 100 °C for 10 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative-HPLC and lyophilized to obtain A6.

[0159] Scheme 2 - General synthesis route II

[0160]

[0161] General synthesis steps of B2

[0162] At 60 °C, ethyl 2-bromoacetate was added to a solution of B1 and K2CO3 in acetone over 10 hours. The reaction mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The concentrated filtrate was obtained as a crude product, which was purified by flash column chromatography on silica gel to obtain B2.

[0163] General synthesis steps of B3

[0164] At 0 °C, Boc2O and DMAP were added to a CH2Cl2 solution of B2. The reaction mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The concentrated filtrate was obtained as a crude product, which was purified by flash column chromatography on silica gel to obtain B3.

[0165] General synthesis steps of B4

[0166] LiOH·H2O was added to a solution of B3 in THF and H2O, and the mixture was stirred at 20 °C for 1 h, then the mixture was stirred at 80 °C for 1 h. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), then poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and then concentrated under reduced pressure to obtain B4.

[0167] General synthesis steps of B6

[0168] CDI was added to a CH3CN solution of B4, and the reaction mixture was stirred at 20 °C for 40 minutes. TLC showed complete consumption of B4. B5 was used for the next step.

[0169] t-BuOK was added to a solution of B5 and A4 in CH3CN, and the mixture was stirred at 80 °C for 1 h. The reaction mixture was poured into water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and then the reaction mixture was concentrated in vacuo to give B6.

[0170] General synthesis steps of B6

[0171] 37% HCl was added to an EtOH solution of B6. The reaction mixture was stirred at 80 °C for 1 h. The reaction solution was concentrated and purified to give A6.

[0172] Scheme 3 - General synthesis of Compound 1

[0173]

[0174] General synthesis steps of C2

[0175] A mixture of C1 (3.68 g, 24.73 mmol), (3,4-dimethoxyphenyl)boronic acid (3 g, 16.49 mmol), Na2CO3 (3.49 g, 32.97 mmol), cyclopentyl(diphenyl)phosphine, dichloropalladium, iron (1.21 g, 1.65 mmol) in dioxane (10 mL) and H2O (1 mL) was degassed and purged with N2 three times, and then the mixture was stirred at 100 °C for 2 h under a N2 atmosphere. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (50 mL × 3), washed with brine (50 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (PE solution with 0 - 35% ethyl acetate) to give white solid C2 (2.57 g, 62.2% yield). K2CO3 (1.10 g, 7.98 mmol) and tert-butyl 2-cyanoacetate (675.76 mg, 4.79 mmol) were added to a solution of C2 (1 g, 3.99 mmol) in DMF (10 mL). The mixture was stirred at 120 °C for 16 h. The reaction mixture was poured into water (10 mL), extracted with ethyl acetate (10 mL × 3), washed with brine (10 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give yellow solid C3 (1.5 g, crude product, ~93% purity).

[0176] General synthesis steps of C3

[0177] ​

[0178] General synthesis steps of C4

[0179] Concentrated HCl (6 mL) was added to a mixture of C3 (1.5 g, ∼93% purity, 3.94 mmol) in EtOH (25 mL), and the mixture was stirred at 80 °C for 40 minutes. The mixture was quenched with saturated NaHCO3 to pH = 7 and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over Na2SO4. The filtrate was concentrated to give the crude product, which was purified by flash column chromatography (petroleum ether solution of 0 - 50% ethyl acetate) to give yellow solid C4 (607 mg, 60.4% yield).

[0180] General synthesis steps of C5

[0181] At 20 °C, pyridine (18.59 mg, 18.97 μL) was added to a solution of C4 (50 mg, 195.87 μmol) in dichloromethane (1 mL). At 40 - 50 °C, 2 - chloroacetyl chloride (22.12 mg, 195.87 μmol) was added dropwise to the reaction mixture. The mixture was stirred at 100 °C for 20 minutes. The reaction mixture was used for the next step without further purification. At 20 °C, pyridine (18.59 mg, 18.97 μL) was added to a solution of C4 (50 mg, 195.87 μmol) in dichloromethane (1 mL). At 40 - 50 °C, 2 - chloroacetyl chloride (22.12 mg, 195.87 μmol) was added dropwise to the reaction mixture. The mixture was stirred at 100 °C for 20 minutes. The reaction mixture was used for the next step without further purification.

[0182] General synthesis steps of Compound 1

[0183] DMF (1 mL) and 2 - fluoroaniline (65.29 mg, 587.61 μmol) were added to a solution of C5 (64.98 mg, 195.87 μmol) from the previous step in dichloromethane. The mixture was stirred at 100 °C for 10 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative - HPLC (column: Waters Xbridge 150×25 5u, mobile phase: 63 - 93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm), and after lyophilization, brown solid compound 1 (3 mg, 3.6% yield, 95% purity) was obtained. DMF (1 mL) and 2 - fluoroaniline (65.29 mg, 587.61 μmol) were added to a solution of C5 (64.98 mg, 195.87 μmol) from the previous step in dichloromethane. The mixture was stirred at 100 °C for 10 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative - HPLC (column: Waters Xbridge 150×25 5u, mobile phase: 63 - 93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm), and after lyophilization, brown solid compound 1 (3 mg, 3.6% yield, 95% purity) was obtained.

[0184] Scheme 4 - General synthesis of Compound 2

[0185]

[0186] General synthesis steps of C7

[0187] C6 (3 g, 15.51 mmol), (3,4 - dimethoxyphenyl)boronic acid (3 g, 16.49 mmol), Na2CO3 (3.29 g, 31.02 mmol), Pd(dppf)Cl2 (1.13 g, 1.55 mmol) were degassed and purged with N2 three times in a mixture of dichloromethane (30 mL) and H2O (5 mL), and then stirred at 80 °C under a N2 atmosphere for 3 hours. The filtrate was concentrated to obtain a crude product, which was purified by flash column chromatography on silica gel (petroleum ether solution of 0 - 40% EtOAc) to give a grayish - white solid C7 (2.76 g, 71.0% yield). To a solution of C7 (2.66 g, 10.61 mmol) in DMF (30 mL) was added K2CO3 (2.93 g, 21.22 mmol) and tert - butyl 2 - cyanoacetate (1.80 g, 12.73 mmol, 1.82 mL). The mixture was stirred at 120 °C for 16 hours. The mixture was quenched with water (20 mL), the pH was adjusted to about 4 with 1 M HCl, and then filtered. The filter cake was dried under reduced pressure to give a brown solid C8 (3.4 g, 90.2% yield).

[0188] General synthesis steps of C8

[0189] C8 (3.4 g, 9.57 mmol) was stirred in a mixture of HCl (3 mL) and EtOH (30 mL) at 90 °C for 40 minutes. The mixture was quenched with saturated NaHCO3 to pH = 8 and extracted with ethyl acetate (100 mL × 3). The combined organic layers were dried over Na2SO4. The filtrate was concentrated to obtain a crude product, which was purified by flash column chromatography on silica gel (petroleum ether solution of 0 - 50% EtOAc) to give a yellow solid C9 (1.77 g, 72.5% yield).

[0190] General synthesis steps of C9

[0191]

[0192] General synthesis steps of C10-2

[0193] ​At 60 °C, ethyl 2-bromoacetate (16.53 g, 99.00 mmol) was added to a solution of C10-1 (10 g, 90.00 mmol, 8.70 mL) and K2CO3 (17.41 g, 125.99 mmol) in acetone (20 mL) over 10 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated under reduced pressure. The concentrated filtrate gave a crude product, which was purified by flash column chromatography on silica gel (petroleum ether solution of 0-10% EtOAc) to give a brown liquid C10-2 (17.3 g, crude product).

[0194] General synthesis steps of C10-3

[0195] At 0 °C, Boc2O (22.13 g, 101.42 mmol, 23.30 mL) and DMAP (9.29 g, 76.06 mmol) were added to a solution of C10-2 (10 g, 50.71 mmol) in CH2Cl2 (100 mL). The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated under reduced pressure. The concentrated filtrate gave a crude product, which was purified by flash column chromatography on silica gel (petroleum ether solution of 0-10% EtOAc) to give a colorless oil C10-3 (2.84 g, 20.0% yield).

[0196] General synthesis steps of C10

[0197] LiOH·H2O (338.73 mg, 8.07 mmol) was added to a solution of C10-3 (1.2 g, 4.04 mmol) in THF (6 mL) and H2O (2 mL), and the mixture was stirred at 20 °C for 1 h, then the mixture was stirred at 80 °C for 1 h. The reaction mixture was adjusted to pH = 4 with HCl (mol / L), then poured into water (10 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over Na2SO4 and then concentrated under reduced pressure to give a brown solid C10 (887 mg, 2.96 mmol, 73.5% yield).

[0198] General synthesis steps of C12

[0199] To a solution of C10 (150 mg, 557.07 μmol) in CH3CN (2 mL) was added CDI (90.33 mg, 557.07 μmol), and the mixture was stirred at 20 °C for 40 minutes. TLC (PE:EtOAc = 3:1) showed that C10 (150 mg, 557.07 μmol) (Rf = 0.3) was completely consumed. C11 (177.89 mg crude product) was used for the next step. To a solution of C11 (177.89 mg, 554.29 μmol) and C9 (141.49 mg, 554.29 μmol) in CH3CN (3 mL) was added t-BuOK (62.20 mg, 554.29 μmol), and the mixture was stirred at 80 °C for 1 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4, and then the reaction mixture was concentrated in vacuo to give yellow solid C12 (358 mg crude product).

[0200] General synthesis steps of Compound 2

[0201] To a solution of C12 (350 mg, 690.98 μmol) in EtOH (3 mL) was added HCl (0.3 mL). The reaction mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated, and the crude product was purified by preparative-HPLC (column: Waters Xbridge 150×25 5u, eluent: 63 - 93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm) to give yellow solid Compound 2 (12.7 mg, 4.5% yield).

[0202] Scheme 5 - General synthesis of Compound 3

[0203]

[0204] General synthesis steps of D2

[0205] To a solution of C10 (100 mg, 371 μmol) in CH3CN (2 mL) was added CDI (60.22 mg, 371 μmol), and the reaction mixture was stirred at 20 °C for 40 minutes. TLC (PE:EtOAc = 3:1) showed that C10 (Rf = 0.3) was completely consumed.

[0206] Transfer half of the above reaction solution to another flask, add D1 (47 mg, 184 μmol) in CH3CN (0.5 mL), then add t-BuOK (20.73 mg, 185 μmol), heat up to 80 °C and stir for 1 h. Adjust the reaction mixture to pH = 4 with HCl (1 mol / L), then pour it into water (10 mL) and extract with EtOAc (10 mL * 3). Wash the combined organic layers with brine (10 mL × 3), dry over Na2SO4, and then concentrate under reduced pressure to obtain D2 (59 mg, crude product, 63.17% yield), which is directly used in the next step.

[0207] General synthesis steps of Compound 3

[0208] Add 37% HCl (0.05 mL) to a solution of D2 (159 mg, 116.71 μmol) in EtOH (0.5 mL). Stir the reaction mixture at 80 °C for 1 h. Concentrate the reaction solution and purify it by preparative-HPLC (column: Waters Xbridge 150×25 5u, eluent: 63 - 93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm) to obtain Compound 3 (22.3 mg, 44.77% yield, 95% purity).

[0209] Scheme 6 - General synthesis of Compound 4

[0210]

[0211] General synthesis steps of E2

[0212] Under N2, add K2CO3 (2 g, 14.47 mmol) and tert-butyl 2-cyanoacetate (1 g, 7.08 mmol) to a solution of E1 (1 g, 6.71 mmol) in DMF (15 mL). Stir the mixture at 25 °C for 16 h. Quench the mixture with water (30 mL) and extract with ethyl acetate (15 mL × 3). Dry the combined organic layers over Na2SO4, filter and concentrate to obtain the crude product E2 (2 g, 85% purity) as a brown oil, which is directly used in the next step without further purification.

[0213] General synthesis steps of E3

[0214] A mixture of E2 (2 g, 6.70 mmol, 85% purity) in HCl (3 mL) (concentrated) and ethanol (15 mL) was stirred at 90 ° C for 40 minutes. The mixture was quenched with saturated NaHCO 3 to pH = 8 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. The residue was purified by flash column chromatography on silica gel (30% ethyl acetate / PE) to give E3 (350 mg, 34.01% yield) as a yellow oil.

[0215] General synthesis steps of E4

[0216] E3 (350 mg, 2.28 mmol) and (3,4-dimethoxyphenyl)boronic acid (414.75 mg, 2.28 mmol) were added to distilled water. To a solution of dapoxetine (5 mL) and H2O (1 mL) was added Pd(dppf)Cl2 (166.76 mg, 227.91 μmol) and Na2CO3 (483.12 mg, 4.56 mmol). The mixture was stirred at 100 ° C under N2 for 2 hours. The mixture was filtered. The filtrate was concentrated to give the crude product, which was purified by flash chromatography on silica gel (0-50% EtOAc in petroleum ether) to give E4 (425 mg, 73% yield) as a yellow solid.

[0217] General synthesis steps of E5

[0218] To a solution of C10 (100 mg, 371.38 μmol) in CH 3 CN (2 mL) was added CDI (60.2 mg, 371.38 μmol) and stirred at 20° C. for 12 hours. To another solution of E4 (47.1 mg, 184.76 μmol) in CH 3 CN (1 mL) was added t-BuOK (20.7 mg, 184.76 μmol) and half of the first reaction solution, and the compound was stirred at 80° C. for 1 h. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), then poured into water (10 mL) and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over Na 2 SO 4 , filtered and concentrated in vacuo to give E5 (93 mg, crude product) as a yellow solid.

[0219] General synthesis steps of Compound 4

[0220] HCl (0.1 mL) was added to a solution of E5 (93 mg, 183.60 μmol) in EtOH (1 mL). The reaction mixture was stirred at 80 °C for 1 h. The mixture was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um, eluent: 43%-53% B (A = water (TFA), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm) and (column: Waters Xbridge 150×25 5u, eluent: 63-93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm) to give the compound 4 as a yellow solid (17.1 mg, 95% purity, 22.9% yield).

[0221] Scheme 7 - General synthesis of Compound 5 and Compound 57

[0222]

[0223] General synthesis steps of F2

[0224] To a solution of F1 (13.42 g, 13.42 mmol) in DMF (5 mL) was added tert-butyl 2-cyanoacetate (2.27 g, 16.11 mmol) and K2CO3 (3.71 g, 26.85 mmol). The resulting mixture was stirred at 120 °C for 12 h. The reaction was diluted with water (10 mL) and adjusted to pH = 6 with HCl (1 M), then extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was combined with another parallel batch, 2 g of F1 was taken, the two batches were combined and purified by column chromatography (SiO2, DCM:MeOH = 10:1) to give F2 as a yellow solid (1.28 g, 6.87% yield, 78% purity) and F2 as a yellow solid (1.07 g, 18.54% yield, 59% purity).

[0225] General synthesis steps of F3

[0226] To a solution of F2 (300 mg, 1.18 mmol, 78% purity) in DMSO (2 mL) and H2O (2 mL) was added NaCl (138 mg, 2.37 mmol). The mixture was stirred at 130 °C for 1 hour. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. 1.25 g of compound 2 was subjected to another parallel reaction, and the two batches of reactants were combined and treated. The combined residue was purified by column chromatography (SiO2, DCM:MeOH = 10:1) to give F3 as a yellow solid (287.6 mg, 48.46% yield).

[0227] General synthesis steps of F4

[0228] To a solution of F3 (100 mg, 0.65 mmol) and Na2CO3 (0.5 mL, 2 M) in DME (1.5 mL) was added (3,4-dimethoxyphenyl)boronic acid (154.05 mg, 0.85 mmol), Pd(dppf)Cl2 (23.82 mg, 0.03 mmol). The mixture was microwave-treated at 120 °C for 30 minutes. The reaction mixture was combined with another batch of 100 mg of parallel reactants and combined for treatment. The combined reaction mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give F4 as a yellow oil (72 mg, 35.52% yield, 82% purity).

[0229] General synthesis steps of F5

[0230] Solution A: To a solution of C10 (80 mg, 0.29 mmol) in CH3CN (0.3 mL) was added CDI (48.17 mg, 0.29 mmol). The mixture was stirred at 25 °C for 5 minutes.

[0231] Solution B: To another solution of F4 (70 mg, 0.27 mmol) in CH3CN (1 mL) was added t-BuOK (30.77 mg, 0.27 mmol), and the mixture was stirred at 80 °C for 3 minutes.

[0232] At 80 °C, mixture A was added dropwise to mixture B. The resulting mixture was stirred at 80 °C for 15 minutes. The reaction mixture was quenched with H2O (5 mL) and extracted with EtOAc (5 mL × 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product F5 (98 mg, crude) as a yellow oil.

[0233] General synthesis steps of Compound 5

[0234] To a solution of F5 (98 mg, 0.19 mmol) in EtOH (1 mL) was added 37% HCl (0.2 mL). The mixture was stirred at 90 °C for 1 h. The reaction mixture was concentrated and then the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 22%-42%, 12 min) to give compound 5 as a white solid (4.8 mg, 4.3% yield over two steps, 99.34% purity).

[0235] General synthesis steps of Compound 57

[0236] A suspension of compound 5 (another batch, 370 mg, 910.41 μmol) in HBr (5 mL, 40% purity) was heated to 120 °C for 10 h. The crude product was purified by preparative-HPLC (column: Boston Green Prime C18 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 12%-42%, 12 min) to give compound 57 as a grey solid (84.6 mg, 24% yield, 98% purity).

[0237] Scheme 8 - General synthesis of Compound 6

[0238]

[0239] General synthesis steps of E7

[0240] Ethyl 2-bromoacetate (1.64 g, 9.80 mmol) was added to a solution of E6 (5 g, 39.19 mmol) and DIEA (5.07 g, 39.19 mmol) in DMF (20 mL), and the mixture was stirred at 100 °C for 10 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated under reduced pressure. The filtrate was concentrated to give the crude product, which was purified by flash column chromatography on silica gel (petroleum ether solution of 0 - 30% EtOAc) to give yellow oil E7 (1.4 g, 33% yield, crude product).

[0241] General synthesis steps of E8

[0242] Boc2O (2.86 g, 13.10 mmol) and DMAP (1.20 g, 9.83 mmol) were added to a solution of E7 (1.4 g, 6.55 mmol) in CH2Cl2 (10 mL), and the mixture was stirred at 0 °C for 20 min, then Boc2O (4.29 g, 19.66 mmol) was added at 0 °C. The reaction mixture was stirred at 20 °C for 12 h. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4. The organic layer was concentrated in vacuo. The residue was purified by flash silica column chromatography (0 - 10% EtOAc in PE). The crude product was then purified by preparative-HPLC (column: Waters Xbridge 150×25 5u, eluent: 63 - 93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector at 220 nm) to give colorless oil E8 (217 mg, 10.34% yield).

[0243] General synthesis steps of E9

[0244] LiOH·H2O (57.24 mg, 1.36 mmol) was added to a solution of E8 (214 mg, 682.02 μmol) in THF (2 mL) and H2O (1 mL). The mixture was stirred at 20 °C for 10 h. The reaction mixture was adjusted to pH = 5 with HCl (1 mol / L), then poured into water (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated under reduced pressure to give yellow oil E9 (170 mg, 85.49% yield).

[0245] General synthesis steps of E11

[0246] To a solution of E9 (80 mg, 279.99 μmol) in CH3CN (2 mL) was added CDI (54.48 mg, 335.99 μmol), and the mixture was stirred at 20 °C for 10 h. TLC (PE:EtOAc = 3:1) showed that E9 (Rf = 0.3) was completely consumed. The reaction mixture of E10 was used for the next step.

[0247] To a solution of E4 (71.46 mg, 279.94 μmol) in CH3CN (3 mL) was added t-BuOK (47.12 mg, 419.91 μmol), and the mixture was stirred at 20 °C for 10 min. Then the reaction mixture of E10 was added, and the reactants were stirred at 80 °C for 1 h. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), then poured into water (5 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over Na2SO4, and then concentrated under reduced pressure to give a yellow solid E11 (189 mg, crude product).

[0248] General synthesis steps of Compound 6

[0249] To a solution of E11 (189 mg, 361.39 μmol) in EtOH (2 mL) was added HCl (0.2 mL, 12 M / L). The reaction mixture was stirred at 80 °C for 1 h. The crude product was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5 um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 41%-71%, 10 min). The reactant was lyophilized to give the white solid compound 6 (2.6 mg, 1.67% yield, 100% purity).

[0250] Scheme 9 - General synthesis of Compound 7

[0251]

[0252] General synthesis steps of G2

[0253] To (3,4-dimethoxyphenyl)boronic acid (930 mg, 5.11 mmol) in dichloro- To a solution of alkane (10 mL) and H2O (2 mL), add G1 (1.01 g, 5.11 mmol), Na2CO3 (1.08 g, 10.22 mmol) and Pd(PPh3)4 (590.54 mg, 0.51 mmol). Stir the mixture at 80 °C for 2 hours. Dilute the reaction mixture with H2O (300 mL) and extract with EtOAC (50 mL × 3). Wash the combined organic layers with brine (50 mL × 3), dry over anhydrous Na2SO4, filter and concentrate under reduced pressure to obtain a residue. Purify the residue by column chromatography (SiO2 (petroleum ether / ethyl acetate = 1 / 1)) to obtain yellow solid G2 (720 mg, 38.23% yield, 69% purity).

[0254] General synthesis steps of G3

[0255] A: To a solution of E9 (350 mg, 1.22 mmol) in CH3CN (3 mL), add CDI (198.63 mg, 1.22 mmol). Stir the mixture at 25 °C for 2 hours to obtain E10 as a yellow solution in CH3CN.

[0256] B: To a solution of G2 (300 mg, 1.18 mmol) in CH3CN (3 mL), add t-BuOK (264.77 mg, 2.36 mmol) and E10 (396.15 mg, 1.18 mmol). Stir the mixture at 80 °C for 1 hour. Combine the reaction mixture with another batch of reaction mixture (100 mg of 2) and process them together. Dilute the combined reaction mixture with H2O (30 mL) and extract with EtOAc (30 mL × 3). Dry the combined organic layers over anhydrous Na2SO4, filter and concentrate under reduced pressure to obtain G3 (779 mg, crude product) as a brown solid.

[0257] General synthesis steps of Compound 7

[0258] To a solution of G3 (670 mg, 1.28 mmol) in EtOH (7 mL), add 37% HCl (1 mL). Stir the mixture at 90 °C for 1 hour. Combine the reaction mixture with another batch of reaction mixture (100 mg from 3), combine the two batches of reaction mixtures and process them together. Concentrate the reaction mixture under reduced pressure to remove the solvent, and then purify by preparative-HPLC (column: BostonGreen ODS 150 × 30 mm × 5um; mobile phase: [water (FA)-ACN]; B%: 7%-37%, 12 min) to obtain compound 7 (237 mg, 43.33% yield, 99.74% purity) as a white solid.

[0259] Scheme 10 - General synthesis of Compound 8

[0260]

[0261] General synthesis steps of E13

[0262] Ethyl 2-bromoacetate (5 g, 29.94 mmol) was added to a solution of E12 (4.38 g, 59.88 mmol) in CH2Cl2 (50 mL), and the mixture was stirred at 25 °C for 12 h. The mixture was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E13 as a yellow oil (3.02 g, 63.35% yield).

[0263] General synthesis steps of E14

[0264] At 0 °C, Et3N (2.78 g, 27.51 mmol) and Boc2O (4.00 g, 18.34 mmol) were added to a solution of E13 (2.92 g, 18.34 mmol) in CH2Cl2 (30 mL). The mixture was stirred at 25 °C for 12 h. The mixture was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E14 as a yellow oil (3.73 g, 78.43% yield).

[0265] General synthesis steps of E15

[0266] LiOH.H2O (1.62 g, 38.56 mmol) was added to a solution of E14 (2 g, 7.71 mmol) in THF (20 mL) and H2O (4 mL), and the mixture was stirred at 60 °C for 2 h. The reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL × 3). The aqueous phase was adjusted to pH 5 with HCl (1 M) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give E15 as a yellow oil (1.5 g, 84.10% yield).

[0267] General synthesis steps of E16

[0268] To a solution of E15 (90.60 mg, 0.39 mmol) in CH3CN (1 mL) was added CDI (76.22 mg, 0.47 mmol), and the mixture was stirred at 25 °C for 2 h. To another solution of E4 (100 mg, 0.39 mmol) in CH3CN (1 mL) was added t-BuOK (87.92 mg, 0.78 mmol), and the mixture was stirred at 80 °C for 5 min. Then the first solution was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 min. The mixture was added to water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give E16 as a white solid (170 mg, 92.62% yield, 100% purity).

[0269] General synthesis steps of Compound 8

[0270] To a solution of E16 (170 mg, 0.36 mmol) in EtOH (5 mL) was added 37% HCl (1 mL). The mixture was stirred at 80 °C for 0.5 h. The mixture was quenched with saturated NaHCO3 to pH = 8 and extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated. The crude product was purified by preparative-HPLC [column: Boston Prime C18 150 × 30 mm × 5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 37%-67%, 10 min], and then dried by lyophilization to give Compound 8 as a white solid (61.3 mg, 46% yield).

[0271] Scheme 11 - General synthesis of Compound 9

[0272]

[0273] General synthesis steps of Compound 9_HCl salt

[0274] A solution of Compound 6 (3 g, 7.09 mmol) in HBr (20 mL, 40% purity) was heated to 130 °C for 10 h, followed by stirring at 140 °C for 10 h. The mixture was filtered and washed with MeOH (20 mL). The filter cake was purified by preparative-HPLC (column: Boston Green ODS 150 × 30 mm × 5um; mobile phase: [water (HCl)-ACN]; B%: 23%-43%, 10 min) to give Compound 9_HCl salt as a yellow solid (1.64 g, 58.85% yield, 100% purity, HCl).

[0275] General synthesis steps of Compound 9_free form

[0276] To a solution of Compound 9_HCl salt (50 mg, 126.64 μmol) in H2O (5 mL) was added Na2CO3 (26.85 mg, 253.29 μmol). The mixture was stirred at 20 °C for 10 h. The mixture was filtered and washed with H2O (10 mL). The filter cake was collected and dried by lyophilization to give Compound 9 in free form as a yellow solid (39 mg, 78.00% yield, 99% purity).

[0277] ""

[0278]

[0279] Scheme 12 - General synthesis of Compound 10

[0280] To a solution of E9 (1 g, 3.50 mmol) in CH3CN (5 mL) was added CDI (567.50 mg, 3.50 mmol), and the mixture was stirred at 25 °C for 2 h. In another flask, to a solution of E3 (500 mg, 3.26 mmol) in CH3CN (5 mL) was added t-BuOK (730.69 mg, 6.51 mmol) and then the first solution. The mixture was stirred at 80 °C for 1 h. The mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated, filtered and concentrated under reduced pressure to give E17 (1.54 g, crude product), which was used in the next step without further purification.

[0281] General synthesis steps of E17

[0282] To a solution of E17 (1 g, 2.37 mmol) in EtOH (10 mL) was added HCl (2.5 mL, 37% purity), and the reaction was heated to 90 °C and maintained for 1 h. The solvent was removed in vacuo, and the residue was basified to pH = 8 with aqueous NaHCO3. The mixture was extracted with CH2Cl2 (30 mL × 3). The organic layer was washed with brine (30 mL), dried over sodium sulfate and concentrated. The residue was triturated with a PE / EtOAc (5 / 1, 10 mL) solution and then filtered. The solid was dried in vacuo to give E18 as a black solid (300 mg, 39% yield).

[0283] General synthesis steps of E18

[0284] To a solution of E18 (50 mg, 155.69 μmol) and 1,3-benzodioxol-5-ylboronic acid (31 mg, 187 μmol) in dioxane (1 mL) and H2O (0.2 mL) were added Pd(dppf)Cl2, CH2Cl2 (12.71 mg, 15.57 μmol), and Na2CO3 (33 mg, 311.37 μmol). The reaction was degassed and refilled with N2 three times, then heated to 100 °C and maintained for 4 h. The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (20 mL), dried over sodium sulfate, and concentrated. The crude product was purified by preparative-TLC (100% EtOAc) to obtain 30 mg of the crude product. The crude product was purified by preparative-HPLC [column: O-Phenomenex C18 150×30 mm×5 μm; mobile phase: [water (NH3·H2O + NH4HCO3)-ACN]; B%: 33% - 63%, 10 min] to obtain Compound 10 as a white solid (4.3 mg, 6.3% yield, 93% purity).

[0285] General synthesis steps of Compound 10

[0286]

[0287] Scheme 13 - General synthesis of Compound 15 and 18

[0288] To a solution of E19 (500 mg, 3.69 mmol, HCl) in THF (10 mL) was added TEA (932.55 mg, 9.22 mmol) and ethyl 2-bromoacetate (677.19 mg, 4.06 mmol). Then the mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with water (30 mL) and extracted with EtOAc (30 mL × 3). The organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by Combi Flash (SiO2, 50% to 60% EtOAc / PE) to obtain E20 as a colorless oil (460 mg, 67.36% yield).

[0289] General synthesis steps of E20

[0290] ​To a solution of E20 (460 mg, 2.48 mmol) in CH2Cl2 (10 mL) was added Et3N (502.50 mg, 4.97 mmol) and Boc2O (812.86 mg, 3.72 mmol), and the mixture was stirred at 25 °C for 2 h. The mixture was diluted with CH2Cl2 (50 mL) and washed with 1N HCl (30 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to give E21 (700 mg, crude product) as a colorless oil.

[0291] General synthesis steps of E21

[0292] To a solution of E21 (700 mg, 2.45 mmol) in THF (10 mL) was added a solution of LiOH·H2O (154.38 mg, 3.68 mmol) in water (5 mL), and the mixture was stirred at 60 °C for 2 h. Water (10 mL) was added to the mixture, and the mixture was treated with 1N HCl to adjust the pH to 2, and then extracted with EtOAc (20 mL × 2). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated to give E22 (450 mg, 71.29% yield) as a colorless oil.

[0293] General synthesis steps of E22

[0294] At 20 °C, CDI (69.87 mg, 0.43 mmol) was added to a solution of E22 (110.88 mg, 0.43 mmol) in MeCN (2 mL), and the mixture was stirred for 30 min. At 20 °C, t-BuOK (43.96 mg, 0.39 mmol) was added to another solution of E4 (100 mg, 0.39 mmol) in MeCN (2 mL), then heated to 80 °C, and the first solution was added. The reaction mixture was stirred at 80 °C for 0.5 h. The reaction was diluted with water (10 mL), acidified to pH = 5 with 1N HCl, and extracted with EtOAc (30 mL × 3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give E23 (190 mg, crude) as a yellow solid.

[0295] General synthesis steps of E23

[0296] HCl (0.3 mL) was added to a solution of E23 (190 mg, 0.38 mmol) in EtOH (2 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; B%: 40%-70%, 14 min) to give Compound 15 as a yellow solid (90 mg, 12.93% yield, 98.11% purity).

[0297] General synthesis steps of Compound 15

[0298] A solution of Compound 15 (35 mg, 0.09 mmol) in HBr (4 mL, 40% purity) was stirred at 120 °C for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (HCl)-ACN]; B%: 18%-38%, 10 min) to give Compound 18 as a yellow solid (5.3 mg, 15.00% yield, 92.78% purity).

[0299] General synthesis steps of Compound 18

[0300]

[0301] Scheme 14 - General synthesis of Compound 16

[0302] Cs2CO3 (1.42 g, 4.35 mmol) was added to a solution of E24 (500 mg, 2.90 mmol), E24-1 (560 mg, 2.90 mmol), Pd2(dba)3 (132.66 mg, 144.87 μmol) and Xantphos (83.82 mg, 144.87 μmol) in dioxane (5 mL). The mixture was degassed and recharged with nitrogen three times. The reaction was heated to 110 °C and maintained for 1 h. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (30 mL×3), dried over sodium sulfate and concentrated. The residue was purified by silica gel chromatography (PE / EtOAc = 2 / 1) to give E25 as a yellow solid (340 mg, 33.85% yield).

[0303] General synthesis steps of E25

[0304] ​A solution of E25 (310 mg, 941.25 μmol) in THF (0.5 mL) was added to a suspension of LiAlH4 (53.59 mg, 1.41 mmol) in THF (5 mL), and the mixture was stirred at -10 °C for 0.5 h. The reaction was quenched by adding water, drop by drop, and then a 15% NaOH solution, drop by drop. The mixture was diluted with a DCM / methanol (5 / 1, 20 mL) mixture and filtered. The filtrate was concentrated to give E26 as a yellow solid (450 mg, 95.19% yield, 60% purity).

[0305] General synthesis steps of E26

[0306] SOCl2 (98.70 mg, 829.63 μmol) was added to a solution of E26 (50 mg, 165.93 μmol) in DCM (1 mL), and the reaction mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give E27 as a brown solid (51 mg, crude product).

[0307] General synthesis steps of E27

[0308] TBAF (1 M, 318.96 uL) was added to a solution of E27 (51 mg, 159.48 μmol) and TMSCN (31.64 mg, 318.96 μmol) in THF (1 mL), and the reaction mixture was stirred at 25 °C for 16 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel chromatography (PE with 56% ethyl acetate) to give E28 as a yellow solid (23 mg, 46.47% yield).

[0309] General synthesis steps of E28

[0310] CDI (13.22 mg, 81.52 μmol) was added to a solution of E9 (23.29 mg, 81.52 μmol) in CH3CN (1 mL), and the reaction was stirred at 20 °C for 10 min.

[0311] t-BuOK (9.15 mg, 81.52 μmol) was added to another solution of E28 (23 mg, 74.11 μmol) in CH3CN (1 mL), and the reaction was heated to 80 °C. Then the first solution mentioned above was added. The reaction mixture was stirred at 80 °C for 15 min. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), then poured into water (5 mL), and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E29 as a brown solid (44 mg, crude product).

[0312] General synthesis steps of E29

[0313] HCl (0.5 mL, 12 mol / L purity) was added to a solution of E29 (40 mg, 69.20 μmol) in EtOH (5 mL). The reaction mixture was stirred at 80 °C for 1 hour. The crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (HCl)-ACN]; B%: 30%-50%, 10 min) to obtain compound 16 as a yellow solid (4.9 mg, 13.49% yield, 100% purity, HCl).

[0314] General synthesis steps of Compound 16

[0315]

[0316] Scheme 15 - General synthesis of Compound 20

[0317] To a solution of E18 (100 mg, 0.31 mmol) in dichloromethane (2 mL) and H2O (0.5 mL) was added (2,4-dimethoxyphenyl)boronic acid (56.66 mg, 0.31 mmol), Pd(dppf)Cl2 (22.78 mg, 0.03 mmol) and Na2CO3 (66.00 mg, 0.62 mmol), and the mixture was stirred at 100 °C for 1 hour. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (HCl)-ACN]; B%: 36%-56%, 10 min) to obtain compound 20 as a yellow solid (50 mg, 34.96% yield, 100% purity, HCl).

[0318] General synthesis steps of Compound 20

[0319]

[0320] Scheme 16 - General synthesis of Compound 21 and Compound 38

[0321] To a solution of E30 (50 mg, 0.229 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (87.3 mg, 0.343 mmol) in dichloromethane To a solution of alkane (2 mL) was added Pd(dppf)Cl2 (16.78 mg, 0.023 mmol) and KOAc (45.01 mg, 0.459 mmol). The mixture was stirred at 100 °C for 1 hour. LC-MS showed that E30 was completely consumed and E31 was formed. To the reaction mixture were added E18 (73.64 mg, 0.229 mmol), Na2CO3 (48.61 mg, 0.459 mmol), Pd(dppf)Cl2 (16.78 mg, 0.023 mmol) and H2O (0.4 mL). The resulting mixture was degassed and refilled with nitrogen 3 times, and then stirred at 100 °C for 1 hour. LC-MS showed that compound 2 was completely consumed and compound 21 was formed. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (HCl)-ACN]; B%: 50%-70%, 10 min) to obtain compound 21 as a yellow solid (14 mg, 98% purity, HCl, 13% yield).

[0322] General synthesis steps of Compound 21

[0323] A solution of compound 21 (20 mg, 0.05 mmol of another batch) in 40% HBr (1.5 mL) was stirred at 80 °C for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. Another parallel reaction (10 mg of compound 21) was carried out, and the crude products of the two batches were combined and purified together. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (HCl)-ACN]; B%: 15%-36%, 10 min) to obtain compound 38 as a white solid (3.8 mg, 19.33% yield, 95.73% purity).

[0324] General synthesis steps of Compound 38

[0325]

[0326] Scheme 17 - General synthesis of Compound 23

[0327] To a solution of E32 (2 g, 10.69 mmol) and Boc2O (11.67 g, 53.47 mmol) in DCM (30 mL) was added NaOH (13.4 mL, 2 N in H2O), and the reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with dichloromethane (70 mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (PE / EtOAc = 5 / 1) to give E33 as a brown solid (3.34 g, 81% yield).

[0328] General synthesis steps of E33

[0329] At 0 °C, to a solution of Cs2CO3 (2.52 g, 7.75 mmol) in DMF (10 mL) was added E33 (1 g, 2.58 mmol), followed by CH3I (1.83 g, 12.91 mmol). The mixture was stirred at 20 °C for 2 h. The mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E34 as a brown solid (1.27 g, crude product, 84% purity).

[0330] General synthesis steps of E34

[0331] To a solution of E34 (100 mg, 240.78 μmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (91.71 mg, 361.17 μmol) in dioxane (2 mL) was added Pd(dppf)Cl2 (17.62 mg, 24.08 μmol) and KOAc (70.89 mg, 722.33 μmol). The mixture was stirred at 100 °C for 2 h. LC-MS showed the formation of E35.

[0332] ​E18 (84.81 mg, 264.07 μmol), Na2CO3 (76.33 mg, 720.18 μmol), Pd(dppf)Cl2 (17.57 mg, 24.01 μmol) and H2O (0.6 mL) were added to the reaction mixture. The resulting mixture was degassed and recharged with N2 three times, and then stirred at 100 °C for 1 hour. LC-MS showed the formation of E36. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a brown solid E36 (220 mg, crude product).

[0333] General synthesis steps of E36

[0334] HCl (0.3 mL, 12 M / L) was added to a solution of E36 (220 mg, 354.20 μmol) in EtOH (3 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was purified by preparative-HPLC (column: BostonPrime C18 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 17%-47%, 12 min) to give the compound 23 as a yellow solid (4.9 mg, 4.8% yield over 3 steps, 100% purity).

[0335] General synthesis steps of Compound 23 Scheme 18 - General synthesis of Compound 25

[0336]

[0337] General synthesis procedure of E37

[0338] To a solution of E24 (3 g, 17.38 mmol) in dodecane (30 mL) were added tert-butyl piperazine-1-carboxylate (3.24 g, 17.38 mmol), Cs2CO3 (11.33 g, 34.77 mmol), Pd2(dba)3 (1.59 g, 1.74 mmol) and Xantphos (1.01 g, 1.74 mmol), and the mixture was stirred at 110 °C for 1 hour. The reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1) to give E37 as a brown solid (1.84 g, 28.57% yield, 87% purity).

[0339] General synthesis procedure of E38

[0340] At -10 °C, a solution of E37 (840 mg, 2.61 mmol) in THF (10 mL) was added dropwise to a solution of LiAlH4 (197.80 mg, 5.21 mmol) in THF (15 mL). The resulting mixture was stirred at -10 °C for 30 minutes. The mixture was quenched with water (0.2 mL), 15% aqueous NaOH solution (0.2 mL), and water (0.6 mL). The mixture was diluted with CH2Cl2 / MeOH (10 / 1, 100 mL), then Na2SO4 was added until the aluminum salt was adsorbed, filtered, and concentrated under reduced pressure to obtain a residue.

[0341] The reaction mixture was combined with another crude product (1 g of E37), and the combined crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to obtain E38 as a yellow solid (365 mg, 21% yield, 90% purity).

[0342] General synthesis procedure of E39

[0343] At 0 °C, SOCl2 (250.59 mg, 2.11 mmol) was added to a solution of E38 (310 mg, 1.05 mmol) in CH2Cl2 (3.5 mL). The reaction mixture was concentrated under reduced pressure to remove CH2CI2. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to obtain E39 as a yellow oil (78 mg, 23.68% yield).

[0344] General synthesis procedure of E40

[0345] To a solution of E39 (78 mg, 0.25 mmol) in THF (1.5 mL) was added TMSCN (49.48 mg, 0.51 mmol) and TBAF (130.40 mg, 0.51 mmol). The mixture was stirred at 25 °C for 16 hours. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to obtain E40 as a yellow oil (52 mg, 56.37% yield, 82% purity).

[0346] General synthesis procedure of E41

[0347] To a solution of E9 (53.87 mg, 0.19 mmol) in CH3CN (1 mL) was added CDI (30.57 mg, 0.19 mmol), and the mixture was stirred at 25 °C for 3 minutes. To another solution of E40 (52 mg, 0.17 mmol) in CH3CN (1 mL) was added t-BuOK (19.23 mg, 0.17 mmol), and the mixture was stirred at 80 °C for 2 minutes and then the first solution was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 minutes. The reaction mixture was diluted with water (10 mL) and then the pH was adjusted to 4 with 1N HCl. The mixture was extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give a residue. The residue was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 26%-56%, 10 min) to give E41 as a yellow solid (20 mg, 20.23% yield, 99% purity).

[0348] General synthesis procedure of E42

[0349] To a solution of E41 (20 mg, 0.04 mmol) in EtOH (1 mL) was added 37% HCl (0.1 mL). The mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent, giving E42 as a yellow oil (13.6 mg, crude product).

[0350] General synthesis procedure of Compound 25

[0351] To a solution of E42 (13.6 mg, 0.04 mmol) and 2-bromo-1,1-dimethoxyethane (18.60 mg, 0.11 mmol) in DMF (1.5 mL) was added K2CO3 (15.21 mg, 0.11 mmol). The mixture was stirred at 100 °C for 16 hours. The reaction mixture was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 30%-60%, 10 min) to give compound 25 as a yellow solid (3.3 mg, 19.41% yield, 99% purity).

[0352] Scheme 19 - General synthesis of Compounds 26 and 27

[0353]

[0354] General synthesis procedure of E43

[0355] To a solution of E34 (200 mg, 481.56 μmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (183.43 mg, 722.33 μmol) in dioxane (4 mL) were added Pd(dppf)Cl2 (35.24 mg, 48.16 μmol) and KOAc (141.78 mg, 1.44 mmol). The mixture was stirred at 100 °C for 2 h. To the reaction mixture were added E3 (81.10 mg, 528.13 μmol), H2O (0.6 mL), Pd(dppf)Cl2 (35.13 mg, 48.01 μmol), and Na2CO3 (152.66 mg, 1.44 mmol). The mixture was stirred at 100 °C for 1 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give E43 as a brown solid (244.2 mg, crude product, 52% purity).

[0356] General synthesis procedure of E44

[0357] 1) To a solution of E15 (136.88 mg, 591.80 μmol) in CH3CN (2 mL) was added CDI (95.96 mg, 591.80 μmol), and the reaction was stirred at 20 °C for 10 min.

[0358] 2) To a solution of E43 (244 mg, 538.00 μmol) in CH3CN (3 mL) was added t-BuOK (66.41 mg, 591.80 μmol), the reaction was heated to 80 °C, and then the first solution was added. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), then poured into water (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E44 as a brown solid (454 mg, crude product, 74% purity).

[0359] General synthesis procedure of Compound 26 and Compound 27

[0360] ​To a solution of E44 (454 mg, 680.86 μmol) in EtOH (3 mL) was added 37% HCl (0.3 mL). The reaction mixture was stirred at 80° C. for 1 hour. The crude product was purified by preparative HPLC (column: Boston Prime C18 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 14%-34%, 14 min) to afford compound 26 (31.9 mg, 96.64% purity) and compound 27 (9.0 mg, 100% purity) as a yellow solid.

[0361] Scheme 20 - General synthesis of Compound 28

[0362]

[0363] General synthesis procedure of Compound 28

[0364] To a distillation reaction mixture of 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (100 mg, 399.84 μmol) and E18 (141.26 mg, 439.83 μmol) To a solution of 1,4-dioxane (3 mL) was added H₂O (0.6 mL), Pd(dppf)Cl₂ (29.26 mg, 39.98 μmol), and Na₂CO₃ (127.14 mg, 1.20 mmol). The mixture was stirred at 100°C for 1 hour. The reaction mixture was diluted with H₂O (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (column: Boston Prime C18 150 x 30 mm x 5 μm; mobile phase: [water (NH₃H₂O + NH₄HCO₃)-ACN]; B%: 26%-56%, 10 min) to obtain compound 28 (45.4 mg, 26.38% yield, 100% purity) as a yellow solid.

[0365] Scheme 21 - General synthesis of Compound 29

[0366]

[0367] General synthesis procedure of Compound 29

[0368] To a distillation reaction of 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (50 mg, 199.92 μmol) and E18 (70.63 mg, 219.91 μmol) To a solution of alkane (1 mL) was added H2O (0.2 mL), Pd(dppf)Cl2 (14.63 mg, 19.99 μmol), and Na2CO3 (63.57 mg, 599.77 μmol). The mixture was stirred at 100 °C for 1 hour. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by preparative-HPLC (column: BostonPrime C18 150×30 mm×5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 29%-59%, 10 min) to give compound 29 as a yellow solid (17.2 mg, 21.04% yield, 100% purity).

[0369] Scheme 22 - General synthesis of Compound 31

[0370]

[0371] General synthesis procedure of Compound 31

[0372] To a solution of E45 (500 mg, 1.95 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (741.05 mg, 2.92 mmol) in di ane (5 mL) was added Pd(dppf)Cl2 (142.35 mg, 0.19 mmol) and KOAc (381.87 mg, 3.89 mmol). The mixture was stirred at 100 °C for 1 hour. More than 1 / 3 of the reaction solution was placed in another flask, and then E18 (187.44 mg, 0.58 mmol), Na2CO3 (144.34 mg, 1.36 mmol), Pd(dppf)Cl2 (42.71 mg, 0.06 mmol), and H2O (1 mL) were added at 25 °C. The mixture was stirred at 100 °C for 1 hour. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 38%-68%, 10 min) to give compound 31 as a white solid (3.6 mg, 1.3% yield, 96.6% purity).

[0373] Scheme 23 - General synthesis of Compound 35

[0374]

[0375] General synthesis procedure of E48

[0376] To a mixture of E47 (500 mg, 3.46 mmol) and (3,4-dimethoxyphenyl)boronic acid (692.38 mg, 3.80 mmol) in dichloromethane (5 mL) and H2O (1 mL) were added Pd(dppf)Cl2 (253.08 mg, 345.88 μmol) and Na2CO3 (1.10 g, 10.38 mmol). The mixture was stirred at 100 °C for 1 hour under a N2 atmosphere. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a brown solid E48 (1.01 g, crude product). At -10 °C, SOCl2 (595.04 μL, 8.20 mmol) was added to a solution of E48 (1.01 g, 4.10 mmol, crude product) in CH2Cl2 (10 mL). The mixture was stirred at 0 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a yellow solid E49 (786 mg, 72% yield).

[0377] General synthesis procedure of E49

[0378] To a solution of E49 (786 mg, 2.97 mmol) and TMSCN (589.17 mg, 5.94 mmol) in THF (5 mL) was added TBAF (1 M, 5.94 mL), and the reaction mixture was stirred at 25 °C for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by flash silica gel chromatography (PE:EtOAc = 1:1) to give a yellow solid E50 (630 mg, 58% yield, 70% purity).

[0379] General synthesis procedure of E50

[0380] 1) To a mixture of E9 (246.24 mg, 861.83 μmol) in CH3CN (2 mL) was added CDI (139.75 mg, 861.83 μmol), and the reaction was stirred at 20 °C for 10 minutes.

[0381] General synthesis procedure of E51

[0382] 1) To a mixture of E9 (246.24 mg, 861.83 μmol) in CH3CN (2 mL) was added CDI (139.75 mg, 861.83 μmol), and the reaction was stirred at 20 °C for 10 minutes.

[0383] 2) t-BuOK (96.71 mg, 861.83 μmol) was added to a solution of E50 (200 mg, 783.48 μmol) in CH3CN (3 mL). The reaction was heated to 80 °C, and then the first mixture solution was added. The reactants were stirred at 80 °C for 15 minutes. The reaction mixture was poured into water (10 mL), adjusted to pH = 4 with 1 M HCl, and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E51 (551 mg, crude product, 60% purity) as a brown solid.

[0384] General synthesis procedure of E52

[0385] Concentrated HCl (0.5 mL) was added to a mixture of E51 (551 mg, 1.05 mmol) in EtOH (5 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E52 (297 mg, 66% yield) as a yellow solid.

[0386] General synthesis procedure of Compound 35

[0387] A suspension of E52 (297 mg, 702.35 μmol) in HBr (5 mL, 40% purity) was heated to 120 °C for 4 hours. The crude product was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 20%-50%, 14 min) to give compound 35 (50 mg, 18% yield, 98% purity) as a yellow solid.

[0388] Scheme 24 - General synthesis of Compound 37

[0389]

[0390] General synthesis procedure of E54

[0391] To a mixture of NaH (71.66 mg, 1.79 mmol, 60% purity) in THF (5 mL) was added E53 (400 mg, 1.79 mmol) and ethyl 2-bromoacetate (299.19 mg, 1.79 mmol). The mixture was stirred at 25 °C for 2 h. The reaction mixture was quenched with H2O (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. Another batch from 100 mg of E53 was combined for purification. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E54 as a yellow oil (256 mg, crude product).

[0392] General synthesis procedure of E55

[0393] To a solution of E54 (256 mg, 0.83 mmol, crude product) in THF (2.5 mL) and H2O (0.5 mL) was added LiOH·H2O (173.63 mg, 4.14 mmol), and the mixture was stirred at 60 °C for 12 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (20 mL). The aqueous phase was adjusted to pH = 5 with 1 M HCl and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give E55 as a yellow oil (147 mg, crude product).

[0394] General synthesis procedure of E56

[0395] A: To a mixture of E55 (145.46 mg, 0.52 mmol) in CH3CN (1 mL) was added CDI (83.85 mg, 0.52 mmol), and the mixture was stirred at 25 °C for 3 min.

[0396] B: To a solution of E4 (120 mg, 0.47 mmol) in CH3CN (1 mL) was added t-BuOK (52.75 mg, 0.47 mmol), and the mixture was stirred at 80 °C for 2 min, then the mixture from step A was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 min. The residue was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give E56 as a yellow solid (220 mg, crude product).

[0397] General synthesis procedure of Compound 37

[0398] To a solution of E56 (220 mg, 0.42 mmol) in EtOH (2 mL) was added concentrated HCl (0.2 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Prime C18 150×30 mm×5um; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; B%: 36%-66%, 10 min) to give the compound 37 as a yellow solid (17.9 mg, 10% yield, 98% purity).

[0399] Scheme 25 - General synthesis of Compound 42

[0400]

[0401] General synthesis procedure of E58

[0402] To a mixture of Cs2CO3 (23.42 g, 71.89 mmol) in DMF (20 mL) was added E57 (5 g, 35.94 mmol). The mixture was stirred at 0 °C for 10 min. Bromo(methoxy)methane (4.94 g, 39.54 mmol) was added to the mixture at 0 °C and the mixture was stirred at 20 °C for 2 h. The mixture was quenched with saturated NH4Cl (30 mL), diluted with water (30 mL) and extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (30 mL×3), dried over Na2SO4, concentrated under reduced pressure, and the residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E58 as a yellow oil (3.99 g, 60% yield).

[0403] General synthesis procedure of E59

[0404] To a mixture of E58 (1 g, 5.46 mmol) in MeOH (12 mL) and H2O (4 mL) were added Fe (1.52 g, 27.30 mmol) and NH4Cl (2.92 g, 54.60 mmol). The mixture was stirred at 60 °C for 1 h. The reaction mixture was filtered and washed with H2O (20 mL). The filtrate was extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (30 mL×3), then dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give E59 as a brown oil (712 mg, 85% yield).

[0405] General synthesis procedure of E60

[0406] 1) To a solution of E59 (510 mg, 3.33 mmol) in Boc2O (2.18 g, 9.99 mmol) was added K2CO3 (920.30 mg, 6.66 mmol) and the mixture was stirred at 100°C for 2 h.

[0407] The mixture was quenched with saturated NH4Cl (20 mL), then poured into water (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 5:1) to give E60 (193 mg, 93% purity) as a colorless oil and E60a (435 mg, 79% purity) as a yellow oil.

[0408] 2) To a solution of E60a (435 mg, 1.23 mmol) in MeOH (10 mL) was added KCO (510.34 mg, 3.69 mmol) and the reaction was stirred at 65° C. for 10 hours. The mixture was quenched with saturated NHCl (20 mL), diluted with water (20 mL), and extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL×3), dried over NaSO, and concentrated under reduced pressure to afford E60 (208 mg, 66% yield) as a brown oil.

[0409] General synthesis procedure of E61

[0410] To a suspension of NaH (189.49 mg, 4.74 mmol, 60% purity) in THF (10 mL) was added E60 (400 mg, 1.58 mmol) at 0°C, followed by ethyl 2-bromoacetate (290.10 mg, 1.74 mmol). The mixture was stirred at 20°C for 2 h. The mixture was quenched with saturated NH4Cl (20 mL), diluted with water (20 mL), and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (20 mL x 3), dried over Na2SO4, and concentrated under reduced pressure to afford E61 (465 mg, 69% yield, 80% purity) as a colorless oil.

[0411] General synthesis procedure of E62

[0412] To a solution of E61 (465 mg, 1.37 mmol) in THF (8 mL) and H2O (6 mL) was added LiOH.H2O (114.98 mg, 2.74 mmol). The mixture was stirred at 20 °C for 10 h. The reaction mixture was poured into water (10 mL), adjusted to pH = 5 with 1 M HCl, and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give E62 as a yellow oil (283 mg, 66% yield).

[0413] General synthesis procedure of E63

[0414] 1) To a mixture of E62 (90 mg, 289.08 μmol) in CH3CN (2 mL) was added CDI (46.87 mg, 289.08 μmol), and the reaction was stirred at 20 °C for 10 min.

[0415] 2) To a mixture of E4 (67.09 mg, 262.80 μmol) in CH3CN (2 mL) was added t-BuOK (32.44 mg, 289.08 μmol), the reactants were heated to 80 °C, then the first reaction mixture was added, and the reactants were stirred at 80 °C for an additional 15 min. The reaction mixture was poured into water (10 mL), adjusted to pH = 4 with 1 M HCl, and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E63 as a brown solid (183 mg, 66% yield, 52% purity).

[0416] General synthesis procedure of Compound 42

[0417] To a mixture of E63 (180 mg, 328.12 μmol) in EtOH (5 mL) was added concentrated HCl (0.5 mL). The reaction mixture was stirred at 80 °C for 1 h. The crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (HCl)-ACN]; B%: 28%-43%, 10 min) to give Compound 42 as a brick red solid (36.7 mg, 25.% yield, 100% purity, HCl salt).

[0418] Scheme 26 - General synthesis of Compound 43

[0419]

[0420] General synthesis procedure of E65

[0421] At 0 °C, DIEA (8.21 g, 63.49 mmol, 11.1 mL) was added to a mixture of E64 (3 g, 15.87 mmol) in THF (10 mL). After stirring for 10 minutes at 0 °C, bromo(methoxy)methane (4.36 g, 34.92 mmol) was added to the mixture at 0 °C, and the mixture was stirred at 20 °C for 10 h. The mixture was quenched with saturated NH4Cl (20 mL), diluted with water (20 mL), and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E65 as a yellow oil (3.67 g, 83% yield).

[0422] General synthesis procedure of E66

[0423] To a mixture of E65 (1.8 g, 6.50 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (2.47 g, 9.74 mmol) in dioxane (10 mL) was added Pd(dppf)Cl2 (475.29 mg, 649.56 μmol) and KOAc (1.91 g, 19.49 mmol). The mixture was stirred at 100 °C for 1 h under a N2 atmosphere. The mixture was poured into water (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E66 as a yellow oil (1.68 g, 79% yield).

[0424] General synthesis procedure of E67

[0425] To a mixture of E66 (500 mg, 1.54 mmol) and E3 (260.55 mg, 1.70 mmol) in To a mixture of alkane (10 mL) and H2O (2 mL) was added Pd(dppf)Cl2 (112.86 mg, 154.24 μmol) and Na2CO3 (490.43 mg, 4.63 mmol). The mixture was stirred at 100 °C for 1 hour. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E67 as a brown oil (378 mg, 71% yield, 92% purity).

[0426] General synthesis procedure of E68

[0427] 1) To a mixture of E62 (80.78 mg, 259.47 μmol) in CH3CN (2 mL) was added CDI (46.28 mg, 285.42 μmol), and the reaction was stirred at 20 °C for 10 minutes.

[0428] 2) To a mixture of E67 (90 mg, 285.42 μmol) in CH3CN (2 mL) was added t-BuOK (32.03 mg, 285.42 μmol), the reaction was heated to 80 °C, and then the first reaction mixture was added. The reaction was stirred at 80 °C for 15 minutes. The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1M HCl, and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E68 as a brown solid (190 mg, 75% yield, 63% purity).

[0429] General synthesis procedure of Compound 43

[0430] To a mixture of E68 (190 mg, 312.17 μmol) in EtOH (5 mL) was added concentrated HCl (0.5 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 16%-36%, 10 min) to give compound 43 as a brick red solid (25.5 mg, 19% yield, 96% purity, HCl salt).

[0431] Scheme 27 - General synthesis of Compounds 56 and 65

[0432]

[0433] General synthesis procedure of C13

[0434] 1) CDI (48.21 mg, 0.3 mmol) was added to a mixture of E22 (76.51 mg, 0.3 mmol) in CH3CN (1 mL). The mixture was stirred at 25 °C for 3 minutes.

[0435] 2) t-BuOK (30.33 mg, 0.27 mmol) was added to a mixture of C4 (69 mg, 0.27 mmol) in CH3CN (1 mL). The mixture was stirred at 80 °C for 2 minutes. The first-step reaction mixture was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 minutes. The residue was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give C13 as an orange solid (173 mg, crude product).

[0436] General synthesis procedure of Compound 56

[0437] Concentrated HCl (0.5 mL) was added to a solution of C13 (173 mg, 0.35 mmol) in EtOH (2 mL). The mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with EtOAc at 25 °C for 30 minutes to give Compound 56 as a brown solid (120 mg, crude product). 60 mg of the crude product was used in the next step without further purification. The remaining 60 mg of the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; B%: 12%-42%, 12 min) to give Compound 56 as a yellow solid (18.4 mg, 13% yield, 99% purity).

[0438] General synthesis procedure of Compound 65

[0439] A suspension of Compound 56 (60 mg, 0.15 mmol) in HBr (2 mL, 40% purity) was stirred at 120 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; B%: 15%-45%, 12 min) to give Compound 65 as a yellow solid (15.8 mg, 28% yield, 98% purity).

[0440] Scheme 28 - General synthesis of Compounds 63 and 64

[0441]

[0442] General synthesis procedure of E70

[0443] To a mixture of E69 (1 g, 6.17 mmol, 729.93 μL) and Boc2O (4.04 g, 18.52 mmol, 4.25 mL) was added K2CO3 (1.71 g, 12.34 mmol). The mixture was stirred at 100 °C for 16 h. The mixture was diluted with water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with PE (30 mL) at 25 °C for 30 min to give E70 as a white solid (1.71 g, 76% yield).

[0444] General synthesis procedure of E71

[0445] To a mixture of E70 (1.70 g, 4.69 mmol) in MeOH (20 mL) was added K2CO3 (1.30 g, 9.39 mmol). The mixture was stirred at 80 °C for 12 h. The reaction mixture was diluted with H2O (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to give E71 as a yellow oil (590 mg, 48% yield).

[0446] General synthesis procedure of E72

[0447] To a mixture of NaH (90.02 mg, 2.25 mmol, 60% purity) in THF (6 mL) was added E71 (590 mg, 2.25 mmol) and ethyl 2-bromoacetate (375.88 mg, 2.25 mmol, 248.93 μL). The mixture was stirred at 25 °C for 2 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give E72 as a yellow oil (914 mg, crude product).

[0448] General synthesis procedure of E73

[0449] To a mixture of E72 (914 mg, 2.62 mmol) in THF (10 mL) was added LiOH.H2O (220.29 mg, 5.25 mmol) and H2O (2 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with H2O (10 mL) and extracted with EtOAc (20 mL). The aqueous phase was adjusted to pH = 5 with 1 M HCl and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give E73 (643 mg, crude product) as a yellow oil.

[0450] General synthesis procedure of E74

[0451] 1) To a mixture of E73 (206.95 mg, 0.65 mmol) in CH3CN (2 mL) was added CDI (104.81 mg, 0.65 mmol), and the mixture was stirred at 25 °C for 3 min.

[0452] 2) To a mixture of E4 (150 mg, 0.59 mmol) in CH3CN (2 mL) was added t-BuOK (65.94 mg, 0.59 mmol), and the mixture was stirred at 80 °C for 2 min. The first mixture was added dropwise at 80 °C, and the resulting mixture was stirred at 80 °C for 15 min. The residue was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give E74 (307 mg, crude product) as an orange solid.

[0453] General synthesis procedure of Compound 63

[0454] To a mixture of E74 (307 mg, 0.55 mmol) in EtOH (3 mL) was added concentrated HCl (0.6 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with EtOAc at 25 °C for 30 min to give Compound 63 (129 mg, crude product) as a brown solid. Portion 1: 70 mg of the crude product was used in the next step without further purification. Portion 2: The remaining 59 mg of the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 37%-67%, 12 min) to give Compound 63 (19.6 mg, 8% yield, 99% purity) as an orange solid.

[0455] General synthesis procedure of Compound 64

[0456] A mixture of Compound 63 (70 mg, 0.15 mmol) and HBr (2 mL, 40% purity) was stirred at 120 °C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 27%-57%, 12 min) to give Compound 64 as a yellow solid (23 mg, 34% yield, 98% purity).

[0457] Scheme 29 - General synthesis of Compounds 14 and 33

[0458]

[0459] E76 General synthesis procedure

[0460] Ethyl 2-bromoacetate (5 g, 29.94 mmol) was added to a solution of E75 (5.10 g, 59.88 mmol) in CH2Cl2 (50 mL). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E76 as a yellow oil (3.33 g, 65% yield).

[0461] General synthesis procedure of E77

[0462] Boc2O (4.24 g, 19.45 mmol) and Et3N (2.95 g, 29.17 mmol) were added to a solution of E76 (3.33 g, 19.45 mmol) in CH2Cl2 (30 mL). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E77 (1 g, 19% yield).

[0463] E78 General synthesis procedure

[0464] A solution of LiOH.H2O (115.98 mg, 2.76 mmol) in water (2 mL) was added to a solution of E77 (500 mg, 1.84 mmol) in THF (6 mL). The mixture was stirred at 60 °C for 1 h. Water (10 mL) was added to the mixture, and the mixture was treated with 1N HCl to adjust the pH to 2, then extracted with EtOAc (20 mL×2). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give E78 as a yellow solid (440 mg, 98% yield).

[0465] General synthesis procedure of E79

[0466] At 20 °C, CDI (69.87 mg, 430.91 μmol) was added to a solution of E78 (104.84 mg, 430.91 μmol) in MeCN (2 mL), and the mixture was stirred for 5 minutes. At 20 °C, t-BuOK (43.96 mg, 391.74 μmol) was added to a second solution of E4 (100 mg, 391.74 μmol) in MeCN (2 mL), and then the mixture was heated to 80 °C, and the first solution was added. The reaction mixture was stirred at 80 °C for 0.5 hour. The reaction was diluted with water (10 mL), acidified to pH = 5 with 1N HCl, and extracted with EtOAc (20 mL × 2). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated to give E79 as a brown solid (200 mg, 62% yield, 58.5% purity), which was used directly without purification.

[0467] General synthesis procedure of Compound 14

[0468] HCl (0.5 mL, 37%) was added to a solution of E79 (200 mg, 243.47 μmol, 58.5% purity) in EtOH (5 mL). The reaction was heated to 80 °C and maintained for 1 hour. The reaction mixture was concentrated. The residue was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5um; mobile phase: [water (HCl)-ACN]; B%: 30%-60%, 10 min) to give Compound 14 as a yellow solid (60 mg, 63% yield, 100% purity).

[0469] General synthesis procedure of Compound 33

[0470] A mixture of Compound 14 (20 mg, 0.05 mmol) and HBr (4 mL, 40% purity) was stirred at 125 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; B%: 8%-38%, 12 min) to give Compound 33 as a yellow solid (5.7 mg, 31% yield, 100% purity).

[0471] Scheme 30 - General synthesis of Compounds 66 and 69

[0472]

[0473] General synthesis procedure of E81

[0474] To a mixture of E80 (1 g, 8.22 mmol) in THF (10 mL) was added Et3N (2.08 g, 20.56 mmol, 2.86 mL) and ethyl 2-bromoacetate (1.51 g, 9.05 mmol, 1.0 mL). The mixture was stirred at 25 °C for 12 h. The mixture was quenched with saturated NH4Cl (30 mL), diluted with water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4, concentrated under reduced pressure to give E81 as a yellow oil (654 mg, 26% yield, 56% purity).

[0475] General synthesis procedure of E82

[0476] At 0 °C, to a mixture of E81 (630 mg, 3.68 mmol) in CH2Cl2 (10 mL) was added Boc2O (1.61 g, 7.36 mmol) and Et3N (558.43 mg, 5.52 mmol, 768 uL). The reaction mixture was stirred at 20 °C for 12 h. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give E82 as a brown oil (1.6 g, crude product).

[0477] General synthesis procedure of E83

[0478] To a mixture of E82 (1.6 g, 5.90 mmol) in THF (8 mL) and H2O (6 mL) was added LiOH.H2O (494.83 mg, 11.79 mmol). The mixture was stirred at 20 °C for 10 h. The reaction mixture was poured into water (10 mL) and adjusted to pH = 5 with 1 M HCl, extracted with EtOAc (20 mL × 3), the combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E83 as a yellow oil (829 mg, 57% yield).

[0479] General synthesis procedure of E84

[0480] 1) To a mixture of E83 (300 mg, 1.23 mmol) in CH3CN (3 mL) was added CDI (219.93 mg, 1.36 mmol), and the reactants were stirred at 20 °C for 10 min.

[0481] 2) t-BuOK (152.20 mg, 1.36 μmol) was added to a mixture of E4 (314.76 mg, 1.23 μmol) in CH3CN (3 mL). The reaction was heated to 80 °C, then the first-step reaction mixture was added, and the resulting mixture was stirred at 80 °C for 15 minutes. The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1 M HCl, and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give E84 as a yellow solid (534 mg, 63% yield, 71% purity).

[0482] General synthesis procedure of Compound 66

[0483] Concentrated HCl (0.5 mL) was added to a mixture of E84 (534 mg, 1.11 mmol) in EtOH (5 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was triturated with ethyl acetate (5 mL) to give Compound 66 as a brown solid (598 mg, crude product). 100 mg of the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 26%-to 56%, 14 min) to give Compound 66 as a yellow solid (24.6 mg, 100% purity).

[0484] General synthesis procedure of Compound 69

[0485] A suspension of Compound 66 (498 mg, 1.31 mmol) in HBr (5 mL, 40% purity) was stirred at 120 °C for 4 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 16%-to 46%, 12 min) to give Compound 69 as a grey solid (58.9 mg, 12% yield, 100% purity).

[0486] Scheme 31 - General synthesis of Compound 70

[0487]

[0488] General synthesis procedure of E85

[0489] 1) CDI (1.05 g, 6.45 mmol) was added to a mixture of E22 (1.66 g, 6.45 mmol) in CH3CN (10 mL), and the mixture was stirred at 25 °C for 3 minutes.

[0490] 2) t-BuOK (657.61 mg, 5.86 mmol) was added to a mixture of E3 (900 mg, 5.86 mmol) in CH3CN (5 mL), and the mixture was stirred at 80 °C for 2 minutes. The reaction mixture from the first step was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 minutes. The residue was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give a brown solid E85 (2.33 g, crude product).

[0491] General synthesis procedure of E86

[0492] Concentrated HCl (1.5 mL) was added to a mixture of E85 (2.33 g, 5.93 mmol) in EtOH (7.5 mL). The mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with EtOAc at 25 °C for 30 minutes to give a yellow solid E86 (1.3 g, 65% yield, 87% purity).

[0493] General synthesis procedure of E88

[0494] E87 (5 g, 24.87 mmol) was added to a mixture of DIEA (12.86 g, 99.49 mmol) in THF (30 mL), and the mixture was stirred at 0 °C for 10 minutes. Then, at 0 °C, bromo(methoxy)methane (3.73 g, 29.85 mmol) was added to the mixture. The mixture was stirred at 20 °C for 10 hours. The mixture was quenched with saturated NH4Cl (20 mL), then poured into water (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give a yellow oil E88 (6.9 g, 84% yield, 75% purity).

[0495] General synthesis procedure of E89

[0496] To a mixture of E88 (2 g, 8.16 mmol) in CH2Cl2 (20 mL) was added m-CPBA (1.99 g, 9.79 mmol, 85% purity). The reaction mixture was stirred at 20 °C for 16 h. NaOH (20 mL, 10% purity) was added to the mixture and stirred at 20 °C for 0.5 h. The reaction mixture was poured into water (20 mL) and adjusted to pH = 4 with 1 M HCl, and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with saturated NaHCO3 (40 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 5:1) to give E89 as a yellow oil (1.14 g, 60% yield).

[0497] General synthesis procedure of E90

[0498] To a mixture of E89 (500 mg, 2.15 mmol) in DMF (6 mL) was added 3-bromooxetane (1.47 g, 10.73 mmol) and K2CO3 (444.77 mg, 3.22 mmol). The mixture was stirred at 100 °C for 5 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1) to give E90 as a yellow oil (365 mg, 59% yield).

[0499] General synthesis procedure of E91

[0500] To a mixture of E90 (365 mg, 1.26 mmol) in di ane (4 mL) was added 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (480.87 mg, 1.89 mmol), Pd(dppf)Cl2 (92.37 mg, 0.13 mmol), and KOAc (247.79 mg, 2.52 mmol). The mixture was stirred at 100 °C for 2 h under N2 atmosphere. The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1) to give E91 as a yellow oil (388 mg, crude product).

[0501] General synthesis procedure of E92

[0502] To the di-reaction of E86 (76 mg, 0.26 mmol) and E91 (100 mg, 0.30 mmol) To a mixture of 2-(4-oxo-1-oxo-2-nitropropene)-2-nitropropene (5 mL) was added Pd(dppf)Cl2, CH2Cl2 (21.20 mg, 0.02 mmol), Na2CO3 (55.03 mg, 0.52 mmol), and H2O (0.1 mL). The mixture was stirred at 100°C under an N2 atmosphere for 3 hours. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (10 mL x 2), dried over Na2SO4, filtered, and concentrated to give the crude product E92 (150 mg, 70% purity) as a brown gum.

[0503] General synthesis procedure of Compound 70

[0504] To a mixture of E92 (130 mg, 0.28 mmol) in CH2Cl2 (0.6 mL) was added TFA (2 mL). The mixture was stirred at 25 ° C for 1 hour. The reaction mixture was concentrated under reduced pressure. The reaction mixture was adjusted to pH = 7-8 with saturated NaHCO3, then extracted with DCM (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: C18-1 150 × 30 mm × 5 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 27%-47%, 13 min) to give compound 70 (6.1 mg, 5% yield, 97% purity) as an off-white solid.

[0505] Scheme 32 - General synthesis of Compound 73

[0506]

[0507] General synthesis procedure of Compound 73

[0508] To the di- To a mixture of alkane (1 mL) and H2O (0.2 mL) was added 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (51.26 mg, 0.2 mmol), Pd(dppf)Cl2 (15.00 mg, 0.02 mmol), and Na2CO3 (65.17 mg, 0.6 mmol). The mixture was stirred at 100 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; B%: 22%-52%, 12 min) to give compound 73 as a yellow solid (0.9 mg, 1% yield, 100% purity).

[0509] Scheme 33 - General synthesis of Compounds 77 and 79

[0510]

[0511] General synthesis procedure of F6

[0512] 1) To a mixture of E22 (222.63 mg, 865.18 mmol) in CH3CN (3 mL) was added CDI (140.29 mg, 865.18 mmol). The reactants were stirred at 20 °C for 10 minutes.

[0513] 2) To a mixture of F4 (200.78 mg, 786.52 μmol) in CH3CN (3 mL) was added t-BuOK (97.08 mg, 865.18 μmol). The reaction was heated to 80 °C, and then the first-step reaction mixture was added. The reactants were stirred at 80 °C for 15 minutes. The reaction mixture was poured into water (10 mL), adjusted to pH = 4 with 1 M HCl, and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over Na2SO4, and then concentrated under reduced pressure to give F6 as a brown solid (447 mg, 56% yield).

[0514] General synthesis procedure of Compound 77

[0515] To a mixture of F6 (447 mg, 903.79 μmol) in EtOH (5 mL) was added concentrated HCl (0.5 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with ethyl acetate (5 mL) to give compound 77 as a brown solid (478 mg, crude product). 378 mg of the crude product was used for the next step.

[0516] Purify 100 mg of the crude product by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 18%-48%, 12 min) to obtain Compound 77 as a yellow solid (8.9 mg, 8.90% yield, 100% purity).

[0517] General synthesis procedure of Compound 79

[0518] Heat a suspension of Compound 77 (378 mg, 958.26 μmol) in HBr (5 mL, 40% purity) to 120 °C for 4 h. Concentrate the reaction mixture under reduced pressure. Purify the crude product by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 8%-32%, 14 min) to obtain Compound 79 as a yellow solid (7.6 mg, 2% yield, 97% purity).

[0519] Scheme 34 - General Synthesis of Compound 80 and Compound 81

[0520]

[0521] General Synthesis Procedure of D3

[0522] 1) Add CDI (70.14 mg, 0.43 mmol) to a mixture of E22 (111.32 mg, 0.4 mmol) in CH3CN (1 mL). Stir the mixture at 25 °C for 3 min.

[0523] 2) Add t-BuOK (44.13 mg, 0.39 mmol) to a mixture of D1 (100 mg, 0.39 mmol) in CH3CN (1 mL). Stir the mixture at 80 °C for 2 min. Add the first-step reaction mixture dropwise at 80 °C and stir the resulting mixture at 80 °C for 15 min. Adjust the residue to pH = 4 with 1 M HCl, then extract with EtOAc (20 mL×3). Wash the combined organic layers with brine (20 mL×3), dry over anhydrous Na2SO4, filter and concentrate to obtain D3 as an orange solid (210 mg, crude product).

[0524] General Synthesis Procedure of Compound 80

[0525] Concentrated HCl (0.4 mL) was added to a mixture of D3 (210 mg, 0.44 mmol) in EtOH (2 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with EtOAc (5 mL) at 25 °C for 30 min to give Compound 80 as a brown solid (120 mg, crude product). 90 mg of the crude product was used in the next step without further purification. 30 mg of the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 20%-50%, 12 min) to give Compound 80 as a yellow solid (12.5 mg, 100% purity).

[0526] General Synthesis Procedure of Compound 81

[0527] A solution of Compound 80 (90 mg, 0.2 mmol) in HBr (1 mL, 40% purity) was stirred at 120 °C for 12 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 15%-45%, 12 min) to give Compound 81 as a yellow solid (23.3 mg, 28% yield, 100% purity).

[0528] Scheme 35 - General Synthesis of Compound 82 and Compound 83

[0529]

[0530] General Synthesis Procedure of C15

[0531] 1) CDI (69.87 mg, 0.43 mmol) was added to a mixture of C14 (154.45 mg, 0.43 mmol) in CH3CN (1 mL). The mixture was stirred at 25 °C for 3 min.

[0532] 2) t-BuOK (43.96 mg, 0.39 mmol) was added to a mixture of C4 (100 mg, 0.39 mmol) in CH3CN (1 mL). The mixture was stirred at 80 °C for 2 min. The reaction mixture of the first step was added dropwise at 80 °C, and the resulting mixture was stirred at 80 °C for 15 min. The mixture was adjusted to pH = 4 with 1 M HCl and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over anhydrous Na2SO4, filtered, and concentrated to give C15 as an orange solid (210 mg, crude product).

[0533] General Synthesis Procedure of Compound 82

[0534] To a mixture of C15 (210 mg, 0.35 mmol) in EtOH (2 mL) was added concentrated HCl (0.4 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure. The crude product was triturated with EtOAc (5 mL) at 25 °C for 30 min to give the compound 82 as a brown solid (99 mg, crude product). 70 mg of the crude product was used in the next step without further purification. 29 mg of the crude product was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 5%-35%, 10 min) to give the compound 821 as a yellow solid (9 mg, 100% purity).

[0535] General Synthesis Procedure of Compound 83

[0536] A suspension of the compound 82 (70 mg, 0.17 mmol) in HBr (2 mL, 40% purity) was stirred at 120 °C for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 0%-22%, 10 min) to give the compound 83 as an off-white solid (13.1 mg, 20% yield, 99% purity).

[0537] Scheme 36 - General Synthesis of Compound 99

[0538]

[0539] General Synthesis Procedure of E93

[0540] To a mixture of E89 (500 mg, 2.15 mmol) in DMF (5 mL) were added 1-bromo-2-methoxyethane (298.19 mg, 2.15 mmol, 202 μL), Cs2CO3 (1.40 g, 4.29 mmol) and KI (356.14 mg, 2.15 mmol). The mixture was stirred at 70 °C for 12 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL×3), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E93 as a yellow oil (430 mg, 69% yield).

[0541] General Synthesis Procedure of E94

[0542] To a mixture of E93 (430 mg, 1.48 mmol) in dichloromethane (4 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (450.07 mg, 1.77 mmol), KOAc (434.85 mg, 4.43 mmol), and Pd(dppf)Cl2 (108.07 mg, 0.15 mmol). The mixture was stirred at 100 °C for 2 h under a N2 atmosphere. The reaction mixture of E94 (499 mg, crude product) was used in the next step without further purification.

[0543] General Synthesis Procedure of E95

[0544] To a solution of E94 (499 mg, 1.48 mmol) in dichloromethane (5 mL) and H2O (1 mL) was added E86 (431.96 mg, 1.48 mmol), Pd(dppf)Cl2 (107.96 mg, 0.15 mmol), and Na2CO3 (312.77 mg, 2.95 mmol). The mixture was stirred at 100 °C for 2 h under a N2 atmosphere. The mixture was quenched with H2O (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E95 as a brown solid (170 mg, 22% yield, 90% purity).

[0545] General Synthesis Procedure of Compound 99

[0546] To a solution of E95 (170 mg, 0.36 mmol) in CH2Cl2 (0.5 mL) was added TFA (2 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150 × 30 mm × 5 um; mobile phase: [water (FA)-ACN]; gradient: 30% - 60% B for 10 min) to give compound 99 as a yellow solid (50.1 mg, 33% yield, 100% purity).

[0547] Scheme 37 - General Synthesis of Compound 103

[0548]

[0549] General Synthesis Procedure of E96

[0550] ​​To a mixture of E89 (500 mg, 2.15 mmol) in DMF (5 mL) was added K2CO3 (593.01 mg, 4.29 mmol) and 2-bromoethanol (268.10 mg, 2.15 mmol, 152 μL). The mixture was stirred at 100 °C for 2 h. The reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E96 as a colorless oil (263 mg, 44% yield).

[0551] General Synthesis Procedure of E97

[0552] To a mixture of E96 (263 mg, 0.95 mmol) in di ane (3 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborolane) (289.21 mg, 1.14 mmol), Pd(dppf)Cl2 (69.44 mg, 0.09 mmol) and KOAc (279.43 mg, 2.85 mmol). The mixture was stirred at 100 °C for 2 h under N2 atmosphere. The reaction mixture E97 (308 mg, crude product) was used in the next step without further purification.

[0553] General Synthesis Procedure of E98

[0554] To a solution of E97 (308 mg, 0.95 mmol) in di ane (3 mL) and H2O (0.6 mL) was added E86 (278.15 mg, 0.95 mmol), Na2CO3 (201.40 mg, 1.90 mmol) and Pd(dppf)Cl2 (69.52 mg, 0.09 mmol). The mixture was stirred at 100 °C for 2 h under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E98 as a brown solid (180 mg, 20% yield, 49% purity).

[0555] General Synthesis Procedure of Compound 103

[0556] TFA (2 mL) was added to a solution of E98 (180 mg, 0.39 mmol) in CH2Cl2 (0.5 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; gradient: 14%-44% B for 12 min) to give Compound 103 as a white solid (8.8 mg, 5% yield, 96% purity).

[0557] Scheme 38 - General Synthesis of Compound 104

[0558]

[0559] General Synthesis Procedure of E100

[0560] Ethyl 2-bromoacetate (423.89 mg, 2.54 mmol, 280.72 μL) and Et3N (385.26 mg, 3.81 mmol, 529.94 μL) were added to a solution of E99 (500 mg, 3.17 mmol, HCl) in CH2Cl2 (10 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (20 mL×3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give E100 as a yellow oil (228 mg, 35% yield).

[0561] General Synthesis Procedure of E101

[0562] Boc2O (240.14 mg, 1.10 mmol, 252.78 μL) and Et3N (167.01 mg, 1.65 mmol, 229.72 μL) were added to a solution of E100 (228 mg, 1.10 mmol) in CH2Cl2 (3 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give E101 as a colorless oil (250 mg, 74% yield).

[0563] General Synthesis Procedure of E102

[0564] To a solution of E101 (250 mg, 0.81 mmol) in THF (3 mL) and H2O (0.6 mL) was added LiOH.H2O (170.68 mg, 4.07 mmol). The mixture was stirred at 25 °C for 12 h. The reaction mixture was adjusted to pH = 4 with 1N HCl and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give a white solid E102 (220 mg, crude product).

[0565] General Synthesis Procedure of E103

[0566] 1) To a mixture of E102 (219.21 mg, 0.78 mmol) in CH3CN (2 mL) was added CDI (127.27 mg, 0.78 mmol), and the mixture was stirred at 25 °C for 3 min.

[0567] 2) To a mixture of E67 (225 mg, 0.71 mmol) in CH3CN (2 mL) was added t-BuOK (80.07 mg, 0.71 mmol), and the mixture was stirred at 80 °C for 2 min. The first-step reaction mixture was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 min. The residue was adjusted to pH = 4 with 1M HCl and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give a brown solid E103 (390 mg, crude product).

[0568] General Synthesis Procedure of Compound 104

[0569] To a solution of E103 (390 mg, 0.68 mmol) in EtOH (4 mL) was added concentrated HCl (1 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: Boston GreenODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 35%-65% B for 12 min) to give a brown solid compound 104 (19.5 mg, 7% yield, 99% purity).

[0570] Scheme 39 - General Synthesis of Compound 105

[0571]

[0572] General Synthesis Procedure of F7

[0573] To a mixture of F3 (500 mg, 3.26 mmol) and 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (895.71 mg, 3.58 mmol) in dioxane (5 mL) was added H2O (0.5 mL), Pd(dppf)Cl2 (238.24 mg, 325.59 μmol), and Na2CO3 (690.18 mg, 6.51 mmol). The mixture was stirred at 100 °C for 3 h under a N2 atmosphere. The reaction mixture was poured into water (15 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 1:1) to give the crude product. The crude product was purified by preparative-HPLC (column: Boston Prime C18 150 × 30 mm × 5 um; mobile phase: [water (FA)-ACN]; gradient: 21%-51% B for 12 min) to give F7 as a yellow solid (82 mg, 100% purity).

[0574] General Synthesis Procedure of F8

[0575] 1) To a mixture of E22 (96.21 mg, 373.89 μmol) in CH3CN (2 mL) was added CDI (60.63 mg, 373.89 μmol), and the reaction was stirred at 20 °C for 10 min.

[0576] 2) To a mixture of F7 (82 mg, 339.90 μmol) in CH3CN (2 mL) was added t-BuOK (41.95 mg, 373.89 μmol), the reaction was heated to 80 °C, then the first-step reaction mixture was added, and the reaction was stirred at 80 °C for 15 min. The reaction mixture was poured into water (20 mL) and adjusted to pH = 4 with 1M HCl, and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give F8 as a brown solid (138 mg, 38% yield).

[0577] General Synthesis Procedure of Compound 105

[0578] ​To a mixture of F8 (138 mg, 287.17 μmol) in EtOH (10 mL) was added concentrated HCl (1 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; gradient: 15%-45% B for 12 minutes) to give Compound 105 as a yellow solid (25.5 mg, 22% yield, 96% purity).

[0579] Scheme 40 - General Synthesis of Compound 107

[0580]

[0581] General Synthesis Procedure of E104

[0582] To a mixture of E89 (300 mg, 1.29 μmol) in THF (10 mL) were added tert-butyl (2-hydroxyethyl)(methyl)carbamate (248.11 mg, 1.42 mmol) and PPh3 (506.43 mg, 1.93 mmol), and then DIAD (390.43 mg, 1.93 mmol) was added to the mixture at 0 °C, and the mixture was stirred at 20 °C for 10 hours. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (20 mL×2), dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by silica gel flash column chromatography (0-30% EtOAc in PE) to give E104 as a colorless oil (337 mg, 47% yield, 70% purity).

[0583] General Synthesis Procedure of E105

[0584] To a mixture of E104 (300 mg, 768.70 μmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (195.20 mg, 768.70 μmol) in dioxane (3 mL) were added Pd(dppf)Cl2 (56.25 mg, 76.87 μmol) and KOAc (226.33 mg, 2.31 mmol). The mixture was stirred at 100 °C for 1 hour. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (20 mL×2), dried over Na2SO4, filtered and concentrated to give E105 as a brown oil (115 mg, 24% yield, 70% purity).

[0585] General Synthesis Procedure of E106

[0586] To a mixture of E105 (115 mg, 262.96 μmol) and E86 (84.68 mg, 289.25 μmol) in dichloromethane (3 mL) was added Pd(dppf)Cl2 (19.24 mg, 26.30 μmol), Na2CO3 (55.74 mg, 525.91 μmol), and H2O (0.3 mL). The mixture was stirred at 100 °C for 1 h under a N2 atmosphere. The mixture was diluted with ethyl acetate (15 mL) and filtered, and the filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by flash column chromatography on silica gel (0 - 30% EtOAc in PE) to give E106 as a yellow oil (39 mg, 18% yield, 70% purity).

[0587] General Synthesis Procedure of Compound 107

[0588] To a suspension of E106 (39 mg, 68.70 μmol) in EtOH (5 mL) was added concentrated HCl (0.5 mL). The reaction mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 10% - 40% B for 12 min) to give compound 107 as a yellow solid (2.4 mg, 8% yield, 100% purity).

[0589] Scheme 41 - General Synthesis of Compound 110

[0590]

[0591] General Synthesis Procedure of C16

[0592] To a solution of C1 (2.46 g, 16.52 mmol) in dichloromethane ​To a solution of alkane (30 mL) and H2O (6 mL) was added 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (3.18 g, 12.71 mmol), Na2CO3 (2.69 g, 25.42 mmol) and Pd(dppf)Cl2 (929.85 mg, 1.27 mmol). The mixture was stirred at 100 °C for 2 h. The reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1) to give C16 as a white solid (2.5 g, crude product).

[0593] General Synthesis Procedure of C17

[0594] To a solution of C16 (1.5 g, 6.34 mmol) in THF (15 mL) was added DIEA (3.28 g, 25.35 mmol, 4.42 mL) and bromo(methoxy)methane (950.49 mg, 7.61 mmol, 620.83 μL). The mixture was stirred at 20 °C for 12 h. The mixture was quenched with saturated NaHCO3 to pH = 8 and extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, DCM:MeOH = 10:1) to give C17 as a white solid (785 mg, 17% yield, 38% purity).

[0595] General Synthesis Procedure of C18

[0596] To a solution of C17 (500 mg, 1.78 mmol) in DMF (5 mL) was added tert-butyl 2-cyanoacetate (301.74 mg, 2.14 mmol, 305.71 μL) and K2CO3 (984.73 mg, 7.12 mmol). The mixture was stirred at 120 °C for 16 h. The crude reaction mixture on page ES20772-487 (735 mg scale) of the notebook was combined with ES20772-488 for workup. The combined reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give C18 as a yellow solid (940 mg, crude product).

[0597] General Synthesis Procedure of C19

[0598] HCl (2 mL) was added to a solution of C18 (940 mg, 2.44 mmol) in EtOH (10 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was triturated with EtOAc (2 mL) at 25 °C for 30 min to give C19 as a yellow solid (560 mg, 79.0% yield, 83% purity).

[0599] General Synthesis Procedure of C20

[0600] 1) CDI (192.23 mg, 1.19 mmol) was added to a mixture of E22 (305.06 mg, 1.19 mmol) in CH3CN (2 mL), and the mixture was stirred at 25 °C for 3 min.

[0601] 2) t-BuOK (120.94 mg, 1.08 mmol) was added to a mixture of C19 (260 mg, 1.08 mmol) in CH3CN (2 mL), and the mixture was stirred at 80 °C for 2 min. The first-step reaction was added dropwise at 80 °C. The resulting mixture was stirred at 80 °C for 15 min. The residue was adjusted to pH = 4 with 1N HCl and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (we20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated to give C20 as a brown solid (670 mg, crude product).

[0602] General Synthesis Procedure of Compound 110

[0603] Concentrated HCl (1 mL) was added to a solution of C20 (670 mg, 1.39 mmol) in EtOH (5 mL). The mixture was stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product on page ES20772 - 493 of the notebook was combined with ES20772 - 498 for further purification. The residue was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5 um; mobile phase: [water (FA)-ACN]; gradient: 10% - 40% B for 12 min) to give compound 110 as a yellow solid (54.5 mg, 10% yield, 96% purity).

[0604] Scheme 42 - General Synthesis of Compound 111

[0605]

[0606] General Synthesis Procedure of E107

[0607] At 0 °C, NaBH4 (322.05 mg, 8.51 mmol) was added to a solution of E107-1 (1 g, 5.68 mmol) in MeOH (10 mL), and the reaction mixture was stirred at 20 °C for 12 h. The mixture was quenched with NH4Cl (30 mL), then poured into water (30 mL) and extracted with EtOAc (30 mL × 3). The organic layer was washed with brine (30 mL × 3), dried over Na2SO4, filtered and concentrated to give E107 as a brown solid (974 mg, 96% yield).

[0608] ​

[0609] To a solution of E89 (1.1 g, 4.72 mmol) in THF (15 mL) was added E107 (925.32 mg, 5.19 mmol) and PPh3 (1.86 g, 7.08 mmol), and then DIAD (1.43 g, 7.08 mmol) was added to the mixture at 0 °C, and the mixture was stirred at 20 °C for 10 h. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (20 mL × 2), dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by flash silica gel column chromatography (0 - 30% EtOAc in PE) to give E108 as a colorless oil (617 mg, 33% yield).

[0610] ​

[0611] To a solution of E108 (617 mg, 1.57 mmol) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (438.24 mg, 1.73 mmol) in di ane (5 mL) were added Pd(dppf)Cl2 (114.80 mg, 156.89 μmol) and KOAc (307.95 mg, 3.14 mmol). The mixture was stirred at 100 °C under N2 for 1 h. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (20 mL × 2), dried over Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by flash silica gel chromatography (PE:EA = 5:1) to give E109 as a colorless oil (495 mg, 71% yield).

[0612] ​

[0613] Pd / C (100 mg, 10% purity) was added to a mixture of E109 (495 mg, 1.12 mmol) in MeOH (10 mL). The reaction mixture was stirred at 20 °C under H2 (15 psi) for 10 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give E110 (365 mg, 64% yield, 70% purity) as a colorless oil.

[0614] ​

[0615] To a solution of E110 (100 mg, 285.54 μmol) and E86 (83.60 mg, 285.54 μmol) in dichloromethane (5 mL) was added Pd(dppf)Cl2 (20.89 mg, 28.55 μmol), Na2CO3 (60.53 mg, 571.08 μmol) and H2O (0.5 mL). The mixture was stirred at 100 °C under N2 for 1 h. The mixture was diluted with ethyl acetate (15 mL) and filtered, and the organic layer was washed with water (20 mL × 2), dried over Na2SO4, filtered and concentrated to give E111 (199 mg, 58% yield, 40% purity) as a brown solid.

[0616] ​

[0617] TFA (2 mL) was added to a solution of E111 (199.00 mg, 414.10 μmol) in CH2Cl2 (5 mL). The reaction mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 22%-52% B for 12 min) to give Compound 111 (4 mg, 2% yield, 100% purity) as a yellow solid.

[0618] ​

[0619]

[0620] ​

[0621] To a solution of F3 (100 mg, 651.17 μmol) and G1-3 (232.20 mg, 716.29 μmol) in dichloromethane (5 mL) was added Pd(dppf)Cl2 (20.89 mg, 28.55 μmol), Na2CO3 (60.53 mg, 571.08 μmol) and H2O (0.5 mL). The mixture was stirred at 100 °C under N2 for 1 h. The mixture was diluted with ethyl acetate (15 mL) and filtered, and the organic layer was washed with water (20 mL × 2), dried over Na2SO4, filtered and concentrated to give E111 (199 mg, 58% yield, 40% purity) as a brown solid. To a solution of alkane (5 mL) was added Pd(dppf)Cl2 (47.65 mg, 65.12 μmol), Cs2CO3 (424.33 mg, 1.30 mmol) and H2O (0.5 mL). The mixture was stirred at 100 °C for 1 hour. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (20 mL × 2), dried over Na2SO4, filtered and concentrated to give a residue. The residue was purified by flash silica gel chromatography (PE:EA = 3:1) to give F9 as a brown solid (204 mg, crude product).

[0622] ​

[0623] 1) To a solution of E9 (203.33 mg, 711.65 μmol) in CH3CN (3 mL) was added CDI (115.39 mg, 711.65 μmol), and the reaction was stirred at 20 °C for 10 minutes.

[0624] 2) To a solution of F9 (204 mg, 646.96 μmol) in CH3CN (3 mL) was added t-BuOK (79.86 mg, 711.65 μmol), the reaction was heated to 80 °C, then the first solution was added, and the reaction was stirred at 80 °C for 15 minutes.

[0625] The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1N HCl, then extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, and then concentrated under reduced pressure to give F10 as a brown solid (565 mg, crude product).

[0626] ​

[0627] To a solution of F10 (565 mg, 135.67 μmol) in EtOH (10 mL) was added concentrated HCl (1 mL). The reaction mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 4%-34% B for 12 minutes) to give compound 114 as a yellow solid (9.1 mg, 96% purity).

[0628] ​

[0629]

[0630] ​

[0631] To a solution of 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1.5 g, 6.00 mmol) in THF (30 mL) was added MOMBr (899.36 mg, 7.20 mmol) at 0 °C for 30 minutes, and then DIEA (3.10 g, 23.99 mmol) was added dropwise at 0 °C. The resulting mixture was stirred at 20 °C for 10 hours. LCMS showed that the desired product was not detected. TLC indicated that 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol was completely consumed and a new spot was formed.

[0632] The reaction mixture was quenched with NH4Cl (20 mL), then poured into water (20 mL), and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E112 as a colorless oil (1.8 g, crude product).

[0633] ​

[0634] To a solution of E112 (1.8 g, 6.12 mmol) and E86 (1.79 g, 6.12 mmol) in dodecane (10 mL) were added Pd(dppf)Cl2 (447.76 mg, 611.93 μmol), Na2CO3 (1.30 g, 12.24 mmol), and H2O (1 mL). The mixture was stirred at 100 °C under N2 for 1 hour.

[0635] The mixture was diluted with ethyl acetate (15 mL) and filtered. The combined organic layers were washed with water (20 mL × 2), dried over Na2SO4, and concentrated under reduced pressure to give compound 116 as a brown solid (3.88 g, 74.68% yield, 50% purity).

[0636] The crude product (400 mg) was purified by preparative-HPLC (column: Boston Green ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 36%-66% B for 12 minutes) to give compound 116 as a yellow solid (52.8 mg, 100% purity).

[0637] ​

[0638]

[0639] ​

[0640] To a solution of E113 (10 g, 77.79 mmol) in DMF (150 mL) was added DIEA (30.16 g, 233.36 mmol, 40.65 mL) and ethyl 2-bromoacetate (14.29 g, 85.56 mmol, 9.47 mL). The mixture was stirred at 100 °C for 10 h. The reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2 (petroleum ether / ethyl acetate = 3 / 1 to 1 / 1)) to give E114 as a yellow oil (3.64 g, 19.6% yield, 90% purity).

[0641] ​

[0642] To a solution of E114 (1 g, 4.66 mmol) in THF (15 mL) and H2O (3 mL) was added LiOH·H2O (977.50 mg, 23.29 mmol). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with H2O (20 mL) and extracted with EtOAc (50 mL). The aqueous phase was adjusted to pH = 5 with HCl (1 N) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give E115 as a white solid (320 mg, crude product).

[0643] ​

[0644] 1) To a mixture of E115 (318.96 mg, 1.71 mmol) in CH3CN (5 mL) was added CDI (277.17 mg, 1.71 mmol), and the mixture was stirred at 25 °C for 3 min.

[0645] 2) To a mixture of E67 (490 mg, 1.55 mmol) in CH3CN (5 mL) was added t-BuOK (348.74 mg, 3.11 mmol), and the mixture was stirred at 80 °C for 2 min.

[0646] At 80 °C, the reactant of step 1 was added dropwise to the reaction mixture of step 2, and the resulting mixture was stirred at 80 °C for 15 minutes. The residue was adjusted to pH = 4 with 1N HCl, and then extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give yellow solid E117 (700 mg, crude product).

[0647] ​

[0648] 37% HCl (2 mL) was added to a solution of E117 (700 mg, 1.45 mmol, crude product) in EtOH (10 mL). The mixture was stirred at 80 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by preparative-HPLC (column: BostonGreen ODS 150×30 mm×5um; mobile phase: [water (FA)-ACN]; gradient: 10%-40% B for 12 minutes), and then by preparative-HPLC (column: Phenomenex Gemini NX 150×30 mm, 5 μm; mobile phase: [water (NH4HCO3)-ACN]; gradient: 13%-43% B for 11 minutes) to give white solid compound 119 (0.9 mg, 100% purity).

[0649] ​

[0650] ​

[0651] The halide sensor YFP F46L / H148Q / I152L is quenched by halide ions such as iodide. When this YFP is expressed in cells, it is expressed in the cytoplasm.

[0652] To measure the activity of H723R-PDS, PANC-1 cells were stably transfected with the halide sensor YFP-F46L / H148Q / I152L and H723R-PDS, and H723R-PDS has membrane trafficking problems due to misfolding.

[0653] If the drug used to treat the cells is a corrector of H723R-PDS, then H723R-PDS will be normally transported to the plasma membrane.

[0654] At this time, when treated with a high-concentration iodide solution outside the cells, iodide enters the cells due to the Cl- / I- exchange activity of pantethine, reducing the fluorescence of the halide sensor YFP.

[0655] Protocol for YFP assay

[0656] Cells for high-throughput screening

[0657] PANC-1 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin. For high-throughput screening, PANC-1 cells were stably transfected with human H723R-pantethein and the halide sensor YFP-F46L / H148Q / I152L.

[0658] ​

[0659] PANC-1 cells expressing human H723R-pantethein and YFP-F46L / H148Q / I152L were seeded at a density of 2×10 4 cells per well in a 96-well microplate and grown at 37 °C (90% humidity, 5% CO2) for 20 - 24 hours. These cells were then treated with 50 μl of growth medium containing the test compound and incubated for 20 - 24 hours. For the YFP quenching assay, each well of the 96-well plate was washed twice with 200 μl of PBS and filled with 50 μl of HEPES-buffered solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM D-glucose, 10 mM HEPES; adjusted to pH 7.4 with NaOH). After incubation at 37 °C for 10 minutes, the 96-well plate was transferred to a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany) for fluorescence measurement. The H723R-pantethein-mediated I - influx of each well was measured individually by recording fluorescence (excitation at 495 ± 15 nm, emission at 520 ± 20 nm) every 400 ms for 1 second (baseline). Then, 50 μL of NaI-substituted HEPES-buffered solution (NaI instead of NaCl) was added in 1 second using a liquid syringe, and YFP fluorescence was recorded every 400 ms for 10 seconds. After the iodide injection, the initial iodide influx rate was determined from the initial slope of the fluorescence by non-linear regression.

[0660] Table 1 shows the measurement results of the Cl - / I - exchange activity of pantethein for the selected compounds of the present invention. It is expressed as EC 50 , with the following symbols: A = EC 50 less than 5 μM; B = EC 50 greater than 5 μM but less than 10 μM; C = EC 50 greater than 10 μM. 0]

[0661] Table 1. YFP assay

[0662]

[0663] Continued Table 1

[0664]

[0665] ​

[0666] Principle of Surface Biotinylation Assay

[0667] Surface biotinylation is a commonly used technique that specifically isolates plasma membrane proteins using modified biotin in a form that readily binds to proteins. Sulfo-NHS-SS-biotin is a commonly used reagent in surface biotinylation due to its impermeability and water solubility. At low temperatures, the extracellular domain of membrane proteins covalently binds to the reactive biotin ester. These proteins are separated from the whole lysate through the biotin-streptavidin reaction. During this process, we can verify the increasing rate of the expression level of mutant pantellin in the plasma membrane.

[0668] Protocol of Surface Biotinylation Assay

[0669] Culture Panc1 cells in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics 亲代 、Panc1 hPDS-WT and Panc1 hPDS-H723R cells. After 24 hours of inoculation into a 6-well plate, treat the cells for 24 hours as indicated. Wash the cells in PBS and incubate them on ice with a PBS solution of 0.3 mg / ml biotin. After 35 minutes, add a PBS solution of 1% BSA, D.W mixture (1:1) to the cells and incubate the cells for 10 minutes. Wash the cells and lyse them in lysis buffer. Sonicate the lysate for 20 seconds without boiling. After centrifugation, collect the supernatant and quantify it by BCA analysis. Add the quantified supernatant to PBS with 10% avidin magnetic beads and incubate overnight at 4°C. After incubation, repeat the following steps 4 times: i) centrifuge, ii) remove the supernatant, iii) wash with lysis buffer. After the last centrifugation and removal of the supernatant, add sample buffer mixed with reducing buffer to the biotin-avidin mixture. Perform elution in a biological oscillator at 38°C for 40 minutes and collect the supernatant. Separate the supernatant by SDS-PAGE and transfer it to an Immobilon membrane. After incubation in a 5% BSA solution to prevent non-specific binding, incubate the membrane with a specific antibody overnight. Then, wash the membrane with TBST buffer and incubate it with an HRP-conjugated anti-rabbit or anti-mouse antibody for 1 hour. ECL buffer and iBright are used for visualization of protein bands. Quantify the intensity of the corresponding protein bands by densitometry using the NIH ImageJ program.

[0670] Table 2 shows the percentage of expression of the selected compounds of the present invention as glycosylated pantethine relative to wild-type hPDS on the H723R-hPDS surface. It is expressed as % expression and has the following symbols: A = % expression greater than 100%; B = % expression greater than 50% but less than 100%; C = % expression less than 50%.

[0671] Table 2. Surface biotinylation assay

[0672]

[0673] ​

[0674] Protocol for PTI assay

[0675] Cl - / HCO3 - Measurement of Cl

[0676] Intracellular pH (pH i ) in PANC-1 cells was measured using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) according to a previously reported protocol. Briefly, cells were incubated with 2 μM BCECF acetoxymethyl ester for 5 minutes and then perfused with HCO3 - buffer [containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM d-glucose, 5 mM Hepes, and 25 mM NaHCO3 (pH 7.4)].

[0677] On the recording device, BCECF fluorescence was recorded at excitation wavelengths of 490 nm and 440 nm with a resolution of 2 / s. Cl - was removed from the buffer containing HCO3 - (25 mM HCO3 - and 5% CO2), and Cl i / HCO3 - exchange activity was estimated from the initial rate of pH - increase.

[0678] pHi calibration was performed using a standard pH solution containing 150 mM KCl and 5 μM nigericin. The intrinsic buffering capacity (βi) was calculated by measuring ΔpHi in response to 5 to 40 mM NH4Cl pulses in the absence of Na + solution. Since the βi value is essentially unaffected by transfection with plasmids encoding WT-pantethin or H723R-pantethin, Clˉ / HCO3 exchange activity is expressed as ΔpH units / minute without compensating for the buffering capacity.

[0679] Table 3 shows the Cl of pantethine of the selected compounds of the present invention - / HCO3 - Measurement results of the exchange activity. Expressed as exchange % with the following symbols: A = exchange % greater than 50%; B = exchange % less than 50%.

[0680] Table 3. PTI determination

[0681]

[0682] ​

[0683] Principle of PDC determination

[0684] The experimental method is based on a multi-step process designed to study the effects of drug treatment on nasal epithelial cells. Initially, nasal tissue is collected and processed to isolate epithelial cells by tissue lysis. Then, these cells are cultured and maintained under air-liquid interface (ALI) conditions for a long time. Thereafter, drug treatment involving human IL-4 and pantethine-correcting drugs is administered, and protein harvest is performed for subsequent analysis. The expression of "hR1 (YONSEI ENTChoi Laboratory)" of pantethine antibody is confirmed by Western blot analysis after drug treatment. In addition, immunofluorescence staining is performed on Transwell membranes from different drug concentrations, and confocal microscopy is performed on the resulting frozen sections using the primary antibody "hR2 (YONSEI ENT Choi Laboratory)". This comprehensive method allows visualization and analysis of pantethine expression in the basal and apical regions of nasal epithelial cells, providing insights into the effects of drug treatment on target cell components.

[0685] Protocol of PDC determination

[0686] Measurement of pantethine expression in nasal epithelial cells of patients by Western blot and confocal microscopy

[0687] 1. Tissue collection and cell isolation

[0688] Nasal tissue is collected from patients and placed in DMEM:F12 (Lonza catalog number. 12-719F) supplemented with 1% penicillin-streptomycin in transfer medium. After removing red blood cells with PBS, the tissue is subjected to tissue lysis with 1% protease in transfer medium at 37 °C for 1 hour to isolate epithelial cells.

[0689] 2. Cell culture

[0690] The obtained epithelial cells were cultured to passage 1 in bronchial epithelial cell medium (BEGM Bulletkit) (Lonza catalog number CC3170) supplemented with 150 mg / ml BSA and EGF (BD catalog number 354001). When the cells reached approximately 90% confluence, the cells were detached using 0.25% trypsin-EDTA and seeded onto a 12-well Transwell plate (Costar catalog number 3450) containing a 1:1 mixture of DMEM medium (Lonza catalog number 12-707F) and medium supplemented with bronchial epithelial cell medium for air-liquid interface (ALI) culture.

[0691] 3. Air-liquid interface culture

[0692] ALI culture was initiated by adding 1 ml of medium to the bottom and seeding 0.5 ml of cells onto the membrane. When the cells on the membrane reached 90% confluence, the bottom was filled with medium supplemented with 50 nM retinoic acid (RA, SIGMA catalog number R2625), and ALI culture was performed for 7 - 14 days.

[0693] 4. Drug treatment and protein harvest

[0694] After ALI culture, the cells were treated with 10 μg / ml human IL-4 for 24 hours, and then treated with the panthrin corrector drugs "Compound 9, Compound 18, and Compound 105" at concentrations of 0.1, 0.3, 1, 3, and 10 μM for 24 hours. After drug treatment, the cells adherent to the Transwell membrane were harvested by scraping into protein buffer (iNtRON catalog number 17081). Protein quantification (30 μg) was performed for each sample, and the expression of "hR1 (YONSEIENT custom antibody)" of panthrin antibody was confirmed by Western blot analysis.

[0695] 5. Immunofluorescence staining

[0696] For immunofluorescence staining, the Transwell membranes from each drug treatment concentration were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes. Subsequently, they were cut into three pieces using a microtome, embedded in OCT compound, and processed into 5-μm frozen sections. Immunostaining was performed using the primary antibody "hR2 (YONSEIENT custom antibody)", and the panthrin expression in the basal and apical regions of the epithelial cells was visualized using a confocal microscope (Carl Zeiss, LSM700).

[0697] The results of the above experiments are as ​ and ​ shown.

[0698] ​ Shows the results of Western blot analysis of patient epithelial cells derived from patient nasal epithelial cells treated with compound 9.

[0699] ​ . (A, B) The B-form (which represents a specific non-glycosylated precursor form of pantellin) showed little obvious change in response to treatment with compound 9; however, a significant dose-dependent increase was observed in the C-form (which represents fully glycosylated functional pantellin), particularly at the high concentration of 10 μM. These observations extrapolated to patient-derived nasal epithelial cells, indicating a concentration-dependent effect of compound 9 treatment on C-type expression in cells carrying the H724R mutation.

[0700] ​ Shows the results of immunofluorescence staining of compounds 9, 18, and 105.

[0701] ​ . (A) This figure illustrates the results of immunofluorescence staining of epithelial cells under different conditions. In the vehicle and IL-4 groups, there was no pantellin expression, indicated by the absence of fluorescence. In contrast, treatment with compound 9, compound 18, and compound 105 at 10 μM showed clear red fluorescence, indicating enhanced pantellin expression localized to the epithelial cell membrane. Scale bar = 20 μm. (B) This figure is a quantitative representation comparing the mean fluorescence intensity levels between the vehicle, IL-4, and compound 9, compound 18, and compound 105 (10 μM) groups. Statistical significance indicates a significant difference in pantellin expression intensity between different experimental conditions. *p < 0.05.

[0702] The specific compounds prepared by the general procedure shown above are described in Table 4 below.

[0703] Table 4. Summary of the structures and corresponding characteristics of compounds 1 - 120.

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

Claims

1. A compound of general formula I and its pharmaceutically acceptable salts: X 1 independently selected from CH, C-Z and N each time it appears; X 2 independently selected from CH, C-Z, and N each time it appears; X 3 independently selected from CH, C-Z and N each time it appears; X 4 independently selected from CH, C-Z, and N each time it appears; X 5 independently selected from CH, C-Z, and N each time it appears; n is independently selected from 0, 1, and 2 each time it appears; R 1 independently selected from the group consisting of: hydrogen; C1-C6 alkyl; C1-C6 alkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 cycloalkyl; C3-C10 cycloalkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 heterocycloalkyl; C3-C10 heterocycloalkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C6-C12 aryl; C6-C12 aryl substituted with one or more of halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C12 heteroaryl; C3-C12 heteroaryl substituted with one or more of halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; each occurrence independently Z is any structure of group A below; Y 1 , Y 2 and Y 3 are each independently selected from CH and N at each occurrence; R 2 and R 3 is independently selected, each time it appears, from the group consisting of hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 and any one of which is optionally substituted; R 4 and R 5 is independently selected, each time it appears, from the group consisting of hydrogen, C1-C6 alkyl, and C3-C10 cycloalkyl, any of which is optionally substituted; R 6 and R 7 is independently selected, each time it appears, from the group consisting of hydrogen; C1-C4 haloalkyl; C1-C6 alkyl; C1-C6 alkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 8 and NR 8 R 9 and C1-C6 alkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and NR 8 R 9 and C3-C10 cycloalkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and NR 8 R 9 and C3-C10 heterocycloalkyl substituted with one or more of halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR R 8 and R 9 is independently selected, each time it appears, from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, and C1-C4 haloalkyl, any of which is optionally substituted.

2. The compound according to claim 1 and its pharmaceutically acceptable salts, wherein, Z is any structure of group B below: wherein, R 2 and R 3 are as defined in claim 1; and Y 1 、Y 2 and Y 3 as defined in claim 1.

3. The compound according to claims 1-2 and its pharmaceutically acceptable salts, wherein, Z is R 2 and R 3 as defined in claim 1; and Y 1 , Y 2 and Y 3 as defined in claim 1.

4. The compound according to any one of claims 1-3 and its pharmaceutically acceptable salts, which has the general formula II, Among them, R 1 、R 2 and R 3 as defined in claim 1; and X 1 、X 2 、X 3 and X 4 as defined in claim 1; and n is as defined in claim 1.

5. The compound according to any one of the preceding claims and its pharmaceutically acceptable salts, wherein, R 1 independently selected from the group consisting of hydrogen; C1-C6 alkyl; C1-C6 alkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 cycloalkyl; C3-C10 cycloalkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 ; C3-C10 heterocycloalkyl; C3-C10 heterocycloalkyl substituted with one or more of halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 each time it appears.

6. The compound according to any one of the preceding claims and its pharmaceutically acceptable salts, wherein, R 1 independently selected from the group consisting of C6-C12 aryl; C6-C12 aryl substituted with one or more of halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 at each occurrence; C3-C12 heteroaryl; C3-C12 heteroaryl substituted with one or more of halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and NR 6 R 7 at each occurrence.

7. The compound according to any one of the preceding claims and its pharmaceutically acceptable salts, wherein, R 2 and R 3 is independently selected from the group consisting of OR 6 and NR 6 R 7 each time it appears 8. The compound according to any one of the preceding claims and its pharmaceutically acceptable salts, which has one of formula 1 - formula 120 as shown below:

9. A composition comprising at least one compound according to any one of claims 1-8 as an active ingredient, and at least one pharmaceutically acceptable carrier, excipient, and / or diluent.

10. Use of the compound according to any one of claims 1-8 or the composition according to claim 9 as a pharmaceutical active agent, preferably in the method of treating Pendred syndrome or its related diseases.

11. Use of the compound according to any one of claims 1-8 in the method of preventing and / or treating the following diseases: hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, volume depletion, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or sinusitis.

12. A method for preventing, ameliorating, or treating Pendred syndrome or its related diseases, comprising the step of administering a compound according to any one of claims 1-8.

13. A method for preventing, ameliorating, or treating hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, volume depletion, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or sinusitis, the method comprising the step of administering a compound according to any one of claims 1-8.

14. Use of a compound according to any one of claims 1-8 for preventing, ameliorating, or treating Pendred syndrome or its related diseases.

15. Use of a compound according to any one of claims 1-8 for preventing, ameliorating or treating hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, volume depletion, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders or sinusitis.

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