Pharmaceutically active compounds, compositions, and methods for modulating pendrin

Abstract

The present invention relates to a novel compound represented by the following chemical formula 1 as a pendrin corrector, and a composition for preventing or treating pendrin syndrome and related diseases, which contains the compound as an active ingredient. [Formula 1] TIFF2026501619000117.tif43168

Description

[Technical Field]

[0001] The present invention relates to a novel compound as a pendrin corrector and a composition containing the same as an active ingredient for preventing or treating Pendred syndrome and related diseases. [Background technology]

[0002] Pendrin is an anion exchange channel protein encoded by SLC26A4 (PDS), a member of the SLC26A family. It is expressed in the apical cell membrane and transports Cl - , HCO3 - , O.H. - , I - It mediates the transport of ions as well as formate, nitrate, and thiocyanate. Pendrin is significantly expressed in the inner ear, thyroid, and kidney, although expression can be induced in other tissues under certain conditions. In the inner ear, pendrin is expressed in the endolymphatic sac and hair cells. Pendrin defects due to gene mutations result in endolymph acidification and Ca 2+ This leads to decreased resorption and induces auditory sensory transmission defects, including Pendred syndrome (Bassot, C., et al. 2017; Biochimie. 132: 109-120).

[0003] Several types of pharmacological modulators have been reported to directly interact with target dysfunctional proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR), restoring ion channel function of ion transporters. Among these modulators, correctors (also known as pharmacological chaperones) and potentiators are the main types of modulators that restore the function of specific membrane transporters. Correctors or pharmacological chaperones help mutant polypeptides fold properly as functional transporter proteins. Thus, correctors enable mutant proteins to form functionally intact structures, allowing them to pass through the trans-Golgi network with appropriate post-translational modifications and be targeted to the plasma membrane. In contrast, enhancers are modulators that stabilize proteins or increase the conductance of cell surface transporter proteins, thereby improving their channel-gating function (Collawn, JF, et al., 2014; Am. J. Physiol. Lung Cell. Mol. Physiol. 307: L431-L434). Numerous compounds using this strategy have been developed and are commercially available for specific diseases, such as cystic fibrosis, caused by genetic defects in CFTR (Lopes-Pacheco, M., 2020; Front. Pharmacol.). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Bassot, C., et al. 2017; Biochimie. 132: 109-120 [Non-patent document 2] Collawn, JF, et al., 2014; Am. J. Physiol. Lung Cell. Mol. Physiol. 307: L431-L434 [Non-patent document 3] Lopes-Pacheco, M., 2020; Front. Pharmacol. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention is based on the discovery that certain compounds have the potential to act as pendrin correctors and prevent, ameliorate, or treat Pendred syndrome and related disorders. [Means for solving the problem]

[0006] In one aspect, the present invention provides a compound represented by the following chemical formula 1, an optical isomer thereof, a mixture of the two isomers thereof, a precursor thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof: [ka] X 1 are each independently selected from CH, CZ, and N; X 2 are each independently selected from CH, CZ, and N; X 3 are each independently selected from CH, CZ, and N; X 4 are each independently selected from CH, CZ, and N; X 5 are each independently selected from CH, CZ, and N; each n is independently selected from 0, 1, and 2; R 1 are each independently hydrogen, C1-C6 alkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 heterocycloalkyl substituted with one or more of: C6-C12 aryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C6-C12 aryl substituted with one or more of: C3-C12 heteroaryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C12 heteroaryl substituted with one or more of: Z is any one of the structures in Group A below; [ka] Y 1 , Y 2 , and Y 3 are each independently selected from CH and N; R 2 and R 3 are each independently hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7each of which is optionally substituted; R 4 and R 5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C10 cycloalkyl, each of which is optionally substituted; R 6 and R 7 are each independently hydrogen, C1-C4 haloalkyl, C1-C6 alkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9 C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9 C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, =O, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9 C3-C10 heterocycloalkyl substituted with one or more of: R 8 and R 9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, and C1-C4 haloalkyl, each of which is optionally substituted; and pharmaceutically acceptable salts thereof.

[0007] In another aspect, the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating Pendred syndrome or a related disorder, comprising one or more of the above compounds, one or more optical isomers thereof, one or more mixtures of the two isomers thereof, one or more precursors thereof, one or more pharmaceutically acceptable salts thereof, or one or more solvates thereof as an active ingredient. In additional aspects, the present invention provides compositions comprising a compound represented by Chemical Formula 1 or a mixture thereof, and compositions comprising a compound represented by Chemical Formula 1 or a mixture thereof together with a pharmaceutically acceptable carrier.

[0008] In a further aspect, the present invention provides the use of a compound of Formula 1 and pharmaceutical compositions thereof as a pendrin corrector.

[0009] In a further aspect, the present invention provides use of a compound represented by Chemical Formula 1, a mixture thereof, and a pharmaceutical composition thereof as an active ingredient of a health functional food for preventing or ameliorating Pendred syndrome or related disorders.

[0010] In a further aspect, the present invention provides the use of the compound and its pharmaceutical composition as a pendrin corrector for preventing or ameliorating pendrin syndrome or related diseases as an active ingredient in a health functional food. [Effects of the Invention]

[0011] According to the present invention, the novel compound can act as a pendrin corrector, and as a result, can be usefully used as a composition for preventing, treating, or ameliorating pendrin syndrome or diseases related thereto. [Brief explanation of the drawings]

[0012] [Figure 1] Western blot analysis of patient-derived epithelial cells treated with compound 9 is shown. (A, B) The B-form, representing a specific non-glycosylated precursor form of pendrin, showed little noticeable change upon treatment with compound 9; however, a notable dose-dependent increase was observed in the C-form, representing fully glycosylated functional pendrin, particularly at a high concentration of 10 μM. These observations, extrapolated to patient-derived nasal epithelial cells, suggest a concentration-dependent effect of compound 9 treatment on C-form expression in cells harboring the H724R mutation. [Figure 2]Immunofluorescence staining results for compounds 9, 18, and 105 are shown. (A) shows the results of immunofluorescence staining of epithelial cells under various conditions. No fluorescence was observed in the control and IL-4 groups, indicating the absence of pendrin expression. Conversely, treatment with 10 μM of compounds 9, 18, and 105 showed clear red fluorescence, indicating increased pendrin expression localized to the epithelial cell membrane. Scale bar = 20 μm. (B) shows a quantitative comparison of the mean fluorescence intensity levels between the control group, IL-4, and compounds 9, 18, and 105 (10 μM) groups. Statistical significance indicated significant differences in pendrin expression intensity between experimental conditions. *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this specification belongs. As used herein, the following terms have the meanings stated below unless otherwise specified.

[0014] Unless otherwise specifically indicated or clear from the context, the term "or" as used herein is to be understood as inclusive.

[0015] Unless otherwise specifically stated or clear from the context, the term "about" as used herein is understood to mean within a general range of acceptance in the technical field, for example, within two standard deviations of the mean. "About" is understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0016] The terms "active agent," "drug," and "pharmaceutical dosage form" are used interchangeably herein and refer to a chemical compound or compound that induces a desired pharmacological effect (e.g., reduced inflammation) when administered to a subject by any means described herein (e.g., any animal, including a human or non-human animal).

[0017] As used herein, the term "additive" can refer to any additional component that can be added to the compositions and formulas described herein. For example, if the additional component is pharmaceutically acceptable for the particular condition being treated, the additive can include an excipient (e.g., one or more excipients), an antioxidant (e.g., one or more antioxidants), a stabilizer (e.g., one or more stabilizers), a preservative (e.g., one or more preservatives), a pH adjuster and / or buffer (e.g., one or more pH adjusters and / or buffers), a tonicity adjuster (e.g., one or more tonicity adjusters), a thickener (e.g., one or more thickeners), a suspending agent (e.g., one or more suspending agents), a binder (e.g., one or more binders), a viscosity-increasing agent (e.g., one or more viscosity-increasing agents), etc. Excipients can also include processing agents, drug delivery modifiers, and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, and ion exchange resins, and combinations of two or more of these.

[0018] As used herein, the term "administration" refers to oral, suppository, topical, intravenous, parenteral, transtympanic, intratympanic, intracochlear, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, intravitreal, or subcutaneous administration, or sustained release devices such as implantation of mini-osmotic pumps into the subject.

[0019] Administration is by any route, including parenteral and transmucosal (eg, oral, intranasal, pulmonary, rectal, intrabuccal, vaginal, ocular, and transdermal).

[0020] The terms "analog" and "derivative" are used interchangeably herein to refer to a compound that has the same core as a parent compound but differs from the parent compound in the bond order, the presence or presence of one or more atoms and / or groups of atoms, and combinations thereof. A derivative may differ from the parent compound, for example, in one or more substituents present on the core, which may include one or more atoms, functional groups, or substructures. A derivative may also differ from the parent compound in the bond order between atoms within the core. In general, a derivative can be predicted, at least theoretically, to form from the parent compound through chemical and / or physical processes.

[0021] As used herein, "antioxidant" can refer to a man-made or natural substance that can prevent or slow certain types of damage and / or oxidation.

[0022] Antioxidants are found in many foods, including fruits and vegetables, and can be used as dietary supplements. Exemplary antioxidants include beta-carotene, lutein, lycopene, selenium, vitamin A, vitamin C, and vitamin E. Other antioxidants known to those skilled in the art can also be used. The antioxidants described herein can be used in any suitable amount.

[0023] "Co-administration" means that a compound or composition described herein is administered simultaneously, immediately before or after the administration of an additional therapeutic or activating agent or additive described herein. The compounds or compositions of the invention can be administered alone or co-administered to a patient. Co-administration is understood to include simultaneous or sequential administration of compounds individually or in combination (one or more compounds or agents). If desired, agents may be combined with other active substances.

[0024] As used herein, terms such as "comprise," "contain," and "have" can have the meanings associated with them and can mean "comprise," etc. Also, "consisting essentially of" or "consist essentially of" can have the meanings associated with them, and such terms are open-ended and allow for the presence of more than what is recited, except when the presence of more than what is recited changes the basic or novel characteristics of what is recited. However, examples of prior art are excluded.

[0025] As used herein, "co-administration" includes at least partial overlapping of time periods. For example, when two formulations (e.g., any formulation or class of formulations having a physiologically active agent described herein) are administered simultaneously, these administrations occur within a particular desired time period. The administration of the formulations can begin and end on the same day. Also, the administration of one formulation can precede the administration of a second formulation, as long as both formulations are administered at least once on the same day. Similarly, the administration of one dosage form can extend beyond the administration of a second dosage form, as long as both dosage forms are administered at least once or more on the same day. Co-administration of physiologically active agents / dosage forms does not require that they be taken at the same time every day to be included.

[0026] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired biological effect, such as a beneficial result, including a clinical result. Thus, the "effective amount" will vary depending on the context in which it is applied. The effective amount may vary depending on factors known in the art, such as the disease state, age, sex, and weight of the individual being treated. Several divided doses may be administered daily, or the dose may be proportionally reduced depending on the exigencies of the therapeutic situation. Alternatively, the compositions / dosage forms of the present invention may be administered as frequently as necessary to achieve a therapeutic dose.

[0027] As used herein, the term "intermittent administration" includes a period during which a dosage form is administered (which may be considered a "first administration period"), a subsequent period during which the dosage form is not taken or is taken at a lower dose (which may be considered a "drug holiday period"), and a subsequent period during which the dosage form is again administered (which may be considered a "second administration period"). Generally, the dosage level of the dosage form during the second administration period is the same as the dose administered during the first administration period, although it may be increased or decreased as medically indicated.

[0028] As used herein, a "liquid" is a dosage form comprised of a composition in a liquid state. A liquid is pourable and flows and behaves within a container at room temperature. A liquid exhibits Newtonian or pseudoplastic flow behavior.

[0029] In one embodiment, "semi-liquid" as used herein can have all the properties of liquids and other dosage forms (i.e., suspensions, oils, solutions, creams, gels, jellies, etc.).

[0030] As used herein, the term "ointment" can refer to a thickened liquid or semi-liquid dosage form used in the therapeutic treatment of a disease, syndrome, or disorder.

[0031] As used herein, "pharmaceutically acceptable carrier" includes all physiologically suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The type of carrier can be selected depending 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 formulations for pharmaceutically active substances is well known in the art. Insofar as any conventional media or formulation is incompatible with the composition (e.g., Formula 1, derivatives or analogs of Formula 1, or pharmaceutically acceptable salts, solvents, hydrates, or polymorphs thereof, as described herein), its use is contemplated in the compositions of the present invention.

[0032] As used herein, a "pharmaceutical carrier" or "carrier" can additionally include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals at the volumes and concentrations specified. Physiologically acceptable carriers are sometimes aqueous pH buffered solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, 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 dextrins; chelating agents, such as EDTA; sugar-alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or Tween (Tween 1). TM ), polyethylene glycol (PEG), Pluronics TM )®. Also, "pharmaceutically acceptable" means that the material is approved or likely to be approved by a federal or state government regulatory agency or agencies in a country other than the United States, or is listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, especially humans.

[0033] The term "pharmaceutically acceptable salt or complex" refers to a salt or complex represented by the specific formula 1 below. Examples of such salts include, but are not limited to, base addition salts formed by reacting a compound represented by formula 1 with an organic or inorganic base, such as the hydroxide, carbonate, or bicarbonate of a metal cation selected from the group consisting of alkali metals (e.g., sodium, potassium, or lithium) and alkaline earth metals (e.g., calcium or magnesium), or with a primary, secondary, or tertiary alkylamine. Amine salts derived from methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, morpholine, N-methyl-D-glutamine, N,N'-bis(phenylmethyl)-1,2-ethanediamine, tromethamine, ethanolamine, diethanolamine, ethylenediamine, N-methylmorpholine, procaine, piperidine, piperazine, and the like, are considered within the scope of the present invention.

[0034] Additionally, as used herein, "salts" or "salt forms" or "pharmaceutically acceptable salts" can include base addition salts (salts having a free carboxyl group or other anionic group) derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. The salts are formed from acid addition salts having any free cationic group, typically with inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, or organic acids such as formic acid, acetic acid, citric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts of the present invention can include amine salts formed by protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the present invention can also include amine salts formed by protonation of an amino group with suitable organic acids such as p-toluenesulfonic acid, acetic acid, and the like.

[0035] As used herein, the term "pH agent" or "buffer" can refer to a compound or buffer useful as a pH adjusting agent, including, but not limited to, glycerol buffer, citrate buffer, borate buffer, acetate buffer, gluconate buffer, phosphate buffer, or citrate-phosphate buffer. The pH agent or buffer can be used in any suitable amount.

[0036] The term "preservative" as used herein can refer to a substance or chemical that prevents undesired changes in the compounds, compositions, or formulas described herein. Suitable preservatives include, for example, benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, edetate disodium sorbate, onamo m polyquat, cetyl bromide, cetylpyridinium chloride, benzyl bromide, EDTA, phenylmercuric nitrate, phenylmercuric acetate, merthiolate, phenylmercuric acetate and borate, polymyxin B sulfate, methyl and propylparaben, tertiary ammonium chloride, sodium benzoate, sodium propionate, sodium perborate, and other substances or combinations thereof known to those skilled in the art. Preservatives can be used in appropriate amounts.

[0037] As used herein, "prevent" or "prevention" and other grammatically similar terms include preventing the onset, occurrence, obstruction, or avoidance of a disease or disease symptom, as well as a reduction in the incidence of the symptom. Prevention may be complete prevention (i.e., no symptoms at all) or partial prevention (i.e., fewer symptoms are observed than without treatment). This term also includes a prophylactic effect. To prevent a disease or disorder, the compositions can be administered to patients at risk of developing a particular disease or to patients who exhibit one or more physiological symptoms, but who are not necessarily diagnosed with a disease.

[0038] Ranges provided herein are intended to abbreviate all values ​​within that range. For example, a range of 1 to 10 is understood to include all numbers or combinations of numbers in subranges selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as well as all intermediate decimal points between the integers recited above, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. In connection with subranges, "superimposed subranges" extending from one of the endpoints of the range are specifically contemplated.

[0039] For example, overlapping subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0040] Ranges may be expressed herein by "about" one particular value and / or by "about" another particular value. When such a range is expressed, another aspect includes the one particular value and / or the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is also understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint. Also, throughout this application, data are provided in a variety of formats, and it is understood that such data represents endpoints, starting points, and ranges for all combinations of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, then values ​​between 10 and 15, as well as values ​​greater than or equal to 10, greater than or equal to 15, less than 15, less than 15, and less than 15, are considered to be disclosed. It is also understood that each unit between two specified units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0041] Additional excipients contemplated for use in practicing the present disclosure are available to those of skill in the art.

[0042] A "semi-solid gel" according to the present disclosure is a semi-solid. The apparent viscosity of a semi-solid dosage form can increase with concentration.

[0043] As used herein, "sequential administration" includes administration of two dosage forms (e.g., a compound or composition described herein) separately on the same day or not on the same day (e.g., on consecutive days).

[0044] A "solution" according to the present disclosure may be a clear, homogeneous liquid dosage form containing one or more chemicals dissolved in a miscible solvent or solvent mixture. Because the drug substance molecules in a solution are uniformly dispersed, the use of a solution as a dosage form generally ensures a uniform dosage upon administration and provides greater accuracy when the solution is diluted or otherwise mixed.

[0045] As used herein, the term "solvent" refers to an aqueous or non-aqueous liquid solvent. The choice of solvent will depend, inter alia, on the solubility and mode of administration of the composition. Aqueous solvents may consist solely of water or 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 include short-chain organic alcohols such as methanol, ethanol, and propanol, short-chain ketones such as acetone, and polyalcohols such as glycerol.

[0046] "Subject" or "patient" means a human or non-human animal, such as a mammal. "Subject" includes all animals, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cows, fish, and birds. A human subject can be referred to as a patient.

[0047] As used herein, a "suspension" is a liquid dosage form that contains solid particles dispersed in a liquid vehicle.

[0048] As used herein, "viscosity" refers to the resistance of a fluid to flow. Viscosity agents may be used herein, and may include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, other substances known to those skilled in the art, or combinations thereof.

[0049] The term "weight percentage" or "%(w / w)" refers to the percentage of a component in solution, calculated based on the weight of the component and solvent. For example, a 1% (w / w) solution of a component may be 1 g of the component dissolved in 100 g of solvent. The term "volume percentage" or "%(v / v)" refers to the percentage of a component in solution, calculated based on the volume of the component and solvent. For example, a 1% (v / v) solution of a component may be 1 ml of the component dissolved in 100 ml of solvent. The term "weight / volume percentage" or "%(w / v)" refers to the percentage of a component in 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 be 1 g of the component dissolved in 100 ml of solvent.

[0050] As used herein, the term "syndrome" refers to a disease characterized by a set of symptoms that occur consistently together or are related to one another. A syndrome (e.g., acute respiratory distress syndrome) can be a set of medical signs and symptoms that are interrelated and sometimes associated with a specific disease. A disease, on the other hand, can be a health condition with a clearly defined cause. However, a syndrome (Greek for "running together") can produce a variety of symptoms without a clear cause. This can indicate the possibility of an underlying disease or the possibility of developing a disease.

[0051] As used herein, the terms "treat" or "treatment" and other grammatical equivalents include, but are not limited to, the alleviation, attenuation, amelioration, or prevention of a disease, disorder (e.g., acute respiratory distress syndrome) or symptom, prevention of additional symptoms, amelioration or prevention of the underlying metabolic cause of a symptom, and inhibition of a disease or disorder (e.g., halting the onset of a disease or disorder, alleviating a disease or disorder, regressing a disease or disorder, alleviating a disease or disorder caused by a disease or disorder, or cessation of a disease or disorder symptom), and are intended to include prevention. This term additionally includes achieving a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit refers to the eradication or amelioration of the underlying disorder being treated. Alternatively, a therapeutic benefit is achieved through the eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, even if the patient still suffers from the underlying disorder.

[0052] The term "health functional food" refers to a food or food supplement manufactured or processed from raw materials, functional ingredients, active pharmaceutical ingredients or additives that are useful for improving, nourishing and / or preserving the physiological functions of the human body.

[0053] The term "PDS" may also be used for the pendrin protein (PDS) encoded by the gene SLC26A4.

[0054] Unless otherwise limited by the definition of an individual substituent, all substituents should be understood to be optionally substituted.

[0055] 2.Compound One aspect of the present invention provides a compound represented by the following chemical formula 1, an optical isomer thereof, a mixture of two isomers thereof, a precursor thereof, a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0056] [ka] X 1 are each independently selected from CH, CZ, and N; X 2are each independently selected from CH, CZ, and N; X 3 are each independently selected from CH, CZ, and N; X 4 are each independently selected from CH, CZ, and N; X 5 are each independently selected from CH, CZ, and N; each n is independently selected from 0, 1, and 2; R 1 are each independently hydrogen; C1-C6 alkyl; halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl; halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen; halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 heterocycloalkyl substituted with one or more of: C6-C12 aryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl; C6-C12 aryl; halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7C6-C12 aryl substituted with one or more of: C3-C12 heteroaryl; and halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C12 heteroaryl substituted with one or more of: Z is any one of the structures in Group A below; [ka] Y 1 , Y 2 , and Y 3 are each independently selected from CH and N; R 2 and R 3 are each independently hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 any of which is optionally substituted; R 4 and R 5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C10 cycloalkyl, any of which is optionally substituted; R 6 and R 7 are each independently hydrogen; C1-C4 haloalkyl; C1-C6 alkyl; halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9 C1-C6 alkyl substituted with one or more selected from: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, ═O, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9C3-C10 cycloalkyl substituted with one or more selected from: C3-C10 heterocycloalkyl; and halogen, C1-C6 alkyl, ═O, C1-C4 haloalkyl, OR 8 and N.R. 8 R 9 C3-C10 heterocycloalkyl substituted with one or more selected from: R 8 and R 9 are each 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; and pharmaceutically acceptable salts thereof.

[0057] In a preferred embodiment, in Formula 1: Z is any one of the structures in Group B below; [ka] where R 2 and R 3 is as defined in claim 1, Y 1 , Y 2 , and Y 3 is as defined in claim 1.

[0058] In a preferred embodiment, in Formula 1: Z is [ka] and where R 2 and R 3 is as defined above; and Y 1 , Y 2 , and Y 3 is as defined above.

[0059] In a preferred embodiment, the compound of Formula 1 has the general formula II: [ka] where R 1 , R 2 , and R 3 is as defined above; and X 1 , X 2 , X 3 , and X 4 is as defined above; and n is as defined above.

[0060] In a preferred embodiment, in Formula 1, R 1 are each independently hydrogen; C1-C6 alkyl; halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl; halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl; halogen, C1-C6 alkyl, =O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C10 heterocycloalkyl substituted with one or more of:

[0061] In a preferred embodiment, in Formula 1, R 1 each independently represents C6-C12 aryl; halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR6 and N.R. 6 R 7 C6-C12 aryl substituted with one or more of: C3-C12 heteroaryl; halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR 6 and N.R. 6 R 7 C3-C12 heteroaryl substituted with one or more of:

[0062] In a preferred embodiment, in Formula 1, R 2 and R 3 are each independently OR 6 and N.R. 6 R 7 is selected from the group consisting of:

[0063] In a preferred embodiment, the compound of Formula 1 has one of the following formulas 1) to 120):

[0064] TIFF2026501619000009.tif209168TIFF2026501619000010.tif197168TIFF2026501619000011.tif202168TIFF2026501619000012.tif198168TIFF202 6501619000013.tif208168TIFF2026501619000014.tif213168TIFF2026501 619000015.tif206168TIFF2026501619000016.tif216168TIFF20265016190 00017.tif202168TIFF2026501619000018.tif208168TIFF2026501619000019.tif223168TIFF2026501619000020.tif202168TIFF2026501619000021.t if217168TIFF2026501619000022.tif215168TIFF2026501619000023.tif213168TIFF2026501619000024.tif200168TIFF2026501619000025.tif144168

[0065] 3. Composition / Formulation In yet 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.

[0066] In a preferred embodiment, the composition may be for use as a pharmaceutically active agent, preferably in a method for treating a disease.

[0067] The present invention includes pharmaceutical compositions comprising the compounds described herein and dosage forms suitable for administering the compounds described herein. Pharmaceutical composition dosage forms suitable for administration by any medically acceptable means are included. Pharmaceutical compositions may include a pharmaceutically acceptable excipient or carrier and a pharmaceutically acceptable compound (composition) suitable for the administration means.

[0068] The compounds described herein may be in dosage forms (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 adjusters and / or buffers (e.g., one or more pH adjusters and / or buffers), tonicity adjusters (e.g., one or more tonicity adjusters), thickeners (e.g., one or more thickeners), suspending agents (e.g., one or more suspending agents), binders (e.g., one or more binders), viscosity-increasing agents (e.g., one or more viscosity-increasing agents), etc., which may be provided as pharmaceutically acceptable additional ingredients for the particular condition being treated. In some embodiments, the dosage forms may include a combination of the additional ingredients described herein (e.g., two, three, four, five, six, seven, eight, or more additional ingredients). In some embodiments, additives can include, for example, processing agents, drug delivery modifiers, and enhancers such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-beta-cyclodextrin, polyvinylpyrrolidone, low melting point waxes, and ion exchange resins, and combinations of two or more thereof.

[0069] 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 ed. (2003) and 21st ed. (2005), which are incorporated herein by reference.

[0070] The dosage forms of the compositions described herein may be suitable for inhalation, nasal spray, intravenous injection, intramuscular injection, intravitreal injection, and oral administration, which may consist of ointments or solutions, suspensions, semi-liquids, semi-solids, gels, semi-solid gels, jellies, oils, ointments, oils, tablets, liquids, and creams. Tablet forms 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, flavorings, dyes, disintegrants, and pharmaceutically suitable carriers. Capsules may contain the compounds together with suitable excipients, or the compounds may be used alone as a shell. All such formulations may be administered alone or in combination, intermittently, sequentially, or simultaneously.

[0071] 4. Administration The compositions of the present invention include, but are not limited to, compositions that can be administered orally, parenterally, sublingually, cutaneously, rectally, transmucosally, topically, transtympanically, intratympanically, intracochlearly, by inhalation, buccally, or intranasally, or any combination thereof. Parenteral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intradural, and intraarterial administration. The compositions of the present invention can also be administered in the form of an implant, which allows for sustained release of the composition and slow, controlled intravenous administration.

[0072] The single or multiple doses administered to an individual may vary depending on a variety of factors, including pharmacokinetic properties, patient condition and characteristics (sex, age, weight, health status, size), severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect.

[0073] According to one embodiment of the present invention, the compounds according to the present invention and pharmaceutical dosage forms thereof can be administered alone or together with adjuvants useful in the treatment of respiratory disorders or diseases. According to another embodiment of the present invention, the compounds according to the present invention and pharmaceutical dosage forms thereof can be administered together with radiation therapy.

[0074] The present invention includes the administration of a compound according to the present invention or a pharmaceutical formulation thereof, which is administered to a subject in a therapeutically effective amount prior to, or sequentially with, the administration of another therapeutic or adjunctive agent (e.g., polytherapy) useful in treating Pendred Syndrome. Compounds according to the present invention or pharmaceutical formulations administered with adjunctive agents can be administered in the same or different compositions and by the same or different routes of administration.

[0075] In one embodiment, a patient according to the present invention may be a patient suffering from Pendred syndrome or a disorder related thereto, such as hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, hypovolemia, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis of the liver, bone abnormalities, cochlear malformation, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or sinusitis.

[0076] 5. Uses of the present invention In another embodiment, the present invention provides a use of a compound represented by Formula 1, a mixture of said compounds, or a pharmaceutical composition thereof for the prevention, amelioration, or treatment of Pendred syndrome or a related disorder.

[0077] In another embodiment, the present invention provides a use of the compound represented by Formula 1 and pharmaceutical compositions thereof as a pendrin corrective agent.

[0078] In another embodiment, the present invention provides for use in treating one or more Pendred syndrome-related disorders selected from the group consisting of hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, hypovolemia, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis of the liver, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, and rhinitis.

[0079] In another embodiment, the present invention provides a use of a compound represented by Chemical Formula 1, a mixture of said compounds, or a pharmaceutical composition thereof as an active ingredient of a health functional food for the prevention or amelioration of Pendred syndrome or a related disorder.

[0080] In another embodiment, the present invention provides a use of the compound represented by Chemical Formula 1 as a pendrin inhibitor for preventing or ameliorating Pendred syndrome or related disorders, and a pharmaceutical composition thereof as an active ingredient in a health functional food.

[0081] Other aspects and advantages of the present invention will become apparent to those skilled in the art upon consideration of the detailed description and drawings. [Example]

[0082] [Example] This specification describes non-limiting examples of detailed experiments that do not limit the overall experiment. The description of the present invention described herein is for illustrative purposes only, and it should be understood by those with ordinary skill in the art that the present invention relates to that it can be easily modified to other specific fields or forms without changing the technical idea or essential characteristics of the present invention. The present invention is presented by way of example in the following examples, but is not limited thereto.

[0083] References cited herein are incorporated herein by reference in their entirety. The present invention is not limited to the specific embodiments described herein, which are intended as single examples of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be encompassed within the scope of the appended claims.

[0084] [ka]

[0085] General procedure for the synthesis of A2 A mixture of A1, A1-1, Na2CO3, cyclopentyl(diphenyl)phosphane, dichloropalladium, and iron in dioxane and HO was degassed, washed with nitrogen (N2) three times, and then stirred at 100 °C under a nitrogen atmosphere for 2 h. The reaction mixture was poured into water and extracted with ethyl acetate, then washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography to give A2.

[0086] General procedure for the synthesis of A3 To a solution of A2 in DMF was added K2CO3 and tert-butyl 2-cyanoacetate. The mixture was stirred at 120 °C for 16 h. The reaction mixture was poured into water and extracted with ethyl acetate, then washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give A3.

[0087] General procedure for the synthesis of A4 A mixture of A3 in ethanol was added with concentrated HCl and stirred at 80 °C for 40 min. The mixture was quenched with saturated NaHCO3 at pH 7 and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4. The filtrate was concentrated to give the crude product, which was purified by flash chromatography on silica gel A4.

[0088] General procedure for the synthesis of A5 To a solution of A4 in dioxane was added pyridine at 20° C. 2-Chloroacetyl chloride was added dropwise to the reaction mixture at 40-50° C. The mixture was stirred at 100° C. for 20 minutes. The reaction mixture was used in the next step without further purification.

[0089] General procedure for the synthesis of A6 To the solution of A5 in dioxane from the previous step, DMF and B1 were added. The mixture was stirred at 100 °C for 10 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by prep-HPLC and lyophilized to give A6.

[0090] [ka]

[0091] General procedure for the synthesis of B2 Ethyl 2-bromoacetate was added to a solution of B1 and K2CO3 in acetone at 60 °C for 10 h. 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 filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography to give B2.

[0092] General procedure for the synthesis of B3 To a solution of B2 in CH2Cl2 was added Boc2O and DMAP at 0 °C. 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 filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography to give B3.

[0093] General procedure for the synthesis of B4 LiOH·H2O was added to a solution of B3 in THF and H2O, and the mixture was stirred at 20 °C for 1 h, followed by stirring at 80 °C for 1 h. The pH of the reaction mixture was adjusted to 4 with HCl (1 mol / L), poured into water, and extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give B4.

[0094] General procedure for the synthesis of B6 CDI was added to a solution of B4 in CH3CN, and the reaction mixture was stirred at 20°C for 40 minutes. TLC analysis showed that B4 was completely consumed. B5 was used in the next step.

[0095] To a solution of B5 and A4 in CH3CN, t-BuOK was added 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 and dried over Na2SO4. The reaction mixture was then concentrated in vacuo to give B6.

[0096] General procedure for the synthesis of A6 To a solution of B6 in ethanol was added 37% HCl. The reaction mixture was stirred at 80°C for 1 hour. The reaction solution was concentrated and purified to give A6.

[0097] [ka]

[0098] General procedure for the synthesis of C2 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)phosphane, dichloropalladium, and iron (1.21 g, 1.65 mmol) were dissolved in dioxane (10 mL) and HO (1 mL). After degassing and rinsing with N2 three times, the mixture was stirred at 100 °C under a N2 atmosphere for 2 h. The reaction mixture was poured into water (10 mL), extracted with ethyl acetate (50 mL x 3), washed with brine (50 mL x 3), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (0-35% ethyl acetate on PE) to give C2 (2.57 g, 62.2% yield) as a white solid.

[0099] General procedure for the synthesis of C3 To a solution of C2 (1 g, 3.99 mmol) in DMF (10 mL) was added K2CO3 (1.10 g, 7.98 mmol) and tert-butyl 2-cyanoacetate (675.76 mg, 4.79 mmol). The mixture was stirred at 120 °C for 16 h. The reaction mixture was poured into water (10 mL) and extracted with ethyl acetate (10 mL × 3), washed with brine (10 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give a yellow solid, C3 (1.5 g, crude, ~93% purity).

[0100] General procedure for the synthesis of C4 A mixture of C3 (1.5 g, approximately 93% purity, 3.94 mmol) in ethanol (25 mL) was added to concentrated HCl (6 mL) and stirred at 80 °C for 40 min. The mixture was quenched with saturated NaHCO to pH 7 and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over NaSO. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0–50% ethyl acetate in petroleum ether) to give C4 (607 mg, 60.4% yield) as a yellow solid.

[0101] General procedure for the synthesis of C5 To a solution of C4 (50 mg, 195.87 μmol) in dioxane (1 mL) was added pyridine (18.59 mg, 18.97 μL) at 20° C. 2-Chloroacetyl chloride (22.12 mg, 195.87 μmol) was added dropwise to the reaction mixture at 40-50° C. The mixture was stirred at 100° C. for 20 minutes. The reaction mixture was used in the next step without further purification.

[0102] General procedure for the synthesis of compound 1 To a solution of C5 (64.98 mg, 195.87 μmol) from the previous step in dioxane was added DMF (1 mL) and 2-fluoroaniline (65.29 mg, 587.61 μmol). The mixture was stirred at 100 °C for 10 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by prep-HPLC (column: Waters Xbridge 150*25 5 μl, column: 63-93% B (A = water (0.05% ammonia hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector 220 nm) and lyophilized to give compound 1 (3 mg, 3.6% yield, 95% purity) as a brown solid.

[0103] [ka]

[0104] General procedure for the synthesis of C7 C6 (3 g, 15.51 mmol), (3,4-dimethoxyphenyl)boronic acid (3 g, 16.49 mmol), Na2CO3 (3.29 g, 31.02 mmol), and Pd(dppf)Cl2 (1.13 g, 1.55 mmol) were dissolved in dioxane (30 mL) and HO (5 mL). The mixture was degassed and washed with N2 three times, then stirred at 80 °C under N2 atmosphere for 3 h. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0–40% EtOAc in petroleum ether) to give C7 (2.76 g, 71.0% yield) as an off-white solid.

[0105] General procedure for the synthesis of C8 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 h. The mixture was quenched with water (20 mL), the pH was adjusted to approximately 4 with 1 M HCl, and then filtered. The filter cake was dried under vacuum to give a brown solid, C8 (3.4 g, 90.2% yield).

[0106] General procedure for the synthesis of C9 A mixture of C8 (3.4 g, 9.57 mmol) in HCl (3 mL) and EtOH (30 mL) was stirred at 90 °C for 40 min. 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 give the crude product, which was purified by silica gel flash chromatography (0–50% EtOAc in petroleum ether) to give C9 (1.77 g, 72.5% yield) as a yellow solid.

[0107] General procedure for the synthesis of C10-2 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) was added ethyl 2-bromoacetate (16.53 g, 99.00 mmol) at 60 °C for 10 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0–10% EtOAc in petroleum ether) to give C10-2 (17.3 g, crude product) as a brown liquid.

[0108] General procedure for the synthesis of C10-3 To a solution of C10-2 (10 g, 50.71 mmol) in CHCl (100 mL) were added BocO (22.13 g, 101.42 mmol, 23.30 mL) and DMAP (9.29 g, 76.06 mmol) at 0 °C. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (30 mL). The combined organic layers were washed with brine (30 mL), dried over NaSO, and concentrated under reduced pressure. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0-10% EtOAc in petroleum ether) to give C10-3 (2.84 g, 20.0% yield) as a colorless oil.

[0109] General procedure for the synthesis of C10 To a solution of C10-3 (1.2 g, 4.04 mmol) in THF (6 mL) and HO (2 mL), LiOH.HO (338.73 mg, 8.07 mmol) was added and stirred at 20 °C for 1 h. The mixture was then stirred at 80 °C for 1 h. The pH of the reaction mixture was adjusted to 4 with HCl (1 mol / L), then poured into water (10 mL), and extracted with EtOAc (50 mL*3). The combined organic layer was washed with brine (50 mL*3), dried over NaSO, and concentrated under reduced pressure to give C10 (887 mg, 2.96 mmol, 73.5% yield) as a brown solid.

[0110] General procedure for the synthesis of C12 To a solution of C10 (150 mg, 557.07 μmol) in CH3CN (2 mL), CDI (90.33 mg, 557.07 μmol) was added and stirred at 20 °C for 40 min. TLC (PE: EtOAc = 3:1) showed complete consumption of C10 (150 mg, 557.07 μmol) (Rf = 0.3). C11 (177.89 mg crude) was used in 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), t-BuOK (62.20 mg, 554.29 μmol) was added and stirred at 80 °C for 1 h. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (20 mL * 3). After the combined organic layers were washed with brine (30 mL*3) and dried over Na2SO4, the reaction mixture was concentrated in vacuo to give C12 (crude 358 mg) as a yellow solid.

[0111] General procedure for the synthesis of compound 2 To a solution of C12 (350 mg, 690.98 μmol) in ethanol (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 Prep-HPLC (Column: Waters Xbridge 150*25 5u, Table: 63-93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), Flow rate: 25 mL / min, UV detector 220 nm) to give compound 2 (12.7 mg, Yield: 4.5%) as a yellow solid.

[0112] [ka]

[0113] General procedure for the synthesis of D2 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 min. TLC (PE: EtOAc = 3:1) confirmed that C10 (Rf = 0.3) was completely consumed.

[0114] Half of the reaction solution was transferred to another bottle, and a solution of D1 (47 mg, 184 umol) dissolved in CH3CN (0.5 mL) was added, followed by the addition of t-BuOK (20.73 mg, 185 umol), which was then heated to 80°C and stirred for 1 hour.

[0115] 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 concentrated under reduced pressure to give D2 (59 mg, crude, yield 63.17%), which was used directly in the next step.

[0116] General procedure for the synthesis of compound 3 To a solution of D2 (59 mg, 116.71 μmol) in ethanol (0.5 mL) was added 37% HCl (0.05 mL). The reaction mixture was stirred at 80 °C for 1 h. The reaction solution was concentrated and purified by Prep-HPLC (Column: Waters Xbridge 150*25 5u, Table: 63-93%B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), Flow rate: 25 mL / min, UV detector 220 nm) to give compound 3 (22.3 mg, Yield: 44.77%, Purity: 95%).

[0117] [ka]

[0118] General procedure for the synthesis of E2 To a solution of E1 (1 g, 6.71 mmol) in DMF (15 mL) was added K2CO3 (2 g, 14.47 mmol) and tert-butyl 2-cyanoacetate (1 g, 7.08 mmol) under a nitrogen atmosphere. The mixture was stirred at 25 °C for 16 h. The mixture was quenched with water (30 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give crude E2 (2 g, 85% purity) as a brown oil, which was used directly in the next step without further purification.

[0119] General procedure for the synthesis of E3 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 min. The mixture was quenched with saturated NaHCO to pH 8 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by silica gel flash column chromatography (30% ethyl acetate in PE) to give E3 (350 mg, 34.01% yield) as a yellow oil.

[0120] General procedure for the synthesis of E4 To a solution of E3 (350 mg, 2.28 mmol) and (3,4-dimethoxyphenyl)boronic acid (414.75 mg, 2.28 mmol) in dioxane (5 mL) and water (1 mL) was added Pd(dppf)Cl2 (166.76 mg, 227.91 mmol) and Na2CO3 (483.12 mg, 4.56 mmol). The mixture was stirred at 100 °C under a nitrogen atmosphere for 2 h. The mixture was filtered. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0–50% EtOAc in petroleum ether) to give E4 (425 mg, 73% yield) as a yellow solid.

[0121] General procedure for the synthesis of E5 To a solution of C10 (100 mg, 371.38 μmol) in CH3CN (2 mL), CDI (60.2 mg, 371.38 μmol) was added and stirred at 20 °C for 12 h. To another solution of E4 (47.1 mg, 184.76 μmol) in CH3CN (1 mL), t-BuOK (20.7 mg, 184.76 μmol) and half of the first reaction mixture were added, and the mixture was stirred at 80 °C for 1 h. The pH of the reaction mixture was adjusted to 4 with HCl (1 mol / L), poured into water (10 mL), and extracted with ethyl acetate (10 mL × 3). The combined organic layer was washed with brine (10 mL × 3), dried over Na2SO4, filtered, and concentrated in vacuo to give E5 (crude 93 mg) as a yellow solid.

[0122] General procedure for the synthesis of compound 4 To a solution of E5 (93 mg, 183.60 μmol) in ethanol (1 mL) was added HCl (0.1 mL). The reaction mixture was stirred at 80 °C for 1 h. The mixture was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm, column: 43%-53% B (A = water (TFA), B = acetonitrile), flow rate: 25 mL / min, UV detector 220 nm) and (column: Waters Xbridge 150*25 5 μm, column: 63-93% B (A = water (0.05% ammonium hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector 220 nm) to give compound 4 (17.1 mg, purity 95%, yield 22.9%) as a yellow solid.

[0123] [ka]

[0124] General procedure for the synthesis of F2 To a solution of F1 (2 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 mixture was diluted with water (10 mL), adjusted to pH 6 with HCl (1 M), and 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 to extract 2 g of F1. The two batches were then purified by column chromatography (SiO2, DCM:MeOH = 10:1) to give F2 (1.28 g, 6.87% yield, 78% purity) as a yellow solid and F2 (1.07 g, 18.54% yield, 59% purity) as a yellow solid.

[0125] General procedure for the synthesis of F3 To a solution of F2 (300 mg, 1.18 mmol, 78% purity) dissolved in DMSO (2 mL) and HO (2 mL) was added NaCl (138 mg, 2.37 mmol). The mixture was stirred at 130 °C for 1 h. The reaction mixture was diluted with HO (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. Another parallel reaction was carried out on 1.25 g of compound 2, and two batch reactions were carried out together. The combined residue was purified by column chromatography (SiO2, DCM:MeOH = 10:1) to give F3 (287.6 mg, 48.46% yield) as a yellow solid.

[0126] General procedure for the synthesis of F4 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) and Pd(dppf)Cl2 (23.82 mg, 0.03 mmol). The mixture was heated in a microwave at 120 °C for 30 min. The reaction mixture was combined with additional 100 mg parallel reaction batches for workup. 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 (72 mg, 35.52% yield, 82% purity) as a yellow oil.

[0127] General procedure for the synthesis of F5 Solution A: CDI (48.17 mg, 0.29 mmol) was added to a solution of C10 (80 mg, 0.29 mmol) in CH3CN (0.3 mL), and the mixture was stirred at 25 °C for 5 min.

[0128] 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 stirred at 80 °C for 3 min.

[0129] Mixture A was added dropwise to mixture B at 80° C., and the resulting mixture was stirred at 80° C. for 15 minutes. The reaction mixture was quenched with HO (5 mL) and extracted with EtOAc (5 mL × 3). The combined organic layers were dried over anhydrous NaSO, filtered, and then concentrated under reduced pressure to give crude F5 (98 mg, crude) in the form of a yellow oil.

[0130] General procedure for the synthesis of compound 5 To a solution of F5 (98 mg, 0.19 mmol) in ethanol (1 mL) was added 37% HCl (0.2 mL). The mixture was stirred at 90 °C for 1 h. After concentrating the reaction mixture, the crude product was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 22%-42%, 12 min) to obtain compound 5 (4.8 mg, two-step yield 4.3%, purity 99.34%) as a white solid.

[0131] General procedure for the synthesis of compound 57 Compound 5 (370 mg, 910.41 μmol in another batch) was suspended in HBr (5 mL, 40% purity) and then heated at 120° C. for 10 h. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; B%: 12%-42%, 12 min) to give compound 57 (84.6 mg, 24% yield, 98% purity) as a gray solid.

[0132] [ka]

[0133] General procedure for the synthesis of E7 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) 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 NaSO, and concentrated under reduced pressure. The filtrate was concentrated to give the crude product, which was purified by silica gel flash chromatography (0–30% EtOAc in petroleum ether) to give E7 (1.4 g, 33% yield, crude) as a yellow oil.

[0134] General procedure for the synthesis of E8 To a solution of E7 (1.4 g, 6.55 mmol) in CHCl (10 mL), BocO (2.86 g, 13.10 mmol) and DMAP (1.20 g, 9.83 mmol) were added at 0 °C for 20 min, followed by the addition of BocO (4.29 g, 19.66 mmol) 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 layer was washed with brine (30 mL × 3) and dried over NaSO. The organic layer was concentrated in vacuo. The residue was purified by silica gel flash column chromatography (0–10% EtOAc in PE). The crude product was then purified by Prep-HPLC (column: Waters Xbridge 150*25 5u, column: 63-93%B (A = water (0.05% ammonia hydroxide v / v)), B = acetonitrile), flow rate: 25 mL / min, UV detector 220 nm) to give E8 (217 mg, 10.34% yield) as a colorless oil.

[0135] General procedure for the synthesis of E9 To a solution of E8 (214 mg, 682.02 μmol) in THF (2 mL) and HO (1 mL) was added LiOH.HO (57.24 mg, 1.36 mmol). 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 NaSO, and concentrated under reduced pressure to give E9 (170 mg, 85.49% yield) as a yellow oil.

[0136] General procedure for the synthesis of E11 To a solution of E9 (80 mg, 279.99 μmol) in CH3CN (2 mL), CDI (54.48 mg, 335.99 μmol) was added 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 in the next step.

[0137] To a solution of E4 (71.46 mg, 279.94 μmol) in CH3CN (3 mL), t-BuOK (47.12 mg, 419.91 μmol) was added and stirred at 20 °C for 10 min. Then, the reaction mixture of E10 was added and the reaction was stirred at 80 °C for 1 h. The reaction mixture was adjusted to pH = 4 with HCl (1 mol / L), poured into water (5 mL), and extracted with EtOAc (10 mL × 3). The combined organic layer was washed with brine (10 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give E11 (189 mg, crude) as a yellow solid.

[0138] General procedure for the synthesis of compound 6 To a solution of E11 (189 mg, 361.39 μmol) in ethanol (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 Prep-HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 41%-71%, 10 min) to give compound 6 (2.6 mg, yield 1.67%, purity 100%) as a white solid.

[0139] [ka]

[0140] General procedure for the synthesis of G2 To a solution of (3,4-dimethoxyphenyl)boronic acid (930 mg, 5.11 mmol) in dioxane (10 mL) and water (2 mL), G1 (1.01 g, 5.11 mmol), Na2CO3 (1.08 g, 10.22 mmol), and Pd(PPh3)4 (590.54 mg, 0.51 mmol) were added and stirred at 80 °C for 2 h. The reaction mixture was diluted with water (300 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 G2 (720 mg, 38.23% yield, 69% purity) as a yellow solid.

[0141] General procedure for the synthesis of G3 A: To a solution of E9 (350 mg, 1.22 mmol) in CHCN (3 mL) was added CDI (198.63 mg, 1.22 mmol). The mixture was stirred at 25 °C for 2 h to give E10 as a yellow solution in CHCN.

[0142] B: To a solution of G2 (300 mg, 1.18 mmol) in CH3CN (3 mL) was added t-BuOK (264.77 mg, 2.36 mmol) and E10 (396.15 mg, 1.18 mmol). The mixture was stirred at 80 °C for 1 h. The reaction mixture was combined with another batch of the reaction mixture (2 was added in 100 mg portions) and worked up together. The combined reaction mixture was diluted with HO (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give G3 (779 mg, crude) as a brown solid.

[0143] General procedure for the synthesis of compound 7 To a solution of G3 (670 mg, 1.28 mmol) in ethanol (7 mL) was added 37% HCl (1 mL). The mixture was stirred at 90 °C for 1 h. The reaction mixture was combined with another batch of the reaction mixture (extracted with 100 mg of 3), and the two batches of reaction mixtures were combined and worked up together. The reaction mixture was concentrated under reduced pressure to remove the solvent, and then purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 7%-37%, 12 min) to give compound 7 (237 mg, yield 43.33%, purity 99.74%) as a white solid.

[0144] [ka]

[0145] General procedure for the synthesis of E13 Ethyl 2-bromoacetate (5 g, 29.94 mmol) was added to a solution of E12 (4.38 g, 59.88 mmol) in CHCl (50 mL), and the mixture was stirred for 12 h at 25° C. The mixture was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E13 (3.02 g, 63.35% yield) in the form of a yellow oil.

[0146] General procedure for the synthesis of E14 To a solution of E13 (2.92 g, 18.34 mmol) in CHCl (30 mL) was added EtN (2.78 g, 27.51 mmol) and BocO (4.00 g, 18.34 mmol) at 0 °C. The mixture was stirred at 25 °C for 12 h. The mixture was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E14 (3.73 g, 78.43% yield) as a yellow oil.

[0147] General procedure for the synthesis of E15 To a solution of E14 (2 g, 7.71 mmol) in THF (20 mL) and HO (4 mL) was added LiOH.HO (1.62 g, 38.56 mmol), and the mixture was stirred at 60 °C for 2 h. The reaction mixture was diluted with HO (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 NaSO, filtered, and concentrated under reduced pressure to give E15 (1.5 g, 84.10% yield) as a yellow oil.

[0148] General procedure for the synthesis of E16 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 (170 mg, 92.62% yield, 100% purity) as a white solid.

[0149] General procedure for the synthesis of compound 8 To a solution of E16 (170 mg, 0.36 mmol) in ethanol (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 NaHCO to pH = 8 and extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150 * 30 mm * 5 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3) - ACN]; B%: 37% - 67%, 10 min) and then lyophilized to give compound 8 (61.3 mg, 46% yield) as a white solid.

[0150] [ka]

[0151] General procedure for the synthesis of compound 9 HCl salt A solution of compound 6 (3 g, 7.09 mmol) in HBr (20 mL, 40% purity) was heated at 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 Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (HCl)-ACN]; B%: 23%-43%, 10 min) to give compound 9_HCl salt (1.64 g, 58.85% yield, 100% purity, HCl) as a yellow solid.

[0152] General procedure for the synthesis of compound 9 - free form To a solution of 9-HCl salt (50 mg, 126.64 μmol) in HO (5 mL) was added NaCO (26.85 mg, 253.29 μmol). The mixture was stirred at 20 °C for 10 h. The mixture was filtered and washed with HO (10 mL). The filter cake was collected and lyophilized to give compound 9-free form (39 mg, 78.00% yield, 99% purity) as a yellow solid.

[0153] [ka]

[0154] General procedure for the synthesis of E17 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), followed by 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, concentrated, and then filtered and concentrated under reduced pressure to give E17 (1.54 g, crude), which was used in the next step without further purification.

[0155] General procedure for the synthesis of E18 To a solution of E17 (1 g, 2.37 mmol) in ethanol (10 mL) was added HCl (2.5 mL, 37% purity), and the reaction was heated at 90 °C for 1 h. The solvent was removed in vacuo, and the residue was basified with aqueous NaHCO to pH 8, and then the mixture was extracted with CHCl (30 mL × 3). The organic layer was washed with brine (30 mL), dried over sodium sulfate, and concentrated. The residue was triturated with PE / EtOAc (5 / 1, 10 mL) solution and filtered. The solid was dried in vacuo to give E18 (300 mg, 39% yield) as a black solid.

[0156] General procedure for the synthesis of compound 10 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 water (0.2 mL) was added Pd(dppf)Cl2·CHCl2 (12.71 mg, 15.57 μmol) and NaCO3 (33 mg, 311.37 μmol). The reaction was degassed and flushed with nitrogen (N2) three times before heating at 100 °C 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 prep-TLC (100% EtOAc) to give 30 mg of crude product. The crude product was purified by prep-HPLC [column: O-Phenomenex C18 150*10mm*5um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 33%-63%, 10 min] to give a white solid, compound 10 (4.3 mg, yield 6.3%, purity 93%).

[0157] [ka]

[0158] General procedure for the synthesis of E20 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), followed by stirring 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 NaSO, and then concentrated. The residue was purified by Combi Flash (SiO, 50%–60% EtOAc in PE) to give E20 (460 mg, 67.36% yield) as a colorless oil.

[0159] General procedure for the synthesis of E21 To a solution of E20 (460 mg, 2.48 mmol) in CHCl (10 mL) were added EtN (502.50 mg, 4.97 mmol) and BocO (812.86 mg, 3.72 mmol), and the mixture was stirred at 25 °C for 2 h. The mixture was diluted with CHCl (50 mL) and washed with 1 N HCl (30 mL). The organic layer was dried over NaSO, filtered, and concentrated to give E21 (700 mg, crude) in the form of a colorless oil.

[0160] General procedure for E22 synthesis To a solution of E21 (700 mg, 2.45 mmol) in THF (10 mL) was added a solution of LiOH·HO (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, and the mixture was treated with 1 N HCl to adjust the pH to 2, followed by extraction with EtOAc (20 mL × 2). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated to give E22 (450 mg, 71.29% yield) as a colorless oil.

[0161] General procedure for the synthesis of E23 To a solution of E22 (110.88 mg, 0.43 mmol) in MeCN (2 mL) was added CDI (69.87 mg, 0.43 mmol) at 20 °C and stirred for 30 min. To another solution of E4 (100 mg, 0.39 mmol) in MeCN (2 mL) was added t-BuOK (43.96 mg, 0.39 mmol) at 20 °C, followed by heating to 80 °C and the addition of the first solution. The reaction mixture was stirred at 80 °C for 0.5 h. The reaction mixture was diluted with water (10 mL), acidified to pH = 5 with 1 N 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 product) as a yellow solid.

[0162] General procedure for the synthesis of compound 15 To a solution of E23 (190 mg, 0.38 mmol) in EtOH (2 mL) was added HCl (0.3 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 40%-70%, 14 min) to produce compound 15 (90 mg, 12.93% yield, 98.11% purity) as a yellow solid.

[0163] General procedure for the synthesis of compound 18 A solution of compound 15 (35 mg, 0.09 mmol) dissolved in HBr (4 mL, 40% purity) was stirred at 120 °C for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (HCl)-ACN]; B%: 18%-38%, 10 min) to give compound 18 (5.3 mg, yield 15.00%, purity 92.78%) as a yellow solid.

[0164] [ka]

[0165] General procedure for E25 synthesis 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 mmol), and Xantphos (83.82 mg, 144.87 mmol) in dioxane (5 mL) was added Cs2CO3 (1.42 g, 4.35 mmol). The mixture was degassed and refilled with nitrogen gas three times. The reaction was heated at 110 °C 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 (340 mg, 33.85% yield) as a yellow solid.

[0166] General procedure for the synthesis of E26 A solution of E25 (310 mg, 941.25 μmol) in THF (0.5 mL) was added to a suspension of LiAlH (53.59 mg, 1.41 mmol) in THF (5 mL) at −10°C for 0.5 h. The reaction was quenched by adding one drop of water and one drop of 15% NaOH solution, diluted with a DCM / methanol (5 / 1, 20 mL) mixture, and filtered. The filtrate was concentrated to give E26 (450 mg, 95.19% yield, 60% purity) as a yellow solid.

[0167] General procedure for the synthesis of E27 To a solution of E26 (50 mg, 165.93 umol) in DCM (1 mL) was added SOCl2 (98.70 mg, 829.63 umol), and the reaction mixture was stirred for 1 h at 20 °C. The reaction mixture was concentrated under reduced pressure to give E27 (51 mg, crude) as a brown solid.

[0168] General procedure for the synthesis of E28 To a solution of E27 (51 mg, 159.48 μmol) and TMSCN (31.64 mg, 318.96 μmol) in THF (1 mL) was added TBAF (1 M, 318.96 μL), 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 in ethyl acetate 56%) to give E28 (23 mg, 46.47% yield) as a yellow solid.

[0169] General procedure for the synthesis of E29 To a solution of E9 (23.29 mg, 81.52 umol) in CH3CN (1 mL) was added CDI (13.22 mg, 81.52 umol) and the reaction was stirred at 20°C for 10 min.

[0170] To another solution of E28 (23 mg, 74.11 μmol) dissolved in CH3CN (1 mL) was added t-BuOK (9.15 mg, 81.52 μmol), and the reaction mixture was heated at 80 °C, followed by the addition of the first solution. The reaction mixture was stirred at 80 °C for 15 min. The reaction mixture was adjusted to pH = 4 using HCl (1 mol / L), 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 concentrated under reduced pressure to give E29 (44 mg, crude) as a brown solid.

[0171] General synthesis of compound 16 To a solution of E29 (40 mg, 69.20 μmol) in ethanol (5 mL) was added HCl (0.5 mL, purity 12 mol / L). The reaction mixture was stirred at 80 °C for 1 h. The crude product was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water (HCl)-ACN]; B%: 30%-50%, 10 min) to give compound 16 (4.9 mg, yield 13.49%, purity 100%, HCl) as a yellow solid.

[0172] [ka]

[0173] General procedure for the synthesis of compound 20 To a solution of E18 (100 mg, 0.31 mmol) in dioxane (2 mL) and HO (0.5 mL), (2,4-dimethoxyphenyl)boronic acid (56.66 mg, 0.31 mmol), Pd(dppf)Cl (22.78 mg, 0.03 mmol), and NaCO (66.00 mg, 0.62 mmol) were added, and the mixture was stirred at 100 °C for 1 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30mm*5um; mobile phase: [water (HCl)-ACN]; B%: 36%-56%, 10 min) to give a yellow solid, compound 20 (50 mg, yield 34.96%, purity 100%, HCl).

[0174] [ka]

[0175] General procedure for the synthesis of compound 21 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 dioxane (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 h. LC-MS analysis showed that E30 was completely consumed and E31 was produced. To the reaction mixture was 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 HO (0.4 mL). The resulting mixture was degassed and refilled with N2 three times and then stirred at 100 °C for 1 h. LC-MS analysis indicated the complete consumption of compound 2 and the formation of compound 21. The reaction mixture was diluted with HO (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 Green ODS 150*30mm*5um; mobile phase: [water (HCl)-ACN]; B%: 50%-70%, 10 min) to give a yellow solid, compound 21 (14 mg, purity 98%, HCl salt, yield 13%).

[0176] General procedure for the synthesis of compound 38 A solution of compound 21 (20 mg, 0.05 mmol) 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 batch (10 mg of compound 21) was run in parallel, and the crude products of the two batches were combined and purified together. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water (HCl)-ACN]; B%: 15%-36%, 10 min) to give compound 38 (3.8 mg, 19.33% yield, 95.73% purity) as a white solid.

[0177] [ka]

[0178] General procedure for the synthesis of E33 To a solution of E32 (2 g, 10.69 mmol), BocO (11.67 g, 53.47 mmol) in DCM (30 mL) was added NaOH (13.4 mL, 2N HO), 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 NaSO, 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 (3.34 g, 81% yield) as a brown solid.

[0179] General procedure for the synthesis of E34 To a solution of CsCO (2.52 g, 7.75 mmol) in DMF (10 mL) was added E33 (1 g, 2.58 mmol) at 0 °C, followed by CHI (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 NaSO, and concentrated under reduced pressure to give E34 (1.27 g, crude, 84% purity) as a brown solid.

[0180] General procedure for the synthesis of E36 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)Cl (17.62 mg, 24.08 μmol) and KOAc (70.89 mg, 722.33 μmol). The mixture was stirred at 100° C. for 2 hours. LC-MS showed the formation of E35.

[0181] To the reaction mixture was added E18 (84.81 mg, 264.07 μmol), Na2CO3 (76.33 mg, 720.18 μmol), Pd(dppf)Cl2 (17.57 mg, 24.01 μmol), and HO (0.6 mL). The resulting mixture was evacuated and refilled with nitrogen (N2) three times, then stirred at 100 °C for 1 h. LC-MS analysis showed the formation of E36. The reaction mixture was diluted with HO (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 E36 (220 mg, crude) as a brown solid.

[0182] General procedure for the synthesis of compound 23 To a solution of E36 (220 mg, 354.20 μmol) in EtOH (3 mL) was added HCl (0.3 mL, 12 M / L). The reaction 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 purified by Prep-HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (FA)-ACN]; B%: 17%-47%, 12 min) to give compound 23 (4.9 mg, three-step yield 4.8%, purity 100%) as a yellow solid.

[0183] [ka]

[0184] General procedure for the synthesis of E37 To a solution of E24 (3 g, 17.38 mmol) in dioxane (30 mL), 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) were added, and the mixture was stirred at 110 °C for 1 h. The reaction mixture was diluted with HO (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 (1.84 g, 28.57% yield, 87% purity) as a brown solid.

[0185] General procedure for the synthesis of E38 A solution of E37 (840 mg, 2.61 mmol) in THF (10 mL) was added dropwise to a solution of LiAlH (197.80 mg, 5.21 mmol) in THF (15 mL) at −10° C. The resulting mixture was stirred at −10° C. for 30 min. The mixture was quenched with water (0.2 mL), 15% aqueous NaOH (0.2 mL), and water (0.6 mL). The mixture was diluted with CHCl / MeOH (10 / 1, 100 mL), and then NaSO was added until the aluminum salts were adsorbed. The mixture was filtered and concentrated under reduced pressure to give a residue.

[0186] The reaction mixture was combined with other crude products (1 g of E37 was distilled), and the combined crude products were purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give a yellow solid, E38 (365 mg, 21% yield, 90% purity).

[0187] General procedure for the synthesis of E39 To a solution of E38 (310 mg, 1.05 mmol) in CHCl (3.5 mL) was added SOCl (250.59 mg, 2.11 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove CHCl. ​​The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E39 (78 mg, 23.68% yield) as a yellow oil.

[0188] General procedure for the synthesis of E40 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 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E40 (52 mg, 56.37% yield, 82% purity) as a yellow oil.

[0189] General procedure for the synthesis of E41 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 min. To a 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 min. Then, the first solution was added dropwise at 80 °C, and the resulting mixture was stirred at 80 °C for 15 min. The reaction mixture was diluted with water (10 mL), and the pH was adjusted to 4 with 1 N 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 prep-HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (NH3H2O ​​+ NH3HCO4)-ACN]; B%: 26%-56%, 10 min) to obtain E41 (20 mg, yield 20.23%, purity 99%) in the form of a yellow solid.

[0190] General procedure for the synthesis of E42 To a solution of E41 (20 mg, 0.04 mmol) in ethanol (1 mL) was added 37% HCl (0.1 mL). The mixture was stirred at 80° C. for 1 h. The reaction mixture was concentrated under reduced pressure to remove the solvent and give E42 (13.6 mg, crude) as a yellow oil.

[0191] General procedure for the synthesis of compound 25 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 h. The reaction mixture was purified by preparative HPLC (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 30%-60%, 10 min) to give compound 25 (3.3 mg, 19.41% yield, 99% purity) as a yellow solid.

[0192] [ka]

[0193] General procedure for the synthesis of E43 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) was added Pd(dppf)Cl (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 was added E3 (81.10 mg, 528.13 μmol), HO (0.6 mL), Pd(dppf)Cl (35.13 mg, 48.01 μmol), and NaCO (152.66 mg, 1.44 mmol). The mixture was stirred at 100° C. for 1 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL×3), dried over anhydrous NaSO, filtered, and then concentrated under reduced pressure to give E43 (244.2 mg, crude, 52% purity) as a brown solid.

[0194] General procedure for the synthesis of E44 1) To a solution of E15 (136.88 mg, 591.80 umol) dissolved in CH3CN (2 mL) was added CDI (95.96 mg, 591.80 umol) and the reaction was stirred at 20°C for 10 minutes.

[0195] 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), and the reaction was heated at 80 °C before the first solution was added. The reaction mixture was adjusted to pH = 4 using 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 E44 (454 mg, crude, 74% purity) as a brown solid.

[0196] General procedure for the synthesis of compounds 26 and 27 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 h. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (FA)-ACN]; B%: 14%-34%, 14 min) to give compound 26 (31.9 mg, purity 96.64%) as a yellow solid and compound 27 (9.0 mg, purity 100%) as a yellow solid.

[0197] [ka]

[0198] General procedure for the synthesis of compound 28 To a solution 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) dissolved in dioxane (3 mL) was added HO (0.6 mL), Pd(dppf)Cl (29.26 mg, 39.98 μmol), and NaCO (127.14 mg, 1.20 mmol).

[0199] The mixture was stirred at 100°C for 1 hour. The reaction mixture was diluted with HO (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 NaSO, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [water (NHHO + NHHCO)-ACN]; B%: 26%-56%, 10 min) to give compound 28 (45.4 mg, yield 26.38%, purity 100%) as a yellow solid.

[0200] [ka]

[0201] General procedure for the synthesis of compound 29 To a solution 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) in dioxane (1 mL) was added HO (0.2 mL), Pd(dppf)Cl (14.63 mg, 19.99 μmol), and NaCO (63.57 mg, 599.77 μmol). The mixture was stirred at 100 °C for 1 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 29%-59%, 10 min) to give compound 29 (17.2 mg, 21.04% yield, 100% purity) as a yellow solid.

[0202] [ka]

[0203] General procedure for the synthesis of compound 31 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 dioxane (5 mL) was added Pd(dppf)Cl (142.35 mg, 0.19 mmol) and KOAc (381.87 mg, 3.89 mmol). The mixture was stirred at 100 °C for 1 h. One-third of the above reaction solution was transferred to another flask, and E18 (187.44 mg, 0.58 mmol), Na2CO3 (144.34 mg, 1.36 mmol), Pd(dppf)Cl2 (42.71 mg, 0.06 mmol), and HO (1 mL) were added at 25 °C. The mixture was stirred at 100 °C for 1 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layer was 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 prep-HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 38%-68%, 10 min) to give compound 31 (3.6 mg, 1.3% yield, 96.6% purity) as a white solid.

[0204] [ka]

[0205] General procedure for the synthesis of E48 To a solution of E47 (500 mg, 3.46 mmol) and (3,4-dimethoxyphenyl)boronic acid (692.38 mg, 3.80 mmol) in dioxane (5 mL) and HO (1 mL) was added Pd(dppf)Cl (253.08 mg, 345.88 mmol) and NaCO (1.10 g, 10.38 mmol). The mixture was stirred at 100 °C under a N atmosphere for 1 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give E48 (1.01 g, crude product) as a brown solid.

[0206] General procedure for the synthesis of E49 To a solution of E48 (1.01 g, 4.10 mmol, crude product) in CHCl (10 mL) was added SOCl (595.04 μL, 8.20 mmol) at −10° C. The mixture was stirred at 0° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give E49 (786 mg, 72% yield) as a yellow solid.

[0207] General procedure for the synthesis of E50 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 h. 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 E50 (630 mg, 58% yield, 70% purity) as a yellow solid.

[0208] General procedure for the synthesis of E51 1) To a mixture of E9 (246.24 mg, 861.83 umol) in CH3CN (2 mL) was added CDI (139.75 mg, 861.83 umol) and stirred at 20°C for 10 minutes.

[0209] 2) To a solution of E50 (200 mg, 783.48 μmol) in CH3CN (3 mL) was added t-BuOK (96.71 mg, 861.83 μmol), and the reaction was heated at 80 °C before the first mixture was added. The reaction was stirred at 80 °C for 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 concentrated under reduced pressure to give E51 (551 mg, crude product, 60% purity) as a brown solid.

[0210] General procedure for the synthesis of E52 To a mixture of E51 (551 mg, 1.05 mmol) 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 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 NaSO, and then concentrated under reduced pressure to give E52 (297 mg, 66% yield) as a yellow solid.

[0211] General procedure for the synthesis of compound 35 E52 (297 mg, 702.35 μmol) was suspended in HBr (5 mL, 40% purity) and the suspension was heated at 120° C. for 4 h. The crude product was purified by Prep-HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: [water (FA)-ACN]; B%: 20%-50%, 14 min) to give compound 35 (50 mg, 18% yield, 98% purity) as a yellow solid.

[0212] [ka]

[0213] General procedure for the synthesis of E54 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 HO (30 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. Another batch obtained from 100 mg of E53 was combined for purification. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E54 (256 mg, crude product) as a yellow oil.

[0214] General procedure for the synthesis of E55 To a solution of E54 (256 mg, 0.83 mmol, crude) in THF (2.5 mL) and HO (0.5 mL) was added LiOH·HO (173.63 mg, 4.14 mmol), and the mixture was stirred at 60 °C for 12 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (20 mL). The aqueous layer 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 NaSO, filtered, and concentrated under reduced pressure to give E55 (147 mg, crude) as a yellow oil.

[0215] General procedure for the synthesis of E56 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.

[0216] B: To a solution of E4 (120 mg, 0.47 mmol) in CHCN (1 mL) was added t-BuOK (52.75 mg, 0.47 mmol), and the mixture was stirred at 80 °C for 2 min, followed by the dropwise addition of the mixture from Step A 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 NaSO, filtered, and concentrated to give E56 (220 mg, crude) as a yellow solid.

[0217] General procedure for the synthesis of compound 37 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 prep-HPLC (column: Boston Prime C18 150*30 mm*5 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; B%: 36%-66%, 10 min) to give compound 37 (17.9 mg, 10% yield, 98% purity) as a yellow solid.

[0218] [ka]

[0219] General procedure for the synthesis of E58 To a mixture of CsCO (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 stirred at 20 °C for 2 h. The mixture was quenched with saturated NHCl (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 NaSO, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 3:1) to give E58 (3.99 g, 60% yield) as a yellow oil.

[0220] General procedure for the synthesis of E59 To a mixture of E58 (1 g, 5.46 mmol) in MeOH (12 mL) and HO (4 mL) was 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 HO (20 mL). The filtrate 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 in vacuo to give E59 (712 mg, 85% yield) as a brown oil.

[0221] General procedure for the synthesis of E60 1) To a solution of E59 (510 mg, 3.33 mmol) in BocO (2.18 g, 9.99 mmol), KCO (920.30 mg, 6.66 mmol) was added, and the mixture was stirred at 100 °C for 2 h. The mixture was quenched with saturated NH Cl (20 mL), 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 Na SO , 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.

[0222] 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 h. 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 give E60 (208 mg, 66% yield) as a brown oil.

[0223] General procedure for the synthesis of E61 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 the addition of 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 × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give E61 (465 mg, 69% yield, 80% purity) as a colorless oil.

[0224] General procedure for the synthesis of E62 To a solution of E61 (465 mg, 1.37 mmol) in THF (8 mL) and HO (6 mL) was added LiOH·HO (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) and adjusted to pH = 5 with 1 M HCl, followed by extraction 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 give E62 (283 mg, 66% yield) as a yellow oil.

[0225] General procedure for the synthesis of E63 1) To a mixture of E62 (90 mg, 289.08 umol) in CH3CN (2 mL) was added CDI (46.87 mg, 289.08 umol) and the reaction was stirred at 20 °C for 10 min.

[0226] 2) To a mixture of E4 (67.09 mg, 262.80 μmol) in CHCN (2 mL) was added t-BuOK (32.44 mg, 289.08 μmol), and the reaction was heated to 80 °C. The first reaction mixture was added and 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 NaSO, and concentrated under reduced pressure to give E63 (183 mg, 66% yield, 52% purity) as a brown solid.

[0227] General procedure for the synthesis of compound 42 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 Prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water (HCl)-ACN]; B%: 28%-43%, 10 min) to give compound 42 (36.7 mg, 25% yield, 100% purity, HCl salt) as a brick-colored solid.

[0228] [ka]

[0229] General procedure for E65 synthesis To a mixture of E64 (3 g, 15.87 mmol) in THF (10 mL) was added DIEA (8.21 g, 63.49 mmol, 11.1 mL) at 0 °C. After stirring at 0 °C for 10 min, bromo(methoxy)methane (4.36 g, 34.92 mmol) was added at 0 °C and 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 (3.67 g, 83% yield) as a yellow oil.

[0230] General procedure for the synthesis of E66 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) dissolved in dioxane (10 mL) was added Pd(dppf)Cl2 (475.29 mg, 649.56 mmol) and KOAc (1.91 g, 19.49 mmol). The mixture was stirred at 100 °C under a N2 atmosphere for 1 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 concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE: EtOAc = 3:1) to give E66 (1.68 g, 79% yield) in the form of a yellow oil.

[0231] General procedure for the synthesis of E67 To a mixture of E66 (500 mg, 1.54 mmol) and E3 (260.55 mg, 1.70 mmol) in dioxane (10 mL) and water (2 mL) was added Pd(dppf)Cl2 (112.86 mg, 154.24 mmol) and Na2CO3 (490.43 mg, 4.63 mmol). The mixture was stirred at 100 °C for 1 h. The reaction mixture was diluted with HO (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 (378 mg, 71% yield, 92% purity) as a brown oil.

[0232] General procedure for the synthesis of E68 1) To a mixture of E62 (80.78 mg, 259.47 umol) in CH3CN (2 mL) was added CDI (46.28 mg, 285.42 umol) and the reaction was stirred at 20 °C for 10 min.

[0233] 2) To a mixture of E67 (90 mg, 285.42 μmol) and CH3CN (2 mL) was added t-BuOK (32.03 mg, 285.42 μmol), and the reaction was heated to 80 °C before the first reaction mixture was added. The reaction was stirred at 80 °C for 15 min. The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1 M HCl, followed by extraction 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 E68 (190 mg, 75% yield, 63% purity) as a brown solid.

[0234] General procedure for the synthesis of compound 43 To a mixture of E68 (190 mg, 312.17 μmol) and 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. The crude product was purified by Prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; B%: 16%-36%, 10 min) to give compound 43 (25.5 mg, 19% yield, 96% purity, HCl salt) as a brick-colored solid.

[0235] [ka]

[0236] General procedure for the synthesis of C13 1) To a mixture of E22 (76.51 mg, 0.3 mmol) in CHCN (1 mL) was added CDI (48.21 mg, 0.3 mmol). The mixture was stirred at 25° C. for 3 minutes.

[0237] 2) To a mixture of C4 (69 mg, 0.27 mmol) in CH3CN (1 mL) was added t-BuOK (30.33 mg, 0.27 mmol). The mixture was stirred at 80 °C for 2 min. The first-stage 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 1 M HCl and extracted with EtOAc (10 mL × 3). The combined organic layer was washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give C13 (173 mg, crude product) as an orange solid.

[0238] General procedure for the synthesis of compound 56 To a solution of C13 (173 mg, 0.35 mmol) in EtOH (2 mL) was added concentrated HCl (0.5 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 compound 56 (120 mg, crude product) as a brown solid. 60 mg of the crude product was used in the next step without further purification. The remaining 60 mg of crude product was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 12%-42%, 12 min) to give compound 56 (18.4 mg, 13% yield, 99% purity) as a yellow solid.

[0239] General procedure for the synthesis of compound 65 A suspension of compound 56 (60 mg, 0.15 mmol) in 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 15%-45%, 12 min) to give compound 65 (15.8 mg, 28% yield, 98% purity) as a yellow solid.

[0240] [ka]

[0241] General procedure for the synthesis of E70 To a mixture of E69 (1 g, 6.17 mmol, 729.93 uL) in BocO (4.04 g, 18.52 mmol, 4.25 mL) was added KCO (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) and dried over anhydrous NaSO, then filtered and concentrated under reduced pressure. The residue was triturated with PE (30 mL) at 25 °C for 30 min to give E70 (1.71 g, 76% yield) as a white solid.

[0242] General procedure for the synthesis of E71 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 HO (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 (590 mg, 48% yield) as a yellow oil.

[0243] General procedure for the synthesis of E72 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 HO (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give E72 (914 mg, crude) as a yellow oil.

[0244] General procedure for the synthesis of E73 To a mixture of E72 (914 mg, 2.62 mmol) in THF (10 mL) was added LiOH.HO (220.29 mg, 5.25 mmol) and HO (2 mL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc (20 mL). The aqueous layer 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 NaSO, filtered, and concentrated under reduced pressure to give E73 (643 mg, crude product) as a yellow oil.

[0245] General procedure for the synthesis of E74 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.

[0246] 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-stage 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 1 M HCl and extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give E74 (307 mg, crude product) as an orange solid.

[0247] General procedure for the synthesis of compound 63 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) as a brown solid.

[0248] Part 1: 70 mg of crude product was used in the next step without further purification.

[0249] Part 2: The remaining 59 mg of crude product was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water (FA)-ACN]; B%: 37%-67%, 12 min) to give compound 63 (19.6 mg, 8% yield, 99% purity) as an orange-brown solid.

[0250] General procedure for the synthesis of compound 64 A mixture of compound 63 (70 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 27%-57%, 12 min) to give compound 64 (23 mg, 34% yield, 98% purity) as a yellow solid.

[0251] [ka]

[0252] General steps for E76 synthesis To a solution of E75 (5.10 g, 59.88 mmol) in CHCl (50 mL) was added ethyl 2-bromoacetate (5 g, 29.94 mmol). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E76 (3.33 g, 65% yield) as a yellow oil.

[0253] General steps for E77 synthesis To a solution of E76 (3.33 g, 19.45 mmol) in CHCl (30 mL) was added BocO (4.24 g, 19.45 mmol) and EtN (2.95 g, 29.17 mmol). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E77 (1 g, 19% yield).

[0254] General process for E78 synthesis To a solution of E77 (500 mg, 1.84 mmol) in THF (6 mL) was added a solution of LiOH·HO (115.98 mg, 2.76 mmol) in water (2 mL), and the mixture was stirred at 60 °C for 1 h. Water (10 mL) was added, and the mixture was treated with 1 N HCl to adjust the pH to 2, followed by extraction with EtOAc (20 mL × 2). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated to give E78 (440 mg, 98% yield) as a yellow solid.

[0255] General steps for E79 synthesis To a solution of E78 (104.84 mg, 430.91 μmol) in MeCN (2 mL) was added CDI (69.87 mg, 430.91 μmol) at 20 °C and stirred for 5 min. To a second solution of E4 (100 mg, 391.74 μmol) in MeCN (2 mL) was added t-BuOK (43.96 mg, 391.74 μmol) at 20 °C, followed by heating to 80 °C and the addition of the first solution. The reaction mixture was stirred at 80 °C for 0.5 h. The reaction mixture was diluted with water (10 mL), acidified to pH = 5 with 1 N 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 (200 mg, 62% yield, 58.5% purity) as a brown solid, which was used immediately without purification.

[0256] General synthesis of compound 14 To a solution of E79 (200 mg, 243.47 μmol, purity 58.5%) in EtOH (5 mL) was added HCl (0.5 mL, 37%). The reaction was heated at 80 °C for 1 h. The reaction mixture was concentrated. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (HCl)-ACN]; B%: 30%-60%, 10 min) to give compound 14 (60 mg, yield 63%, purity 100%) as a yellow solid.

[0257] General synthesis of compound 33 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 8%-38%, 12 min) to give compound 33 (5.7 mg, 31% yield, 100% purity) as a yellow solid.

[0258] [ka]

[0259] General procedure for the synthesis of E81 To a mixture of E80 (1 g, 8.22 mmol) dissolved in THF (10 mL) was added EtN (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 NH Cl (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 Na SO , and concentrated under reduced pressure to give E81 (654 mg, 26% yield, 56% purity) as a yellow oil.

[0260] General procedure for the synthesis of E82 To a mixture of E81 (630 mg, 3.68 mmol) dissolved in CHCl (10 mL) was added BocO (1.61 g, 7.36 mmol) and EtN (558.43 mg, 5.52 mmol, 768 μL) 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 NaSO, filtered, and concentrated under reduced pressure to give E82 (1.6 g, crude product) in the form of a brown oil.

[0261] General procedure for the synthesis of E83 To a mixture of E82 (1.6 g, 5.90 mmol) dissolved in THF (8 mL) and HO (6 mL) was added LiOH HO (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, followed by extraction 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 give E83 (829 mg, 57% yield) as a yellow oil.

[0262] General procedure for the synthesis of E84 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 reaction was stirred at 20 °C for 10 min.

[0263] 2) To a mixture of E4 (314.76 mg, 1.23 μmol) in CH3CN (3 mL) was added t-BuOK (152.20 mg, 1.36 μmol). The reaction was heated at 80 °C, and then the step-1 reaction mixture was added. The resulting reaction mixture was stirred at 80 °C for 15 min. The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1 M HCl, followed by extraction 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 E84 (534 mg, 63% yield, 71% purity) as a yellow solid.

[0264] General procedure for the synthesis of compound 66 To a mixture of E84 (534 mg, 1.11 mmol) 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. The residue was triturated with ethyl acetate (5 mL) to give compound 66 (598 mg, crude product) as a brown solid. 100 mg of the crude product was purified by Prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; B%: 26%-56%, 14 min) to give compound 66 (24.6 mg, 100% pure) as a yellow solid.

[0265] General procedure for the synthesis of compound 69 A suspension of compound 66 (498 mg, 1.31 mmol) in HBr (5 mL, 40% purity) was stirred at 120 °C for 4 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 16%-46%, 12 min) to give compound 69 (58.9 mg, 12% yield, 100% purity) as a gray solid.

[0266] [ka]

[0267] General procedure for E85 synthesis 1) To a mixture of E22 (1.66 g, 6.45 mmol) in CH3CN (10 mL) was added CDI (1.05 g, 6.45 mmol), and the mixture was stirred at 25 °C for 3 min.

[0268] 2) To a mixture of E3 (900 mg, 5.86 mmol) in CH3CN (5 mL) was added t-BuOK (657.61 mg, 5.86 mmol), and the mixture was stirred at 80 °C for 2 min. The first-stage 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 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 E85 (2.33 g, crude product) as a brown solid.

[0269] General synthesis process of E86 To a mixture of E85 (2.33 g, 5.93 mmol) in EtOH (7.5 mL) was added concentrated HCl (1.5 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 was triturated with EtOAc at 25 °C for 30 min to give E86 (1.3 g, 65% yield, 87% purity) as a yellow solid.

[0270] General steps for E88 synthesis To a mixture of DIEA (12.86 g, 99.49 mmol) and THF (30 mL), E87 (5 g, 24.87 mmol) was added and stirred at 0 °C for 10 min. Then, bromo(methoxy)methane (3.73 g, 29.85 mmol) was added at 0 °C. The mixture was stirred at 20 °C for 10 h. The mixture was quenched with saturated NH4Cl (20 mL), 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 E88 (6.9 g, 84% yield, 75% purity) as a yellow oil.

[0271] General steps for E89 synthesis To a mixture of E88 (2 g, 8.16 mmol) and CHCl (20 mL) was added m-CPBA (1.99 g, 9.79 mmol, 85% purity). The reaction was stirred at 20 °C for 16 h. NaOH (20 mL, 10% purity) was added 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, followed by extraction with EtOAc (40 mL × 3). The combined organic layers were washed with saturated NaHCO (40 mL × 3), dried over NaSO, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (PE:EtOAc = 5:1) to give E89 (1.14 g, 60% yield) as a yellow oil.

[0272] General process for E90 synthesis To a mixture of E89 (500 mg, 2.15 mmol) dissolved 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 HO (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 (365 mg, 59% yield) as a yellow oil.

[0273] General steps for E91 synthesis To a mixture of E90 (365 mg, 1.26 mmol) and dioxane (4 mL), 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)Cl (92.37 mg, 0.13 mmol), and KOAc (247.79 mg, 2.52 mmol) were added. The mixture was stirred at 100 °C under a nitrogen atmosphere for 2 h. The reaction mixture was diluted with HO (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=3 / 1) to give E91 (388 mg, crude product) in the form of a yellow oil.

[0274] General steps for E92 synthesis To a mixture of E86 (76 mg, 0.26 mmol) and E91 (100 mg, 0.30 mmol) in dioxane (5 mL) was added Pd(dppf)Cl2·CHCl2 (21.20 mg, 0.02 mmol), NaCO3 (55.03 mg, 0.52 mmol), and HO (0.1 mL). The mixture was stirred at 100 °C under a nitrogen atmosphere for 3 h. The mixture was diluted with ethyl acetate (15 mL) and filtered. The organic layer was washed with water (10 mL × 2), dried over NaSO4, filtered, and concentrated to give crude E92 (150 mg, 70% purity) as a brown gum.

[0275] General synthesis of compound 70 To a mixture of E92 (130 mg, 0.28 mmol) and CHCl (0.6 mL) was added TFA (2 mL). The mixture was stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was adjusted to pH 7-8 with saturated NaHCO and then extracted with DCM (10 mL × 3). The combined organic layer was washed with brine (10 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: C18-1 150*30 mm*5 um; mobile phase: [water (NHHCO)-ACN]; B%: 27%-47%, 13 min) to give 70 (6.1 mg, 5% yield, 97% purity) as a white solid.

[0276] [ka]

[0277] General procedure for the synthesis of compound 73 To a mixture of E86 (60 mg, 0.2 mmol) in dioxane (1 mL) and water (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 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 22%-52%, 12 min) to give compound 73 (0.9 mg, 1% yield, 100% purity) as a yellow solid.

[0278] [ka]

[0279] General procedure for the synthesis of F6 1) To a mixture of E22 (222.63 mg, 865.18 mmol) in CHCN (3 mL) was added CDI (140.29 mg, 865.18 mmol). The reaction was stirred at 20° C. for 10 minutes.

[0280] 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 at 80 °C, followed by the addition of the first-step reaction mixture. The reaction was stirred at 80 °C for 15 min. The reaction mixture was poured into water (10 mL) and adjusted to pH = 4 with 1 M HCl, followed by extraction 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 F6 (447 mg, 56% yield) as a brown solid.

[0281] General procedure for the synthesis of compound 77 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 h. 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 (478 mg, crude) as a brown solid. 378 mg of the crude product was used in the next step.

[0282] 100 mg of the crude product was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 18%-48%, 12 min) to give compound 77 (8.9 mg, 8.90% yield, 100% purity) as a yellow solid.

[0283] General procedure for the synthesis of compound 79 A suspension of compound 77 (378 mg, 958.26 μmol) in HBr (5 mL, 40% purity) was heated at 120 °C for 4 h. The reaction mixture was concentrated under reduced pressure. The crude product was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 8%-32%, 14 min) to give compound 79 (7.6 mg, 2% yield, 97% purity) as a yellow solid.

[0284] [ka]

[0285] General procedure for the synthesis of D3 1) To a mixture of E22 (111.32 mg, 0.4 mmol) in CHCN (1 mL) was added CDI (70.14 mg, 0.43 mmol). The mixture was stirred at 25° C. for 3 minutes.

[0286] 2) To a mixture of D1 (100 mg, 0.39 mmol) in CH3CN (1 mL) was added t-BuOK (44.13 mg, 0.39 mmol). The mixture was stirred at 80 °C for 2 min. The first-stage 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 1 M HCl and then extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give D3 (210 mg, crude product) as an orange solid.

[0287] General procedure for the synthesis of compound 80 To a mixture of D3 (210 mg, 0.44 mmol) and 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 to remove the solvent. The crude product was triturated with EtOAc (5 mL) at 25 °C for 30 min to give compound 80 (120 mg, crude product) as a brown solid. 90 mg of the crude product was used in the next step without further purification. 30 mg of the crude product was purified by prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 um; mobile phase: [water (FA)-ACN]; B%: 20%-50%, 12 min) to give compound 80 (12.5 mg, 100% pure) as a yellow solid.

[0288] General procedure for the synthesis of compound 81 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 15%-45%, 12 min) to give compound 81 (23.3 mg, 28% yield, 100% purity) as a yellow solid.

[0289] [ka]

[0290] General procedure for the synthesis of C15 1) To a mixture of C14 (154.45 mg, 0.43 mmol) and CH3CN (1 mL) was added CDI (69.87 mg, 0.43 mmol). The mixture was stirred at 25 °C for 3 min.

[0291] 2) To a mixture of C4 (100 mg, 0.39 mmol) and CH3CN (1 mL) was added t-BuOK (43.96 mg, 0.39 mmol). The mixture was stirred at 80 °C for 2 min. The first-stage reaction mixture was added dropwise at 80 °C. 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 (210 mg, crude) as an orange solid.

[0292] General procedure for the synthesis of compound 82 To a mixture of C15 (210 mg, 0.35 mmol) and 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 compound 82 (99 mg, crude) as a brown solid. 70 mg of the crude product was used in the next step without further purification. 29 mg of the crude product was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 5%-35%, 10 min) to give compound 82 (9 mg, 100% pure) as a yellow solid.

[0293] General procedure for the synthesis of compound 83 A suspension of compound 82 (70 mg, 0.17 mmol) dissolved 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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; B%: 0%-22%, 10 min) to give compound 83 (13.1 mg, 20% yield, 99% purity) as a white solid.

[0294] [ka]

[0295] General procedure for the synthesis of E93 To a mixture of E89 (500 mg, 2.15 mmol) in DMF (5 mL) was added 1-bromo-2-methoxyethane (298.19 mg, 2.15 mmol, 202 μL), CsCO (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 HO (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E93 (430 mg, 69% yield) as a yellow oil.

[0296] General procedure for the synthesis of E94 To a mixture of E93 (430 mg, 1.48 mmol) in dioxane (4 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (450.07 mg, 1.77 mmol), KOAc (434.85 mg, 4.43 mmol), and Pd(dppf)Cl (108.07 mg, 0.15 mmol). The mixture was stirred at 100 °C under a N atmosphere for 2 h. The reaction mixture E94 (499 mg, crude) was used in the next step without further purification.

[0297] General procedure for the synthesis of E95 To a solution of E94 (499 mg, 1.48 mmol) in dioxane (5 mL) and HO (1 mL) was added E86 (431.96 mg, 1.48 mmol), Pd(dppf)Cl (107.96 mg, 0.15 mmol), and NaCO (312.77 mg, 2.95 mmol). The mixture was stirred at 100 °C for 2 h under a N atmosphere. The mixture was quenched with HO (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. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1) to give E95 (170 mg, 22% yield, 90% purity) as a brown solid.

[0298] General procedure for the synthesis of compound 99 To a solution of E95 (170 mg, 0.36 mmol) in CHCl (0.5 mL) was added TFA (2 mL). The mixture was stirred at 25° C. for 1 hour. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; gradient: 30%-60% B over 10 min) to give compound 99 (50.1 mg, 33% yield, 100% purity) as a yellow solid.

[0299] [ka]

[0300] General procedure for the synthesis of E96 To a solution of E89 (500 mg, 2.15 mmol) in DMF (5 mL) was added K2CO3 (593.01 mg, 4.29 mmol) and 2-bromoethan-1-ol (268.10 mg, 2.15 mmol, 152 μL). The mixture was stirred at 100 °C for 2 h. The reaction mixture was diluted with HO (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 (263 mg, 44% yield) as a colorless oil.

[0301] General procedure for the synthesis of E97 To a solution of E96 (263 mg, 0.95 mmol) in dioxane (3 mL) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (289.21 mg, 1.14 mmol), Pd(dppf)Cl (69.44 mg, 0.09 mmol), and KOAc (279.43 mg, 2.85 mmol). The mixture was stirred at 100 °C under a N atmosphere for 2 h. The reaction mixture E97 (308 mg, crude) was used in the next step without further purification.

[0302] General procedure for the synthesis of E98 To a solution of E97 (308 mg, 0.95 mmol) in dioxane (3 mL) and HO (0.6 mL) was added E86 (278.15 mg, 0.95 mmol), NaCO (201.40 mg, 1.90 mmol), and Pd(dppf)Cl (69.52 mg, 0.09 mmol). The mixture was stirred at 100 °C under a N atmosphere for 2 h. The reaction mixture was diluted with HO (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E98 (180 mg, 20% yield, 49% purity) as a brown solid.

[0303] General procedure for the synthesis of compound 103 To a solution of E98 (180 mg, 0.39 mmol) in CHCl (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 prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 um; mobile phase: [water (FA)-ACN]; gradient: 14%-44% B, 12 min) to give compound 103 (8.8 mg, 5% yield, 96% purity) as a white solid.

[0304] [ka]

[0305] General procedure for the synthesis of E100 To a solution of E99 (500 mg, 3.17 mmol, HCl) in CHCl (10 mL) were added ethyl 2-bromoacetate (423.89 mg, 2.54 mmol, 280.72 μL) and EtN (385.26 mg, 3.81 mmol, 529.94 μL). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with H0 (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1) to give E100 (228 mg, 35% yield) as a yellow oil.

[0306] General procedure for the synthesis of E101 To a solution of E100 (228 mg, 1.10 mmol) in CHCl (3 mL) was added BocO (240.14 mg, 1.10 mmol, 252.78 μL) and EtN (167.01 mg, 1.65 mmol, 229.72 μL). 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 (SiO, petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give E101 (250 mg, 74% yield) as a colorless oil.

[0307] General procedure for the synthesis of E102 To a solution of E101 (250 mg, 0.81 mmol) in THF (3 mL) and HO (0.6 mL) was added LiOH·HO (170.68 mg, 4.07 mmol). The mixture was stirred at 25 °C for 12 h. The pH of the reaction mixture was adjusted to pH 4 with 1 N HCl and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated to give E102 (220 mg, crude) as a white solid.

[0308] General procedure for the synthesis of E103 1) To a mixture of E102 (219.21 mg, 0.78 mmol) and CH3CN (2 mL) was added CDI (127.27 mg, 0.78 mmol), and the mixture was stirred at 25 °C for 3 minutes.

[0309] 2) To a mixture of E67 (225 mg, 0.71 mmol) and CH3CN (2 mL), t-BuOK (80.07 mg, 0.71 mmol) was added, and the mixture was stirred at 80 °C for 2 min. The reaction mixture from step 1 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 then extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give a brown solid, E103 (390 mg, crude).

[0310] General procedure for the synthesis of compound 104 To a solution of E103 (390 mg, 0.68 mmol) in ethanol (4 mL) was added concentrated hydrochloric acid (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 prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; gradient: 35%-65% B, 12 min) to give compound 104 (19.5 mg, 7% yield, 99% purity) as a brown solid.

[0311] [ka]

[0312] General procedure for the synthesis of F7 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 HO (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 under a nitrogen atmosphere for 3 h. 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 Prep-HPLC (column: Boston Prime C18 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; gradient: 21%-51% B, 12 min) to give F7 (82 mg, 100% pure) as a yellow solid.

[0313] General procedure for the synthesis of F8 1) To a mixture of E22 (96.21 mg, 373.89 umol) in CH3CN (2 mL) was added CDI (60.63 mg, 373.89 umol) and the reaction was stirred at 20°C for 10 minutes.

[0314] 2) To a mixture of F7 (82 mg, 339.90 μmol) in CH3CN (2 mL), t-BuOK (41.95 mg, 373.89 μmol) was added and the reaction mixture was heated to 80 °C. The first-stage reaction mixture was then added and the reaction mixture was stirred at 80 °C for 15 min. The reaction mixture was poured into water (20 mL), adjusted to pH 4 with 1 M HCl, and extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (20 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give F8 (138 mg, 38% yield) as a brown solid.

[0315] General procedure for the synthesis of compound 105 To a mixture of F8 (138 mg, 287.17 μmol) and 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 Prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; gradient: 15%-45% B, 12 min) to give compound 105 (25.5 mg, 22% yield, 96% purity) as a yellow solid.

[0316] [ka]

[0317] General procedure for the synthesis of E104 To a mixture of E89 (300 mg, 1.29 mmol) dissolved in THF (10 mL), tert-butyl(2-hydroxyethyl)(methyl)carbamate (248.11 mg, 1.42 mmol) and PPh3 (506.43 mg, 1.93 mmol) were added, followed by DIAD (390.43 mg, 1.93 mmol) at 0 °C and stirring 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 silica gel flash column chromatography (0-30% EtOAc in PE) to give E104 (337 mg, 47% yield, 70% purity) as a colorless oil.

[0318] General procedure for the synthesis of E105 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) was 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 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 E105 (115 mg, 24% yield, 70% purity) as a brown oil.

[0319] General procedure for the synthesis of E106 To a mixture of E105 (115 mg, 262.96 μmol) and E86 (84.68 mg, 289.25 μmol) in dioxane (3 mL) was added Pd(dppf)Cl (19.24 mg, 26.30 μmol), NaCO (55.74 mg, 525.91 μmol), and HO (0.3 mL). The mixture was stirred at 100 °C for 1 h under a nitrogen atmosphere. The mixture was diluted with ethyl acetate (15 mL) and filtered. The filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by silica gel flash column chromatography (0–30% EtOAc in PE) to give E106 (39 mg, 18% yield, 70% purity) as a yellow oil.

[0320] General procedure for the synthesis of compound 107 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 Prep-HPLC (column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; gradient: 10%-40% B, 12 min) to give compound 107 (2.4 mg, 8% yield, 100% purity) as a yellow solid.

[0321] [ka]

[0322] General procedure for the synthesis of C16 To a solution of C1 (2.46 g, 16.52 mmol) in dioxane (30 mL) and HO (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), NaCO (2.69 g, 25.42 mmol), and Pd(dppf)Cl (929.85 mg, 1.27 mmol). The mixture was stirred at 100 °C for 2 h. The reaction mixture was diluted with HO (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic layers were washed with brine (50 mL × 3), dried over anhydrous NaSO, 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 a white solid, C16 (2.5 g, crude).

[0323] General procedure for the synthesis of C17 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 NaHCO to pH = 8 and extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by column chromatography (SiO, DCM:MeOH = 10:1) to give C17 (785 mg, 17% yield, 38% purity) as a white solid.

[0324] General procedure for the synthesis of C18 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 laptop page ES20772-487 (735 mg scale) was adapted for workup as ES20772-488. The combined reaction mixture was poured into water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layer was 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 a yellow solid, C18 (940 mg, crude product).

[0325] General procedure for the synthesis of C19 To a solution of C18 (940 mg, 2.44 mmol) in EtOH (10 mL) was added HCl (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 (2 mL) at 25 °C for 30 min to give C19 (560 mg, 79.0% yield, 83% purity) as a yellow solid.

[0326] General procedure for the synthesis of C20 1) To a mixture of E22 (305.06 mg, 1.19 mmol) and CH3CN (2 mL) was added CDI (192.23 mg, 1.19 mmol), and the mixture was stirred at 25°C for 3 minutes.

[0327] 2) To a mixture of C19 (260 mg, 1.08 mmol) and CH3CN (2 mL), t-BuOK (120.94 mg, 1.08 mmol) was added, and 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 N HCl and then extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (20 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated to give C20 (670 mg, crude) as a brown solid.

[0328] General procedure for the synthesis of compound 110 To a solution of C20 (670 mg, 1.39 mmol) in ethanol (5 mL) was added concentrated HCl (1 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 of laptop page ES20772-493 was combined with ES20772-498 for further purification. The residue was purified by prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; gradient: 10%-40% B, 12 min) to give compound 110 (54.5 mg, 10% yield, 96% purity) as a yellow solid.

[0329] [ka]

[0330] General procedure for the synthesis of E107 To a solution of E107-1 (1 g, 5.68 mmol) in MeOH (10 mL) was added NaBH (322.05 mg, 8.51 mmol) at 0 °C, and the reaction was stirred at 20 °C for 12 h. The mixture was quenched with NH Cl (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 Na SO , filtered, and concentrated to give E107 (974 mg, 96% yield) as a brown solid.

[0331] General procedure for the synthesis of E108 To a solution of E89 (1.1 g, 4.72 mmol) in THF (15 mL), E107 (925.32 mg, 5.19 mmol) and PPh3 (1.86 g, 7.08 mmol) were added, followed by DIAD (1.43 g, 7.08 mmol) at 0 °C. 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 silica gel flash column chromatography (0-30% EtOAc in PE) to give E108 (617 mg, 33% yield) as a colorless oil.

[0332] General procedure for the synthesis of E109 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 dioxane (5 mL) was 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 a nitrogen atmosphere 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 (495 mg, 71% yield) as a colorless oil.

[0333] General procedure for the synthesis of E110 To a mixture of E109 (495 mg, 1.12 mmol) and MeOH (10 mL) was added Pd / C (100 mg, 10% purity). The reaction mixture was stirred at 20 °C for 10 h under H (15 psi). 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.

[0334] General procedure for the synthesis of E111 To a solution of E110 (100 mg, 285.54 μmol) and E86 (83.60 mg, 285.54 μmol) in dioxane (5 mL) was added Pd(dppf)Cl (20.89 mg, 28.55 μmol), NaCO (60.53 mg, 571.08 μmol), and HO (0.5 mL). The mixture was stirred at 100 °C under a nitrogen atmosphere 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 NaSO, filtered, and concentrated to give E111 (199 mg, 58% yield, 40% purity) as a brown solid.

[0335] General procedure for the synthesis of compound 111 To a solution of E111 (199.00 mg, 414.10 μmol) in CHCl (5 mL) was added TFA (2 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 Prep-HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (FA)-ACN]; gradient: 22%-52% B, 12 min) to give compound 111 (4 mg, 2% yield, 100% purity) as a yellow solid.

[0336] [ka]

[0337] General procedure for the synthesis of F9 To a solution of F3 (100 mg, 651.17 μmol) and G1-3 (232.20 mg, 716.29 μmol) in dioxane (5 mL) was added Pd(dppf)Cl2 (47.65 mg, 65.12 μmol), Cs2CO3 (424.33 mg, 1.30 mmol), and HO (0.5 mL). The mixture was stirred at 100 °C 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 a residue. The residue was purified by flash silica gel chromatography (PE:EA = 3:1) to give F9 (204 mg, crude) as a brown solid.

[0338] General procedure for the synthesis of F10 1) To a solution of E9 (203.33 mg, 711.65 umol) in CH3CN (3 mL) was added CDI (115.39 mg, 711.65 umol) and the reaction was stirred at 20°C for 10 minutes.

[0339] 2) To a solution of F9 (204 mg, 646.96 umol) in CH3CN (3 mL) was added t-BuOK (79.86 mg, 711.65 umol) and the reaction was heated to 80 °C, after which the first solution was added and the reaction was stirred at 80 °C for 15 minutes.

[0340] The reaction mixture was poured into water (10 mL), adjusted to pH 4 with 1N HCl, and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over NaSO, and concentrated under reduced pressure to give a brown solid, F10 (565 mg, crude).

[0341] General procedure for the synthesis of compound 114 To a solution of F10 (565 mg, 135.67 umol) in ethanol (10 mL) was added concentrated hydrochloric acid (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 Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; gradient: 4%-34% B, 12 min) to give compound 114 (9.1 mg, purity 96%) as a yellow solid.

[0342] [ka]

[0343] General procedure for the synthesis of E112 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 min, followed by the dropwise addition of DIEA (3.10 g, 23.99 mmol) at 0 °C. The resulting mixture was stirred at 20 °C for 10 h. LCMS analysis showed no detectable desired product. TLC analysis showed complete consumption of 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol with the formation of one new spot.

[0344] The reaction mixture was quenched with NH4Cl (20 mL), 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 (1.8 g, crude) as a colorless oil.

[0345] General procedure for the synthesis of compound 116 To a solution of E112 (1.8 g, 6.12 mmol) and E86 (1.79 g, 6.12 mmol) in dioxane (10 mL) was added Pd(dppf)Cl (447.76 mg, 611.93 μmol), NaCO (1.30 g, 12.24 mmol), and HO (1 mL). The mixture was stirred at 100 °C under a nitrogen atmosphere for 1 h.

[0346] The mixture was diluted with ethyl acetate (15 mL) and filtered. The combined organic layers were washed with water (20 mL×2), dried over NaSO, and then concentrated under reduced pressure to give compound 116 (3.88 g, 74.68% yield, 50% purity) as a brown solid.

[0347] The crude product (400 mg) was purified by Prep-HPLC (column: Boston Green ODS 150*30 mm*5 um; mobile phase: [water (FA)-ACN]; gradient: 36%-66% B in 12 min) to give a yellow solid, Compound 116 (52.8 mg, purity 100%).

[0348] [ka]

[0349] General procedure for the synthesis of E114 To a solution of E113 (10 g, 77.79 mmol) in DMF (150 mL) were 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 HO (100 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (100 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 3 / 1 to 1 / 1) to give E114 (3.64 g, 19.6% yield, 90% purity) as a yellow oil.

[0350] General procedure for the synthesis of E115 To a solution of E114 (1 g, 4.66 mmol) dissolved in THF (15 mL) and HO (3 mL) was added LiOH.HO (977.50 mg, 23.29 mmol). The mixture was stirred at 25 °C for 12 h. The reaction mixture was diluted with HO (20 mL) and extracted with EtOAc (50 mL). The aqueous layer was adjusted to pH = 5 with HCl (1 N) and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give E115 (320 mg, crude product) as a white solid.

[0351] General procedure for the synthesis of E117 1) To a mixture of E115 (318.96 mg, 1.71 mmol) dissolved in CH3CN (5 mL) was added CDI (277.17 mg, 1.71 mmol) and stirred at 25°C for 3 minutes.

[0352] 2) To a mixture of E67 (490 mg, 1.55 mmol) dissolved in CH3CN (5 mL) was added t-BuOK (348.74 mg, 3.11 mmol) and stirred at 80°C for 2 minutes.

[0353] The first-stage reaction mixture was added dropwise to the second-stage reaction mixture at 80°C. 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 x 3). The combined organic layers were washed with brine (50 mL x 3), dried over anhydrous NaSO, filtered, and concentrated to give E117 (700 mg, crude product) as a yellow solid.

[0354] General procedure for the synthesis of compound 119 To a solution of E117 (700 mg, 1.45 mmol, crude product) dissolved in EtOH (10 mL) was added 37% HCl (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 prep-HPLC (column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water(FA)-ACN]; gradient: 10%-40% B over 12 min) and then repurified by prep-HPLC (column: Phenomenex Gemini NX 150 × 30 mm, 5 μm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 13%-43% B over 11 min) to give compound 119 (0.9 mg, 100% pure) as a white solid.

[0355] Experimental Example 1 - YFP Assay

[0356] Principles for the YFP assay The halide sensor YFP F46L / H148Q / I152L is quenched by halide ions such as iodine, and when expressed intracellularly, it is also expressed in the cytoplasm.

[0357] To measure H723R-PDS activity, PANC-1 cells were stably transfected with the halide sensor YFP-F46L / H148Q / I152L and H723R-PDS, which is defective in membrane trafficking due to misfolding.

[0358] When the drug treated cells is a corrective agent for H723R-PDS, H723R-PDS translocates to the cell membrane normally.

[0359] In this case, if a high concentration of iodine solution is applied to the outside of the cells, iodine flows into the cells due to the Cl- / I- exchange activity of pendrin, and the fluorescence of the halide sensor YFP decreases.

[0360] Protocol for the YFP assay

[0361] High-throughput screening cells PANC-1 cells were cultured in Dulbecco's Modified Eagle's Medium containing 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-pendrin and the halide sensor YFP-F46L / H148Q / I152L.

[0362] YFP fluorescence quenching analysis procedure PANC-1 cells expressing human H723R-Pendrin and YFP-F46L / H148Q / I152L were plated at 2 x 10 cells per well in a 96-well microplate. 4 The cells were plated at a density of 1 / 3 cells per well and incubated at 37°C (90% humidity, 5% CO2) for 20–24 hours. The cells were then treated with 50 μl of culture medium containing the test compound and incubated for 20–24 hours. For YFP quenching assays, each well of a 96-well plate was washed twice with 200 μl of PBS and filled with 50 μl of HEPES buffer solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM D-glucose, 10 mM HEPES; pH 7.4, NaOH). After 10 minutes of incubation at 37°C, the 96-well plate was transferred to a FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany) for fluorescence assays. Each well was individually assayed for H723R-pendrin-mediated I-influx by recording fluorescence (excitation at 495 ± 15 nm, emission at 520 ± 20 nm) every 400 ms for 1 s (baseline). Subsequently, 50 μL of NaI-substituted HEPES buffer solution (NaI replaces NaCl) was added at 1 s intervals using a liquid injector, and YFP fluorescence was measured for 10 s at 400 ms intervals. The initial iodine influx rate was determined from the initial slope of fluorescence after iodine injection using nonlinear regression analysis.

[0363] Table 1 shows the measured Cl- / I-exchange activity of pendrin for selected compounds of the invention, which is expressed as EC using the following symbols: 50A=EC 50 <5uM; B=EC 50 Over 5uM and under 10uM; C=EC 50 Exceeds 10uM.

[0364] [Table 1-1] [Table 1-2]

[0365] Experimental Example 2 - Surface Biotinylation Assay

[0366] Principle of surface biotinylation analysis Surface biotinylation is a commonly used technique for specifically isolating plasma membrane proteins using modified biotin in a form that is easily bound to proteins. Sulfo-NHS-SS-biotin is a commonly used reagent for surface biotinylation due to its impermeability and water solubility. Under low-temperature conditions, the extracellular domains of plasma membrane proteins covalently bind to reactive biotin esters. These proteins are isolated from whole cell lysates through the biotin-streptavidin reaction. Through this process, the rate at which the expression level of mutant pendrin increases on the plasma membrane can be determined.

[0367] Surface Biotinylation Analysis Protocol Panc1 parental , Panc1 hPDS-WT , and Panc1 hPDS-H723RCells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics. After 24 hours in a 6-well plate, the cells were treated as indicated for 24 hours. The cells were washed with PBS and incubated on ice with 0.3 mg / ml biotin in PBS. After 35 minutes, 1% BSA and DW mixture (1:1) was added to PBS and the cells were incubated for 10 minutes. The cells were washed and lysed with lysis buffer. The lysate was sonicated for 20 seconds without boiling. After centrifugation, the supernatant was collected and quantified via BCA analysis. The quantified supernatant was added to 10% avidin beads in PBS and incubated overnight at 4°C. After incubation, the following steps were repeated four times: i) centrifugation, ii) supernatant removal, and iii) washing with lysis buffer. After the final centrifugation and supernatant removal, sample buffer mixed with reducing buffer was added to the biotin-avidin mixture. Elution was performed for 40 minutes on a bioshaker at 38°C, and the supernatant was collected. The supernatant was separated by SDS-PAGE and transferred to an Immobilon membrane. To prevent nonspecific binding, the membrane was incubated with a 5% BSA solution, followed by overnight incubation with specific antibodies. The membrane was then washed with TBST buffer and incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies for 1 hour. Protein bands were visualized using ECL buffer and iBright. The intensity of each protein band was quantified by densitometry analysis using the NIH ImageJ program.

[0368] Table 2 shows the expression ratio of the glycosylated form of pendrin on the surface of H723R-hPDS for selected compounds of the present invention compared to wild-type hPDS. The expression ratio is expressed as an expression ratio using the following symbols: A = expression ratio over 100%; B = expression ratio over 50% but less than 100%; C = expression ratio less than 50%.

[0369] [Table 2]

[0370] Experimental Example 3 - PTI Assay

[0371] Protocol for PTI assay

[0372] Cl - / HCO3 - Measurement of exchange activity Intracellular pH (pH i The measurements were performed 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 a 2 μM BCECF acetoxymethyl ester solution for 5 min, followed by HCO3 - The organs were perfused with a buffer solution 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).

[0373] BCECF fluorescence was measured using a recording device with a resolution of 2 / s at excitation wavelengths of 490 nm and 440 nm. - The exchange activity is HCO3 - A buffer solution containing 25 mM HCO3 (containing 5% CO2) - ) to Cl - The initial pH generated when i It was estimated from the rate of increase.

[0374] pH i Corrections were made using a standard pH solution containing 150 mM KCl and 5 μM nigericin. The intrinsic buffering capacity (βi) was calculated by measuring the ΔpHi in response to a 5-40 mM NH4Cl pulse in a Na+-free solution. Transfection with plasmids encoding WT-pendrin or H723R-pendrin did not significantly affect βi values, so Cl - / HCO3 - Exchange activity was expressed as ΔpH units / min without correction for buffer capacity.

[0375] Table 3 shows the Cl of pendrin for selected compounds of the present invention. - / HCO3 -The exchange activity measurements are shown, which are expressed as percent exchange using the following symbols: A = exchange rate above 50%; B = exchange rate below 50%.

[0376] [Table 3]

[0377] Experimental Example 4 - Patient-Derived Cell-Based (PDC) Assay

[0378] Principle for the assay The experimental approach is based on a multi-step process to investigate the effects of drug treatment on nasal epithelial cells. First, nasal tissue is harvested and epithelial cells are isolated through tissue lysis. These cells are then cultured and exposed to air-liquid interface (ALI) conditions for extended periods.

[0379] Subsequently, drug treatments containing human IL-4 and pendrin-modifying drugs were administered, and proteins were harvested for subsequent analysis. Expression of the pendrin antibody hR1 (Yonsei ENT Choi Lab.) was confirmed by Western blot analysis after drug treatment. Various drug concentrations were also immunofluorescently stained on Transwell membranes, and the resulting frozen sections were observed under a confocal microscope using the primary antibody hR2 (Yonsei Otolaryngology, Choi Jin-sil Lab.). This comprehensive methodology allowed visualization and analysis of pendrin expression in both the basal and apical regions of nasal epithelial cells, providing insight into the effects of drug treatment on target cellular components.

[0380] Protocol for PDC analysis

[0381] Measurement of pendrin expression in patient nasal epithelial cells by Western Blot and confocal microscopy

[0382] 1. Tissue Harvesting and Cell Isolation Nasal tissue samples were collected from patients and immersed in DMEM:F12 medium (Lonza, Cat. No. 12-719F) containing 1% penicillin-streptomycin. After removing red blood cells with PBS, the tissue was digested with 1% protease solution in the transfer media at 37°C for 1 hour, resulting in the isolation of epithelial cells.

[0383] 2.Cell culture The acquired epithelial cells were cultured in the BEGM Bullet Kit (Lonza Cat No. CC3170) supplemented with 150 mg / ml BSA and EGF (BD Cat No. 354001) in a standard manner until the first passage. When the cells reached approximately 90% cell density, they were detached using 0.25% trypsin-EDTA and distributed to 12-well Transwell plates (Costar Cat No. 3450) containing a 1:1 mixture of DMEM medium (Lonza Cat No. 12-707F) and BEGM Bullet Kit supplemented medium for air-liquid interface (ALI) culture.

[0384] 3. Air-Liquid Interface Culture ALI culture was initiated by placing 1 ml of medium on the bottom of the membrane and dispensing 0.5 ml of cells onto the membrane. When the cells on the membrane reached 90% confluency, the bottom was filled with culture medium supplemented with 50 nM retinoic acid (RA, SIGMA Cat No. R2625), and ALI culture was continued for 7–14 days.

[0385] 4. Drug Treatment and Protein Harvesting After ALI culture, cells were treated with 10 μg / ml human IL-4 for 24 hours, followed by treatment with pendrin-modifying drugs (compounds 9, 18, and 105) at concentrations of 0.1, 0.3, 1, 3, and 10 μM for 24 hours. After drug treatment, cells attached to the Transwell membrane were harvested by scraping into protein buffer (iNtRON Cat No. 17081). Protein quantification (30 μg) was performed for each sample, and expression of the pendrin antibody (hR1, custom-made antibody) was confirmed by Western blot analysis.

[0386] 5. Immunofluorescence Staining For immunofluorescence staining, Transwell membranes containing each drug treatment were fixed in 4% paraformaldehyde (PFA) at room temperature for 10 minutes. They were then cut into three sections using a microtome, embedded in OCT solution, and processed into 5-μm frozen sections. Immunostaining was performed using the primary antibody hR2 (YONSEI ENT ​​custom antibody), and pendrin expression was visualized in the basal and apical regions of epithelial cells using a confocal microscope (Carl Zeiss, LSM700).

[0387] The results of the experiment are shown in FIGS.

[0388] FIG. 1 shows the results of Western blot analysis of epithelial cells from a patient treated with Compound 9, inferred from the patient's nasal epithelial cells.

[0389] Figure 1. (A, B) Form B, a specific non-glycosylated precursor of pendrin, showed little noticeable change in response to compound 9 treatment. However, a significant dose-dependent increase in form C, a fully glycosylated functional pendrin, was observed, particularly at a high concentration of 10 μM. These observations, extrapolated to patient-derived nasal epithelial cells, suggest that compound 9 treatment has a concentration-dependent effect on form C expression in cells harboring the H724R mutation.

[0390] FIG. 2 shows the immunofluorescence staining results of compounds 9, 18, and 105.

[0391] Figure 2. (A) This figure shows the immunofluorescence staining results of epithelial cells under different conditions. The vehicle and IL-4 groups showed no fluorescence, indicating the absence of pendrin expression. Conversely, treatment with Compound 9, Compound 18, and Compound 105 at 10 μM concentrations showed bright red fluorescence, indicating increased pendrin expression locally on the epithelial cell membrane. Scale bar = 20 μm. (B) This graph shows a quantitative comparison of the mean fluorescence intensity levels among the vehicle, IL-4, Compound 9, Compound 18, and Compound 105 (10 μM) groups. Statistical significance indicates significant differences in pendrin expression intensity among the experimental conditions. *p<0.05.

[0392] Specific compounds prepared by the general procedures set out above are set forth in Table 4 below.

[0393] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20

Claims

1. Compounds having the general formula I and pharmaceutically acceptable salts thereof: 【Chemistry 1】 X １ are each independently selected from CH, CZ, and N; X ２ are each independently selected from CH, CZ, and N; X ３ are each independently selected from CH, CZ, and N; X ４ are each independently selected from CH, CZ, and N; X ５ are each independently selected from CH, CZ, and N; n is independently selected from 0, 1, and 2; R １ are each independently hydrogen, C1-C6 alkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C3-C10 heterocycloalkyl substituted with one or more of: C6-C12 aryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C6-C12 aryl substituted with one or more of: C3-C12 heteroaryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C3-C12 heteroaryl substituted with one or more of: Z is any one of the structures in Group A below; 【Chemistry 2】 Y １ , Y ２ , and Y ３ are each independently selected from CH and N; R ２ and R ３ are each independently hydrogen, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ each of which is optionally substituted; R ４ and R ５ are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, and C3-C10 cycloalkyl, each of which is optionally substituted; R ６ and R ７ are each independently hydrogen, C1-C4 haloalkyl, C1-C6 alkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ８ and N.R. ８ R ９ C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, ═O, C1-C4 haloalkyl, OR ８ and N.R. ８ R ９ C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, ═O, C1-C4 haloalkyl, OR ８ and N.R. ８ R ９ C3-C10 heterocycloalkyl substituted with one or more of: R ８ and R ９ are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, and C1-C4 haloalkyl, each of which is optionally substituted.

2. In this case, Z is selected from any of the structures in Group B below; 【Transformation 3】 R ２ and R ３ is as defined in claim 1, Y １ , Y ２ , and Y ３ 2. The compound of claim 1, wherein: is as defined in claim 1, and pharmaceutically acceptable salts thereof.

3. At this time, Z is 【Chemistry 4】 and R ２ and R ３ is as defined in claim 1; Y １ , Y ２ , and Y ３ 3. The compound of claim 1 or claim 2, and pharmaceutically acceptable salts thereof, wherein:

4. A compound according to any one of claims 1 to 3, having the general formula II, and pharmaceutically acceptable salts thereof; 【Transformation 5】 At this time, R １ , R ２ , and R ３ is as defined in claim 1; X １ , X ２ , X ３ , and X ４ is as defined in claim 1; n is as defined in claim 1.

5. The R １ are each independently hydrogen, C1-C6 alkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C1-C6 alkyl substituted with one or more of: C3-C10 cycloalkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ C3-C10 cycloalkyl substituted with one or more of: C3-C10 heterocycloalkyl, halogen, C1-C6 alkyl, ═O, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ The compound according to any one of claims 1 to 4, and a pharmaceutically acceptable salt thereof, wherein the compound is a C3-C10 heterocycloalkyl substituted with one or more of the following:

6. The R １ are each independently C6-C12 aryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, C6-C12 aryl, OR ６ and N.R. ６ R ７ C6-C12 aryl substituted with one or more of: C3-C12 heteroaryl, halogen, C1-C6 alkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, C1-C4 haloalkyl, OR ６ and N.R. ６ R ７ The compound according to any one of claims 1 to 5, and a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of C3-C12 heteroaryl substituted with one or more of:

7. The R ２ and R ３ are each independently OR ６ and N.R. ６ R ７ The compound according to any one of claims 1 to 6, and a pharmaceutically acceptable salt thereof, which is selected from the group consisting of:

8. The compound of any one of claims 1 to 7, having one of the following chemical formulas 1 to 120, and pharmaceutically acceptable salts thereof.

9. A composition comprising one or more compounds according to any one of claims 1 to 8 as active ingredients and one or more pharmaceutically acceptable carriers, excipients and / or diluents.

10. A compound according to any one of claims 1 to 8 or a composition according to claim 9 for use as a pharmaceutically active agent or in the treatment of Pendred syndrome or its related disorders.

11. 9. The compound of any one of claims 1 to 8 for use in a method for the prevention and / or treatment of hearing loss, enlarged vestibular aqueduct, goiter, hypertension, hypokalemia, hypothyroidism, hypochloremic alkalosis, renal tubular acidosis, hypovolemia, hypovolemia, edema, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rhinitis, sinusitis, cirrhosis of the liver, bone abnormalities, cochlear malformations, chronic obstructive pulmonary disease, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchitis, speech disorders, or rhinitis.

12. A method for preventing, ameliorating, or treating Pendred Syndrome or a related disorder, comprising administering a compound according to any one of claims 1 to 8.

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

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

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

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