Substituted phenylbenzenesulfonamide derivatives and their uses

Abstract

Substituted phenylbenzenesulfonamide compounds and compositions thereof for modulating TRPML1 are provided herein. In some embodiments, the compounds and compositions are provided for the treatment of neurodegenerative diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 420,369, filed on 28 October 2022, which is incorporated in its entirety by reference. Throughout this application, various publications, patents, and / or patent applications are referenced. The disclosures of these publications, patents, and / or patent applications are incorporated in their entirety by reference to provide a more complete description of the prior art relevant to this disclosure.

[0002] field This disclosure generally relates to substituted phenylbenzenesulfonamide derivative compounds and compositions for treating, for example, neurodegenerative diseases, including compounds, compositions, methods for preparing them, and their uses. [Background technology]

[0003] Transient receptor potentials (TRPs) are multifunctional signaling molecules involved in sensory and cellular physiology. The 28 members of the mammalian TRP channel superfamily are divided into six subfamilies, one of which is mucolipin (TRPML1-3). Mucolipin TRP channel subfamily 1 (TRPML1) is the main Ca25 channel localized to the lysosome membrane. 2+ It is an release channel (Zeevi et al., Biochim.Biophys.Acta 2007, 1772, 851-858). Lysosomal Ca released through the TRPML1 channel. 2+TRPML1 promotes the dephosphorylation and subsequent nuclear translocation of the transcription factor EB (TFEB), which increases the transcription of genes that promote autophagy and lysosome biosynthesis (Tedeschi et al., Cell 2019, 8, 1216). Due to its important regulatory function and ability to remove pathogenic molecules, TRPML1 has attracted attention as a potential target for lysosomal storage diseases, metabolic diseases, cardiovascular diseases, inflammatory disorders, immunological disorders, cancer, aging, and neurodegenerative diseases (Krogsaeter et al., Cell Calcium 2022, 103, 102553 and Park et al., Front. Cell Dev. Biol. 2022, 10, 811701). For example, TRPML1 has been shown to regulate α-synuclein exocytosis in dopaminergic neurons in a mouse model of Parkinson's disease (Tsunemi et al., J. Neurosci. 2019, 39, 5760-5772). These studies aim to identify novel small molecule agonists that activate TRPML1 and may be useful for treating diseases and disorders related to lysosomes and autophagy. [Overview of the project]

[0004] This embodiment can be better understood by the detailed description and reference to examples intended to illustrate non-limiting embodiments.

[0005] Equation (I)

[0006] [ka] (In the formula, X is either N or H. R 1 is selected from H and optionally substituted alkyl groups, R 2 This is selected from optionally substituted alkyl, optionally substituted cycloalkyl, aryl and optionally substituted heterocyclyl, Or, when X is H, R 1 and R 2 do not exist, R 3 is H, -CH3 or optionally substituted C2-C6 alkyl, or or, R 1 and R 3 together with the atom to which they are attached form a 5- to 7-membered ring, each R 4 is independently H, halo, cyano or optionally substituted alkyl,<� R 5a and R 5b are each independently H or optionally substituted alkyl, provided that R 2 is not pyrrolidine) A compound of ) or a pharmaceutically acceptable salt thereof is disclosed.

[0007] In another aspect, a compound selected from the compounds disclosed herein or a pharmaceutically acceptable salt thereof is provided herein.

[0008] In a further aspect, a pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient is provided herein.

[0009] Another aspect provides a method of modulating mucolipin TRP channel subfamily 1 (TRPML1), the method comprising contacting TRPML1 with an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

[0010] A related aspect provides a method of treating a disease associated with TRPML1, the method comprising administering to a subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.

[0011] In another embodiment, the use of a compound disclosed herein or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for modulating TRPML1 in a subject is provided herein.

[0012] In another embodiment, the use of the compounds disclosed herein or pharmaceutically acceptable salts thereof in the manufacture of a medicament for treating a disease associated with TRPML1 in a subject is provided herein.

[0013] In related embodiments, pharmaceutical formulations comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier are provided herein. [Modes for carrying out the invention]

[0014] definition As used herein, the terms “comprising” and “including” are interchangeable. The terms “comprising” and “including” are interpreted as specifying the presence of the described characteristics or components, but do not preclude the presence or addition of one or more characteristics or components or groups thereof. In addition, the terms “comprising” and “including” are intended to include examples encompassed by the term “consisting.” Consequently, the term “consisting” can be used in place of the terms “comprising” and “including” to provide more specific embodiments of the present invention.

[0015] The term "consisting of" means that the subject has at least 90%, 95%, 97%, 98%, or 99% of the described properties or components that constitute it. In another embodiment, the term "consisting of" excludes any other properties or components from the scope of any subsequent description, except those that are not essential to the technical effect to be achieved.

[0016] As used herein, the term “or” is interpreted as an inclusive “or” meaning any one or any combination thereof. Thus, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C.” Exceptions to this definition arise only when any combination of elements, functions, steps, or actions is in any way inherently mutually exclusive.

[0017] In embodiments for carrying out the present invention, any range of concentration, percentage, ratio, or integer is understood, unless otherwise specified, to include any integer within the enumerated range and, if applicable, fractions thereof (e.g., one-tenth and one-hundredth of an integer). Similarly, any range of numbers enumerated herein with respect to any physical properties, such as polymer subunits, size, or thickness, is understood, unless otherwise specified, to include any integer within the enumerated range. As used herein, “about” and “approximately” mean ±20%, ±10%, ±5%, or ±1% of the indicated range, value, or structure, unless otherwise specified.

[0018] The "alkyl" group consists of 1 to 10 carbon atoms (C1 to C1). 10Alkyl alkyl groups are saturated, partially saturated, or unsaturated linear or branched acyclic hydrocarbons having typically 1 to 8 carbon atoms (C1-C8 alkyl), or in some embodiments, 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 1 to 3 carbon atoms (C1-C3 alkyl), or 2 to 6 carbon atoms (C2-C6 alkyl). In some embodiments, alkyl groups are monovalent. Examples of monovalent alkyl groups include, but are not limited to, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2(CH3), -CH2(CH2)2CH3, -CH2CH(CH3)CH3, -CH2(CH2)3CH3, -CH2(CH2)4CH3, -CH2(CH2)5CH3, -CH2(CH2)6CH3, etc. In some embodiments, alkyl groups are divalent. Examples of divalent alkyl groups include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2(CH2)2CH2-, -CH2CH(CH3)CH2-, -CH2(CH2)3CH2-, -CH2(CH2)4CH2-, -CH2(CH2)5CH2-, and -CH2(CH2)6CH2-. In some embodiments, the alkyl group is a saturated alkyl group. Typical saturated alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl, while saturated branched-chain alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, -tert-pentyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl, and -2,3-dimethylbutyl. In some embodiments, the alkyl group is an unsaturated alkyl group, also called an alkenyl or alkynyl group. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3). The alkyl group may be substituted or unsubstituted.Where an alkyl group described herein is said to be “substituted,” it may be substituted with any substituent(s) as found in the exemplary compounds and embodiments disclosed herein, as well as with halogens, hydroxyl, alkoxy, cycloalkyloxy, aryloxy, heterocyclyloxy, heteroaryloxy, heterocycloalkyloxy, cycloalkylalkyloxy, aralkyloxy, heterocyclylalkyloxy, heteroarylalkyloxy, heterocycloalkylalkyloxy, oxo(=O), amino, alkylamino, cycloalkylamino, arylamino, heterocyclylamino, heteroarylamino, heterocycloalkylamino, cycloalkylalkylamino, aralkylamino, heterocyclylalkylamino, heteroaralkylamino, heterocycloalkylalkylamino, acylamino, sulfonylamino, oxime, hydroxylamino, hydrazine, hydrazide, hydrazono, azide, nitro, thio(-SH), alkylthio, =S, sulfinyl, sulfonyl, aminosulfonyl, acyl, formyl, carboxy, ester, carbamate, amide, cyano, or -B(OH)2. In certain embodiments, where an alkyl group described herein is referred to as “substituted,” it may be substituted with any substituent(s) as found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo, or fluoro); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; aminocarbonyl; acylamino; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; oxime; hydroxylamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; -B(OH)2; or -O(alkyl)aminocarbonyl.

[0019] A "cycloalkyl" group is a group consisting of 3 to 10 carbon atoms (C3-C) having a single ring or multiple fused or bridging rings. 10A cycloalkyl group is a saturated or partially saturated cyclic alkyl group of the cycloalkyl group. In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms (C3 to C8 cycloalkyl groups), while in other embodiments, the number of ring carbon atoms ranges from 3 to 5 (C3 to C5 cycloalkyl groups), 3 to 6 (C3 to C6 cycloalkyl groups), or 3 to 7 (C3 to C7 cycloalkyl groups). In some embodiments, the cycloalkyl group is a saturated cycloalkyl group. Examples of such saturated cycloalkyl groups include single ring structures, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., or multiple ring structures or cross-linked ring structures, e.g., 1-bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, etc. In other embodiments, the cycloalkyl group is an unsaturated cycloalkyl group. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl. Cycloalkyl groups can be substituted or unsubstituted. Examples of such substituted cycloalkyl groups include cyclohexanol.

[0020] A "heterocyclyl" is a non-aromatic cycloalkyl group in which 1 to 4 of the ring carbon atoms are independently substituted with heteroatoms. In some embodiments, a heterocyclyl group comprises 3 to 10 ring members, while other such groups comprise 3 to 5, 3 to 6, or 3 to 8 ring members. In some embodiments, a heterocyclyl group comprises 1 to 3 heteroatoms, while other such groups comprise 1 to 2 heteroatoms or 1 heteroatom. Heterocyclyls can also be bonded to other groups at any ring atom (i.e., any carbon or heteroatom of the heterocycle). Heterocyclyl groups can be substituted or unsubstituted. Heterocyclyl groups encompass saturated and partially saturated ring systems. Furthermore, the term heterocyclyl is intended to encompass any non-aromatic ring containing at least one heteroatom, in which the ring may condense into an aryl or heteroaryl ring regardless of its bonding to other parts of the molecule. This clause also includes bridging polycyclic systems containing heteroatoms. Representative examples of heterocyclyl groups include, but are not limited to, azilidinyl, azetidinyl, azepanil, pyrrolidyl, imidazolidinyl (e.g., imidazolidine-4-onyl or imidazolidine-2,4-dionyl), pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranil, piperidyl, piperazinyl (e.g., piperazine-2-onyl), morpholinyl, thiomorpholinyl, tetrahydropyranil (e.g., tetrahydro-2H-pyranil), tetrahydrothiopyranil, oxathianil, dithianil, 1,4-dioxaspiro[4.5]decanyl, homopiperazinyl, quinuclidyl, or tetrahydropyrimidine-2(1H)-one. Typical substituted heterocyclyl groups may be monosubstituted or substituted more than once, for example, but not limited to, pyridyl or morpholinyl groups, which are 2, 3, 4, 5, or 6-substituted, or 2-substituted with various substituents, e.g., those listed below. In some embodiments, when a heterocyclyl group described herein is said to contain only heteroatom X, it exclusively contains at least one heteroatom X and does not contain any other heteroatoms.For example, “heterocyclil containing only N” describes a heterocyclil containing 1, 2, 3 or more nitrogen heteroatoms and not containing other heteroatoms, such as oxygen or sulfur. In some embodiments, when a heterocyclil group described herein is said to contain at least one heteroatom X, it may contain at least one heteroatom X and may or may not contain additional heteroatoms of different types. For example, “heterocyclil containing at least N” describes a heterocyclil containing 1, 2, 3 or more nitrogen heteroatoms together with 0, 1, 2, 3 or more oxygen heteroatoms and / or 1, 2, 3 or more sulfur heteroatoms.

[0021] The "aryl" group consists of 6 to 14 carbon atoms (C6-C) having a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthryl). 14 It is an aromatic carbon ring of aryl groups. In some embodiments, the aryl group contains 6 to 14 carbon atoms in the ring portion of the group (C6 to C6). 14 (aryl), 6-12 others (C6-C) 12 Aryl) or even 6 to 10 carbon atoms (C6 to C6) 10 It contains aryl groups. Specific aryl groups include phenyl, biphenyl, and naphthyl. The aryl group may be substituted or unsubstituted. The phrase "aryl group" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0022] A "heteroaryl" group is an aromatic ring system having 1 to 4 heteroatoms as ring atoms in the heteroaromatic ring system, with the remainder being carbon atoms. In some embodiments, the heteroaryl group contains 3 to 10 ring atoms in the ring portion of the group, while others contain 6 to 9 or even 6 to 10 atoms. In some embodiments, the heterocyclyl group has 1 to 3 heteroatoms, while other such groups have 1 to 2 heteroatoms or 1 heteroatom. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, benzoisoxazolyl (e.g., benzo[d]isoxazolyl), thiazolyl, pyrrolyl, pyridadinyl, pyrimidyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl (e.g., indolyl-2-onyl or isoindoline-1-onyl), azaindolyl (pyrrolopyridyl or 1Hpyrrolo[2,3b]pyridyl), indazolyl, benzimidazolyl (e.g., 1Hbenzo[d]imidazolyl) Examples include imidazopyridyl (e.g., azabenzimidazolyl or 1Himidazo[4,5b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl (e.g., 1Hbenzo[d][1,2,3]triazolyl), benzoxazolyl (e.g., benzo[d]oxazolyl), benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, prinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl (e.g., 3,4-dihydroisoquinoline-1(2H)-onyl), tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups may be substituted or unsubstituted.

[0023] "Halogen" or "halo" refers to fluorine, chlorine, bromine, or iodine.

[0024] The "oxo" group is an "=O" group that is bonded to a carbon atom.

[0025] Where a group described herein, with the exception of alkyl groups, is said to be "substituted," it may be substituted with some appropriate substituent(s). Examples of exemplary substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogens (chloro, iodo, bromo, or fluoro); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amino; alkylamino; carboxy; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfinyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; urethane; oxime; hydroxylamine; alkoxyamine; aralkoxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxygen (=O); -B(OH)2, -O(alkyl)aminocarbonyl; monocyclic, Alternatively, cycloalkyls that are condensed or uncondensed polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocyclyls that may be monocyclic or condensed or uncondensed polycyclic (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiadinyl); monocyclic or condensed or uncondensed polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridadinyl, pyrimidyl, benzimidazolyl, benzothiophenyl, or benzofuranyl) aryloxys; aralkyloxys; heterocyclyloxys; and heterocyclylalkoxys.

[0026] "Depending on the circumstances" or "depending on the circumstances" means that the event or situation described below may or may not occur, and that the description includes both cases in which the event or situation occurs and cases in which it does not occur. For example, "a heterocyclyl group which may be substituted with an alkyl group" means that an alkyl group may or may not be present, and that the description includes both cases in which the heterocycloalkyl group is substituted with an alkyl group and cases in which the heterocycloalkyl group is not substituted with an alkyl group.

[0027] Polymers or similar non-specific structures obtained by defining substituents with an infinite number of further substituents (e.g., substituted aryls having a substituted alkyl that is itself substituted with a substituted aryl group and further substituted with a substituted heteroalkyl group) are not intended to be included herein. Similarly, the above definitions are not intended to include unacceptable substitution patterns (e.g., a methyl group substituted with five fluorine atoms or a heteroaryl group having two adjacent oxygen ring atoms). Such unacceptable substitution patterns are well known to those skilled in the art.

[0028] Embodiments of this disclosure include pharmaceutically acceptable salts, tautomers, isotopic substitutions and stereoisomers of the compounds provided herein, such as compounds of formulas (I), (II), (III), (IV), and (V), as well as the compounds in Table 1.

[0029] As used herein, the term “pharmaceutically acceptable salt” refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base, including inorganic acids and bases, as well as organic acids and bases. Preferred pharmaceutically acceptable base addition salts of the compounds disclosed herein include, but are not limited to, metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts made from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Suitable non-toxic acids include, but are not limited to, inorganic and organic acids such as acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethensulfonic acid, formic acid, fumaric acid, furic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucoic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric acid, hydrobromic acid, maleic acid, phosphoric acid, sulfuric acid, and methanesulfonic acid. Therefore, examples of specific salts include hydrochloride, formate, and mesylate salts. Other information is well known in the relevant technical field; see, for example, Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th eds., Mack Publishing, Easton PA (1995).

[0030] As used herein, and unless otherwise noted, the terms “stereoisomer” or “stereoisomerically pure” mean one stereoisomer of a particular compound that substantially has no other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center substantially has no symmetrical enantiomers of the compound. A stereoisomerically pure compound having two chiral centers substantially has no other diastereomers of the compound. Typical stereoisomerically pure compounds include one stereoisomer of the compound in more than about 80% by weight and less than about 20% by weight of other stereoisomers of the compound, one stereoisomer of the compound in more than about 90% by weight and less than about 10% by weight of other stereoisomers of the compound, one stereoisomer of the compound in more than about 95% by weight and less than about 5% by weight of other stereoisomers of the compound, or one stereoisomer of the compound in more than about 97% by weight and less than about 3% by weight of other stereoisomers of the compound. The compounds disclosed herein may contain chiral centers and may arise as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms, including mixtures thereof, are included within the embodiments disclosed herein.

[0031] The use of stereoisomerically pure forms of the compounds disclosed herein, as well as the use of mixtures of these forms, are encompassed in the embodiments disclosed herein. For example, mixtures containing equivalences or non-equal amounts of enantiomers of a particular compound may be used in the methods and compositions disclosed herein. These isomers may be synthesized asymmetrically or resolved using standard techniques such as chiral columns or chiral resolution agents.For example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972); Todd, M., Separation Of Enantiomers : Synthetic Methods (Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim, Germany, 2014); Toda, F., Enantiomer Separation: Fundamentals and Practical Methods (Springer Science & Business Media, 2007); See G. Chiral Separation Techniques: A Practical Approach (John Wiley & Sons, 2008); and Ahuja, S., Chiral Separation Methods for Pharmaceutical and Biotechnological Products (John Wiley & Sons, 2011).

[0032] It should also be noted that the compounds disclosed herein may include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compound is isolated as either the E or Z isomer. In other embodiments, the compound is a mixture of the E and Z isomers.

[0033] "Tautomers" refer to isomeric forms of a compound that are in equilibrium with each other. The concentrations of these isomeric forms may vary depending on the environment in which the compound is found, for example, whether the compound is in a solid state or in an organic solution or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other.

[0034] [ka]

[0035] As will be readily apparent to those skilled in the art, a wide range of functional groups and other structures can exhibit tautomerism, and all tautomers of the compounds disclosed herein are within the scope of this disclosure.

[0036] It should also be noted that the compounds disclosed herein may contain unnatural proportions of atomic isotopes in one or more atoms. For example, a compound may contain tritium. 3 H), Iodine-125( 125 I), Sulfur 35 ( 35 S) or carbon-14 ( 14 It can be radiolabeled with radioactive isotopes such as C, or for example, deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15They may be enriched with the isotope of N). As used herein, “isotope-substituted compound” is an isotope-enriched compound. The term “isotope-enriched” refers to an atom having an isotope composition other than the natural isotope composition of that atom. “Isotope-enriched” may also refer to a compound containing at least one atom having an isotope composition other than the natural isotope composition of that atom. The term “isotope composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotope-enriched compounds are useful as therapeutic agents, e.g., cancer treatment agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo contrast agents. All isotope variations of compounds as described herein, whether radioactive or not, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isotope-substituted compounds of the compounds disclosed herein are provided, for example, isotope-substituted compounds enriched with deuterium, carbon-13, and / or nitrogen-15. As used herein, “deuterated” means that at least one hydrogen (H) is converted into deuterium (D or 2 This refers to a compound in which deuterium (represented by H) is substituted, meaning that the compound is concentrated with deuterium in at least one part.

[0037] It is understood that, independently of the stereoisomer or isotopic composition, each compound disclosed herein may be provided in any form of the pharmaceutically acceptable salts discussed herein. Similarly, it is understood that the isotopic composition may vary independently of the stereoisomer composition of each compound referred to herein. Furthermore, although the isotopic composition is limited to the elements present in each compound or its salt disclosed herein, it may vary independently of the selection of the pharmaceutically acceptable salt of each compound.

[0038] Please note that if there is a discrepancy between the illustrated structure and its name, the illustrated structure will take precedence.

[0039] As used herein, "activation" means a method by which a biomolecule becomes responsive, active, or effective in performing its function. In one embodiment, the biomolecule is a signaling molecule. In one embodiment, the biomolecule is TRPML1.

[0040] As used herein, the term "agonist" refers to a molecule that can bind to a receptor, activate it, and produce a biological response.

[0041] As used herein, "modulation" means a method of altering the activity of another biomolecule, such that the activity may be increased or decreased.

[0042] The terms “treatment” or “treatment” of a disease or disorder are used interchangeably herein and mean, in the subject, 1) preventing at least one symptom or preventing the recurrence of at least one symptom, 2) preventing the disease or at least one symptom or halting its progression, or 3) improving or alleviating the disease or at least one symptom thereof. Where used herein, “treatment” or “treatment” refers to a method for obtaining a beneficial or desired outcome, including a clinical outcome. For the purposes of this disclosure, beneficial or desired outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms resulting from a disease or disorder; reducing the severity of a disease or disorder; stabilizing a disease or disorder (e.g., preventing or delaying the worsening of a disease or disorder); delaying the onset or recurrence of a disease or disorder; delaying or slowing the progression of a disease or disorder; improving the condition of a disease or disorder; achieving (partial or complete) remission of a disease or disorder; reducing the dose of one or more other drugs required to treat a disease or disorder; enhancing the effect of another drug used to treat a disease or disorder; delaying the progression of a disease or disorder; improving quality of life; and / or extending the survival of the subject. Reduction of the pathological consequences of a disease or disorder is also included in “treatment.” The methods of the present invention aim to achieve one or more of these treatments.

[0043] In relation to the compounds disclosed herein, the term “effective amount” means an amount capable of treating the disorder, disease, condition or symptoms thereof disclosed herein.

[0044] The terms “subject” or “patient,” as used herein, include animals such as cattle, monkeys, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, mammals in one embodiment, and humans in another embodiment. In one embodiment, the subject is a human at risk of having TRPML1-mediated disease or its symptoms.

[0045] Various features of the present invention may be described in the context of a single embodiment, but features can also be provided individually or in any suitable combination. Conversely, the present invention may be described herein in the context of a separate embodiment for clarity, but the present invention can also be implemented in a single embodiment.

[0046] compound In one embodiment, equation (I)

[0047] [ka] Compounds or pharmaceutically acceptable salts thereof (In the formula, X is either N or H. R 1 is selected from H and optionally substituted alkyl groups, R 2 This is selected from optionally substituted alkyl, optionally substituted cycloalkyl, aryl and optionally substituted heterocyclyl, Alternatively, when X is H, R 1 and R 2 It does not exist. R 3 is H, -CH3, or a C2-C6 alkyl group which may be substituted. Or, R 1 and R 3 These, together with the atoms to which they are bonded, form a 5-7 membered ring. Each R 4These are independently H, halo, cyano, or optionally substituted alkyl, R 5a and R 5b Each is independently H or an optionally substituted alkyl, However, R 2 (Provided that it is not pyrrolidine) However, this is provided herein.

[0048] In some embodiments, X is N or H. In some embodiments, X is N. In some embodiments, X is H.

[0049] In some embodiments, R 1 is selected from H and optionally substituted alkyl groups. In some embodiments, R 1 is selected from H and optionally substituted C1-C6 alkyl groups. In some embodiments, R 1 is selected from C1-C6 alkyl groups which may be substituted with H and a 3-6 member heterocycline containing a halo, oxo, or at least O. In some embodiments, R 1 R is selected from C1-C3 alkyl groups which may be substituted with H and a 4-6 member heterocycline containing a halo, oxo, or at least O. In some embodiments, R 1 is selected from C1-C3 alkyl groups which may be substituted with H and a 4-6 member heterocycline containing F, Cl, Br, I, oxo, or at least O. In some embodiments, R 1 This is selected from C1-C3 alkyl groups, which may be substituted with 4-6 membered heterocyclines containing only H and F, oxo, or O.

[0050] In some embodiments, R 1 H is H.

[0051] In some embodiments, R 1 R is an alkyl which may be substituted. In some embodiments, R 1R is a C1-C6 alkylalkyl which may be substituted. In some embodiments, 1 is a C1-C6 alkyl which may be substituted with a halo, oxo, or a 3-6 member heterocycline containing at least O. In some embodiments, R 1 is a C1-C6 alkyl which may be substituted with a 4-6 member heterocycline containing F, Cl, Br, I, oxo, or at least O. In some embodiments, R 1 is a C1-C3 alkyl which may be substituted with a 4-6 member heterocycline containing F, Cl, Br, I, oxo, or at least O. In some embodiments, R 1 is a C1-C3 alkyl which may be substituted with a 4-6 member heterocycline containing only F, oxo, or O. In some embodiments, R 1 teeth

[0052] [ka] That is the case.

[0053] In some embodiments, R 2 R is selected from optionally substituted alkyl, optionally substituted aryl, and optionally substituted heterocyclyl. In some embodiments, R 2 This is selected from optionally substituted C1-C6 alkyl groups, optionally substituted C3-C6 cycloalkyl groups, 6-membered aryl groups, optionally substituted heterocyclines containing at least N, and optionally substituted heterocyclines containing at least O. In some embodiments, R 2This is selected from a C1-C6 alkyl group that may be substituted with a C1-C6 alkyl group, a C3-C6 cycloalkyl group that may be substituted with a C1-C6 alkyl group, a C3-C6 cycloalkyl group that may be substituted with a C1-C6 alkyl group, a C3-C6 cycloalkyl group that may be substituted with a C3-C6 alkyl group, a C3-C6 cycloalkyl group that may be substituted with a C3-C6 alkyl group, and a C1-C6 alkyl group that may be substituted with a C3-C6 alkyl group that contains at least O. In some embodiments, R 2 This is a C1-C5 alkyl group which may be substituted, and a C which may be substituted. 4~6 Selected from cycloalkyls, 6-membered aryls, 6-membered heterocyclines containing at least N which may be substituted with C1-C3 alkyls, -C(O)O(C1-C6 alkyls), -C(O)(C1-C6 alkyls), or -C(O)(C1-C6 cycloalkyls), and 3- to 10-membered heterocyclines containing at least O which may be substituted with C1-C3 alkyls. In some embodiments, R 2 This is selected from optionally substituted C1-C5 alkyl groups, optionally substituted C4-C6 cycloalkyl groups, 6-membered aryl groups, 6-membered heterocyclines containing only N, optionally substituted with -C(O)O(C1-C4 alkyl groups), -C(O)(C1-C4 alkyl groups), or -C(O)(C1-C4 cycloalkyl groups), and 4- to 9-membered heterocyclines containing at least O, optionally substituted with C1-C3 alkyl groups. In some embodiments, R 2 This is selected from optionally substituted C1-C5 alkyl, optionally substituted C4-C6 cycloalkyl, phenyl, a 6-membered heterocycline containing only N, which may be substituted with C1-C3 alkyl, -C(O)O(C1-C4 alkyl), -C(O)(C1-C4 alkyl) or -C(O)(C1-C4 cycloalkyl), and a 4- to 9-membered heterocycline containing only O, which may be substituted with C1-C3 alkyl. In some embodiments, R 2This is selected from 6-membered heterocyclines that may contain only phenyl, C1-C3 alkyl, -C(O)O(C1-C4 alkyl), -C(O)(C1-C4 alkyl), or -C(O)(C1-C4 cycloalkyl), and 4- to 9-membered heterocyclines that contain only O and may be substituted with -CH3.

[0054] In some embodiments, R 2 R is selected from alkyl groups that may be substituted. In some embodiments, R 2 R is selected from C1-C6 alkyl groups, which may be substituted. In some embodiments, R 2 R is selected from C1-C5 alkyl groups, which may be substituted. In some embodiments, 2 teeth

[0055] [ka] [ka] That is the case.

[0056] In some embodiments, R 2 R is a cycloalkyl which may be substituted. In some embodiments, R 2 C may be substituted. 3~6 It is cycloalkyl. In some embodiments, R 2 C may be substituted. 4~6 It is cycloalkyl. In some embodiments, R 2 teeth

[0057] [ka] That is the case.

[0058] In some embodiments, R 2 is an arrow. In some embodiments, R 2is a 6-membered aryl. In some embodiments, R 2 It is phenyl.

[0059] In some embodiments, R 2 is a heterocycline which may be substituted. In some embodiments, R 2 is a heterocycline which may be substituted, containing at least N. In some embodiments, R 2 is a 4-6 member heterocyclyl which contains at least N and may be substituted with C1-C6 alkyl, -C(O)O(C1-C6 alkyl), -C(O)(C1-C6 alkyl), or -C(O)(C1-C6 cycloalkyl). In some embodiments, R 2 is a six-membered heterocycline containing at least N, which may be substituted with C1-C6 alkyl, -C(O)O(C1-C6 alkyl), -C(O)(C1-C6 alkyl), or -C(O)(C1-C6 cycloalkyl). In some embodiments, R 2 is a six-membered heterocycline containing at least N, which may be substituted with C1-C3 alkyl, -C(O)O(C1-C4 alkyl), -C(O)(C1-C4 alkyl) or -C(O)(C1-C4 cycloalkyl). In some embodiments, R 2 is a 6-membered heterocycline containing only N, which may be substituted with C1-C3 alkyl, -C(O)O(C1-C4 alkyl), -C(O)(C1-C4 alkyl), or -C(O)(C1-C4 cycloalkyl). In some embodiments, R 2 teeth

[0060] [ka] That is the case.

[0061] In some embodiments, R 2 is a heterocycline which contains at least O and may be substituted. In some embodiments, R 2is a 3- to 10-member heterocyclyl optionally substituted with C1-C6 alkyl and containing at least O. In some embodiments, R 2 is a 3- to 10-member heterocyclyl optionally substituted with C1-C3 alkyl and containing at least O. In some embodiments, R 2 is a 4- to 9-member heterocyclyl optionally substituted with C1-C3 alkyl and containing at least O. R 2 is a 4- to 9-member heterocyclyl optionally substituted with C1-C3 alkyl and containing only O. In some embodiments, R 2 is a 4- to 9-member heterocyclyl optionally substituted with -CH3 and containing only O. In some embodiments, R 2 is

[0062]

Chemical formula

[0063] In some embodiments, R 3 is H, -CH3 or optionally substituted C2-C6 alkyl. In some embodiments, R 3 is H, -CH3 or optionally substituted C2 alkyl. In some embodiments, R 3 is H. In some embodiments, R 3 is -CH3. In some embodiments, R 3 is optionally substituted C2-C6 alkyl. In some embodiments, R 3 is optionally substituted C2 alkyl.

[0064] In some embodiments, R 1 and R 3 together with the atom to which they are attached form a 5- to 7-member ring. In some embodiments, R 1 and R 3 together with the atom to which they are attached form a 5- to 6-member ring.

[0065] In some embodiments, R4 is H, halo, cyano, or optionally substituted alkyl. In some embodiments, R 4 is H, halo, cyano, or a C1-C6 alkyl which may be substituted. In some embodiments, R 4 is H, F, Cl, Br, I, cyano or possibly substituted C1-C3 alkyl. In some embodiments, R 4 is a C1-C3 alkyl which may be substituted with H, F, Cl, cyano, or halo. In some embodiments, R 4 is a C1-C3 alkyl which may be substituted with H, F, Cl, cyano, or F. In some embodiments, R 4 is -CH3 which may be substituted with H, F, Cl, cyano, or one or more F. In some embodiments, R 4 is H. In some embodiments, R 4 is a halo. In some embodiments, R 4 is F, Cl, Br, or I. In some embodiments, R 4 is a halo. In some embodiments, R 4 is F or Cl. In some embodiments, R 4 is cyano. In some embodiments, R 4 R is an alkyl which may be substituted. In some embodiments, R 4 R is a C1-C6 alkyl which may be substituted. In some embodiments, 4 R is a C1-C3 alkyl which may be substituted. In some embodiments, 4 R is a C1-C3 alkyl which may be substituted with a halo. In some embodiments, R 4 is a C1-C3 alkyl which may be substituted with F. In some embodiments, R 4 This is a CH3 which may be substituted with one or more F atoms.

[0066] In some embodiments, R 5a and R 5b Each is independently H or an alkyl group which may be substituted. In some embodiments, R 5a and R5b Each is independently H or a C1-C6 alkyl group which may be substituted. In some embodiments, R 5a and R 5b Each is independently H or a C1-C3 alkyl group which may be substituted. In some embodiments, R 5a and R 5b Each is independently H or may be substituted -CH3. In some embodiments, R 5a and R 5b Both are -CH3.

[0067] In some embodiments, the compound of formula (I) is of formula (II).

[0068] [ka] (In the formula, R 1 , R 2 and R 4 (This is as described in equation (I)). It is a compound of [the compound].

[0069] In some embodiments, the compound of formula (I) is of formula (III).

[0070] [ka] (In the formula, R 1 and R 2 (This is as described in equation (I)). It is a compound of [the compound].

[0071] In some embodiments, the compound of formula (I) is of formula (IV)

[0072] [ka] (In the formula, R 2 and R 4 (This is as described in equation (I)). It is a compound of [the compound].

[0073] In some embodiments, the compound of formula (I) is of formula (V)

[0074] [ka] (In the formula, R 2 (This is as described in equation (I)). It is a compound of [the compound].

[0075] In the descriptions herein, it is understood that any description, variation, embodiment, or aspect of a part can be combined with any description, variation, embodiment, or aspect of any other part, just as any combination of descriptions is specifically and individually listed. For example, any description, variation, embodiment, or aspect of X in formula (I) provided herein can be combined with any combination of R, just as any combination of X in formula (I) is specifically and individually listed. 1 , R 2 , R 3 and R 4 All descriptions, variations, embodiments, or aspects of formula (I) can be combined with any other description, variation, embodiment, or aspect of formula (I). It is also understood that, where appropriate, all descriptions, variations, embodiments, or aspects of formula (I) are equally applicable to and described with respect to any other formulas detailed herein, just as all descriptions, variations, embodiments, or aspects are listed separately and individually for each formula. For example, all descriptions, variations, embodiments, or aspects of formula (I) are equally applicable to any of the formulas detailed herein, such as formulas (II), (III), (IV), and (V), and are described equally, just as all descriptions, variations, embodiments, or aspects are listed separately and individually for each formula.

[0076] In some embodiments, compounds are provided that are selected from the compounds in Table 1 or pharmaceutically acceptable salts thereof. Certain compounds described in the present disclosure, including Table 1, can exist as specific stereoisomers and / or as non-stereochemical forms, but any and all of the stereochemical forms, including any enantiomeric or diastereomeric form, and any tautomeric or other form, of any of the compounds of the present disclosure (e.g., compounds of Formulas (I), (II), (III), (IV), and (V), and those in Table 1) are described herein.

[0077]

Table 1-1

[0078]

Table 1-2

[0079]

Table 1-3

[0080]

Table 1-4

[0081]

Table 1-5

[0082]

Table 1-6

[0083]

Table 1-7

[0084]

Table 1-8

[0085] Table 1-9

[0086] Table 1-10

[0087] Table 1-11

[0088] Table 1-12

[0089] Table 1-13

[0090] Table 1-14

[0091] Table 1-15

[0092] Table 1-16

[0093] Table 1-17

[0094] Table 1-18

[0095] Table 1-19

[0096] Table 1-20

[0097] Table 1-21

[0098] Table 1-22

[0099] Table 1-23

[0100] Table 1-24

[0101] Table 1-25

[0102] Table 1-26

[0103] Table 1-27

[0104] Table 1-28 or a pharmaceutically acceptable salt thereof. In one embodiment, tautomers, stereoisomers, and deuterated forms of any of the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) are included.

[0105] In this specification, it is understood that combinations of substituents and / or variables in the illustrated formulas are permissible only if such contributions result in a stable compound.

[0106] Furthermore, all compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) existing in free base or acid form can be converted to pharmaceutically acceptable salts thereof by treatment with appropriate inorganic or organic bases or acids using methods known to those skilled in the art. Salts of the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) can be converted to their free base or acid form by standard techniques.

[0107] Synthesis method The compounds described herein can be prepared using conventional organic synthesis and commercially available starting materials, as well as methods provided herein. For example, and without limitation, the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), and those in Table 1) can be prepared as outlined in the examples described herein. It should be noted that those skilled in the art will know how to modify the procedures described in the exemplary examples to obtain the desired products.

[0108] How to use Embodiments of the present disclosure provide a method for modulating TRPML1 in a subject requiring it, comprising the step of administering to the subject an effective amount of a compound of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1). Modulation of TRPML1 (e.g., inhibition or activation) can be evaluated and demonstrated by a wide range of methods known in the art. Published assays can be used to determine whether and to what extent TRPML1 has been modulated.

[0109] In one embodiment, a method for preparing TRPML1 is provided herein, comprising the step of contacting TRPML1 with an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, the compounds of the Disclosure activate TRPML1. In some embodiments, the compounds of the Disclosure are agonists of TRPML1.

[0110] In some embodiments, the compounds of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) adjust the activity of TRPML1 by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) increase the activity of TRPML1 by approximately 1-100%, 5-100%, 10-100%, 15-100%, 20-100%, 25-100%, 30-100%, 35-100%, 40-100%, 45-100%, 50-100%, 55-100%, 60-100%, 65-100%, and 70%. Adjust to ~100%, 75~100%, 80~100%, 85~100%, 90~100%, 95~100%, 5~95%, 5~90%, 5~85%, 5~80%, 5~75%, 5~70%, 5~65%, 5~60%, 5~55%, 5~50%, 5~45%, 5~40%, 5~35%, 5~30%, 5~25%, 5~20%, 5~15%, 5~10%, 10~90%, 20~80%, 30~70%, or 40~60%.

[0111] In certain embodiments of the present disclosure, a method is also provided for activating TRPML1 in a subject requiring such activation, comprising the step of administering an effective amount of the compounds of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to the subject. Activation of TRPML1 can be evaluated and demonstrated by a wide range of methods known in the art. Published assays, including cell-based assays, can be used to determine whether and to what extent TRPML1 has been activated.

[0112] TRPML1 activation can be measured by measuring intracellular calcium influx using the FLIPR Tetra instrument in cells overexpressing TRPML1 on the plasma membrane. Activation can also be measured in cells overexpressing a calcium-sensitive Gcamp protein tag on wild-type TRPML1. Activation can also be measured using various electrophysiological measurements, such as patch-clamping on enlarged lysosomes overexpressing Trpml1.

[0113] In one embodiment, a method for activating TRPML1 is provided herein, comprising the step of contacting TRPML1 with an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, the compounds of the Disclosure partially activate TRPML1. In some embodiments, the compounds of the Disclosure fully activate TRPML1.

[0114] In some embodiments, the compounds of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) activate TRPML1 by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the compounds of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) contain approximately 1-100%, 5-100%, 10-100%, 15-100%, 20-100%, 25-100%, 30-100%, 35-100%, 40-100%, 45-100%, 50-100%, 55-100%, 60-100%, 65-100%, and 70-1% TRPML1. Activates at 00%, 75-100%, 80-100%, 85-100%, 90-100%, 95-100%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-90%, 20-80%, 30-70%, or 40-60%.

[0115] In another embodiment, a method is provided herein for treating a TRPML1-related disease in a subject requiring its use, comprising the step of administering to the subject an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, a method is provided herein for treating a TRPML1-related disease in a subject requiring its use, comprising the step of administering to the subject an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0116] In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects predisposed to TRPML1-related diseases prevents the subjects from developing any symptoms of TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who do not yet show symptoms of TRPML1-related diseases prevents the subjects from developing any symptoms of TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need reduces the severity of TRPML1-related diseases in the subjects. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need stabilizes TRPML1-related disease (prevents or delays the progression of TRPML1-related disease). In some embodiments, administering the compound of formula (I) to subjects in need delays the onset or recurrence of TRPML1-related disease. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need slows the progression of TRPML1-related disease. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need results in partial remission of TRPML1-related disease. In some embodiments, administration of the compounds of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects requiring it results in complete remission of TRPML1-related diseases.In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need reduces the dose of one or more other medications required to treat TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need enhances the effect of other medications used to treat TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need slows the progression of TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need improves the quality of life of subjects with TRPML1-related diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects requiring such intervention extends the survival time of subjects with TRPML1-related diseases. In some embodiments, TRPML1-related diseases include neurodegenerative diseases, lysosomal storage diseases, Charcot-Marie-Tooth disease, mitochondrial diseases, renal diseases, metabolic diseases, cardiovascular diseases, inflammatory disorders, immunological disorders, cancer, or aging. In some embodiments, neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0117] In one embodiment, a method is provided herein for preventing a subject predisposed to a TRPML1-related disease from developing any symptoms of the TRPML1-related disease, comprising the step of administering a compound of the disclosed compound (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to the subject. In some embodiments, a method is provided herein for treating a subject that has not yet shown symptoms of a TRPML1-related disease from developing any symptoms of the TRPML1-related disease, comprising the step of administering a compound of the disclosed compound (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to the subject. In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0118] In some embodiments, a method is provided herein for reducing the severity of a TRPML1-related disease in a subject, comprising the step of administering a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to the subject. In some embodiments, a method is provided herein for stabilizing a TRPML1-related disease in a subject, comprising the step of administering a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to the subject. In some embodiments, the method prevents the worsening of a TRPML1-related disease. In some embodiments, the method delays the worsening of a TRPML1-related disease. In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0119] In another embodiment, a method for delaying the onset or recurrence of a TRPML1-related disease is provided herein, comprising the step of administering a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to a subject. In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-related dementia, Huntington's disease, or Alzheimer's disease (AD).

[0120] In some embodiments, a method is provided herein for slowing the progression of a TRPML1-related disease in a subject, comprising the step of administering a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to the subject. In some embodiments, the method results in partial remission of a TRPML1-related disease. In some embodiments, the method results in complete remission of a TRPML1-related disease. In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0121] In further embodiments, methods are provided herein for reducing the dose of one or more other drugs required to treat a TRPML1-related disease in a subject, comprising the step of administering the subject a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, methods are provided herein for enhancing the effect of another drug used to treat a TRPML1-related disease in a subject, comprising the step of administering the subject a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1). In some embodiments, the TRPML1-related disease is a neurodegenerative disease, a lysosomal storage disease, Charcot-Marie-Tooth disease, a mitochondrial disease, a renal disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging. In some embodiments, the neurodegenerative disease is Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0122] Methods for delaying the progression of TRPML1-related diseases in subjects, comprising the step of administering compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) to subjects, are also provided herein. In some embodiments, the method improves the quality of life of subjects having TRPML1-related diseases. In some embodiments, the method extends the survival time of subjects having TRPML1-related diseases. In some embodiments, TRPML1-related diseases are neurodegenerative diseases, lysosomal storage diseases, Charcot-Marie-Tooth disease, mitochondrial diseases, renal diseases, metabolic diseases, cardiovascular diseases, inflammatory disorders, immunological disorders, cancer, or aging. In some embodiments, neurodegenerative diseases are Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0123] In another embodiment, a method is provided herein for treating TRPML1-related symptoms caused by disease in a subject requiring such treatment, comprising the step of administering to the subject an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1). In some embodiments, a method is provided herein for treating TRPML1-related symptoms caused by disease in a subject requiring such treatment, comprising the step of administering to the subject an effective amount of a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1). In some embodiments, administering a compound of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to a subject predisposed to a disease causing TRPML1-related symptoms prevents the subject from developing any TRPML1-related symptoms. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who do not yet exhibit TRPML1-related symptoms of a disease causing TRPML1-related symptoms prevents the subjects from developing any TRPML1-related symptoms. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who require it reduces the severity of TRPML1-related symptoms caused by the disease in the subjects. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who require it stabilizes TRPML1-related symptoms of the disease (prevents or delays the worsening of TRPML1-related symptoms). In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects requiring such treatment delays the onset or recurrence of TRPML1-related symptoms caused by the disease.In some embodiments, administration of the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need slows the progression of TRPML1-related symptoms caused by the disease. In some embodiments, administration of the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need results in partial remission of the disease causing TRPML1-related symptoms. In some embodiments, administration of the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need results in complete remission of the disease causing TRPML1-related symptoms. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who require them reduces the dose of one or more other medications required to treat diseases that cause symptoms associated with TRPML1. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who require them enhances the effect of other medications used to treat symptoms associated with TRPML1 in diseases. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects who require them delays the progression of diseases that cause symptoms associated with TRPML1. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need improves the quality of life of subjects with diseases that cause symptoms associated with TRPML1. In some embodiments, administering the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) to subjects in need extends the survival time of subjects with diseases that cause symptoms associated with TRPML1. In some embodiments, the disease is associated with TRPML1.In some embodiments, TRPML1-associated diseases include neurodegenerative diseases, lysosomal storage diseases, Charcot-Marie-Tooth disease, mitochondrial diseases, renal diseases, metabolic diseases, cardiovascular diseases, inflammatory disorders, immunological disorders, cancer, or aging. In some embodiments, neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0124] In some embodiments, the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) are useful for treating diseases selected from Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-associated dementia, Huntington's disease, or Alzheimer's disease (AD).

[0125] Pharmaceutical composition and route of administration The compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) can be administered orally, topically, or parenterally to subjects in conventional forms of preparations, such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions.

[0126] The compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) can be administered orally, topically, or parenterally to subjects in conventional forms of preparations, such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable formulations are generally formulated using conventional organic or inorganic additives, such as excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate, or calcium citrate), lubricants (e.g., magnesium stearate, light anhydrous silicic acid, talc, or sodium lauryl sulfate), fragrances (e.g., citric acid, menthol, glycine, or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methylparaben, or propylparaben), stabilizers (e.g., citric acid, sodium citrate, or acetic acid), and suspending agents (e.g., methylcellulose, polyvinylpyrrolidone). It can be prepared using pyrroliclone (or aluminum stearate), a dispersant (e.g., hydroxypropyl methylcellulose), a diluent (e.g., water), and a base wax (e.g., cocoa butter, white petrolatum, or polyethylene glycol). The effective amount of the compound of formula (I) in the pharmaceutical composition may be at a level that produces the desired effect, and may be, for example, about 0.005 mg / kg of body weight to about 10 mg / kg of body weight in a unit dose for both oral and parenteral administration.

[0127] The doses of the compounds of this disclosure administered to subjects (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) can vary somewhat and are left to the discretion of healthcare professionals.

[0128] The compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) can be administered orally for convenience. In one embodiment, when administered orally, the compounds of this disclosure are administered with food and water. In another embodiment, the compounds of this disclosure are dispersed in water or juice (e.g., apple juice or orange juice) or any other liquid and administered orally as a solution or suspension.

[0129] The compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) may also be administered intradermally, intramuscularly, intraperitoneally, transdermally, intravenously, subcutaneously, nasally, epidurally, sublingually, intracerebrally, vaginally, transdermally, rectally, mucosally, by inhalation, or topically to the ear, nose, eye, or skin. The mode of administration is at the discretion of the healthcare professional and depends in part on the site of the medical condition.

[0130] In one embodiment, capsules containing the compounds of the Disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) and having no further carriers, excipients, or vehicles are provided herein.

[0131] In another embodiment, compositions comprising an effective amount of a compound of the present disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) and a pharmaceutically acceptable carrier or vehicle, wherein the pharmaceutically acceptable carrier or vehicle may include excipients, diluents, or mixtures thereof, are provided herein. In one embodiment, the composition is a pharmaceutical composition.

[0132] The compositions may be in the form of tablets, chewable tablets, capsules, solutions, parenteral solutions, lozenges, suppositories, and suspensions. The compositions may be formulated to contain a daily dose or a convenient fraction of a daily dose in a dose unit that may be a single tablet or capsule or a convenient amount of liquid. In one embodiment, the solution is prepared from a water-soluble salt, e.g., hydrochloride. In general, all compositions are prepared according to methods known in medicinal chemistry. Capsules may be prepared by mixing the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those in Table 1) with a suitable carrier or diluent and filling a capsule with an accurate amount of the mixture. Common carriers and diluents include, but are not limited to, inert powdered substances, e.g., many different types of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars, e.g., fructose, mannitol, and sucrose, cereal flour, and similar edible powders.

[0133] Tablets can be prepared by direct compression, wet granulation, or dry granulation. These formulations typically incorporate not only the compound but also diluents, binders, lubricants, and disintegrants. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts such as sodium chloride, and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include, for example, substances such as starch and gelatin, and sugars such as lactose, fructose, and glucose. Natural and synthetic gums are also suitable, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone. Polyethylene glycol, methylcellulose, and waxes also serve as binders.

[0134] Lubricants are necessary in tablet formulation to prevent the tablet and punch from sticking to the mold. Lubricants can be selected from slippery solids such as talc, magnesium stearate and calcium stearate, stearic acid and hydrogenated vegetable oil. Tablet disintegrants are substances that expand when wet, causing the tablet to disintegrate and release the compound. These include starch, clay, cellulose, algin and gum. More specifically, for example, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp and carboxymethylcellulose, as well as sodium lauryl sulfate can be used. Tablets can be coated with flavorings and sugars such as sealants, or film-forming protecting agents to modify the disintegration properties of the tablet. Compositions can also be formulated as chewable tablets by using a substance such as mannitol in the formulation.

[0135] When it is desired to administer the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), as well as those listed in Table 1) as suppositories, typical bases can be used. Cocoa butter is a conventional suppository base, and its melting point can be slightly increased by the addition of wax. Water-miscible suppository bases containing polyethylene glycol of various molecular weights are widely used.

[0136] The effects of the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), and those listed in Table 1) can be delayed or extended by appropriate formulation. For example, slowly dissolving pellets of the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), and those listed in Table 1) can be prepared and incorporated into tablets or capsules, or as slow-release implantable devices. This technique also includes producing pellets with several different dissolution rates and filling capsules with mixtures of pellets. The tablets or capsules can be coated with a film that can withstand dissolution for a predictable period. Even parenteral preparations can be made long-acting by dissolving or suspending the compounds of this disclosure (e.g., compounds of formulas (I), (II), (III), (IV), and (V), and those listed in Table 1) in an oily or emulsified vehicle that allows for slow dissolution in serum. [Examples]

[0137] The following examples are provided for illustrative purposes only, not limitation. Compounds are named using an automated naming tool provided in ChemDraw (Cambridgesoft) that generates systematic names for chemical structures under the support of the Kahn-Ingold-Prelogue order rule for stereochemistry. Those skilled in the art can modify the procedures described in the exemplary examples to obtain the desired products.

[0138] Salts of the compounds described herein can be prepared by standard methods such as including an acid (e.g., TFA, formic acid, or HCl) in the mobile phase during chromatographic purification, or stirring the product with an acid solution (e.g., aqueous HCl solution) after chromatographic purification.

[0139] The following abbreviations may be relevant to this application.

[0140] [Table 2]

[0141] Synthesis example Analysis method Unless otherwise noted, all reagents were used without further purification. 1 1H NMR spectra were obtained in DMSO-d6, CD3OD, or CDCl3 at room temperature using a Bruker 300 MHz or Agilent 400 MHz instrument. If more than one conformational isomer was detected, the chemical shift for the most abundant one was recorded. 1 The chemical shifts of the 1H NMR spectra were recorded in parts per million (ppm) on a delta scale from the internal standard of the residual solvent. The splitting patterns are represented as follows: s, single line; d, double line; t, triple line; q, quadruple line; m, multiple line; br, broad line. The LC-MS and HPLC conditions are shown below.

[0142] The following is a typical LC method for LC / MS.

[0143] General LC method A Column: Agilent Zorbax XDB C18 4.6×50mm, 3.5μm

[0144] Mobile phase A: 0.1% formic acid in water

[0145] Mobile phase B: MeOH

[0146] Flow rate: 1.0 mL / min, Run time: 2 minutes gradient (20% to 90% B), then 3 minutes at 90% B, Column temperature: 30°C.

[0147] General LC method B Column: X Select CSH C18 2.5um; 3.0×50mm

[0148] Mobile phase A: 0.05% FA + 5% ACN in water

[0149] Mobile phase B: 0.05% FA in ACN

[0150] Flow rate: 1.2 mL / min, column temperature: 50°C

[0151] Gradient program (time / B%): 0 / 2, 0.2 / 2, 2.2 / 98, 3 / 98, 3.2 / 2, 4 / 2

[0152] General LC method C Column: X-Bridge BEH C18 (50mm x 3.0mm, 2.5μm)

[0153] Mobile phase A: 2.5 mM ammonium bicarbonate in water + 5% ACN

[0154] Mobile phase B: 100%ACN

[0155] Flow rate: 1.2 mL / min, Column temperature: 50°C

[0156] Gradient program (B%): 0.0 / 0, 1.4 / 100, 2.4 / 100, 2.6 / 0, 3.0 / 0

[0157] The following is a common HPLC method.

[0158] General HPLC method A Column: Agilent SB-C18 4.6×150mm, 3.5μm

[0159] Mobile phase A: 0.02% TFA in water

[0160] Mobile phase B: MeOH

[0161] Flow rate: 1.0 mL / min, execution time: 0.5 min at 10% B, 9.5 min gradient (10% to 90% B), then 10 min at 90% B, temperature: 30°C.

[0162] General HPLC method B Column: X-Select CSH C18 (4.6 × 150) mm 5 μm

[0163] Mobile phase A: 0.1% formic acid in water:acetonitrile (95:05)

[0164] Mobile phase B: Acetonitrile

[0165] Flow rate: 1.0.mL / min,

[0166] Gradient program time (minutes) / B concentration: 0.01 / 5, 1.0 / 5, 8.0 / 100, 12.0 / 100, 14.0 / 5, 18.0 / 5

[0167] General HPLC method C Column: X-Bridge C18 (4.6 × 150) mm 5 μm,

[0168] Mobile phase A: 5 mM ammonium bicarbonate in water

[0169] Mobile phase B: Acetonitrile, Injection volume: 5.0 μL,

[0170] Flow rate: 1.0mL / min,

[0171] Gradient program: Time (minutes) / B concentration: 0.01 / 5, 1.0 / 5, 8.0 / 100, 12.0 / 100, 14.0 / 5, 18.0 / 5

[0172] General HPLC method D Column: X-Select CSH C18 (4.6 × 150) mm 5 μm

[0173] Mobile phase A: 0.1% TFA in water,

[0174] Mobile phase B: Acetonitrile, Injection volume: 5.0 μL,

[0175] Flow rate: 1.2mL / min,

[0176] Gradient program: Time (minutes) / B concentration: 0.01 / 5, 1.0 / 5, 8.0 / 100, 12.0 / 100, 14.0 / 5, 18.0 / 5

[0177] General HPLC method E Column: X-Select CSH C18 (4.6 × 150) mm 5u,

[0178] Mobile phase A: 0.02% TFA in water

[0179] Mobile phase B: MeOH

[0180] Flow rate: 1.0mL / min,

[0181] Execution time: 0.5 minutes at 10%B, 9.5 minutes gradient (10% to 90%B), then 10 minutes at 90%B.

[0182] Temperature: 30℃.

[0183] The following is a general preparative LC method.

[0184] General preparative LC method Column: Phenomenex Luna 5u 100A, 21.2 × 250 mm, 5 μm

[0185] Mobile phase A: water

[0186] Mobile phase B: MeOH

[0187] Flow rate: 10 mL / min, Runtime: 1 minute at 20%B gradient, 30 minutes gradient (20% to 80%B), then 10 minutes at 90%B gradient, Temperature: Ambient temperature

[0188] Preparation of common intermediates The reference to a specific intermediate compound with a number such as 1 or 2 is specific to the example in which it is described. Therefore, multiple examples may refer to the same intermediate compound number, e.g., 1 or 2, but the chemical structure of the compound will differ in each example.

[0189] [Example I-1] Synthesis of common intermediate 1

[0190] [ka]

[0191] Common intermediate 1 was prepared according to the procedure described in International Publication No. 2017 / 141049 and International Publication No. 2018 / 005713.

[0192] [Example I-2] Synthesis of common intermediate 2

[0193] [ka]

[0194] To a solution of benzene-1,2-diamine (620 mg, 5.74 mmol) in THF (50 mL), common intermediate 1 (1.30 g, 4.59 mmol) and pyridine (1.82 g, 22.96 mmol) were added. The reaction mixture was stirred overnight at 65°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (50 mL) and extracted with siRNA (70 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The resulting solid was purified by silica gel chromatography (DCM = 100%) to obtain 1.3 g of common intermediate 2. The yield was 63.8%.

[0195] LCMS[M+H] + C 14 H 18 The calculated value for N3O4S2 is 356.07; the measured value is 356.

[0196] [Example I-3] Synthesis of common intermediate 3

[0197] [ka]

[0198] N-methyl-2-nitroaniline (1)

[0199] [ka]

[0200] To a solution of 1-fluoro-2-nitrobenzene (SM) (5 g, 35.46 mmol) in EtOH (37.5 mL), 33% MeNH2 in ethanol (8 mL, 85.2 mmol) was slowly added at 0°C. The resulting reaction mixture was heated at 80°C and stirred for 16 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under vacuum. The crude product was dissolved in Depositphotos (50 mL) and washed with water (2 × 100 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 5.1 g of the title compound. The yield was 95%.

[0201] LCMS[M+H] + The calculated value for C7H9N2O2 is 153.06; the measured value is 153.11.

[0202] Tert-butylmethyl(2-nitrophenyl)carbamate(2)

[0203] [ka]

[0204] To a stirred solution of N-methyl-2-nitroaniline (1) (5.6 g, 36.84 mmol) in THF (56 mL), DMAP (449 mg, 3.68 mmol) and Boc2O (25 mL, 110.52 mmol) at room temperature were added. The resulting reaction mixture was refluxed for 16 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (50 mL) and extracted with siRNA (2 × 100 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography with elution of 10-20% siRNA in heptane to obtain 4.5 g of the title compound. The yield was 48%.

[0205] 1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 7.8 Hz, 1H), 7.79 - 7.68 (m, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 3.21 (s, 3H), 1.22 (s, 9H); LCMS [M-Boc+H] + Calculated value of C7H9N2O2: 153.06; Measured value: 153.2.

[0206] Tert-butyl(2-aminophenyl)(methyl)carbamate(3)

[0207] [ka]

[0208] To a stirred solution of tert-butylmethyl(2-nitrophenyl)carbamate (2) (4.5 g, 17.85 mmol) in EtOH:H2O (60 mL, 5:1), Zn powder (11.6 g, 178.5 mmol) and NH4Cl (9.5 g, 177.6 mmol) were added at 0°C. The reaction mixture was heated at 80°C and stirred for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was diluted with water (100 mL) and extracted with siRNA (3 × 100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 3.2 g of the title compound. The yield was 80.6%.

[0209] 1 H NMR (400 MHz, DMSO-d6) δ 6.99 - 6.83 (m, 2H), 6.69 (d, J = 7.8 Hz, 1H), 6.50 (t, J = 7.1 Hz, 1H), 3.32 (s, 2H), 2.98 (s, 3H), 1.29 (br s, 9H); LCMS [M-Boc+H] + C7H 11 Calculated value of N2: 123.08; Measured value: 123.2.

[0210] Tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)carbamate(4)

[0211] [ka]

[0212] To a stirred solution of tert-butyl(2-aminophenyl)(methyl)carbamate (3) (2 g, 9.01 mmol) in CH2Cl2 (20 mL), pyridine (1.5 mL, 18.6 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (3 g, 10.6 mmol) were added at 0°C. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (5 mL) and extracted with ELISA (2 × 30 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 3.2 g of the title compound. The yield was 76%.

[0213] 1 LCMS [M-Boc+H] + C 15 H 20 Calculated value of N3O4S2: 370; Measured value: 370.3.

[0214] N1,N1-dimethyl-N4-(2-(methylamino)phenyl)benzene-1,4-disulfonamide (common intermediate 3)

[0215] [ka]

[0216] To a stirred solution of tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)carbamate (4) (1.5 g, 3.19 mmol) in CH2Cl2 (10 mL), 4 M HCl in dioxane (15 mL) was added at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under reduced pressure. The crude product was pulverized with diethyl ether (2 × 10 mL) and dried under vacuum to obtain 1.1 g of the title compound. The yield was 93%.

[0217] 1 H NMR (400 MHz, DMSO-d6) δ 9.80 - 9.43 (br s, 1H), 7.93 - 7.86 (m, 4H), 7.09 - 7.01 (m, 1H), 6.56 (dd, J = 1.5, 7.8 Hz, 1H), 6.50 (dd, J = LCMS [M+H] + C 15 H 20 Calculated value of N3O4S2: 370.08; Measured value: 370.55.

[0218] [Example I-4] Synthesis of common intermediate 3

[0219] [ka]

[0220] Tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)carbamate(1)

[0221] [ka]

[0222] A solution of SM (2 g, 9.01 mmol) in CH2Cl2 (20 mL) was added at 0°C to pyridine (1.5 mL, 18.6 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (3 g, 10.6 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (5 mL) and extracted with ELISA (2 × 30 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 3.2 g of the title compound. The yield was 76%.

[0223] 1 LCMS [M-Boc+H] + C 15 H 20 Calculated value of N3O4S2: 370; Measured value: 370.3

[0224] N1,N1-dimethyl-N4-(2-(methylamino)phenyl)benzene-1,4-disulfonamide (common intermediate 3)

[0225] [ka]

[0226] A solution of intermediate 1 (1.5 g, 3.19 mmol) in CH2Cl2 (10 mL) was added to 4 M HCl in dioxane (15 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under reduced pressure. The crude product was pulverized with diethyl ether (2 × 10 mL) and dried under vacuum to obtain 1.1 g of the title compound. The yield was 93%.

[0227] 1H NMR (400 MHz, DMSO-d6) δ 9.80 - 9.43 (br s, 1H), 7.93 - 7.86 (m, 4H), 7.09 - 7.01 (m, 1H), 6.56 (dd, J = 1.5, 7.8 Hz, 1H), 6.50 (dd, J = 1.1, 8.3 Hz, 1H), 6.41 (td, J = 1.3, 7.5 Hz, 1H), 5.47 - 5.42 (br s, 1H), 2.62 (s, 6H), 2.57 (s, 3H); LCMS [M+H] + C 15 H 20 Calculated value of N3O4S2: 370; Measured value: 370.55

[0228] [Example I-5] Synthesis of common intermediate 4

[0229] [ka]

[0230] 2-Chloro-N-methyl-6-nitroaniline (1)

[0231] [ka]

[0232] To a solution of 1-chloro-2-fluoro-3-nitrobenzene SM (5 g, 28.57 mmol) in ethanol (10 mL), methylamine (2.6 g, 85.71 mmol) was added at 0°C. The reaction mixture was allowed to cool to room temperature and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30% siRNA in hexane by medium-pressure liquid chromatography to obtain 4 g of the title compound. The yield was 45%.

[0233] Tert-butyl(2-chloro-6-nitrophenyl)(methyl)carbamate(2)

[0234] [ka]

[0235] To a solution of intermediate 1 (1.2 g, 7.05 mmol) in THF (10 mL), DMAP (0.07 g, 0.705 mmol) and Boc anhydrous (5.9 g, 21.15 mmol) were added at 0°C. The reaction mixture was heated to 85°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was concentrated under reduced pressure to obtain 800 mg of crude product. The crude product was used directly in the next step without further purification or analysis.

[0236] Tert-butyl(2-amino-6-chlorophenyl)(methyl)carbamate(3)

[0237] [ka]

[0238] To a solution of intermediate 2 (1 g, 3.48 mmol) in a mixture of ethyl acetate and water (2 mL, 5:1), NH4Cl (1.7 g, 32.01 mmol) and iron powder (1.92 g, 34.8 mmol) were added at room temperature. The reaction mixture was heated to 85°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 800 mg of crude product, which was used in the next step without further purification or analysis.

[0239] Tert-butyl(2-chloro-6-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)carbamate(4)

[0240] [ka]

[0241] A solution of intermediate 3 (280 mg, 0.55 mmol) in DCM (10 mL) was added at room temperature to pyridine (1.5 mL, 10.44 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (1.18 g, 4.17 mmol) for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with DCM and water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30% siRNA in hexane by medium-pressure liquid chromatography to obtain 800 mg of the title compound. The yield was 45%.

[0242] N1-(3-chloro-2-(methylamino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (common intermediate 4)

[0243] [ka]

[0244] A solution of intermediate 4 (280 mg, 0.55 mmol) in CH2Cl2 (10 mL) was added to 4 M HCl in dioxane (10 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was washed with DCM and concentrated under reduced pressure. The crude product was ground with pentane and dried under red light to obtain 130 mg of the title compound. The yield was 59%.

[0245] LCMS[M+H]+ C 15 H 19 The calculated value for ClN3O4S2 is 404.04; the measured value is 404.3.

[0246] [Example I-6] Synthesis of common intermediate 5

[0247] [ka]

[0248] N,2-dimethyl-6-nitroaniline(1)

[0249] [ka]

[0250] To a solution of 2-fluoro-1-methyl-3-nitrobenzene SM (2 g, 12.90 mmol) in DMSO (20 mL), methylamine (1.45 mL, 15.48 mmol) was added at 0°C. The reaction mixture was heated to 80°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 1.07 g, which was used directly in the next step without purification.

[0251] LCMS[M+H]+ C8H 11 Calculated value for N2O2: 167.07; measured value: 167.02.

[0252] Tert-butylmethyl(2-methyl-6-nitrophenyl)carbamate(2)

[0253] [ka]

[0254] To a solution of intermediate 1 (2.2 g, 13.25 mmol) in THF (22 mL), DMAP (0.16 g, 1.32 mmol) and anhydrous Boc (12.5 mL, 39.76 mmol) were added at 0°C. The reaction mixture was heated to 80°C and stirred for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by medium-pressure liquid chromatography and eluted with 80-90% ethyl acetate in heptane to obtain 0.55 g. The yield was 15%.

[0255] LCMS[MH]+ C 13 H 17 Calculated value for N2O4: 265.13; measured value: 265.30.

[0256] Tert-butyl(2-amino-6-methylphenyl)(methyl)carbamate(3)

[0257] [ka]

[0258] To a solution of intermediate 2 (0.55 g, 2.06 mmol) in a mixture of ethyl acetate and water (4:1), NH4Cl (1.1 g, 20.67 mmol) and zinc powder (1.3 g, 20.67 mmol) were added at room temperature. The reaction mixture was heated to 80°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layers were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain 0.4 g of the title compound. The yield was 83%.

[0259] LCMS C 13 H 20 Calculated value for N2O2: 236.15; measured value: [M-Boc+H]+ 137.03.

[0260] Tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)-6-methylphenyl)(methyl)carbamate(4)

[0261] [ka]

[0262] A solution of compound 3 (0.4 g, 1.69 mmol) in DCM (4 mL) was added at 0°C to pyridine (0.4 mL, 5.08 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (0.57 g, 2.04 mmol). The reaction mixture was allowed to rise to room temperature and stirred for 1 hour. After the reaction was complete as shown by TLC, the reaction mixture was extracted with water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by elution with 50-60% ethyl acetate in heptane by medium-pressure liquid chromatography to obtain 0.4 g of the title compound. The yield was 50%.

[0263] N1,N1-dimethyl-N4-(3-methyl-2-(methylamino)phenyl)benzene-1,4-disulfonamide (common intermediate 5)

[0264] [ka]

[0265] A solution of compound 4 (0.4 g, 0.83 mmol) in CH2Cl2 (4 mL) was added to 4 M HCl in dioxane (2 mL) at 0°C. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under reduced pressure. The crude product was ground with pentane and dried under reduced pressure to obtain 0.28 g of the title compound. The yield was 90%.

[0266] LCMS[MH]+ C 16 H 22Calculated value for N3O4S2: 384.10; Measured value: 384.3

[0267] [Example I-7] Synthesis of common intermediate 6

[0268] [ka]

[0269] 2-Fluoro-N-methyl-6-nitroaniline (1)

[0270] [ka]

[0271] To a solution of 1,2-difluoro-3-nitrobenzene SM (1 g, 6.29 mmol) in ethanol (5 mL), methylamine (0.58 g, 18.87 mmol) was added at 0°C. The reaction mixture was allowed to cool to room temperature and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 10% siRNA in hexane by medium-pressure liquid chromatography to obtain 1.2 g of crude, which was used directly in the next step without analysis.

[0272] Tert-butyl(2-fluoro-6-nitrophenyl)(methyl)carbamate(2)

[0273] [ka]

[0274] To a solution of intermediate 1 (1.2 g, 7.05 mmol) in THF (10 mL), DMAP (0.07 g, 0.705 mmol) and Boc anhydrous (5.9 g, 21.15 mmol) were added at 0°C. The reaction mixture was heated to 85°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was concentrated under reduced pressure to obtain 800 mg of crude product. The crude product was used directly in the next step without further purification or analysis.

[0275] Tert-butyl(2-amino-6-fluorophenyl)(methyl)carbamate(3)

[0276] [ka]

[0277] A solution of intermediate 2 (500 mg, 1.85 mmol) in a mixture of ethyl acetate and water (5 mL, 4:1) was added to NH4Cl (294 mg, 5.55 mmol) and zinc powder (1.20 g, 185 mmol) at room temperature. The reaction mixture was heated to 85°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with ethyl acetate and water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30% siRNA in hexane by medium-pressure liquid chromatography to obtain 400 mg of the title compound. The yield was 90%.

[0278] LCMS[M-Boc+H]+ C 12 H 17 Calculated value for FN2O2: 240.13; measured value: 140.9.

[0279] Tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)-6-fluorophenyl)(methyl)carbamate(4)

[0280] [ka]

[0281] A solution of compound 3 (400 mg, 1.66 mmol) in DCM (5 mL) was added at 0°C to pyridine (394 mg, 4.99 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (567 mg, 1.99 mmol). The reaction mixture was allowed to rise to room temperature and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was extracted with water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30% ethyl acetate in hexane by medium-pressure liquid chromatography to obtain 150 mg of the title compound. The yield was 18%.

[0282] N1-(3-fluoro-2-(methylamino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (common intermediate 6)

[0283] [ka]

[0284] A solution of compound 4 (150 mg, 0.30 mmol) in CH2Cl2 (5 mL) was added to 4 M HCl in dioxane (5 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was washed with DCM and concentrated under reduced pressure. The crude product was ground with pentane and dried under reduced pressure to obtain 110 mg of the title compound. The yield was 91%.

[0285] LCMS[M+H] + C 15 H 19 Calculated value for FN3O4S2: 388; Measured value: 388.3.

[0286] [Examples I-8] Synthesis of common intermediate 7

[0287] [ka]

[0288] N1-Cyclohexylbenzene-1,2-diamine(1)

[0289] [ka]

[0290] A solution of N-cyclohexyl-2-nitroaniline SM (1 g, 4.54 mmol) in a mixture of ethanol and water (10 mL, 8:2) was added to Zn powder (1.47 g, 22.7 mmol) and NH4Cl (2.4 g, 45.4 mmol) at 0°C. The reaction mixture was heated to 80°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water and extracted with DCM (2 × 25 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 700 mg of the title compound. The yield was 81%.

[0291] LCMS[M+H]+ C 12 H 19 Calculated value for N2: 191; measured value: 191.37.

[0292] N1-(2-(cyclohexylamino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (common intermediate 7)

[0293] [ka]

[0294] A solution of intermediate 1 (600 mg, 3.16 mmol) in DCM (6 mL) was added at 0°C to pyridine (0.6 mL, 7.9 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride, and to the common intermediate 1 (983 mg, 3.47 mmol). The reaction mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water and extracted with DCM (2 × 25 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by medium-pressure liquid chromatography and eluted with 20% ethyl acetate in heptane to obtain 1 g of the title compound. The yield was 72%.

[0295] LCMS[M+H]+ C 20 H 28 Calculated value for N3O4S2: 438; measured value: 438.4.

[0296] [Example S1] Synthesis of compound 20

[0297] [ka]

[0298] Step 1

[0299] [ka]

[0300] To a solution of common intermediate 2 (300 mg, 0.84 mmol) in MeOH (4 mL) and THF (4 mL), 3,3-dimethylcyclopentanone (1) (188 mg, 1.68 mmol), NaBH3CN (211 mg, 3.36 mmol), and 3 drops of AcOH were added. The reaction mixture was stirred overnight at 50°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (20 mL) and extracted with SiO2 (25 mL x 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting solid was purified by silica gel chromatography (PE:SiO2 = 10:1 to 5:1) to obtain 236 mg of N1-(2-((3,3-dimethylcyclopentyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (2). The yield was 62.3%.

[0301] LCMS[M+H] + C 21 H 30 Calculated value for N3O4S2: 452.16; measured value: 452.

[0302] Step 2

[0303] [ka]

[0304] To a solution of N1-(2-((3,3-dimethylcyclopentyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (2) (250 mg, 0.55 mmol) in MeOH (4 mL) and DCM (4 mL), formaldehyde (180 mg, 2.22 mmol, 37%), NaBH3CN (141 mg, 2.22 mmol) and 2 drops of AcOH were added. The reaction mixture was stirred at ambient temperature for 6 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (20 mL) and extracted with DCM (30 mL x 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 250 mg of crude 4-((3-(3,3-dimethylcyclopentyl)-2,3-dihydro-1H-benzo[d]74imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (3). The crude title compound was used in the next step without purification.

[0305] LCMS[M+H] + C 22 H 30 Calculated value for N3O4S2: 464.16; measured value: 464.

[0306] Step 3

[0307] [ka]

[0308] To a solution of 4-((3-(3,3-dimethylcyclopentyl)-2,3-dihydro-1H-benzo[d]75imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (3) (250 mg, 0.55 mmol) in MeOH (10 mL), NaBH4 (141 mg, 3.72 mmol) was added in four portions over 30 minutes. The reaction mixture was stirred at ambient temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (20 mL) and extracted with siRNA (20 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting oily substance was purified by silica gel chromatography to obtain the impure title compound. The impure title compound was further purified by preparative HPLC to obtain 68.3 mg of N1-(2-((3,3-dimethylcyclopentyl)(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 20). The yield was 26.7%.

[0309] 1 HNMR (300 MHz, CDCl3) δ 8.55 (br s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 6.0 Hz, 1H), 7.18-7.01 (m, 3H), 3.31 (m, 1H), 2.70 (s, 6H), 2.22 (s, 3H), 1.69 (m, 1H), 1.55 (m, 1H), 1.40 (m, 1H), 1.38 (m, 1H), 1.20 (m, 1H), 1.09 (m, 1H), 1.00 (s, 3H), 0.92 (s, 3H).LCMS [M+H] + C 22 H 32 Calculated value of N3O4S2: 466.18; Measured value: 466.

[0310] [Example S2] Synthesis of Compound 27

[0311] [ka]

[0312] Step 1

[0313] [ka]

[0314] Under nitrogen protection, NaH (168.04 mg, 4.20 mmol, 60%) was gradually added over 2 minutes to a solution of 2-nitro-N-phenylaniline (1) (300 mg, 1.40 mmol) in DMF (15 mL). The resulting mixture was stirred at room temperature for 10 minutes, after which iodomethane (397.55 mg, 2.80 mmol) was added to the reaction mixture, and the reaction was continued overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with water (30 mL) and extracted with siRNA (30 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 50:1) to obtain 300 mg (1.31 mmol) of N-methyl-2-nitro-N-phenylaniline (2). The yield was 93.6%.

[0315] LCMS[M+H] + C 13 H 13 Calculated value for N2O2: 229.09; measured value: 229.

[0316] Step 2

[0317] [ka]

[0318] To a solution of N-methyl-2-nitro-N-phenylaniline (2) (300 mg, 1.31 mmol) in IPA (15 mL), Pd / C (50 mg, 10%) was added. The suspension was drained, and the mixture was then refilled three times with hydrogen. The reaction mixture was then stirred overnight under hydrogen (balloon) pressure. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad, and the filtered cake was washed with IPA (10 mL). The combined filtrate was concentrated under reduced pressure to obtain 220 mg of crude N1-methyl-N1-phenylbenzene-1,2-diamine (3). The yield was 84.4%.

[0319] LCMS[M+H] + C 13 H 15 Calculated value for N2: 199; Measured value: 199

[0320] Step 3

[0321] [ka]

[0322] To a solution of N1-methyl-N1-phenylbenzene-1,2-diamine (3) (100 mg, 0.50 mmol) in THF (5 mL), common intermediate 1 (171.74 mg, 0.60 mmol) and pyridine (119.69 mg, 1.51 mmol) were added. The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (20 mL) and extracted with SiO2 (20 mL x 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the impure title compound. The impure product was further purified by preparative HPLC to obtain 77.7 mg of N1,N1-dimethyl-N4-(2-(methyl(phenyl)amino)phenyl)benzene-1,4-disulfonamide (compound 27). The yield was 34.6%.

[0323] 1HNMR (300 MHz, CDCl3) δ 7.87-7.74 (m, 5H), 7.35-7.30 (m, 2H), 7.17-7.05 (m, 4H), 6.84 (t, J = 7.2 Hz, 1H), 6.34 (d, J = 7.8 Hz, 1H), 2.92 (s, 3H), 2.73 (s, 6H). LCMS [M+H] + C 21 H 24 Calculated value of N3O4S2: 446.11; Measured value: 445.9.

[0324] [Example S3] Synthesis of Compound 26

[0325] [ka]

[0326] Step 1

[0327] [ka]

[0328] To a solution of 1-fluoro-2-nitrobenzene (1) (350 mg, 2.48 mmol) in DMF (25 mL), N-methyl-1-phenylmethaneamine (2) (902 mg, 7.44 mmol) and K2CO3 (1028 mg, 7.44 mmol) were added. The reaction mixture was stirred at 100°C for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (50 mL) and extracted with siRNA (40 mL x 2). The combined organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The remaining oily substance was purified by silica gel chromatography (PE:siRNA = 60:1) to obtain 560 mg of N-benzyl-N-methyl-2-nitroaniline (3). The yield was 93.3%.

[0329] 1HNMR (300 MHz, CDCl3) δ 7.76 (d, J = 8.1 Hz, 1H), 7.46-7.20 (m, 6H), 7.07 (d, J = 8.1 Hz, 1H), 6.89 (t, J = 7.8 Hz, 1H), 4.38 (s, 2H), 2.78 (s, 3H).

[0330] Step 2

[0331] [ka]

[0332] To a solution of N-benzyl-N-methyl-2-nitroaniline (3) (400 mg, 1.65 mmol) in saturated aqueous NH4Cl (5 mL), THF (5 mL), and EtOH (5 mL), Fe powder (370 mg, 6.60 mmol) was added in one step. The reaction mixture was stirred at 70°C for 2 hours. After the reaction was complete as shown by TLC, the suspension was filtered to remove any undissolved solids. The filtrate was quenched with water (30 mL) and extracted with siRNA (40 mL × 2). The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 10:1~20:1) to obtain 170 mg of N1-benzyl-N1-methylbenzene-1,2-diamine (4). The yield was 48.6%.

[0333] LCMS[M+H] + C 14 H 17 Calculated value for N2: 213.13; measured value: 213.

[0334] Step 3

[0335] [ka]

[0336] Compound 26 was synthesized using N1-benzyl-N1-methylbenzene-1,2-diamine (4) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0337] 1 HNMR (300 MHz, CDCl3) δ 8.28 (br s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.31-7.26 (m, 3H), 7.18-7.03 (m, 5H), 3.67 (s, 2H), 2.67 (s, 6H), 2.38 (s, 3H). LCMS [M+H] + C 22 H 26 Calculated value of N3O4S2: 460.13; Measured value: 459.8.

[0338] [Example S4] Synthesis of compound 23

[0339] [ka]

[0340] Step 1

[0341] [ka]

[0342] N-(cyclohexylmethyl)-2-nitroaniline (3) was synthesized from the starting materials (1) and (2) in the above scheme according to the procedure of step 1 in the preparation of compound 27.

[0343] LCMS[M+H] + C 13 H 19 Calculated value for N2O2: 235.14; measured value: 235.

[0344] Step 2

[0345] [ka]

[0346] N-(cyclohexylmethyl)-N-methyl-2-nitroaniline (4) was synthesized from N-(cyclohexylmethyl)-2-nitroaniline (3) according to the procedure of Step 1 in the preparation of compound 27.

[0347] LCMS[M+H] + C 14 H 21 Calculated value for N2O2: 249.15; measured value: 249.

[0348] Step 3

[0349] [ka]

[0350] N1-(cyclohexylmethyl)-N1-methylbenzene-1,2-diamine (5) was synthesized from N-(cyclohexylmethyl)-N-methyl-2-nitroaniline (4) according to the procedure of step 2 in the preparation of compound 27.

[0351] LCMS[M+H] + C 14 H 23 Calculated value for N2: 219; measured value: 219

[0352] Step 4

[0353] [ka]

[0354] N1-(2-((cyclohexylmethyl)(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 23) was synthesized from N1-(cyclohexylmethyl)-N1-methylbenzene-1,2-diamine (5) and common intermediate 1 following the procedure of step 3 in the preparation of compound 27.

[0355] 1 HNMR (300 MHz, CDCl3) δ 8.35 (br s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.87 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 7.2 Hz, 1H), 7.12-7.01 (m, 3H), 2.70 (s, 6H), 2.47 (d, J = 7.2 Hz, 2H), 2.32 (s, 3H), 1.72-1.55 (m, 5H), 1.39-1.36 (m, 1H), 1.15-1.13 (m, 3H), 0.83-0.70 (m, 2H).LCMS [M+H] + C 22 H 32 Calculated value of N3O4S2: 466.18; Measured value: 466.3.

[0356] [Example S5] Synthesis of Compound 25

[0357] [ka]

[0358] Step 1

[0359] [ka]

[0360] N-(cyclopentylmethyl)-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0361] LCMS[M+H] + C 12 H 17 Calculated value for N2O2: 221.12; measured value: 221.

[0362] Step 2

[0363] [ka]

[0364] N-(cyclopentylmethyl)-N-methyl-2-nitroaniline (4) was synthesized from N-(cyclopentylmethyl)-2-nitroaniline (3) according to the procedure of Step 1 in the preparation of compound 27.

[0365] LCMS[M+H] + C 13 H 19 Calculated value for N2O2: 235.14; measured value: 235.

[0366] Step 3

[0367] [ka]

[0368] N1-(cyclopentylmethyl)-N1-methylbenzene-1,2-diamine (5) was synthesized from N-(cyclopentylmethyl)-N-methyl-2-nitroaniline (4) according to the procedure of step 2 in the preparation of compound 27.

[0369] LCMS[M+H] + C 13 H 21 Calculated value for N2: 205.16; measured value: 205.

[0370] Step 4

[0371] [ka]

[0372] N1-(2-((cyclopentylmethyl)(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 25) was synthesized from N1-(cyclopentylmethyl)-N1-methylbenzene-1,2-diamine (5) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0373] 1 HNMR (300 MHz, CDCl3) δ 8.40 (br s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.61-7.58 (m, 1H), 7.14-7.01 (m, 3H), 2.71 LCMS [M+H] + C 21 H 30 Calculated value of N3O4S2: 452.16; Measured value: 451.9.

[0374] [Example S6] Synthesis of compound 28

[0375] [ka]

[0376] Step 1

[0377] [ka]

[0378] N-((1r,4r)-4-methylcyclohexyl)-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0379] LCMS[M+H] + C 13 H 19 Calculated value for N2O2: 235.14; measured value: 235.

[0380] Step 2

[0381] [ka]

[0382] N-methyl-N-((1r,4r)-4-methylcyclohexyl)-2-nitroaniline (4) was synthesized from N-((1r,4r)-4-methylcyclohexyl)-2-nitroaniline (3) according to the procedure of Step 1 in the preparation of compound 27.

[0383] LCMS[M+H] + C 14 H 21 Calculated value for N2O2: 249.15; measured value: 249.

[0384] Step 3

[0385] [ka]

[0386] N1-methyl-N1-((1r,4r)-4-methylcyclohexyl)benzene-1,2-diamine (5) was synthesized from N-methyl-N-((1r,4r)-4-methylcyclohexyl)-2-nitroaniline (4) according to the procedure of step 2 in the preparation of compound 27.

[0387] LCMS[M+H] + C 14 H 23 Calculated value for N2: 219.18; measured value: 219.

[0388] Step 4

[0389] [ka]

[0390] N1,N1-dimethyl-N4-(2-(methyl((1r,4r)-4-methylcyclohexyl)amino)phenyl)benzene-1,4-disulfonamide (compound 28) was synthesized from N1-methyl-N1-((1r,4r)-4-methylcyclohexyl)benzene-1,2-diamine (5) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0391] 1 HNMR (300 MHz, CDCl3) δ 8.51 (br, 1H) 7.99 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 7.01-7.16 (m, 3H), 2.70 LCMS [M+H] + C 22 H 32 Calculated value of N3O4S2: 466.18; Measured value: 465.9.

[0392] [Example S7] Synthesis of compound 29

[0393] [ka]

[0394] Step 1

[0395] [ka]

[0396] N1-(2-((4,4-dimethylcyclohexyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (2) was synthesized from starting material (1) and common intermediate 2 according to the procedure of step 1 in the preparation of compound 20.

[0397] LCMS[M+H] + C 22 H 32 Calculated value for N3O4S2: 466; Measured value: 466.

[0398] Step 2

[0399] [ka]

[0400] 4-((3-(4,4-dimethylcyclohexyl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (3) was synthesized from N1-(2-((4,4-dimethylcyclohexyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (2) according to the procedure of step 2 in the preparation of compound 20.

[0401] LCMS[M+H] + C 23 H 32 Calculated value for N3O4S2: 478; Measured value: 478

[0402] Step 3

[0403] [ka]

[0404] N1-(2-((4,4-dimethylcyclohexyl)(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 29) was synthesized from 4-((3-(4,4-dimethylcyclohexyl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (3) according to the procedure of step 3 in the preparation of compound 20.

[0405] 1 HNMR (300 MHz, CDCl3) δ 8.50 (br, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 7.5 Hz, 1H), 7.19-7.01 (m, 3H), 2.70 (s, 6H), 2.38 (m, 1H), 2.39 (s, 3H), 1.51-1.40 (m, 2H), 1.39-1.22 (m, 2H), 1.20-0.98 (m, 4H), 0.86 (s, 3H), 0.84 (s, 3H).LCMS [M+H] + C 23 H 34 Calculated value of N3O4S2: 480.19; Measured value: 479.9.

[0406] [Example S8] Synthesis of Compound 17

[0407] [ka]

[0408] Step 1

[0409] [ka]

[0410] N-(1-methylcyclohexyl)-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0411] LCMS[M+H] + C 13 H 18 Calculated value for N2O2: 235.14; measured value: 235.

[0412] Step 2

[0413] [ka]

[0414] N1-(1-methylcyclohexyl)benzene-1,2-diamine (4) was synthesized from N-(1-methylcyclohexyl)-2-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 27.

[0415] LCMS[M+H] + C 13 H 21 Calculated value for N2: 205.16; measured value: 205.

[0416] Step 3

[0417] [ka]

[0418] N1,N1-dimethyl-N4-(2-((1-methylcyclohexyl)amino)phenyl)benzene-1,4-disulfonamide (5) was synthesized from N1-(1-methylcyclohexyl)benzene-1,2-diamine (4) and common intermediate 1 following the procedure of step 3 in the preparation of compound 27.

[0419] LCMS[M+H] + C 21 H 30 Calculated value for N3O4S2: 452.16; measured value: 452.

[0420] Step 4

[0421] [ka]

[0422] N,N-dimethyl-4-((3-(1-methylcyclohexyl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)benzenesulfonamide (6) was synthesized from N1,N1-dimethyl-N4-(2-((1-methylcyclohexyl)amino)phenyl)benzene-1,4-disulfonamide (5) according to the procedure of step 2 in the preparation of compound 20.

[0423] LCMS[M+H] + C 22 H 30 Calculated value for N3O4S2: 464.16; measured value: 464.

[0424] Step 5

[0425] [ka]

[0426] N1,N1-dimethyl-N4-(2-(methyl(1-methylcyclohexyl)amino)phenyl)benzene-1,4-disulfonamide (compound 17) was synthesized from N,N-dimethyl-4-((3-(1-methylcyclohexyl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)benzenesulfonamide (6) according to the procedure of step 3 in the preparation of compound 20.

[0427] 1HNMR (300 MHz, CD3OD) δ 8.06 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.01 (t, J = 7.8 Hz, 1H), 2.65 (s, 6H), 2.33 (s, 3H), 1.73-1.49 (m, 3H), 1.43-1.21 (m, 3H), 1.19-1.08 (m, 4H), 1.14 (s, 3H).LCMS [M+H] + C 22 H 32 Calculated value of N3O4S2: 466.18; Measured value: 465.7.

[0428] [Example S9] Synthesis of compound 22

[0429] [ka]

[0430] Step 1

[0431] [ka]

[0432] Tert-butyl 4-((2-nitrophenyl)amino)piperidine-1-carboxylate (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0433] LCMS[M+Na] + C 16 H 23 Calculated value for N3O4: 321.17; measured value: 344.

[0434] Step 2

[0435] [ka]

[0436] Tert-butyl 4-(methyl(2-nitrophenyl)amino)piperidine-1-carboxylate (4) was synthesized from tert-butyl 4-((2-nitrophenyl)amino)piperidine-1-carboxylate (3) according to the procedure of step 1 in the preparation of compound 27.

[0437] LCMS[M+Na] + C 17 H 25 Calculated value for N3O4: 335.18; measured value: 358.

[0438] Step 3

[0439] [ka]

[0440] Tert-butyl 4-((2-aminophenyl)(methyl)amino)piperidine-1-carboxylate (5) was synthesized from tert-butyl 4-(methyl(2-nitrophenyl)amino)piperidine-1-carboxylate (4) according to the procedure of step 2 in the preparation of compound 27.

[0441] LCMS[M+H] + C 17 H 28 Calculated value for N3O2: 306.21; measured value: 306.

[0442] Step 4

[0443] [ka]

[0444] Tert-butyl 4-((2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)amino)piperidine-1-carboxylate (6) was synthesized from tert-butyl 4-((2-aminophenyl)(methyl)amino)piperidine-1-carboxylate (5) and common intermediate 1 according to the procedure of step 3 in the preparation of compound 27.

[0445] 1 HNMR (300 MHz, CD3OD) δ 8.06 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 6.6 Hz, 1H), 7.20 (t, J = 6.0 Hz, 1H), 7.16-7.01 (m, 2H), 4.01-3.92 (m, 2H), 2.79-2.54 (m, 3H), 2.65 (s, 6H), 2.32 (s, 3H), 1.71-1.60 (m, 2H), 1.45 (s, 9H), 1.32-1.17 (m. 2H). LCMS [M+H] + C 25 H 37 Calculated value of N4O6S2: 553.21; Measured value: 552.9.

[0446] Step 5

[0447] [ka]

[0448] To a solution of tert-butyl 4-((2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)amino)piperidine-1-carboxylate (6) (900 mg, 1.63 mmol) in DCM (3 mL), HCl / Et2O (6 mL, 5 mol / L) was added. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete as shown by LC-MS, the reaction mixture was concentrated under vacuum to obtain 790 mg of N1,N1-dimethyl-N4-(2-(methyl(piperidine-4-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 22) as the HCl salt. The yield was 99.2%.

[0449] 1 HNMR (300 MHz, CD3OD) δ 8.09 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 6.6 Hz, 1H), 7.26 (d, J = 6.9 Hz, 1H), 7.18-7.05 (m, LCMS [M+H] + C 20 H 29 Calculated value of N4O4S2: 453.16; Measured value: 452.9.

[0450] [Example S10] Synthesis of Compound 21

[0451] [ka]

[0452] Step 1

[0453] [ka]

[0454] To a solution of N1,N1-dimethyl-N4-(2-(methyl(piperidine-4-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 22) (HCl salt, 220 mg, 0.46 mmol) in MeOH (3 mL), aqueous formaldehyde (149 mg, 1.84 mmol, 37 wt%), NaBH3CN (115.6 mg, 1.84 mmol) and one drop of AcOH were added. The reaction mixture was stirred overnight at ambient temperature. After the reaction was complete as shown by TLC, it was quenched with water (20 mL) and extracted with SiO2 (30 mL x 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The resulting oily substance was purified by silica gel chromatography to obtain impure N1,N1-dimethyl-N4-(2-(methyl(1-methylpiperidine-4-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 21). The impure compound was further purified by preparative HPLC to obtain 22.8 mg of purified N1,N1-dimethyl-N4-(2-(methyl(1-methylpiperidine-4-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 21).

[0455] 1 HNMR (300 MHz, CDCl3) δ 8.39 (br s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 7.8 Hz, 1H), 7.14-7.01 (m, 3H), 2.77-2.70 (m, 2H), 2.70(s, 6H), 2.49 (m, 1H), 2.36 (s, 3H), 2.33 (s, 3H), 1.83-1.75 (m, 2H), 1.71-1.53 ​​(m, 2H), 1.41-1.27 (m, 2H).LCMS [M+H] + C 21 H 31 Calculated value of N4O4S2: 467.17; Measured value: 466.8.

[0456] [Example S11] Synthesis of compound 19

[0457] [ka]

[0458] Step 1

[0459] [ka]

[0460] N-methyl-N-(2-nitrophenyl)tetrahydro-2H-pyran-4-amine (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0461] LCMS[M+H] + C 12 H 17 Calculated value for N2O3: 237.12; measured value: 237.

[0462] Step 2

[0463] [ka]

[0464] N1-methyl-N1-(tetrahydro-2H-pyran-4-yl)benzene-1,2-diamine (4) was synthesized from N-methyl-N-(2-nitrophenyl)tetrahydro-2H-pyran-4-amine (3) according to the procedure of step 2 in the preparation of compound 27.

[0465] LCMS[M+H] + C 12 H 19 Calculated value for N2O: 207.14; measured value: 207.

[0466] Step 3

[0467] [ka]

[0468] N1,N1-dimethyl-N4-(2-(methyl(tetrahydro-2H-pyran-4-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 19) was synthesized from N1-methyl-N1-(tetrahydro-2H-pyran-4-yl)benzene-1,2-diamine (4) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0469] 1 HNMR (400 MHz, CDCl3) δ 8.45 (br, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 5.4 Hz, 1H), 7.16-7.14 (m, 2H), 7.07-7.03 (m, 1H), 3.91-3.87 (m, 2H), 3.26 (t, J = 7.8 Hz, 2H), 2.75-2.74 (m, 1H), 2.73 (s, 6H), 2.33(s, 3H), 1.56-1.58 (m, 2H), 1.38-1.25 (m, 2H). LCMS [M+H] + C 20 H 28 Calculated value of N3O5S2: 454.14; Measured value: 454.3.

[0470] [Example S12] Synthesis of Compound 24

[0471] [ka]

[0472] Step 1

[0473] [ka]

[0474] N-cyclopentyl-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0475] LCMS[M+H] + C 11 H 15 Calculated value for N2O2: 207.11; measured value: 207.

[0476] Step 2

[0477] [ka]

[0478] N-cyclopentyl-N-methyl-2-nitroaniline (4) was synthesized from N-cyclopentyl-2-nitroaniline (3) according to the procedure of step 1 in the preparation of compound 27.

[0479] LCMS[M+H] + C 12 H 17 Calculated value for N2O2: 221.12; measured value: 221.

[0480] Step 3

[0481] [ka]

[0482] N1-cyclopentyl-N1-methylbenzene-1,2-diamine (5) was synthesized from N-cyclopentyl-N-methyl-2-nitroaniline (4) according to the procedure of step 2 in the preparation of compound 27.

[0483] LCMS[M+H] + C 12 H 19 Calculated value for N2: 191.15; measured value: 191.

[0484] Step 4

[0485] [ka]

[0486] N1-(2-(cyclopentyl(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 24) was synthesized from N1-cyclopentyl-N1-methylbenzene-1,2-diamine (5) and common intermediate 1 following the procedure of step 3 in the preparation of compound 27.

[0487] 1 HNMR (300 MHz, CDCl3) δ 8.53 (br, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.1 Hz, 1H), 7.20-7.04 (m, 3H), 3.23-3.13 (m, 1H), 2.69 (s, 6H), 2.27 (s, 3H), 1.68-1.48 (m, 6H), 1.26-1.10 (m, 2H). LCMS [M+H] + C 20 H 28 Calculated value of N3O4S2: 438.14; Measured value: 438.3.

[0488] [Example S13] Synthesis of compound 18

[0489] [ka]

[0490] Step 1

[0491] [ka]

[0492] N-(2-nitrophenyl)tetrahydrofuran-3-amine (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0493] 1 HNMR (300 MHz, CDCl3) δ 8.14-8.18 (m, 2H), 7.45 (t, J = 7.5 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 6.69 t, J = 7.5 Hz, 1H), 4.25 (s, 1H), 4.12-3.89 (m, 3H), 3.79 (m, 1H), 2.35 (m, 1H), 1.98 (m, 1H).

[0494] Step 2

[0495] [ka]

[0496] N-methyl-N-(2-nitrophenyl)tetrahydrofuran-3-amine (4) was synthesized from N-(2-nitrophenyl)tetrahydrofuran-3-amine (3) according to the procedure of step 1 in the preparation of compound 27.

[0497] LCMS[M+H] + C 11 H 15 Calculated value for N2O3: 223.10; measured value: 223.

[0498] Step 3

[0499] [ka]

[0500] N1-methyl-N1-(tetrahydrofuran-3-yl)benzene-1,2-diamine (5) was synthesized from N-methyl-N-(2-nitrophenyl)tetrahydrofuran-3-amine (4) according to the procedure of step 2 in the preparation of compound 27.

[0501] LCMS[M+H] + C 11 H 17 Calculated value for N2O: 193.13; measured value: 193.

[0502] Step 4

[0503] [ka]

[0504] N1,N1-dimethyl-N4-(2-(methyl(tetrahydrofuran-3-yl)amino)phenyl)benzene-1,4-disulfonamide (compound 18) was synthesized from N1-methyl-N1-(tetrahydrofuran-3-yl)benzene-1,2-diamine (5) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0505] 1 HNMR (300 MHz, CDCl3) δ 8.41 (br, 1H), 8.03 (d, J = 6.3 Hz, 2H), 7.83 (d, J = 6.3 Hz, 2H), 7.64-7.60 (m, 1H), 7.17-7.12 (m, 2H), 7.08-7.05 (m, 1H), 3.92-3.88 (m, 1H), 3.77-3.72 (m, 1H), 3.61-3.50 (m, 2H), 3.28-3.22 (m, 1H), 2.70 (s, 6H), 2.32 (s, 3H), 1.94-1.88 (m, 1H), 1.65-1.58 (m, 1H). LCMS [M+H] + C 19 H 26 Calculated value of N3S5O2: 440.12; Measured value: 440.3.

[0506] [Example S14] Synthesis of compound 12

[0507] [ka]

[0508] Step 1

[0509] [ka]

[0510] N-cyclohexyl-2-fluoro-N-methyl-6-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26. (The MS reaction was not observed by LC-MS.)

[0511] Step 2

[0512] [ka]

[0513] N1-cyclohexyl-6-fluoro-N1-methylbenzene-1,2-diamine (4) was synthesized from -cyclohexyl-2-fluoro-N-methyl-6-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 27.

[0514] LCMS[M+H] + C 13 H 20 Calculated value for FN2: 223; Measured value: 223.

[0515] Step 3

[0516] [ka]

[0517] N1-(2-(cyclohexyl(methyl)amino)-3-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 12) was synthesized from N1-cyclohexyl-6-fluoro-N1-methylbenzene-1,2-diamine (4) and common intermediate 1 following the procedure of step 3 in the preparation of compound 27.

[0518] 1 HNMR (300 MHz, CDCl3) δ 8.51 (br, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H),7.14-7.07 (m, 1H), 6.74 (t, J = 8.7 Hz, 1H), 2.81-2.71 (m, 1H), 2.71 (s, 6H), 2.57 (s, 3H), 2.09-2.00 (m, 1H), 1.78-1.65 (m, 1H), 1.62-1.55 (m, 1H), 1.26-1.06 (m, 6H), 0.64-0.57 (m, 1H). LCMS [M+H] + C 21 H 29 Calculated value for FN3S2O4: 470.15; Measured value: 470.5.

[0519] [Example S15] Synthesis of Compound 4

[0520] [ka]

[0521] Step 1

[0522] [ka]

[0523] N-cyclohexyl-5-fluoro-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0524] LCMS[M+H] + C 12 H 16 Calculated value for FN2O2: 239.11; measured value: 239.

[0525] Step 2

[0526] [ka]

[0527] N1-cyclohexyl-5-fluorobenzene-1,2-diamine (4) was synthesized from N-cyclohexyl-5-fluoro-2-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 27.

[0528] LCMS[M+H] + C 12 H 18 Calculated value for FN2: 209.14; measured value: 209.

[0529] Step 3

[0530] [ka]

[0531] N1-(2-(cyclohexylamino)-4-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (5) was synthesized from N1-cyclohexyl-5-fluorobenzene-1,2-diamine (4) and common intermediate 1 according to the procedure of step 3 in the preparation of compound 27.

[0532] LCMS[M+H] + C 20 H 27Calculated value for FN3O4S2: 456.13; measured value: 456.

[0533] Step 4

[0534] [ka]

[0535] To a solution of N1-(2-(cyclohexylamino)-4-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (5) (260 mg, 0.57 mmol) in MeOH (6 mL), formaldehyde (2 mL, 33%) was added. The reaction mixture was stirred at ambient temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (20 mL) and extracted with ELISA (10 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to obtain 240 mg of crude 4-((3-cyclohexyl-5-fluoro-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (6).

[0536] LCMS[M+H] + C 21 H 27 Calculated value for FN3O4S2: 468.13; measured value: 468.

[0537] Step 5

[0538] [ka]

[0539] Under nitrogen protection, at 0°C, a solution of crude 4-((3-cyclohexyl-5-fluoro-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (6) (240 mg, 0.51 mmol) in THF (6 mL) was added all at once to BH3-THF (1 M, 6 mL). The reaction mixture was stirred overnight at room temperature. After the reaction was complete as shown by LC-MS, the reaction was quenched with water (15 mL) and extracted with Â(15 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting oily substance was purified by silica gel chromatography to obtain an impure product. The impure product was further purified by preparative HPLC to obtain 83.1 mg of N1-(2-(cyclohexyl(methyl)amino)-4-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 4). The yield was 33.6%.

[0540] 1 HNMR (300 MHz, CDCl3) δ 8.07 (br, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.62 (m, 1H), 6.87-6.81 (m, 2H), 2.71 (s, 6H), 2.42-2.32 (m, 1H), 2.27 (s, 3H), 1.68-1.65 (m, 4H), 1.15-0.85 (m, 6H). LCMS [M+H] + C 21 H 29 Calculated value for FN3S2O4: 470.15; Measured value: 470.3.

[0541] [Example S16] Synthesis of Compound 3

[0542] [ka]

[0543] Step 1

[0544] [ka]

[0545] N-cyclohexyl-3-fluoro-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0546] LCMS[M+H] + C 12 H 16 Calculated value for FN2O2: 239.11; measured value: 239.

[0547] Step 2

[0548] [ka]

[0549] N1-cyclohexyl-3-fluorobenzene-1,2-diamine (4) was synthesized from N-cyclohexyl-3-fluoro-2-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 27.

[0550] LCMS[M+H] + C 12 H 18 Calculated value for FN2: 209.14; measured value: 209.

[0551] Step 3

[0552] [ka]

[0553] N1-(2-(cyclohexylamino)-6-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (5) was synthesized from N1-cyclohexyl-3-fluorobenzene-1,2-diamine (4) and common intermediate 1 following the procedure of step 3 in the preparation of compound 27.

[0554] LCMS[M+H] + C 20 H 27 Calculated value for FN3O4S2: 456.13; measured value: 456.

[0555] Step 4

[0556] [ka]

[0557] 4-((3-cyclohexyl-7-fluoro-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (6) was synthesized from N1-(2-(cyclohexylamino)-6-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (5) following the procedure of step 4 in the preparation of compound 4.

[0558] LCMS[M+H] + C 21 H 27 Calculated value for FN3O4S2: 468.13; measured value: 468.

[0559] Step 5

[0560] [ka]

[0561] N1-(2-(cyclohexyl(methyl)amino)-6-fluorophenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 3) was synthesized from 4-((3-cyclohexyl-7-fluoro-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (6) following the procedure of step 5 in the preparation of compound 4.

[0562] 1HNMR (300 MHz, CDCl3) δ 8.16 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.09-7.02 (m, 1H), 6.98-6.95 (m, 1H), 6.81 (t, J = 9.9 Hz, LCMS [M+H] + C 21 H 29 Calculated value for FN3S2O4: 470.15; Measured value: 470.3.

[0563] [Example S17] Synthesis of Compound 2

[0564] [ka]

[0565] Step 1

[0566] [ka]

[0567] 5-Chloro-N-cyclohexyl-N-methyl-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0568] LCMS[M+H] + C 13 H 18 Calculated value for ClN2O2: 269.10; measured value: 269.

[0569] Step 2

[0570] [ka]

[0571] 5-Chloro-N1-cyclohexyl-N1-methylbenzene-1,2-diamine (4) was synthesized from 5-chloro-N-cyclohexyl-N-methyl-2-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 26.

[0572] LCMS[M+H] + C 13 H 20 Calculated value for ClN2: 239.12; measured value: 239.

[0573] Step 3

[0574] [ka]

[0575] N1-(4-chloro-2-(cyclohexyl(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 2) was synthesized from 5-chloro-N1-cyclohexyl-N1-methylbenzene-1,2-diamine (4) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0576] 1 HNMR (300 MHz, CDCl3) δ 8.26 (br s, 1H), 8.20 (d, J = 8.1 Hz, 2H), 7.92 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.18-7.01 (m, 2H), 2.72 (s, 6H), 2.40-2.37 (m, 1H), 2.32 (s, 3H), 1.68-1.66 (m, 4H), 1.11-0.88 (m, 6H). LCMS [M+H] + C 21 H 29 Calculated value of ClN3S2O4: 486.12; Measured value: 486.3.

[0577] [Example S18] Synthesis of Compound 1

[0578] [ka]

[0579] Step 1

[0580] [ka]

[0581] 4-Chloro-N-cyclohexyl-N-methyl-2-nitroaniline (3) was synthesized from starting materials (1) and (2) according to the procedure of step 1 in the preparation of compound 26.

[0582] LCMS[M+H] + C 13 H 18 Calculated value for ClN2O2: 269.10; measured value: 269.

[0583] Step 2

[0584] [ka]

[0585] 4-chloro-N1-cyclohexyl-N1-methylbenzene-1,2-diamine (4) was synthesized from 4-chloro-N-cyclohexyl-N-methyl-2-nitroaniline (3) according to the procedure of step 2 in the preparation of compound 26.

[0586] LCMS[M+H] + C 13 H 20 Calculated value for ClN2: 239.12; measured value: 239.

[0587] Step 3

[0588] [ka]

[0589] N1-(5-chloro-2-(cyclohexyl(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 1) was synthesized from 4-chloro-N1-cyclohexyl-N1-methylbenzene-1,2-diamine (4) and common intermediate 1, following the procedure of step 3 in the preparation of compound 27.

[0590] 1 HNMR (300 MHz, CDCl3) δ 8.02 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 8.4 Hz, 2H), 7.65 (s, 1H), 6.98-7.04 (m, 2H), 2.72 (s, 6H), 2.34-2.41 (m, 1H), 2.32 (s, 3H), 1.53-1.70 (m, 4H), 0.94-1.08 (m, 6H). LCMS [M+H] + C 21 H 29 Calculated value of ClN3S2O4: 486.12; Measured value: 486.4.

[0591] [Example S19] Synthesis of compound 16

[0592] [ka]

[0593] Step 1

[0594] [ka]

[0595] To a stirred solution of 1-fluoro-2-nitrobenzene (SM1) (500 mg, 3.54 mmol) in DMF (5 mL), K2CO3 (1.2 g, 8.69 mmol) and N-methylpropan-1-amine (310 mg, 4.24 mmol) were added at room temperature. The reaction mixture was heated at 80 °C and stirred for 5 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (2 mL) and extracted with siRNA (2 × 20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 460 mg of N-methyl-2-nitro-N-propylaniline (1). The yield was 67%.

[0596] 1 H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.8 Hz, 1H), 7.44 - 7.32 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.82 (t, J = 7.6 Hz, 1H), 3.12 (t, J = 7.3 Hz, 2H), 2.81 (s, 3H), 1.67 - 1.59 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); LCMS [M+H] + C 10 H 15 Calculated value of N2O2: 195.11; Measured value: 195.3

[0597] Step 2

[0598] [ka]

[0599] To a stirred solution of N-methyl-2-nitro-N-propylaniline (1) (460 mg, 2.37 mmol) in a mixture of EtOH and H2O (12 mL, 5:1), Zn powder (1.54 g, 23.71 mmol) and NH4Cl (1.26 g, 23.71 mmol) were added at 0°C. The reaction mixture was heated at 80°C and stirred for 5 hours. After the reaction was complete as shown by TLC, the reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was diluted with water (10 mL) and extracted with siRNA (2 × 30 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 420 mg of crude N1-methyl-N1-propylbenzene-1,2-diamine (2). This material was used in the next step without any further purification.

[0600] Step 3

[0601] [ka]

[0602] To a stirred solution of N1-methyl-N1-propylbenzene-1,2-diamine (2) (350 mg, 2.13 mmol) in CH2Cl2 (10 mL), pyridine (0.33 mL, 4.08 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (750 mg, 2.64 mmol) were added at 0°C. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (5 mL) and extracted with CH2Cl2 (2 × 30 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30-50% ethyl acetate in heptane by medium-pressure liquid chromatography to obtain 80 mg of N1,N1-dimethyl-N4-(2-(methyl(propyl)amino)phenyl)benzene-1,4-disulfonamide (compound 16). The yield was 9.1%.

[0603] 1H NMR (400 MHz, DMSO-d6) δ 9.22 (br s, 1H), 8.07 - 8.01 (m, 2H), 7.96 - 7.89 (m, 2H), 7.23 (dd, J = 1.4, 7.9 Hz, 1H), 7.16 - 7.12 (m, 1H), 7.08 (dt, J = 1.5, 7.6 Hz, 1H), 7.04 - 6.98 (m, 1H), 2.62 (s, 8H), 2.37 (s, 3H), 1.28 - 1.18 (m, 2H), 0.73 (t, J = 7.4 Hz, 3H); LCMS [M+H] + C 18 H 26 Calculated value of N3O4S2: 412.13; Measured value: 412.2.

[0604] The following compounds were prepared according to the synthesis of compound 16.

[0605] [Table 3-1]

[0606] [Table 3-2]

[0607] [Example S20] Synthesis of compound 13

[0608] [ka]

[0609] Step 1

[0610] [ka]

[0611] To a solution of N1,N1-dimethyl-N4-(2-(methylamino)phenyl)benzene-1,4-disulfonamide (common intermediate 3) (150 mg, 0.406 mmol) in MeOH (2 mL), AcOH (19 mg, 0.32 mmol) and thiophene-3-carbaldehyde (60 mg, 0.528 mmol) were added at room temperature, and the mixture was stirred for 16 hours. The reaction mixture was cooled to 0°C, and NaBH4 (30 mg, 0.079 mmol) was added. The resulting reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated, extracted with ELISA (20 mL x 3), and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 170 mg of a mixture of crude N,N-dimethyl-4-((3-methyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)benzenesulfonamide (1) and N1,N1-dimethyl-N4-(2-(methyl(thiophen-3-ylmethyl)amino)phenyl)benzene-1,4-disulfonamide (compound 13). This mixture was used in the next step without any further purification.

[0612] Step 2

[0613] [ka]

[0614] To a solution of crude N,N-dimethyl-4-((3-methyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)benzenesulfonamide (1) and N1,N1-dimethyl-N4-(2-(methyl(thiophen-3-ylmethyl)amino)phenyl)benzene-1,4-disulfonamide (compound 13) (170 mg, 0.367 mmol) in EtOH (2 mL), NaBH4 (55 mg, 1.45 mmol) was added at room temperature. The resulting reaction mixture was heated at 90 °C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under reduced pressure. The crude product was purified by elution with 20-30% SiO2 in heptane using medium-pressure liquid column chromatography to obtain 50 mg of N1,N1-dimethyl-N4-(2-(methyl(thiophen-3-ylmethyl)amino)phenyl)benzene-1,4-disulfonamide (compound 13). The yield was 29%.

[0615] 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (br s, 1H), 8.05 (d, J = 7.8 Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.41 (br s, 1H), 7.26 (br s, 1H), 7.14 (d, J LCMS [M+H] + C 20 H 24 Calculated value of N3O4S3: 466.09; Measured value: 466.8; HPLC: 98.06%

[0616] The following compounds were prepared according to the synthesis of compound 13.

[0617] [Table 4-1]

[0618] [Table 4-2]

[0619] [Example S21] Synthesis of compound 30

[0620] [ka]

[0621] Step 1

[0622] [ka]

[0623] To a solution of 1-fluoro-2-nitrobenzene (350.0 mg, 2.5 mmol) in DMF (19.0 mL), (1s,4s)-4-methylcyclohexaneaminium chloride (556.9 mg, 3.7 mmol) and K2CO3 (1.0 g, 7.4 mmol) were added. The reaction mixture was stirred at 100°C for 4 hours. After the reaction was complete as shown by TLC, the reaction was quenched with saturated aqueous solution NH4Cl (60.0 mL) and extracted with siRNA (60.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 200:1) to obtain 560.0 mg (2.4 mmol) of the title compound. The yield was 96.4%.

[0624] LCMS[M+H] + C 13 H 19 Calculated value for N2O2: 235.14; measured value: 235.10.

[0625] Step 2

[0626] [ka]

[0627] Under nitrogen protection, at 0°C, NaH (382.4 mg, 9.6 mmol, 60%) was added gradually over 2 minutes to a solution of N-((1s,4s)-4-methylcyclohexyl)-2-nitroaniline (560.0 mg, 2.4 mmol) in DMF (32.0 mL). The resulting mixture was stirred at 0°C for 30 minutes, after which iodomethane (1.4 g, 9.6 mmol) was added dropwise to the reaction over 2 minutes. The reaction was stirred overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with saturated aqueous solution NH4Cl (60.0 mL) and extracted with siRNA (80.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 200:1) to obtain 550.0 mg (2.2 mmol) of the title compound. The yield was 92.7%.

[0628] LCMS[M+H] + C 14 H 21 Calculated value for N2O2: 249.15; measured value: 249.03.

[0629] Step 3

[0630] [ka]

[0631] To a solution of N-methyl-N-((1s,4s)-4-methylcyclohexyl)-2-nitroaniline (280.0 mg, 1.1 mmol) in IPA (18 mL) and THF (18 mL), Pd / C (90 mg, 10%) was added. The suspension was drained and then refilled three times with hydrogen. The reaction mixture was then stirred for 3 hours under a hydrogen atmosphere from a balloon. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad and the filtrate was washed with IPA (10 mL). The combined filtrate was concentrated under reduced pressure to obtain 220.0 mg of the crude title compound. The yield was 89.4%.

[0632] LCMS[M+H] + C 14 H 23 Calculated value for N2: 219.18; measured value: 219.27.

[0633] Step 4

[0634] [ka]

[0635] To a solution of N1-methyl-N1-((1s,4s)-4-methylcyclohexyl)benzene-1,2-diamine (100.0 mg, 0.5 mmol) in THF (5.0 mL), 4-(N,N-dimethylsulfamoyl)benzene-1-sulfonyl chloride (169.0 mg, 0.6 mmol) and pyridine (144.9 mg, 1.8 mmol) were added. The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (20.0 mL) and extracted with RINKAN (25.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the impure title compound. The impure product was further purified by preparative HPLC to obtain 71.6 mg of the title compound. The yield was 33.6%.

[0636] 1 H-NMR (300 MHz, CDCl3) δ 8.42 (brs, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 7.5 Hz, 1H), 7.19-7.00 ( m, 3 H), 2.70 (s, 6H), 2.28 (s, 3H), 1.71-1.63 (m, 1H), 1.44-1.21 (m, 8H), 0.90 (d, J = 6.9 Hz, 3H). LCMS [M+H] + C 22 H 32Calculated value of ClN3S2O4: 466; Measured value: 466.29.

[0637] The following compounds were prepared according to the synthesis of compound 30.

[0638] [Table 5-1]

[0639] [Table 5-2]

[0640] [Table 5-3]

[0641] [Table 5-4]

[0642] [Table 5-5]

[0643] [Example S22] Synthesis of compound 39

[0644] [ka]

[0645] Step 1

[0646] [ka]

[0647] To a solution of 4-(aminomethyl)cyclohexanecarboxylic acid (2.5 g, 15.9 mmol) in MeOH (160.0 mL), sulfurous dichloride (7.6 g, 63.6 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under vacuum to obtain 2.7 g (13.0 mmol) of the title compound. The yield was 81.8%.

[0648] LCMS[M+H] + C9H 18 Calculated value for NO2: 172.13; measured value: 172.25.

[0649] Step 2

[0650] [ka]

[0651] (Boc)2O (4.3 g, 19.5 mmol) was added to a solution of (4-(methoxycarbonyl)cyclohexyl)methaneaminium chloride (2.7 g, 13.0 mmol) in saturated aqueous Na2CO3 (75.0 mL) and HCl (75.0 mL). The reaction mixture was stirred at room temperature for 6 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (60.0 mL) and extracted with HCl (60.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:HCl = 50:1~10:1) to obtain 4.2 g (15.5 mmol) of the title compound. The yield was quantitative.

[0652] LCMS[M+Na] + C 14 H 25 Calculated value for NO4: 271.18; measured value: 294.27.

[0653] Step 3

[0654] [ka]

[0655] Under nitrogen protection, at 0°C, a solution of methyl 4-(((tert-butoxycarbonyl)amino)methyl)cyclohexanecarboxylate (4.2 g, 15.5 mmol) in DMF (150.0 mL) was mixed with NaH (1.2 g, 31.0 mmol, 60%). The resulting mixture was stirred at 0°C for 30 minutes, after which iodomethane (1.2 g, 31.0 mmol) was added to the reactant. The reactant was stirred overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with water (150.0 mL) and extracted with siRNA (100.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 40:1~30:1) to obtain 4.7 g (16.5 mmol) of the title compound. The yield was 106.4%.

[0656] LCMS[M+Na] + C 15 H 27 Calculated value for NO4: 285.19; measured value: 308.20.

[0657] Step 4

[0658] [ka]

[0659] Under nitrogen protection, LDA (18.4 mLg, 36.8 mmol, 2M) was added dropwise to a solution of methyl 4-(((tert-butoxycarbonyl)(methyl)amino)methyl)cyclohexanecarboxylate (3.5 g, 12.3 mmol) in anhydrous THF (100.0 mL) at -72°C. The resulting mixture was stirred at -72°C for 10 minutes, after which iodomethane (2.6 g, 18.4 mmol) was added to the reactant. The reactant was stirred at -72°C for 3 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (100.0 mL) and extracted with siRNA (80.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 20:1) to obtain 2.4 g (8.0 mmol) of the title compound. The yield was 65.4%.

[0660] LCMS[M+Na] + C 16 H 29 Calculated value for NO4: 299.21; measured value: 322.25.

[0661] Step 5

[0662] [ka]

[0663] Under nitrogen protection, at 0°C, DIBAL-H (3.4 g, 24.0 mmol) was added dropwise over 5 minutes to a solution of methyl 4-(((tert-butoxycarbonyl)(methyl)amino)methyl-1-methylcyclohexanecarboxylate (2.4 g, 8.0 mmol) in anhydrous THF (80.0 mL). The reaction mixture was stirred at 0°C for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (100.0 mL), the mixture was filtered through a Celite pad, and the filtrate was extracted with SiO2 (50.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:SiO2 = 8:1) to obtain 1.3 g (4.8 mmol) of the title compound. The yield was 59.8%.

[0664] LCMS[M+Na] + C 15 H 29 Calculated value for NO3: 271.21; measured value: 294.27.

[0665] Step 6

[0666] [ka]

[0667] Under nitrogen protection, at 0°C, a solution of tert-butyl((4-(hydroxymethyl)-4-methylcyclohexyl)methyl)(methyl)carbamate (1.3 g, 4.8 mmol) in DMF (48.0 mL) was added with NaH (383.2 mg, 9.6 mmol, 60%) over 5 minutes. The resulting mixture was stirred at 0°C for 20 minutes, after which iodomethane (1.4 g, 9.6 mmol) was added dropwise to the reaction mixture over 2 minutes. The reaction mixture was warmed to room temperature and stirred overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with water (400.0 mL) and extracted with siRNA (150.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 3:1) to obtain 290.0 mg (1.0 mmol) of the title compound. The yield was 21.2%.

[0668] LCMS[M+Na] + C 16 H 31 Calculated value for NO3: 285.23; measured value: 308.66.

[0669] Step 7

[0670] [ka]

[0671] To a solution of tert-butyl((4-(methoxymethyl)-4-methylcyclohexyl)methyl)(methyl)carbamate (270.0 mg, 1.0 mmol) in DCM (10 mL), TFA (3 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by LC-MS, the reaction mixture was concentrated under reduced pressure to obtain 175.0 mg of the crude title compound. The yield was 100%.

[0672] LCMS[M+H] + C 11 H 24 Calculated value for NO: 186.18; measured value: 185.97.

[0673] Step 8

[0674] [ka]

[0675] To a solution of 1-(4-(methoxymethyl)-4-methylcyclohexyl)-N-methylmethanamine (175.0 mg, 0.9 mmol) in DMAc (9.0 mL), 1-fluoro-2-nitrobenzene (266.5 mg, 1.8 mmol) and K2CO3 (522.1 mg, 3.8 mmol) were added. The reaction mixture was stirred at 130 °C for 5 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (20.0 mL) and extracted with  (15.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE: = 100:1 to 50:1) to obtain 330.0 mg (1.1 mmol) of the title compound. The yield was 114.1%.

[0676] LCMS[M+H] + C 17 H 27 Calculated value for N2O3: 307.15; measured value: 307.15.

[0677] Step 9

[0678] [ka]

[0679] To a solution of N-((4-(methoxymethyl)-4-methylcyclohexyl)methyl)-N-methyl-2-nitroaniline (310.0 mg, 1.0 mmol) in MeOH (10.0 mL), Pd / C (30 mg, 10%) was added. The suspension was drained and then refilled three times with hydrogen. The reaction mixture was then stirred under hydrogen (balloon) for 3 hours. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad and the filtrate cake was washed with MeOH (10 mL). The combined filtrate was concentrated under reduced pressure to obtain 190.0 mg of the crude title compound. The yield was 67.9%.

[0680] LCMS[M+H] + C 17 H 29 Calculated value for N2O: 277.22; measured value: 277.12.

[0681] Step 10

[0682] [ka]

[0683] To a solution of N1-((4-(methoxymethyl)-4-methylcyclohexyl)methyl)-N1-methylbenzene-1,2-diamine (177.0 mg, 0.6 mmol) in THF (6.0 mL), 4-(N,N-dimethylsulfamoyl)benzene-1-sulfonyl chloride (236.2 mg, 0.8 mmol) and pyridine (202.6 mg, 2.6 mmol) were added. The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (15.0 mL) and extracted with RINKAN (10.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the impure title compound. The impure product was further purified by preparative HPLC to obtain 33.8 mg of the title compound. The yield was 10.1%.

[0684] 1H-NMR (300 MHz, CDCl3) δ 8.32 (br, 1H ), 8.01 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.12-7.02 ( m, 3 H), 3.34 (s, 3H), 3.17 (s, 2H), 2.71 (s, 6H), 2.55 (d, J = 6.6 Hz, 2H), 2.31 (s, 3H), 1.65-1.61 (m, 2H), 1.53-1.47 (m, 2H), 1.33-1.17 (m, 2H), 1.03 (d, J = 9.3 Hz, 3H), 0.90 (s, 3H).LCMS [M+H] + C 26 H 38 Calculated value of N3S2O5: 524.22; Measured value: 523.89.

[0685] The following compounds were prepared according to the synthesis of compound 39.

[0686] [Table 6]

[0687] [Example S23] Synthesis of Compound 53

[0688] [ka]

[0689] Step 1

[0690] [ka]

[0691] To a solution of 1,4-difluoro-2-nitrobenzene (330.0 mg, 2.0 mmol) in DMF (15.0 mL), N-methylcyclohexaneamine (352.2 mg, 3.1 mmol) and K2CO3 (1.2 g, 8.3 mmol) were added. The reaction mixture was stirred at 140 °C for 4 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (30.0 mL) and extracted with RINKAN (20.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE=100%) to obtain 500.0 mg (2.0 mmol) of the title compound. The yield was 95.5%.

[0692] LCMS[M+H] + C 13 H 18 Calculated value for FN2O2: 253.13; measured value: 253.31.

[0693] Step 2

[0694] [ka]

[0695] To a solution of N-cyclohexyl-4-fluoro-N-methyl-2-nitroaniline (500.0 mg, 2.0 mmol) in MeOH (19.0 mL), Pd / C (50 mg, 10%) was added. The suspension was drained, and the mixture was refilled three times with hydrogen. The reaction mixture was then stirred for 3 hours under hydrogen from a balloon. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad, and the filtrate cake was washed with MeOH (10 mL). The combined filtrate was concentrated under reduced pressure to obtain 400.0 mg of the crude title compound. The yield was 90.8%.

[0696] LCMS[M+H] + C 13 Calculated value for H20FN2: 223.15; measured value: 223.30.

[0697] Step 3

[0698] [ka]

[0699] To a solution of N1-cyclohexyl-4-fluoro-N1-methylbenzene-1,2-diamine (220.0 mg, 1.0 mmol) in THF (10.0 mL), 4-(N,N-dimethylsulfamoyl)benzene-1-sulfonyl chloride (365.1 mg, 1.3 mmol) and pyridine (234.8 mg, 3.0 mmol) were added. The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (25.0 mL) and extracted with RINKAN (15.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain the impure title compound. The impure product was further purified by preparative HPLC to obtain 80.0 mg of the title compound. The yield was 17.2%.

[0700] 1 H-NMR (300 MHz, CDCl3) δ 8.01 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.39 (dd, J = 2.7Hz, 10.2 Hz, 1H), 7.07 (dd, J = 6 Hz, 9 Hz, 1H), 6.76-6.70 (m, 1H), 2.72 (s, 6H), 2.48-2.37 (m, 1H), 2.33 (s, 3H), 1.71-1.60 (m, 4H), 1.11-0.88 (m, 6H); LCMS [M+H] + C 21 H 29 Calculated value for FN3S2O4: 470.15; Measured value: 470.47.

[0701] The following compounds were prepared according to the synthesis of compound 53.

[0702] [Table 7]

[0703] [Example S24] Synthesis of Compound 52

[0704] [ka]

[0705] Step 1

[0706] [ka]

[0707] To a solution of 1-chloro-3-fluoro-2-nitrobenzene (300.0 mg, 1.7 mmol) in DMF (15.0 mL), cyclohexaneamine (254.0 mg, 2.6 mmol) and K2CO3 (709.0 mg, 5.1 mmol) were added. The reaction mixture was stirred at 140 °C for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (30.0 mL) and extracted with  (30.0 mL × 2). The combined organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The remaining oily substance was purified by silica gel chromatography (PE) to obtain 415.0 mg of the title compound. The yield was 95.3%.

[0708] LCMS[M+H] + C 12 H 16 Calculated value for ClN2O2: 255.08; measured value: 255.30.

[0709] Step 2

[0710] [ka]

[0711] To a solution of 3-chloro-N-cyclohexyl-2-nitroaniline (390.0 mg, 1.5 mmol) in aqueous saturated NH4Cl solution (7.0 mL) and EtOH (7.0 mL), Fe powder (257.0 mg, 4.6 mmol) was added in one step. The reaction mixture was stirred at 70°C for 2 hours. After the reaction was complete as shown by TLC, the suspension was cooled to room temperature and filtered to remove any undissolved solids. The filtrate was quenched with water (15.0 mL) and extracted with ethyl acetate (15.0 mL × 2). The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:ethyl acetate = 50:1) to obtain 243.0 mg of the title compound. The yield was 70.6%.

[0712] LCMS[M+H] + C 12 H 18 Calculated value for ClN2: 225.11; measured value: 225.

[0713] Step 3

[0714] [ka]

[0715] To a solution of 3-chloro-N1-cyclohexylbenzene-1,2-diamine (220.0 mg, 1.0 mmol) in THF (10 mL), 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (416.0 mg, 1.5 mmol) and pyridine (233.0 mg, 2.9 mmol) were added. The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction was quenched with water (10.0 mL) and extracted with  (10.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE: = 10:1~5:1) to obtain 150.0 mg of the title compound. The yield was 32.06%.

[0716] LCMS[M+H] + C 20 H 27 Calculated value for ClN3O4S2: 472.11; measured value: 472.45.

[0717] Step 4

[0718] [ka]

[0719] To a solution of 4-((7-chloro-3-cyclohexyl-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (120.0 mg, 0.3 mmol) in THF (2.5 mL), formaldehyde (15.3 mg, 0.5 mmol, 37%) was added. The reaction mixture was stirred at ambient temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (10.0 mL) and extracted with DCM (10.0 mL × 2). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 100 mg of the crude title compound. The crude title compound was used in the next step without purification.

[0720] LCMS[M+H] + C 21 H 27 Calculated value for ClN3O4S2: 484.11; measured value: 483.99.

[0721] Step 5

[0722] [ka]

[0723] To a solution of 4-((7-chloro-3-cyclohexyl-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (100.0 mg, 0.2 mmol) in THF (2.0 mL), NaBH4 (23.0 mg, 0.8 mmol) was added in four portions over 30 minutes. The reaction mixture was stirred at ambient temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (10.0 mL) and extracted with siRNA (10.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting oily substance was purified by silica gel chromatography to obtain the impure title compound. The impure title compound was further purified by preparative HPLC to obtain 22.7 mg of the title compound. The yield was 22.3%.

[0724] 1 H-NMR (300 MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.08 (s, 3H), 2.86-2.82 (m, 1H), 2.75 (s, 6H), 2.58 (s, 3H), 1.84-1.77(m, 4H), 1.77-1.64(m, 1H), 1.36-1.26(m, 4H), 1.19-1.09(m, 1H). + C 21 H 29 Calculated value of ClN3O4S2: 486; Measured value: 486.02.

[0725] The following compounds were prepared according to the synthesis of compound 52.

[0726] [Table 8-1]

[0727] [Table 8-2]

[0728] [Table 8-3]

[0729] [Table 8-4]

[0730] [Example S25] Synthesis of Compound 87

[0731] [ka]

[0732] Step 1

[0733] [ka]

[0734] To a solution of cyclobutanone (2.5 g, 35.7 mmol) and buta-3-en-1-ol (2.6 g, 35.7 mmol) in DCM (350.0 mL), methanesulfonic acid (6.9 g, 71.3 mmol) was added. The reaction mixture was stirred at room temperature for 2.5 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (300.0 mL) and extracted with DCM (200.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 8:1~5:1) to obtain 6.1 g (27.7 mmol) of the title compound. The yield was 77.6%.

[0735] GCMS[M-OMs] C8H 15 Calculated value for O2: 125.09; measured value: 125.

[0736] Step 2

[0737] [ka]

[0738] To a solution of 5-oxaspiro[3.5]nonane-8-ylmethanesulfonate (6.0 g, 27.2 mmol) in DMSO (270.0 mL), NaN3 (5.3 g, 81.7 mmol) was added. The reaction mixture was stirred at 80°C for 4 hours. After the reaction was complete as shown by TLC, the reaction was quenched with water (500.0 mL) and extracted with siRNA (200.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 80:1 to 60:1) to obtain 3.5 g (27.2 mmol) of the title compound. The yield was 76.9%.

[0739] It could not be detected by either GCMS or LCMS.

[0740] Step 3

[0741] [ka]

[0742] To a solution of 8-azido-5-oxaspiro[3.5]nonane (200.0 mg, 1.2 mmol) in MeOH (12.0 mL), Pd / C (20 mg, 10%) was added. The suspension was drained, and the mixture was then refilled three times with hydrogen. The reaction mixture was then stirred under hydrogen (balloon) for 6 hours. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad, and the filtrate cake was washed with MeOH (10 mL). The combined filtrate was concentrated under reduced pressure to obtain 170.0 mg of the crude title compound. The yield was quantitative.

[0743] LCMS[M+H] + C8H 16 Calculated value for NO: 142.12; measured value: 142.30.

[0744] Step 4

[0745] [ka]

[0746] This compound was synthesized according to step 1 of the synthesis procedure for compound 30.

[0747] LCMS[M+H] + C 14 H 19 The calculated value for N2O3 was 263.13, while the measured value was 263.29.

[0748] Step 5

[0749] [ka]

[0750] This compound was synthesized according to step 2 of the synthesis procedure for compound 30.

[0751] LCMS[M+H] + C 15 H 21 The calculated value for N2O3 was 277.15, while the measured value was 276.98.

[0752] Step 6

[0753] [ka]

[0754] This compound was synthesized according to step 3 of the synthesis procedure for compound 30.

[0755] LCMS[M+H] + C 15 H 23 The calculated value for N2O was 247.17, while the measured value was 247.02.

[0756] Step 7

[0757] [ka]

[0758] This compound was synthesized according to step 4 of the synthesis procedure for compound 30.

[0759] 1 H-NMR (300 MHz, CDCl3) δ 8.45 (br, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 7.5 Hz, 1H), 7.16-7.06 (m, 3H), 3.75-3.71 (m, 1H), 3.32 (t, J = 10.8 Hz, 1H), 2.85-2.73 (m, 1H), 2.71 (s, 6H), 2.35 (s, 3H), 2.12-2.08 (m, 1H), 1.94-1.79 (m, 5H), 1.56-1.45 (m, 2H), 1.38-1.16 (m, 2H).LCMS [M+H] + C 23 H 32 Calculated value of N3O5S2: 494; Measured value: 493.94.

[0760] [Example S26] Synthesis of compound 102

[0761] [ka]

[0762] Step 1

[0763] [ka]

[0764] To a solution of cyclohexaneamine (1.0 g, 10.0 mmol) in DCM (100.0 mL), DIEA (3.9 g, 30.2 mmol) and (Boc)2O (3.3 g, 15.1 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by LC-MS, the reaction was quenched with water (150.0 mL) and extracted with SiO (100.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:SiO = 50:1) to obtain 2.0 g (10.0 mmol) of the title compound. The yield was 99.5%.

[0765] LCMS[M+H] + C 11 H 22 Calculated value for NO2: 200.16; measured value: 200.12.

[0766] Step 2

[0767] [ka]

[0768] Under nitrogen protection, at 0°C, NaH (602.0 mg, 15.0 mmol, 60%) was gradually added over 5 minutes to a solution of tert-butylcyclohexyl carbamate (1.0 g, 5.0 mmol) in DMF (45.0 mL). The resulting mixture was stirred at 0°C for 30 minutes, after which iodomethane-d3 (2.2 g, 15.0 mmol) was added to the reactant. The reactant was stirred overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with water (100.0 mL) and extracted with siRNA (50.0 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 100:1) to obtain 1.0 g (4.6 mmol) of the title compound. The yield was 92.1%.

[0769] LCMS[M+Na]+ C 12 Calculated value for H2D3NO2: 216.19; measured value: 239.11.

[0770] Step 3

[0771] [ka]

[0772] To a solution of tert-butylcyclohexyl (methyl-d3) carbamate (1.0 g, 4.6 mmol) in DCM (30.0 mL), TFA (10.0 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by LC-MS, the reaction mixture was concentrated under reduced pressure to obtain 540.0 mg (4.6 mmol) of the title compound. The yield was 100.0%.

[0773] LCMS[M+H] + C7H 13 Calculated value for D3N: 117.14; measured value: 116.89.

[0774] Step 4

[0775] [ka]

[0776] This compound was synthesized according to step 2 of the synthesis procedure for compound 30.

[0777] LCMS[M+H] + C 13 H 16 Calculated value for D3N2O2: 238.16; measured value: 238.05.

[0778] Step 5

[0779] [ka]

[0780] This compound was synthesized according to step 3 of the synthesis procedure for compound 30.

[0781] LCMS[M+H] + C 13 H 18 Calculated value for D3N2: 208.18; measured value: 208.33.

[0782] Step 6

[0783] [ka]

[0784] This compound was synthesized according to step 4 of the synthesis procedure for compound 30.

[0785] 1 H-NMR (300 MHz, CDCl3) δ 8.56 (br, 1H), 8.00 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 7.5 HZ, 1H), 7.14-7.01 (m, 3H), 2.70 (s, 6H), 2.47-2.43 (m, 1H), 1.67-1.56 (m, 5H), 1.07-0.91 (m, 5H); LCMS [M+H] + C 21 H 27 Calculated value of D3N3O4S2: 455; Measured value: 455.01.

[0786] [Example S27] Synthesis of compound 55

[0787] [ka]

[0788] Step 1

[0789] [ka]

[0790] To a solution of N-methyl-2-nitroaniline (2.0 g, 13.1 mmol) in THF (20.0 mL), DMAP (160.6 mg, 1.3 mmol) and (Boc)2O (8.6 g, 39.4 mmol) were added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete as shown by TLC, the reaction was quenched with water (50.0 mL) and extracted with siRNA (50.0 mL × 2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 200:1 to 30:1) to obtain 3.1 g (12.3 mmol) of the title compound. The yield was 93.5%.

[0791] LCMS[M+Na] + C 12 H 16 Calculated value for N2O4: 252.27; measured value: 275.02.

[0792] Step 2

[0793] [ka]

[0794] To a solution of tert-butylmethyl(2-nitrophenyl)carbamate (1.0 g, 4.0 mmol) in MeOH (40.0 mL), Pd / C (100 mg, 10%) was added. The suspension was drained, and the mixture was refilled three times with hydrogen. The reaction mixture was then stirred under hydrogen (balloon) for 3 hours. After the reaction was complete as shown by TLC, the suspension was filtered through a Celite pad, and the filtrate cake was washed with MeOH (20 mL). The combined filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:siRNA = 10:1 to 5:1) to obtain 830.0 mg (3.7 mmol) of the title compound. The yield was 94.2%.

[0795] 1 H-NMR (300 MHz, CDCl3) δ7.20-6.92 (m, 4H), 6.61 (br, 2H), 3.16 (s, 3H), 1.77-0.88 (br, 9H).

[0796] Step 3

[0797] [ka]

[0798] This compound was synthesized according to step 4 of the synthesis procedure for compound 30.

[0799] 1 H-NMR (300 MHz, CDCl3) δ7.98-7.85 (m, 4H), 7.51-7.42 (m, 1H), 7.30-7.22 (m, 2H), 7.08-7.05 (m, 1H), 2.73 (s, 6H), 2.65 (s, 3H), 1.57-1.33 (m, 9H).

[0800] Step 4

[0801] [ka]

[0802] To a solution of tert-butyl(2-((4-(N,N-dimethylsulfamoyl)phenyl)sulfonamide)phenyl)(methyl)carbamate (0.8 g, 1.7 mmol) in DCM (15.0 mL), TFA (5.0 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by LC-MS, the reaction mixture was concentrated under reduced pressure to obtain 610.0 mg (1.7 mmol) of the title compound. The yield was 96.9%.

[0803] LCMS[M+H] + C 15 H 20Calculated value for N3O4S2: 370.08; measured value: 369.95.

[0804] Step 5

[0805] [ka]

[0806] Thiazole-5-carbaldehyde (453.2 mg, 4.0 mmol) was added to a solution of N1,N1-dimethyl-N4-(2-(methylamino)phenyl)benzene-1,4-disulfonamide (1.0 g, 4.0 mmol) in THF (20.0 mL). The reaction mixture was stirred overnight at 60°C. After the reaction was complete as shown by TLC, the reaction product was concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE:SiO=3:1) to obtain 700 mg (1.5 mmol) of the title compound. The yield was 75.2%.

[0807] 1 H-NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 7.99 (s, 1H), 7.79 (s, 4H), 7.49 (d, J = 7.5 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 6.41 (d, J = 7.2 Hz, 2H), 2.70 (s, 6H), 2.50 (s, 3H).

[0808] Step 6

[0809] [ka]

[0810] This compound was synthesized according to step 5 of the synthesis procedure for compound 52.

[0811] 1H-NMR (300 MHz, d6-DMSO) δ 9.53 (s, 1H), 8.91 (s, 1H),8.05 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.07-6.95 (m, 3H), 4.20 (s, 2H), 2.59 (s, 6H), 2.39 (s, 3H). LCMS [M+H] + C 19 H 23 Calculated value for N4O4S3: 467.08; Measured value: 466.77.

[0812] [Example S28] Synthesis of compound 16

[0813] [ka]

[0814] N-methyl-2-nitro-N-propylaniline (1)

[0815] [ka]

[0816] A solution of 1-fluoro-2-nitrobenzene (SM1) (500 mg, 3.54 mmol) in DMF (5 mL) was added to K2CO3 (1.2 g, 8.69 mmol) and N-methylpropan-1-amine (SM2) (310 mg, 4.24 mmol) at room temperature. The reaction mixture was heated at 80 °C and stirred for 5 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (2 mL) and extracted with ₹ (2 × 20 mL). The combined organic layer was washed with brine, dried over anhydrous sodium 2 SO4, filtered, and concentrated under reduced pressure to obtain 460 mg of the title compound. The yield was 67%.

[0817] 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.8 Hz, 1H), 7.44 - 7.32 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.82 (t, J = 7.6 Hz, 1H), 3.12 (t, J = 7.3 Hz, 2H), 2.81 (s, 3H), 1.67 - 1.59 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); LCMS [M+H] + C 10 H 15 Calculated value of N2O2: 195; Measured value: 195.3

[0818] N1-methyl-N1-propylbenzene-1,2-diamine(2)

[0819] [ka]

[0820] A solution of intermediate 1 (460 mg, 2.37 mmol) in EtOH and H2O (12 mL, 5:1) was added to Zn powder (1.54 g, 23.71 mmol) and NH4Cl (1.26 g, 23.71 mmol) at 0°C. The reaction mixture was heated at 80°C and stirred for 5 hours. After the reaction was complete as shown by TLC, the reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was diluted with water (10 mL) and extracted with SiO2 (2 × 30 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 420 mg of the crude title compound. This material was used in the next step without any further purification.

[0821] N1,N1-dimethyl-N4-(2-(methyl(propyl)amino)phenyl)benzene-1,4-disulfonamide (compound 16)

[0822] [ka]

[0823] A solution of intermediate 2 (350 mg, 2.13 mmol) in CH2Cl2 (10 mL) was added at 0°C to pyridine (0.33 mL, 4.08 mmol) and 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride (common intermediate 1) (750 mg, 2.64 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (5 mL) and extracted with CH2Cl2 (2 × 30 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30-50% ethyl acetate in heptane by medium-pressure liquid chromatography to obtain 80 mg of the title compound. The yield was 9.1%.

[0824] 1 H NMR (400 MHz, DMSO-d6) δ 9.22 (br s, 1H), 8.07 - 8.01 (m, 2H), 7.96 - 7.89 (m, 2H), 7.23 (dd, J = 1.4, 7.9 Hz, 1H), 7.16 - 7.12 (m, 1H), 7.08 (dt, J = 1.5, 7.6 Hz, 1H), 7.04 - 6.98 (m, 1H), 2.62 (s, 8H), 2.37 (s, 3H), 1.28 - 1.18 (m, 2H), 0.73 (t, J = 7.4 Hz, 3H);

[0825] LCMS[M+H] + C 18 H 26 Calculated value for N3O4S2: 412; Measured value: 412.2

[0826] The following compounds were prepared according to the synthesis of compound 16.

[0827] [Table 9]

[0828] [Example S29] Synthesis of compound 13

[0829] [ka]

[0830] N,N-dimethyl-4-((3-methyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)benzenesulfonamide(1)

[0831] [ka]

[0832] A solution of N1,N1-dimethyl-N4-(2-(methylamino)phenyl)benzene-1,4-disulfonamide (common intermediate 3) (150 mg, 0.406 mmol) in MeOH (2 mL) was added to AcOH (19 mg, 0.32 mmol) and thiophene-3-carbaldehyde (SM) (60 mg, 0.528 mmol) at room temperature and stirred for 16 hours. The reaction mixture was cooled to 0°C and NaBH4 (30 mg, 0.079 mmol) was added. The resulting reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated, extracted with SiO4 (20 mL × 3), and washed with water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a mixture of 170 mg of the title compound and intermediate 1. This mixture was used in the next step without any further purification.

[0833] N1,N1-dimethyl-N4-(2-(methyl(thiophen-3-ylmethyl)amino)phenyl)benzene-1,4-disulfonamide (compound 13)

[0834] [ka]

[0835] A mixture of crude intermediate 1 in EtOH (2 mL) and compound 13 (170 mg, 0.367 mmol) was added to NaBH4 (55 mg, 1.45 mmol) at room temperature. The resulting reaction mixture was heated at 90°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated under reduced pressure. The crude product was purified by medium-pressure liquid column chromatography eluting with 20-30% siRNA in heptane to obtain 50 mg of the title compound. The yield was 29%.

[0836] 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (br s, 1H), 8.05 (d, J = 7.8 Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.41 (br s, 1H), 7.26 (br s, 1H), 7.14 (d, J LCMS [M+H] + C 20 H 24 Calculated value of N3O4S3: 466; Measured value: 466.8; HPLC: 98.06%

[0837] The following compounds were prepared according to the synthesis of compound 13.

[0838] [Table 10-1]

[0839] [Table 10-2]

[0840] [Table 10-3]

[0841] [Table 10-4]

[0842] [Table 10-5]

[0843] [Table 10-6]

[0844] [Table 10-7]

[0845] [Example S30] Scheme for the synthesis of compound 105

[0846] [ka]

[0847] N-(3-(1H-pyrazole-1-yl)propyl)-2-nitroaniline(1)

[0848] [ka]

[0849] A solution of 3-(1H-pyrazole-1-yl)propan-1-amine (SM) (200 mg, 1.6 mmol) in DMF (5 mL) was added to K2CO3 (441 mg, 3.20 mmol) and 1-fluoro-2-nitrobenzene (compound A) (270 mg, 1.92 mmol) at 0°C and stirred for 15 minutes. The reaction mixture was heated at 70°C and stirred for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (20 mL) and extracted with siRNA (2 × 25 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 400 mg of the title compound. The crude product was used directly in the next step without further purification and analysis.

[0850] Synthesis of N-(3-(1H-pyrazole-1-yl)propyl)-N-methyl-2-nitroaniline(2)

[0851] [ka]

[0852] A solution of intermediate 1 (500 mg, 2.03 mmol) in DMF (5 mL) was added to NaH (250 mg, 6.09 mmol) and CH3I (577 mg, 4.06 mmol) at 0°C. The reaction mixture was allowed to cool to room temperature and stirred for 16 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with cold water (10 mL) and extracted with ₹ (2 × 25 mL). The combined organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain 528 mg of the title compound. The crude product was used directly in the next step without further purification. LCMS[M+H] + C 13 H 16 Calculated value for N4O2: 260.13; measured value: 261.35.

[0853] N1-(3-(1H-pyrazole-1-yl)propyl)-N1-methylbenzene-1,2-diamine(3)

[0854] [ka]

[0855] A solution of intermediate 2 (528 mg, 2.03 mmol) in a mixture of ethanol and water (10 mL, 4:1) was added to Zn powder (1.32 g, 20.30 mmol) and NH4Cl (1.07 g, 20.30 mmol) at room temperature. The reaction mixture was heated to 85 °C and stirred for 1 hour. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (20 mL) and extracted with DCM (2 × 25 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 450 mg of the title compound. The crude product was used directly in the next step without further purification. LCMS[M+H] + C 13 H 18 Calculated value for N4: 230.15; Measured value: 231.4

[0856] N1-(2-((3-(1H-pyrazole-1-yl)propyl)(methyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 105)

[0857] [ka]

[0858] A solution of compound 3 (450 mg, 1.95 mmol) in DCM (5 mL) was added at 0°C to 4-(N,N-dimethylsulfamoyl)benzenesulfonyl chloride, common intermediate 1 (664 mg, 2.34 mmol), and pyridine (309 mg, 3.91 mmol). The reaction mixture was brought to room temperature and stirred for 16 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water (25 mL) and extracted with DCM (2 × 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. 450 mg of the title compound was obtained from the crude product. The crude product was purified by elution with 50% ethyl acetate in heptane by medium-pressure liquid chromatography to obtain 60 mg of the title compound. The yield was 6%.

[0859] 1 H NMR (400 MHz, DMSO-d6) δ = 8.06 - 8.04 (m, 1H), 8.04 - 8.02 (m, 1H), 7.93 (s, 1H), 7.91 (s, 1H), 7.44 (d, J = 1.3 Hz, 1H), 7.23 (d, J = 1.6 Hz, 1H), 7.22 (d, J = 1.5 Hz, 1H), 7.12 - 6.98 (m, 4H), 6.22 (t, J = 2.0 Hz, 1H), 4.10 (t, J = 6.8 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 2.61 (s, 6H), 2.24 (s, 3H), 1.80 (t, J = 6.9 Hz, 2H); LCMS [M+H] + C 21 H 27 Calculated value of N5O4S2: 477.15; Measured value: 478.39; HPLC: 99.49%

[0860] The following compounds were prepared according to the synthesis of compound 105.

[0861] [Table 11]

[0862] [Example S31] Synthesis of Compound 73

[0863] [ka]

[0864] 4-((4-chloro-3-methyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide(1)

[0865] [ka]

[0866] A solution of thiophene-3-carbaldehyde (SM) (45 mg, 0.40 mmol) in MeOH (5 mL) was added to titanium isopropoxide (113 mg, 0.40 mmol) at 0°C and stirred for 5 minutes. Next, N1-(3-chloro-2-(methylamino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (common intermediate 4) (130 mg, 0.267 mmol) was added to the reaction mixture at room temperature and stirred for 8 hours. The reaction mixture was cooled to 0°C and NaBH4 (19 mg, 0.53 mmol) was added. The resulting reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated and extracted with ELISA and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 170 mg of a crude mixture of the title compound and compound 73. This mixture was used in the next step without any further purification.

[0867] N1-(3-chloro-2-(methyl(thiazole-5-ylmethyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 73)

[0868] [ka]

[0869] A mixture of crude intermediate 1 and compound 73 (100 mg, 0.20 mmol) in EtOH (4 mL) was added to NaBH4 (29 mg, 0.80 mmol) at 0°C. The reaction mixture was heated at 85°C and stirred for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was washed with ethyl acetate and water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30-50% siRNA in hexane by medium-pressure liquid chromatography to obtain 30 mg of the title compound. The yield was 29%.

[0870] 1 H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.93 (s, 1H), 8.05 - 8.00 (m, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.73 (s, 1H), 7.18 - 7.09 (m, 3H), 4.38 (s, 2H), 2.60 (s, 6H), 2.52 (s, 3H); LCMS [M+H] + C 19 H 22 Calculated value of N4O4S3: 501; Measured value: 501.0; HPLC: 99.75%

[0871] The following compounds were prepared according to the synthesis of compound 73.

[0872] [Table 12]

[0873] [Example S32] Synthesis of Compound 72

[0874] [ka]

[0875] 4-((3,4-dimethyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide(1)

[0876] [ka]

[0877] A solution of common intermediate 5 (0.14 g, 0.36 mmol) in MeOH (1.4 mL) was added to titanium isopropoxide (0.15 mL, 0.55 mmol) at 0°C and stirred for 10 minutes. Next, thiazole-5-carbaldehyde compound A (0.05 g, 0.44 mmol) was added to the reaction mixture. The reaction mixture was allowed to cool to room temperature and stirred for 12 hours. The reaction mixture was cooled to 0°C and NaBH4 (0.02 g, 0.55 mmol) was added. The resulting reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was filtered through a Celite bed, the filtrate was concentrated and extracted with DCM and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 0.15 g of compound 1 and compound 72 as a mixture. This mixture was used in the next step without any further purification.

[0878] LCMS[MH]+ C 21 H 22 Calculated value for N3O4S3: 477.09; Measured value: 479.3

[0879] N1,N1-dimethyl-N4-(3-methyl-2-(methyl(thiazole-5-ylmethyl)amino)phenyl)benzene-1,4-disulfonamide (compound 72)

[0880] [ka]

[0881] A mixture of crude intermediate 1 (0.15 g, 0.31 mmol) in EtOH (1.5 mL) was added to NaBH4 (0.059 g, 1.56 mmol) at 0°C. The reaction mixture was heated at 80°C and stirred for 1 hour. After the reaction was complete as shown by TLC, the reaction mixture was evaporated under vacuum and then extracted with ethyl acetate and water. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 80-90% SiO2 in heptane by medium-pressure liquid chromatography to obtain 0.02 g of the title compound. The yield was 17%.

[0882] 1H NMR (400 MHz, DMSO-d6) δ = 10.67 - 10.61 (m, 1H), 9.27 (d, J = 1.8 Hz, 1H), 7.95 - 7.90 (m, 2H), 7.86 - 7.80 (m, 2H), 7.60 (s, 1H), 7.33 - 7.27 (m, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.94 - 6.87 (m, 1H), 2.69 - 2.65 (m, 2H), 2.56 (s, 6H), 2.23 (s, 6H); LCMS [M+H] + C 20 H 25 Calculated value of N4O4S3: 481; Measured value: 481.4; HPLC: 98.82%

[0883] The following compounds were prepared according to the synthesis of compound 72.

[0884] [Table 13]

[0885] [Example S33] Synthesis of compound 70

[0886] [ka]

[0887] 4-((4-fluoro-3-methyl-2-(thiophen-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide(1)

[0888] [ka]

[0889] A solution of thiazole-5-carbaldehyde (compound A) (48 mg, 0.42 mmol) in MeOH (4 mL) was added to titanium isopropoxide (121 mg, 0.42 mmol) at 0°C and stirred for 5 minutes. Next, N1-(3-fluoro-2-(methylamino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (common intermediate 6) (110 mg, 0.28 mmol) was added to the reaction mixture at room temperature and stirred for 8 hours. The reaction mixture was cooled to 0°C and NaBH4 (21 mg, 0.57 mmol) was added. The resulting reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was concentrated and extracted with SiO2 and water. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 100 mg of a crude mixture of the title intermediate 1 and compound 70. This mixture was used in the next step without any further purification.

[0890] N1-(3-fluoro-2-(methyl(thiazole-5-ylmethyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 70)

[0891] [ka]

[0892] A mixture of crude compound 1 and compound 70 (100 mg, 0.20 mmol) in EtOH (5 mL) was added to NaBH4 (30 mg, 0.83 mmol) at 0°C. The reaction mixture was heated at 85°C and stirred for 2 hours. After the reaction was complete as shown by TLC, the reaction mixture was washed with ethyl acetate and water. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by elution with 30-50% siRNA in hexane by medium-pressure liquid chromatography to obtain 30 mg of the title compound. The yield was 30%.

[0893] 1 H NMR (400 MHz, DMSO-d6) δ = 9.63 - 9.51 (m, 1H), 8.93 (s, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 7.9 Hz, 2H), 7.67 (s, 1H), 7.12 (s, 2H), 6.98 - 6.87 (m, 1H), 4.17 (s, 2H), 2.67 (s, 6H), 2.44 (s, 3H); LCMS [M+H] + C 19 H 22 Calculated value of FN4O4S3: 485; Measured value: 485.4; HPLC: 99.80%

[0894] The following compounds were prepared according to the synthesis of compound 70.

[0895] [Table 14]

[0896] [Example S34] Synthesis of Compound 84

[0897] [ka]

[0898] 4-((3-cyclohexyl-2-(oxetan-3-yl)-2,3-dihydro-1H-benzo[d]imidazole-1-yl)sulfonyl)-N,N-dimethylbenzenesulfonamide (SP-1)

[0899] [ka]

[0900] A solution of common intermediate 7 (200 mg, 0.46 mmol) in methanol (4 mL) was added at 0°C to oxetane-3-carbaldehyde compound A (47 mg, 0.55 mmol) and acetic acid (1 mL). The reaction mixture was heated to room temperature and stirred for 16 hours. The reaction mixture was cooled to 0°C and NaBH4 (26 mg, 0.68 mmol) was added gradually. The resulting reaction mixture was heated to room temperature and stirred for 4 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water and extracted with DCM (2 × 25 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain 200 mg of the title compound. The yield was 86%.

[0901] LCMS[M+H]+ C 24 H 32 Calculated value for N3O5S2: 506; Measured value: 506.2.

[0902] N1-(2-(cyclohexyl(oxetane-3-ylmethyl)amino)phenyl)-N4,N4-dimethylbenzene-1,4-disulfonamide (compound 84)

[0903] [ka]

[0904] A solution of SP-1 (200 mg, 0.39 mmol) in ethanol (4 mL) was added to NaBH4 (60 mg, 1.58 mmol) at 0°C. The reaction mixture was heated to 80°C and stirred for 3 hours. After the reaction was complete as shown by TLC, the reaction mixture was diluted with water and extracted with DCM (2 × 25 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by medium-pressure column chromatography and eluted with 30% ethyl acetate in heptane to obtain 15 mg of the title compound. The yield was 7.5%.

[0905] 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.12 (d, J = 8.5 Hz, 3H), 7.96 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 1.5 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 7.11 (dt, J = 1.4, 7.7 Hz, 1H), 7.07 - 7.02 (m, 1H), 4.19 (dd, J = 5.9, 7.8 Hz, 2H), 3.21 - 3.17 (m, 3H), 2.68 - 2.65 (m, 1H), 2.62 (s, 6H), 2.34 - 2.31 (m, 1H), 1.71 - 1.58 (m, 5H), 1.06 - 0.96 (m, 6H); LCMS [M+H] + C 24 H 33 Calculated value of N3O5S2: 507.19; Measured value: 508.2. HPLC: 95.04%.

[0906] The following compounds were prepared according to the synthesis of compound 84.

[0907] [Table 15]

[0908] Biological examples

[0909] [Example B1] Calcium-calcium 6 dye in HEK293 overexpressing TRPML1 plasma membrane variant

[0910] [Example B1.1] material

[0911] Cells: HEK293 / TRPML1-PM clone A5, a clonal cell line containing a mutant TRPML1 (with deletion of both N and C terminal dileucine target motifs) localized to the plasma membrane.

[0912] Culture medium: EMEM (ATCC, catalog number 30-2003), 10% FBS (Hyclone defined, catalog number SH30070.03), 1% pen / strep (Gibco, catalog number 15140-122), 200 mg / ml G418 (Gibco, catalog number 10131-035)

[0913] 0.05% Trypsin: Gibco, Catalog No. 25300-054

[0914] Plate: Perkin Elmer Viewplate, catalog number 6007460, black, optically clear, 384-well plate for tissue culture treatment.

[0915] Calcium dye: Molecular Devices FLIPR Calcium 6 Assay Kit, catalog number R8190 or Thermo Fisher Fluo-4 Direct, catalog number F10471

[0916] Chip: Molecular Devices 384-well FLIPR Tetra black chip, catalog number 9000-0764

[0917] [Example B1.2] method

[0918] HEK293 / TRPML1-PM clone A5 cells were trypsinized from a flask, counted, then diluted in culture medium to 15,000 cells per 25 μL, seeded in a black 384-well view plate, and cultured overnight.

[0919] One vial of calcium-6 dye was diluted with 10 mL of assay buffer (HBSS + 20 mM HEPES pH 7.4), and 25 μL of the dilution was added to each well of the cells.

[0920] After incubation at 37°C for 2 hours, the plate was moved to the FLIPR reading position.

[0921] The compound was diluted to a 5× concentration in EMEM with 0.125% FBS and placed on a 384-well plate according to the desired plate map for the assay, including DMSO alone and a carbachol control, and then the plate was moved to the source 2 position of the FLIPR.

[0922] The chip box was placed in source 1 position within the FLIPR to complete the assay setup.

[0923] Before starting the operation, an initial baseline fluorescence measurement was performed for 10 seconds before adding the compound (excitation at 470–495 nm and emission at 515–575 nm).

[0924] 12.5 μL of 5× test compound, DMSO alone, or carbachol was added via FLIPR, and real-time fluorescence measurements were performed for a further 170 seconds with one reading per second.

[0925] The data was normalized to the baseline by calculating the maximum fluorescence minus the minimum fluorescence, and the response (multiple) to the baseline was plotted logarithmically (inhibitor concentration). EC 50 This was determined using 4-parameter logistic curve fitting in Graphpad Prism 9.

[0926] [Example B2] TFEB translocation assay

[0927] [Example B2.1] reagent

[0928] Rabbit TFEB antibody, Cell Signaling Technology #4240S

[0929] PFA 16%MeOH free, Election Microscopy Sciences #50-980-487

[0930] HCS CellMask Deep Red, ThermoFisher Scientific #H32721

[0931] Hoechst 33342, AbCam #ab228551

[0932] Plate: Perkin Elmer Phenoplate #605730, black, optically clear, tissue culture treated 384-well plate.

[0933] [Example B2.2] Method, Day 1: Cells were seeded in 384WP.

[0934] Cells were seeded in the afternoon. To avoid the edge effect of the outer wall, the outermost row of the plate was not used.

[0935] HeLa cells were detached using trypsin, and the cells were counted using a cell counter.

[0936] 4000 HeLa cells were seeded per well in 50 mL of growth medium (DMEM containing 10% FBS, 1 mM sodium pyruvate, and Pen-Strep).

[0937] [Example B2.3] Method, Day 2: Assay using TRPML1 compound + staining

[0938] The compound was added to the cells using a Tecan D300e dispenser.

[0939] Cells were incubated with the compound (compound range: 80 nM to 10 mM (2 × serial dilutions - 8 dilution steps, 3 replications per condition)) at 37°C for 90 minutes.

[0940] The cells were fixed by adding 16.5 μL of 16% PFA (final concentration 4%) and incubated at room temperature for 15 minutes.

[0941] The cells were washed three times with 1 × 100 μL of PBS-TX (PBS + 0.1% Triton X-100), followed by 2 × 50 μL of PBS-TX.

[0942] The cells were blocked in 30 μL of blocking buffer (PBS-TX + 5% goat serum) for 1 hour and then permeabilized.

[0943] Cells were incubated overnight at 4°C with TFEB antibody (1:200, CST 4240S) in 30 μL of blocking buffer. The plates were kept moist to prevent evaporation.

[0944] [Example B2.4] Method, Day 3: Staining + Imaging

[0945] The cells were washed three times in PBS-TX with 3 × 100 μL using a Biotek EL406 washer.

[0946] Cells were incubated for 1 hour at room temperature in 30 μL of blocking buffer with goat anti-Rb Alexa-Fluor 488 (1:2000), cell mask (1:10,000), and Hoechst (1:10,000). The solution was dispensed using a 5 μL peristaltic cassette (EL406).

[0947] The cells were washed three times in 3 × 100 μL of PBS-TX using an EL406 washer, and then 50 mL of PBS was dispensed using a 5 μL peristaltic cassette (EL406).

[0948] On the same day, cells were imaged on the Operetta CLS high-content imaging system using a 20X water immersion objective (NA 1.0).

[0949] Image analysis was performed using Harmony V4.9 (Perkin Elmer). Briefly, nuclei were segmented from background-corrected images using Hoechst intensity, and cell bodies were segmented using HCS CellMask DeepRed. A sliding parabolic filter was applied to the AlexaFluor 488 channel (TFEB), and the sum of pixel intensities in the nuclear and cytoplasmic regions was then calculated. The ratio of nuclear to cytoplasmic TFEB was used to define TFEB translocation from the cellular matrix to the nuclear compartment. EC50 values ​​were derived by fitting the data to an S-shaped dose-response curve using Prism 9 (Graphpad).

[0950] The data from the assays described in Examples B1 and B2 are summarized in Table 2. EC of 10 μM or less. 50 Compounds exhibiting this value are considered TRPML1 activators.

[0951] [Table 16-1]

[0952] [Table 16-2]

[0953] [Table 16-3]

[0954] All publications cited in this application, including but not limited to granted patents, patent applications, and academic articles, are incorporated herein by reference in their entirety.

[0955] The present invention is described above with reference to the disclosed embodiments, but those skilled in the art will readily understand that the specific experiments detailed are merely illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Therefore, the present invention is limited only by the following claims.

Claims

1. Equation (I) 【Chemistry 1】 Compounds or pharmaceutically acceptable salts thereof (In the formula, X is either N or H, R １ is selected from H and optionally substituted alkyl groups, R ２ This is selected from optionally substituted alkyl, optionally substituted cycloalkyl, aryl and optionally substituted heterocyclyl, Alternatively, when X is H, R １ and R ２ It does not exist. R ３ H, -CH ３ Alternatively, C may be replaced. ２ ~C ６ Is it alkyl? or R １ and R ３ together with the atoms to which they are attached form a 5- to 7-membered ring, Each R ４ These are independently H, halo, cyano, or optionally substituted alkyl, R ５ａ and R ５ｂ Each is independently H or an optionally substituted alkyl, However, R ２ (Provided that it is not pyrrolidine.)

2. The compound according to claim 1, wherein X is N, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein X is H, or a pharmaceutically acceptable salt thereof.

4. R １ A compound according to claim 1 or 2, wherein H is present, or a pharmaceutically acceptable salt thereof.

5. R １ C may be substituted with a halo, oxo, or a 3-6 member heterocycline containing at least O. １ ~C ６ A compound according to claim 1 or 2, wherein the compound is alkyl, or a pharmaceutically acceptable salt thereof.

6. R １ C may be substituted with a 4-6 member heterocycline containing only halo, oxo, or O. １ ~C ３ A compound according to claim 5, wherein the compound is alkyl, or a pharmaceutically acceptable salt thereof.

7. R １ but 【Chemistry 2】 The compound according to claim 6 or a pharmaceutically acceptable salt thereof.

8. R ２ C may be substituted. １ ~C ６ A compound according to any one of claims 1, 2, or 4 to 7, which is alkyl, or a pharmaceutically acceptable salt thereof.

9. R ２ C may be substituted. １ ~C ５ A compound according to claim 8, wherein the compound is alkyl, or a pharmaceutically acceptable salt thereof.

10. R ２ but 【Chemistry 3-1】 【Chemistry 3-2】 The compound according to claim 9 or a pharmaceutically acceptable salt thereof.

11. R ２ C may be substituted. ３ ~C ６ A cycloalkyl compound or a pharmaceutically acceptable salt thereof according to any one of claims 1, 2, or 4 to 7.

12. R ２ C may be substituted. ４ ~C ６ A cycloalkyl compound or a pharmaceutically acceptable salt thereof according to claim 11.

13. R ２ but 【Chemistry 4】 The compound according to claim 12 or a pharmaceutically acceptable salt thereof.

14. R ２ A compound according to any one of claims 1, 2, or 4 to 7, wherein is a six-membered aryl compound, or a pharmaceutically acceptable salt thereof.

15. R ２ The compound according to claim 14 or a pharmaceutically acceptable salt thereof, wherein is phenyl.

16. R ２ However, C １ ~C ６ Alkyl, -C(O)O(C １ ~C ６ Alkyl), -C(O)(C １ ~C ６ Alkyl) or -C(O)(C １ ~C ６ A compound according to any one of claims 1, 2, or 4-7, or a pharmaceutically acceptable salt thereof, which is a six-membered heterocyclyl containing only nitrogen, which may be substituted with a cycloalkyl group.

17. R ２ but 【Transformation 5】 The compound according to claim 16 or a pharmaceutically acceptable salt thereof.

18. R ２ However, -CH ３ A compound according to any one of claims 1, 2, or 4-7, which is a 3- to 10-membered heterocycline containing only oxygen, which may be substituted with oxygen, or a pharmaceutically acceptable salt thereof.

19. R ２ but 【Transformation 6】 The compound according to claim 18 or a pharmaceutically acceptable salt thereof.

20. R ３ A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein is H.

21. R ３ ga-CH ３ A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof.

22. R ３ It may be substituted -CH ２ CH ３ A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof.

23. R １ and R ３ The compound according to any one of claims 1, 2, or 8 to 19, or a pharmaceutically acceptable salt thereof, wherein the compounds together with the atoms to which they are bonded form a five-membered ring.

24. R ４ A compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein is H.

25. R ４ A compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein is F or Cl.

26. R ４ C may be replaced with a halo. １ ~C ３ A compound according to any one of claims 1 to 23, which is alkyl, or a pharmaceutically acceptable salt thereof.

27. R ４ It may be replaced by one or more F - CH ３ The compound according to claim 26 or a pharmaceutically acceptable salt thereof.

28. R ４ A compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, wherein the compound is cyano.

29. R ５ａ and R ５ｂ Each of them independently becomes H or C １ ~C ３ A compound according to any one of claims 1 to 28, which is alkyl, or a pharmaceutically acceptable salt thereof.

30. R ５ａ and R ５ｂ ga-CH ３ The compound according to claim 29 or a pharmaceutically acceptable salt thereof.

31. Formula (II) 【Transformation 7】 A compound according to any one of claims 1, 2, 4-20, 24-28, or 29-30, or a pharmaceutically acceptable salt thereof.

32. Formula (III) 【Transformation 8】 A compound according to any one of claims 1, 2, 4 to 19, 21, 24 or 29 to 30, or a pharmaceutically acceptable salt thereof.

33. Formula (IV) 【Chemistry 9】 A compound according to any one of claims 1, 2, 5-10, 20, 24-28 or 29-30, or a pharmaceutically acceptable salt thereof.

34. Formula (V) 【Chemistry 10】 A compound according to any one of claims 1, 2, 5-10, 21, 24 or 29-30, or a pharmaceutically acceptable salt thereof.

35. A compound selected from the compounds in Table 1 or their pharmaceutically acceptable salts.

36. The compound according to any one of claims 1 to 35 and / or a pharmaceutically acceptable salt thereof, wherein one or more hydrogen atoms bonded to the carbon atom of the compound are replaced by deuterium atoms.

37. A pharmaceutical composition comprising a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

38. A method for modulating mucolipin TRP channel subfamily 1 (TRPML1), comprising the step of contacting TRPML1 with an effective amount of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 37.

39. A method for treating a disease related to TRPML1, comprising the step of administering to a subject an effective amount of a compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 37.

40. The method according to claim 39, wherein the disease is a neurodegenerative disease, a lysosomal storage disease, a metabolic disease, a cardiovascular disease, an inflammatory disorder, an immunological disorder, cancer, or aging.

41. The method according to claim 39, wherein the disease is a neurodegenerative disease.

42. The method according to claim 41, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), HIV-related dementia, Charcot-Marie-Tooth disease, and Huntington's disease.

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