Estrogen receptor β-ligand for the prevention and treatment of multiple sclerosis (MS) and other demyelinating, inflammatory, and neurodegenerative diseases

Abstract

6- or 7-substituted 2-(4-hydroxyphenyl)-2H-indazole-5-ol compounds are estrogen receptor β ligands with immunomodulatory properties that increase oligodendrocyte survival, differentiation, and remyelination. The compounds, compositions, and kits are useful in the treatment of multiple sclerosis and endometriosis.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 488,883, filed on 7 March 2023, which is incorporated herein by reference in its entirety.

[0002] Statements relating to research or development funded by the U.S. government. This invention was created under approval number R01 DK015556, granted by the National Institutes of Health (NIH) with government support. The government has certain rights in this invention.

[0003] Technical field This invention relates to estrogen receptor β-ligands and their use in the treatment of demyelinating diseases and endometriosis. [Background technology]

[0004] Demyelinating diseases are characterized by damage to the myelin sheath of the central nervous system. Once the myelin sheath is damaged, the axons are exposed and unable to effectively transmit nerve impulses. Symptoms include vision loss, muscle weakness, muscle stiffness and spasms, ataxia, sensory alterations, pain, and changes in bladder and bowel function.

[0005] Multiple sclerosis (MS) is the most common autoimmune demyelinating and neurodegenerative disease of the central nervous system (CNS). The cause and treatment of MS are unknown. Experimental autoimmune encephalomyelitis (EAE) is the most common inducible animal model of MS, exhibiting recurrent inflammation, demyelination, and neurodegeneration observed in MS. Many currently approved MS treatments have been developed using the EAE model, including interferon (IFN)β, glatiramer acetate, fingolimod, and the anti-CD20 monoclonal antibody, ocrelizumab. While approved treatments are effective in treating a variety of symptoms and reducing inflammation, they cannot reverse or prevent neurodegeneration or initiate remyelination.

[0006] Accumulated evidence suggests that estrogen possesses both neuroprotective and immunomodulatory properties, making it an attractive candidate for the treatment of MS (Khalaj, 2016). Estrogen distorts the inflammatory T helper (Th)1 response prevalent in MS towards an anti-inflammatory Th2 profile (Cua et al., 1995; Nicot, 2009). Furthermore, preclinical studies have shown that treatment with pregnancy-level placental estrogen hormone estriol attenuated the severity of EAE disease (Jansson and Holmdahl, 1998; Kim et al., 1999). However, despite their great potential for treating MS, endogenous estrogen therapy has several undesirable or harmful side effects (Banks and Canfell, 2009). In addition to feminized male recipients, treatment with endogenous estrogen increases the risk of developing breast and endometrial cancer in females (Banks and Canfell, 2009). Importantly, the carcinogenic effects of estrogen are mediated through estrogen receptor (ER)α, not through ERβ, and targeted therapy of specific ER subtypes may provide the benefits of estrogen therapy while avoiding these side effects (Burns and Korach, 2012).

[0007] Developing and providing new therapeutic agents that can induce intentional remyelination and provide neuroprotection and immune system regulation without unwanted side effects is critically important.

Summary of the Invention

[0008] The present invention provides compounds or pharmaceutically acceptable salts thereof and methods and compositions disclosed herein for treating demyelinating diseases, differentiating oligodendrocyte progenitor cells, or promoting remyelination of demyelinated axons.

[0009] In one aspect, the present invention relates to formula (I)

Chemical formula

[0010] In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0011] In another embodiment, the present invention provides a method for treating a demyelinating disease, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, to a subject in need thereof.

[0012] In another embodiment, the present invention provides a method for promoting the remyelination of demyelinated axons, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, to a subject in need.

[0013] In another embodiment, the present invention provides a method for differentiating oligodendrocyte progenitor cells, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, to a subject in need thereof.

[0014] In another embodiment, the present invention provides a method for treating endometriosis, comprising administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, to a subject in need.

[0015] In another aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, for use in the treatment of demyelinating diseases, or in promoting the remyelination of demyelinated axons, or in the differentiation of oligodendrocyte progenitor cells, or in the treatment of endometriosis.

[0016] In another aspect, the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, in the manufacture of a drug for the treatment of demyelinating diseases, or for promoting the remyelination of demyelinated axons, or for the differentiation of oligodendrocyte progenitor cells, or for the treatment of endometriosis.

[0017] In another embodiment, the present invention provides a kit comprising a compound of formula (I), or a pharmaceutically acceptable salt or composition thereof, and instructions for use. [Brief explanation of the drawing]

[0018] [Figure 1A] Figures 1A-1D show the effects of treatment with 7-substituted indazole compounds 1-5, 7, 9, and 11 on myelin basic protein expression in OL (MBP+-OL, upper two panels) and on total cell number (DAPI nuclear staining, lower two panels) as an indicator of cell proliferation / survival. [Figure 1B] (As stated above.) [Figure 1C] (As stated above.) [Figure 1D] (As stated above.) [Figure 2] Figure 2A shows the effect of treatment with 6-substituted indazole compounds 17-20 on myelin basic protein expression in OL (MBP+-OL). Figure 2B shows the effect of treatment with 6-substituted indazole compounds 17-20 on total cell number (DAPI nuclear staining) as an indicator of cell proliferation / survival. [Figure 3] The results of 2-hour single-point pharmacokinetic (PK) studies of compounds 7, 12, and 14 administered orally (PO) or subcutaneously (SC) are shown. [Figure 4] The results of a 2-hour single-point pharmacokinetic (PK) study of compound 17 administered either orally (PO) or subcutaneously (SC) are shown. [Figure 5]The effects of compounds 5, 7, 12, and 14 on myelin formation in the cuprizon model are shown. The height of each bar graph represents the level of myelin formation, assessed by MOG expression in the white matter track of the corpus callosum region, quantified by immunohistochemical analysis (IHC) after staining with MOG antibody. N: normal diet, DM: demyelination after 6 weeks of cuprizon treatment, SC-Veh: spontaneous remyelination after injection of matrix in saline only (no compounds; recovery is only about 40%). [Figure 6] This shows the effect of compound 17 on myelin formation in the cuprizon model. The height of each bar graph represents the level of myelin formation, assessed by MOG expression in the white matter track of the corpus callosum region, quantified by immunohistochemical analysis (IHC) after staining with MOG antibody. N: normal diet, DM: demyelination after 6 weeks of cuprizon treatment, SC-Veh: spontaneous remyelination after injection of matrix in saline only (no compound; recovery is only about 40%). [Figure 7] The effects of compounds 5 and 7 on uterine weight compared to the media used in non-ovariectomized mice in the Cuprizon model are shown. [Modes for carrying out the invention]

[0019] Detailed explanation 1.Definition As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, for example, as generally exemplified above, or as exemplified by the specific classes, subclasses, and species of the present invention. As described herein, the variables in Formula I include certain groups, such as alkyl and cycloalkyl groups. As those skilled in the art will understand, the substituent combinations envisioned by the present invention are combinations that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, means a compound that is substantially unaltered when subject to conditions that enable their production, detection, and preferably their recovery, purification, and use for one or more purposes disclosed herein. In some embodiments, a stable or chemically feasible compound means a compound that is substantially unaltered when held at a temperature of 40°C or less for at least one week in the absence of moisture or other chemical reaction conditions.

[0020] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative and not intended to be limiting.

[0021] The words “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and their variations are intended, when used herein, to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The singular forms “a,” “an,” and “the” include multiple references unless explicitly indicated otherwise in the context. This disclosure also contemplates other embodiments that “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether expressly or not.

[0022] The modifier "approximately" used in relation to quantity encompasses a defined value and has a meaning indicated by the context (for example, it includes at least the degree of error related to the measurement of a particular quantity). The modifier "approximately" should also be considered to disclose a range defined by the absolute values ​​of two endpoints. For example, the expression "approximately 2 to approximately 4" also discloses the range "2 to 4". The term "approximately" may refer to ±10% of a given number. For example, "approximately 10%" may indicate a range of 9% to 11%, and "approximately 1" may mean 0.9 to 1.1. Other meanings of "approximately," such as rounding, may be obvious from the context; for example, "approximately 1" may also mean 0.5 to 1.4.

[0023] Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are based on the periodic table of elements, CAS versions, Handbook of Chemistry and Physics, 75. thThe identification is based on the inside cover of the ed., and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional parts and reactivity, are described in *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999; and *Smith and March March's Advanced Organic Chemistry*, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This is described in Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of these are incorporated herein by reference.

[0024] As used herein, the term "alkyl" means a straight-chain or branched-chain saturated hydrocarbon. 1~4 The term "alkyl" refers to a linear or branched hydrocarbon containing 1 to 4 carbon atoms. Representative examples of alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0025] As used herein, the term "alkenyl" means a linear or branched hydrocarbon chain containing at least one carbon-carbon double bond.

[0026] As used herein, the term "alkylene" refers to a divalent group derived from a straight-chain or branched-chain saturated hydrocarbon. Representative examples of alkylenes include, but are not limited to, CH2-, -CD2-, -CH2CH2-, -C(CH3)(H)-, -C(CH3)(D)-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0027] The term "halogen" refers to chlorine, bromine, iodine, or fluorine atoms.

[0028] The term "haloalkyl," as used herein, means alkyl, as defined herein, where 1, 2, 3, 4, 5, 6, or 7 hydrogen atoms are substituted with halogens. Representative examples of haloalkyls include, but are not limited to, 2-fluoroethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and 2,2,2-trifluoro-1,1-dimethylethyl.

[0029] The term "fluoroalkyl," as used herein, means an alkyl group as defined herein, where 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms are substituted with fluorine. Representative examples of fluoroalkyls include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyls such as 3,3,3-trifluoropropyl.

[0030] Terms such as "alkyl," "cycloalkyl," "alkylene," and "cycloalkylene" may be preceded by a name indicating the number of atoms present in the group in a particular case (for example, "C"). 1~4 Alkyl," "C 1~4These names are used as is generally understood by those skilled in the art. For example, the subscript number following the notation "C" indicates the number of carbon atoms present in the following group. Thus, "C3 alkyl" is an alkyl group having three carbon atoms (i.e., n-propyl, isopropyl). 1~4 When a range is given, such as "C", the members of the subsequent group may have any number of carbon atoms that fall within the listed range. For example, "C 1~4 "Alkyl" refers to an alkyl group having 1 to 4 carbon atoms, but it can be arranged (i.e., linear or branched).

[0031] Unless otherwise specified, the structures shown herein include the structures of all isomers (e.g., enantiomers, diastereoisomers, and geometric (or stereostructural) forms); for example, the R and S stereostructures for each chiral center, the (Z) and (E) double bond isomers, and the (Z) and (E) stereostructural isomers. Thus, single stereochemical isomers, as well as enantiomeric mixtures, diastereoisomeric mixtures, and geometric (or stereostructural) mixtures of the compound, are included within the scope of the invention. Unless otherwise specified, all tautomers of the compound of the invention are included within the scope of the invention. Thus, the tautomers of the compound of formula I are included within the scope of the invention. Their structures also include, if necessary, the zwitterionic forms of the compound or salt of formula I.

[0032] "Effective dose" or "therapeutic effective dose," as used herein, refers to an amount of an agent or composition or combination of compositions administered that is sufficient to alleviate, to some extent, one or more symptoms of a disease or condition being treated. The result may be a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired modification of the biological system. For example, an "effective dose" for therapeutic use is the amount of a composition containing the compounds disclosed herein required to produce a clinically significant reduction in the symptoms. The appropriate "effective" dose in any individual case may be determined using techniques such as dose escalation studies. That dose may be administered in one or more doses. However, the precise determination of what is considered an effective dose may be based on individual patient factors, including, but are not limited to, the patient's age, size, type or severity of the disease, stage of the disease, route of administration of regenerative cells, type or degree of additional therapy used, ongoing disease progression, and desired type of treatment (e.g., invasive treatment vs. conventional treatment).

[0033] As used herein, “treat,” “treating,” etc., mean the slowing, cessation, or reversal of the progression of a disease or disorder when the composition described herein is provided to a suitable control subject. The term also means the reversal of the progression of such disease or disorder to a point in which cell proliferation is eliminated or significantly reduced. As such, “treating” means the application or administration of the composition described herein to a subject having a disease or symptoms of a disease, wherein the objective is to cure, treat, alleviate, mitigate, modify, treat, remission, improve, or affect the disease or symptoms of a disease.

[0034] The “subject” or “patient” may be human or non-human, and may include, for example, animal strains or species used as a “model system,” such as the mouse model described herein, for research purposes. Similarly, the patient may include either an adult or a juvenile (e.g., a child). Furthermore, the patient may mean any organism, preferably a mammal (e.g., human or non-human), that could benefit from the administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the methods and compositions provided herein, the mammal is human.

[0035] As used herein, the terms “provide,” “administer,” and “introduce” are interchangeable herein and refer to the placement of the compositions of this disclosure into a subject by a method or route that results in at least partial localization of the composition to a desired site. The compositions may be administered by any suitable route that results in delivery to a desired site in the subject.

[0036] 2.Compound A first aspect of the present invention is R 1 ~R 6 The present invention provides a compound or composition of formula (I), or a pharmaceutically acceptable salt thereof, where n is as defined herein.

[0037] The following numbered embodiments of the present invention are disclosed. The first embodiment is denoted as E1, and subsequent embodiments are denoted as E1.1, E2, E2.1, E2.2, E2.3, E3, and so on.

[0038] E1. Equation (I) [ka] (In the formula, R 1 and R 2 These are, independently, hydrogen, halogen, and C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, cyano, OH, -OC 1~4 Alkyl, or -OC 1~4 It is a fluoroalkyl, however, R 1 and R 2 At least one of them is not hydrogen; R 3 is hydrogen, halogen, or C 1~4 It is alkyl; R 4 Hydrogen, halogen, C 1~4 Alkyl, C 1~4 Fluoroalkyl, or C 2~4 It is an alkenil; R 5 OH and R 5 It is replaced by either a meta or para position; R 6 Each instance of occurrence is independent of halogen and C. 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, cyano, OH, -OC 1~4 Alkyl, or -OC 1~4 It is a fluoroalkyl group; and n is 0, 1, or 2. Compounds thereof, or pharmaceutically acceptable salts thereof.

[0039] E1.1.R 1 and R 2 However, independently, hydrogen, halogen, C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, Cyano, -OC 1~4 Alkyl, or -OC 1~4 It is a fluoroalkyl, however, R 1 and R 2 At least one of them is a non-hydrogen compound of E1, or a pharmaceutically acceptable salt thereof.

[0040] E2.R 1 However, halogen, C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, Cyano, -OC 1~4 Alkyl, or -OC 1~4 A fluoroalkyl compound of type E1 or E1.1, or a pharmaceutically acceptable salt thereof.

[0041] E2.1.R 1 However, a halogen, an E2 compound, or a pharmaceutically acceptable salt thereof.

[0042] E2.2.R 1 However, a fluoro, E2.1 compound, or a pharmaceutically acceptable salt thereof.

[0043] E2.3.R 1 However, it is a chloro (choro) compound of E2.1, or a pharmaceutically acceptable salt thereof.

[0044] E3.R 1 However, a compound of E1 or E1.1, which is hydrogen, or a pharmaceutically acceptable salt thereof.

[0045] E4.R 2 However, halogen, C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, cyano, OH, -OC 1~4 Alkyl, or -OC 1~4 A fluoroalkyl compound, either E1 or E2-E3, or a pharmaceutically acceptable salt thereof.

[0046] E4.1.R 2 However, halogen, C 1~4 Alkyl, C 1~4 Fluoroalkyl, C 2~4 Alkenyl, Cyano, -OC 1~4 Alkyl, or -OC 1~4 A fluoroalkyl compound, one of the E1-E4 compounds, or a pharmaceutically acceptable salt thereof.

[0047] E4.2.R 2 is a compound of E4.1 that is a halogen, or a pharmaceutically acceptable salt thereof.

[0048] E4.3.R 2 is a compound of E4.2 that is fluorine, or a pharmaceutically acceptable salt thereof.

[0049] E4.4.R 2 is a compound of E​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​However, a fluoro, E7 compound, or a pharmaceutically acceptable salt thereof.

[0058] E7.2.R 4 However, a chloro compound of E7, or a pharmaceutically acceptable salt thereof.

[0059] E7.3.R 4 However, bromo is a compound of E7, or a pharmaceutically acceptable salt thereof.

[0060] E7.4.R 4 However, it is iodine, an E7 compound, or a pharmaceutically acceptable salt thereof.

[0061] E8.R 4 However, it is hydrogen, one of the compounds E1 to E6, or a pharmaceutically acceptable salt thereof.

[0062] E9.Formula (IA): [ka] As shown, R 5 However, any of the E1-E8 compounds, or a pharmaceutically acceptable salt thereof, is substituted at the meta position.

[0063] E10.Formula (IB): [ka] As shown, R 5 However, any of the E1-E8 compounds, or a pharmaceutically acceptable salt thereof, is substituted at the para position.

[0064] A compound from E1 to E10, or a pharmaceutically acceptable salt thereof, in which E11.n is 1.

[0065] E12.Formula (II): [ka] A compound of E11 having, or a pharmaceutically acceptable salt thereof.

[0066] E13.Formula (III): [ka] A compound of E11 having, or a pharmaceutically acceptable salt thereof.

[0067] E14.R 6 However, at each occurrence, independently, halogen or C 1~4 A compound that is alkyl, belonging to any of the E1-E13 groups, or a pharmaceutically acceptable salt thereof.

[0068] E14.1.R 6 However, each instance is independently a halogen (i.e., the halogens may be the same or different), an E14 compound, or a pharmaceutically acceptable salt thereof.

[0069] E14.2.R 6 However, each instance is fluoro, a compound of E14.1, or a pharmaceutically acceptable salt thereof.

[0070] E14.3.R 6 However, each occurrence is independent of C 1~4 A compound of E14 that is alkyl (i.e., the alkyls may be the same or different), or a pharmaceutically acceptable salt thereof.

[0071] E14.4.R 6 However, each instance is methyl, a compound of E14.3, or a pharmaceutically acceptable salt thereof.

[0072] A compound from E1 to E10, or a pharmaceutically acceptable salt thereof, in which E15.n is 0.

[0073] E16.Formula (IV): [ka] A compound of E15 having, or a pharmaceutically acceptable salt thereof.

[0074] E17.Formula (V): [ka] A compound of E15 having, or a pharmaceutically acceptable salt thereof.

[0075] E18.7-Bromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Bromo-3-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Bromo-3-chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-dibromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Bromo-2-(4-hydroxyphenyl)-3-iodo-2H-indazole-5-ol; 7-Chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-Dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-Chloro-7-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-Chloro-7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Bromo-3-chloro-2-(3-hydroxyphenyl)-2H-indazole-5-ol; 7-Chloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-Dichloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 7-Chloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-Dichloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3-Chloro-6-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-Chloro-6-fluoro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol; 3,6-Dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,6-Dichloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol; 3-Chloro-2-(4-hydroxyphenyl)-2H-indazole-5,7-diol; and 3-Chloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5,7-diol; A compound of type E1 selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof.

[0076] A pharmaceutical composition comprising one of the compounds E1 to E18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0077] E20. A method for treating a demyelinating disease, comprising administering a therapeutically effective amount of any compound from E1 to E18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of E19 to a subject in need thereof.

[0078] E21. Demyelinating disease is multiple sclerosis, as described in E20.

[0079] E22. The method of E21, wherein the multiple sclerosis is primary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis.

[0080] E23. A method for promoting the remyelination of demyelinated axons, comprising administering a therapeutically effective amount of any compound from E1 to E18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of E19 to a subject in need thereof.

[0081] E24. A method for differentiating oligodendrocyte progenitor cells, comprising administering a therapeutically effective amount of any compound E1 to E18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of E19 to a subject requiring such treatment.

[0082] E25. A method for treating endometriosis, comprising administering a therapeutically effective amount of any compound from E1 to E18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of E19 to a subject in need thereof.

[0083] E26. Any compound from E1 to E18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of E19, for use in the treatment of demyelinating diseases, or in promoting the remyelination of demyelinated axons, or in the differentiation of oligodendrocyte progenitor cells, or in the treatment of endometriosis.

[0084] E27. Use of any compound from E1 to E18, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of E19, in the manufacture of a drug for the treatment of demyelinating diseases, or for promoting the remyelination of demyelinated axons, or for the differentiation of oligodendrocyte progenitor cells, or for the treatment of endometriosis.

[0085] A kit comprising one of the compounds E1 to E18, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition E19, and instructions for use thereof.

[0086] Certain compounds can exist in one or more specific geometric, optical, enantiomer, diastereoisomer, epimeric, atropic, stereoisomer, tautomer, stereoconformal, or anomeric forms, for example, but not limited to, cis and trans; E and Z; c, t, and r; endo and exo; R, S, and meso; D and L; d and 1; (+) and ( ); keto, enol, and enolate; syn and anti; syncrinal and anticrinal; α and β; axial and equatorial; boat, chair, twist, envelope, and half-chair; and combinations thereof, and are collectively referred to hereafter as "isomers" (or "isomer forms").

[0087] The compounds may be prepared in racemic form or as individual enantiomers or diastereoisomers by either stereospecific synthesis or resolution. For example, the compounds can be resolved by standard techniques, such as the formation of stereoisomer pairs by salt formation with optically active bases, followed by fractional crystallization and regeneration of the free acids. The compounds can also be resolved by the formation of stereoisomerous esters or amides, followed by chromatographic separation and removal of chiral additives. Alternatively, the compounds can be resolved using a chiral HPLC column. Enantiomers can also be obtained by the kinetic resolution of the racemic compound of the corresponding ester using lipase enzymes.

[0088] Exemplary tautomers include, for example, the following tautomer pairs: keto / enol and imine / enamine.

[0089] In the compounds of formula (I) and any subformula, every "hydrogen" or "H", whether explicitly listed or implied in the structure, is a hydrogen isotope. 1 H (protium) and 2 It includes H (Deuterium).

[0090] In another embodiment, the compound includes an isotopically labeled form. The isotopically labeled form of the compound is identical to the compound, except that one or more atoms of the compound are substituted with one or more atoms having a different atomic mass or mass number than the atoms that normally exist in a higher natural abundance. Examples of isotopes readily available commercially and that can be incorporated into the compound by well-known methods include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, for example, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 18 F and 36 Cl is an example. The isotope-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples, which involve using a suitable isotope-labeled reagent in place of the non-isotope-labeled reagent.

[0091] Isotope-enriched forms of compounds of formula (I), or any subformula, can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying examples, using appropriate isotope-enriching reagents in place of non-isotope-enriching reagents. The degree of isotope enrichment can be characterized as the percentage incorporation of a particular isotope at the isotope-labeled atom (e.g., % deuterium incorporation at the deuterium label).

[0092] 3.How to use method In some embodiments, the compound of formula (I) may reduce pro-inflammatory cytokines and / or increase anti-inflammatory cytokines. In another embodiment, the method of the present invention may include reducing pro-inflammatory cytokines and / or increasing anti-inflammatory cytokines with the compound of formula (I) or a composition thereof. In another embodiment, the method of the present invention may be useful for treating or reducing the severity of a disease or disorder selected from among diseases or disorders associated with increased pro-inflammatory cytokines, and may include administering a therapeutically effective amount of the compound of formula (I) or a composition thereof to a subject in need.

[0093] In some embodiments, compounds of formula (I) can promote the growth, differentiation, or survival of oligodendrocytes. In one embodiment, the method described herein also provides a method for promoting the growth, differentiation, or survival of oligodendrocytes, comprising contacting oligodendrocytes with a compound of formula (I) or a composition thereof.

[0094] In another embodiment, the method of the present invention comprises promoting the proliferation, differentiation, or survival of oligodendrocytes. In yet another embodiment, the method of the present invention is useful for treating or reducing the severity of a disease or disorder selected from among diseases or disorders associated with a lack of oligodendrocyte proliferation, differentiation, or survival, and comprises administering a therapeutically effective amount of a compound of formula (I) or a composition thereof to a subject in need.

[0095] In some embodiments, the compound of formula (I) can increase chemokines involved in the proliferation, differentiation, and survival of oligodendrocyte progenitor cells. In other embodiments, the method of the present invention can increase chemokines involved in the proliferation, differentiation, and survival of oligodendrocyte progenitor cells using the compound of formula (I) or a composition thereof.

[0096] In some embodiments, the compound of formula (I) can suppress the production of chemokines and / or cytokines that promote oligodendrocyte death. In other embodiments, the method of the present invention can reduce the production of chemokines and / or cytokines involved in oligodendrocyte death by using the compound of formula (I) or a composition thereof.

[0097] In another embodiment, the method of the present invention includes promoting myelin formation by contacting nerve cells, oligodendrocyte cells, or oligodendrocyte progenitor cells with a compound of formula (I) or a composition thereof.

[0098] In another embodiment, the method of the present invention is useful for treating or reducing the severity of a disease or disorder selected from demyelinating diseases or conditions, and comprises administering a therapeutically effective amount of a compound of formula (I) or a composition thereof to a subject in need. In one embodiment, the demyelinating disease or condition is a CNS disorder or CNS demyelinating disease as described herein. In one embodiment, the disease is multiple sclerosis.

[0099] In another embodiment, the subject has or is at risk of having multiple sclerosis. The subject with multiple sclerosis may be at any stage of treatment or disease. The subject with multiple sclerosis may have one or more of the following: benign multiple sclerosis, relapsing-remitting multiple sclerosis, inactive relapsing-remitting multiple sclerosis, active relapsing-remitting multiple sclerosis, progressive relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis, clinically isolated syndromes, or clinically defined multiple sclerosis. The subject may be asymptomatic. The subject may have one or more multiple sclerosis-like symptoms, e.g., multiple sclerosis-like symptoms with clinically isolated syndromes or clinically defined multiple sclerosis. The subject may have one or more multiple sclerosis relapses.

[0100] In some embodiments, the subject has a relapsing form of multiple sclerosis, such as relapsing-remitting multiple sclerosis or relapsing secondary progressive multiple sclerosis. In one embodiment, the subject has relapsing-remitting multiple sclerosis and one or more ongoing clinical exacerbations. In another embodiment, the subject has relapsing-remitting multiple sclerosis and one or more asymptomatic activity. In one embodiment, clinical exacerbations or asymptomatic activity can be indicated by white matter lesions using magnetic resonance imaging.

[0101] In one embodiment, clinical exacerbations or asymptomatic activity can be monitored by functional readings such as gait changes (e.g., changes in gait, axial sway), T25W changes, and / or EDSS changes. In another embodiment, clinical exacerbations or asymptomatic activity can be monitored by visual evoked potential assays, visual acuity tests, optic nerve thickness measurements, or myelin-labeled assays.

[0102] Subjects with multiple sclerosis may be treated or at any stage of the disease, and treatment with a compound of formula (I) of the present invention results in improvement of the disease or symptoms. In one embodiment, improvement in the disease or symptoms is demonstrated by a reduction or disappearance of one or more white matter lesions in the brain. In another embodiment, improvement in the disease or symptoms is demonstrated by improved function such as improved gait, improved pace, reduced sway, improved T25W score, or improved EDSS score. In another embodiment, improvement in the disease or symptoms is demonstrated by improvement in visual acuity testing or visual evoked potential assay. In another embodiment, improvement in the disease or symptoms is demonstrated by increased optic nerve thickness. In another embodiment, improvement in the disease or symptoms is demonstrated by increased myelin formation in a myelin labeling assay.

[0103] In another embodiment, the compound of formula (I) of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting myelin regeneration in progressive demyelinating diseases. In one embodiment, the compound of formula (I) of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting myelin regeneration in primary progressive multiple sclerosis. In another embodiment, the compound of formula (I) of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting myelin regeneration in secondary progressive multiple sclerosis. In another embodiment, the compound of formula (I) of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting myelin regeneration in relapsing-remitting multiple sclerosis. In another embodiment, the compound of formula (I) of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting myelin regeneration in progressive relapsing multiple sclerosis.

[0104] In yet another embodiment, the compounds of Formula I of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting remyelination at the cellular level, where oligodendrocyte cells are stimulated to regenerate or differentiate.

[0105] In another embodiment, the compounds of Formula I of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting remyelination at the cellular level, thereby stimulating, regenerating or differentiating oligodendrocyte cells, and thereby treating demyelinating diseases or disorders. In yet another embodiment, the compounds of Formula I of the present invention, as well as the methods, compositions and kits disclosed herein, are useful for promoting remyelination at the cellular level, thereby stimulating remyelination of axons by oligodendrocyte cells, and thereby treating demyelinating diseases or disorders.

[0106] In another embodiment, the present invention provides a method for treating or reducing the severity of a demyelinating disease in a subject, comprising administering an effective amount of a compound of formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. The demyelinating disease may be a demyelinating myelinating disease or a demyelinating white matter atrophy disease. The demyelinating myelinating disease may be multiple sclerosis, Devic's disease, or another inflammatory demyelinating disorder. The demyelinating white matter atrophy disease may be a central nervous system disorder, central pontine myelinolysis, leukodystrophy, or another myelopathy. The demyelinating disease may affect the central nervous system or the peripheral nervous system. Demyelinating disorders of the peripheral nervous system include: Guillain-Barré syndrome and its chronic counterparts; chronic inflammatory demyelinating polyneuropathy; anti-MAG peripheral neuropathy; Charcot-Marie-Tooth disease and its corresponding hereditary neuropathy responsible for pressure palsy; copper deficiency-related conditions (peripheral neuropathy, myelopathy, and rarely optic neuropathy); and progressive inflammatory neuropathy.

[0107] In another aspect, the present invention relates to hearing impairment, optic neuritis, decreased visual acuity, diplopia, nystagmus, ataxia, internuclear ophthalmoplegia, movement and sound phosphenes, afferent pupillary abnormalities, paresis, parenoplegia, parparaplegia, parhemiplegia, quadriplegia, paraplegia, hemiplegia, quadriplegia, spasticity, dysarthria, motor dysfunction, gait dysfunction, muscle atrophy, spasm, muscle cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, gait abnormalities, foot drop, reflex dysfunction, vibration sensation, anesthesia, neuralgia, neuropathic and neurogenic pain, Lhermitte's sign, proprioceptive dysfunction, trigeminal neuralgia, ataxia, and A method is provided for treating, preventing, or relieving one or more symptoms of multiple sclerosis or another neurodegenerative disease, selected from graphic tremor, dysmetria, vestibular ataxia, rotational vertigo, ataxia, dystonia, disability progression, antagonistic repetition failure, frequent urination, bladder spasm, flaccid bladder, detrusor-sphincter dyssynergy, erectile dysfunction, or anorgasmia, comprising administering an effective amount of a compound of formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, together with one or more additional therapeutic agents administered concurrently with, before, or after, treatment with the compound or pharmaceutical composition.

[0108] In another embodiment, the present invention provides a method for treating, preventing or relieving one or more symptoms of endometriosis, comprising administering an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a subject in need.

[0109] Administration As described herein, the compounds of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions as described elsewhere herein, can be administered to such subjects by a variety of methods. In any of the uses or methods described herein, administration may be via a variety of routes known to those skilled in the art, including, but not limited to, oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and / or intraperitoneal administration to subjects requiring it.

[0110] The amount of the compound of the present invention, or a pharmaceutically acceptable salt thereof, required for therapeutic use will vary depending not only on the specific compound or salt selected, but also on the route of administration, the nature and / or symptoms of the estrogen receptor-dependent and / or estrogen receptor-mediated disease or condition being treated, and the patient's age and condition, and ultimately on the discretion of the attending physician or clinician. In the case of administration of a pharmaceutically acceptable salt, the dose may be calculated as free base. As will be understood by those skilled in the art, under certain circumstances, it may be necessary to administer the compounds disclosed herein in amounts exceeding or far exceeding the dose ranges described herein, particularly to effectively and aggressively treat invasive estrogen receptor-dependent and / or estrogen receptor-mediated diseases or conditions.

[0111] In some embodiments, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions may be administered by inhalation, oral administration, or intravenous administration. However, generally, preferred doses are often in the range of about 0.01 mg / kg to about 1000 mg / kg, for example, about 0.05 mg / kg to about 10 mg / kg. For example, preferred doses may be in the range of about 0.10 mg / kg to about 10 mg / kg per body weight / day, for example, about 0.10 mg / kg to about 0.50 mg / kg per recipient's body weight / day, about 0.10 mg / kg to about 1.0 mg / kg per recipient's body weight / day, about 0.15 mg / kg to about 5.0 mg / kg per recipient's body weight / day, or about 0.2 mg / kg to about 4.0 mg / kg per recipient's body weight / day. The compound may be administered in unit dosage forms containing, for example, 1 to 100 mg, 10 to 100 mg, or 5 to 50 mg of the active ingredient / unit dosage form.

[0112] The desired dose may, for convenience, be presented as a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more partial doses per day. The partial dose itself may be further divided into, for example, several separate, well-spaced doses.

[0113] As will be readily apparent to those skilled in the art, the useful in vivo dose and specific mode of administration will vary depending on age, weight, severity of pain, and the mammalian species being treated, the specific compound used, and the specific application in which these compounds are used. Determining the effective dose level, which is the dose level required to achieve the desired result, can be achieved by those skilled in the art using conventional methods, such as human clinical trials, in vivo studies, and in vitro studies. For example, useful doses of the compounds of the present invention, or pharmaceutically acceptable salts thereof, can be determined by comparing their in vitro and in vivo activities in animal models. Such comparisons can be made by comparison with established drugs, such as fulvestrant.

[0114] Dosage and intervals can be individually adjusted to provide a plasma level of the active portion sufficient to maintain the modulating effect, or minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vivo and / or in vitro data. The dose required to achieve the MEC will depend on the individual characteristics and route of administration. However, plasma concentrations can be determined using FIPLC assays or bioassays. Dose intervals can also be determined using the MEC value. The composition should be administered using a regimen that maintains a plasma level above the MEC for a period of 10–90%, preferably 30–90%, and most preferably 50–90%. In the case of topical administration or selective uptake, the effective topical concentration of the drug may not be related to the plasma concentration.

[0115] It should be noted that the attending physician will be aware of when and how to terminate, interrupt, or adjust the administration due to toxicity or organ failure. Conversely, the attending physician will also be aware of adjusting the treatment to a higher level if the clinical response is insufficient (too toxic). The scale of the dose administered in the management of the target disorder will vary depending on the severity of the estrogen receptor-dependent and / or estrogen receptor-mediated disease or condition to be treated, as well as the route of administration. The severity of the estrogen receptor-dependent and / or estrogen receptor-mediated disease or condition may be assessed, for example, in part by standard prognostic assessment methods. Furthermore, the dose, and possibly the frequency of administration, will also vary depending on the individual patient's age, weight, and response. Programs comparable to those discussed above may be used in veterinary medicine.

[0116] The compounds, salts, and compositions disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicity of a particular compound, or a subset of compounds sharing a particular chemical part, can be established by determining in vitro toxicity to cell lines such as mammals, and preferably human cell lines. The results of such tests often predict toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of a particular compound in animal models such as mice, rats, rabbits, dogs, or monkeys can be determined using known methods. The efficacy of a particular compound can be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model for determining efficacy, those skilled in the art may be guided by state-of-the-art techniques for selecting an appropriate model, dose, route of administration, and / or regime.

[0117] A therapeutically effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein may be administered alone or in combination with at least one additional therapeutic agent in a therapeutically effective amount. In some embodiments, the compound or pharmaceutical composition disclosed herein is administered in combination with at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent is administered before or after the administration of the compound or pharmaceutical composition disclosed herein. In some embodiments, the compounds and compositions of the present invention may be administered in combination with one or more of the following: interferon beta-1a, interferon beta-1b, glatiramer acetate, pegylated interferon beta-1a, daclizumab, teriflunomide, fingolimod, dimethyl fumarate, alemtuzumab, mitoxantrone, ocrelizumab, or natalizumab. In some embodiments, the compounds and compositions of the present invention may be administered in combination with one or more of the following: methylprednisolone, prednisone, ACTH, onabotulinum toxin A, desmopressin, tolterodine, oxybutynin, dalifenacin, tamsulosin, terazosin, prazosin, mirabegron, propantheline, trospium chloride, imipramine, solifenacin succinate, dantrolene, baclofen, clonazepam, diazepam, tizanidine, isoniazid, clonazepam, or darfampridine.

[0118] 4. Pharmaceutical compositions In another aspect of the present invention, pharmaceutically acceptable compositions are provided, which comprise any of the compounds described herein and optionally comprise a pharmaceutically acceptable carrier, auxiliary agent, or medium. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or medium.

[0119] The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulation, sugar-coated tablet production, powdering, emulsification, capsule encapsulation, capture, or freeze-drying processes.

[0120] As used herein, the term “pharmaceutically acceptable salt” means a salt that, within the bounds of sound medical judgment, is suitable for use in contact with human and lower animal tissues, does not produce excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable risk-benefit ratio. pharmaceutically acceptable salts are well known in the art. For example, SMBerge, et al., describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed by using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoic acid, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and lac. This includes tobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, valersates, etc. Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N(C) 1~4This invention also includes alkyl) tetrasalts. The present invention also envisions the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water-soluble, oil-soluble, or dispersible products can be obtained by such quaternization. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Furthermore, pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl (e.g., phenyl / substituted phenyl) sulfonates.

[0121] As described herein, the pharmaceutically acceptable compositions of the present invention also include pharmaceutically acceptable carriers, auxiliaries, or media, which, when used herein, include a wide range of solvents, diluents, or other liquid media, dispersions or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., to suit the specific dosage form of the invention. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses a variety of carriers used in the formulation of pharmaceutically acceptable compositions and known techniques for preparing them. Unless any conventional carrier medium is incompatible with the compounds of the present invention, for example, to produce any undesirable biological effect or otherwise interact in a detrimental manner with any other component of the pharmaceutically acceptable composition, its use is intended to fall within the scope of the present invention.Some examples of materials that can act as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, e.g., human serum albumin, buffers, e.g., phosphates, glycine, sorbic acid, or potassium sorbate, partially glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, lanolin, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., carboxymethylcellulose sodium Excipients such as cellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; propylene glycol or polyethylene glycol, etc.; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer, as well as other non-toxic, interchangeable lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants, which may also be present in the composition at the discretion of the formulater.

[0122] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals, depending on the severity of the disease being treated, for example, orally, rectally, parenterally, intrasacrally, vaginally, intraperitoneally, topically (in the form of powders, ointments, or drops), buccally, orally or as a nasal spray.

[0123] Pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), vegetable oils (such as olive oil), injectable organic esters (such as ethyl oleate), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0124] These compositions may also contain auxiliary agents such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action can be ensured by encapsulation of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride. Sustained absorption of injectable pharmaceutical dosage forms can be achieved by encapsulation of absorption-delaying agents such as aluminum monostearate and gelatin.

[0125] In some cases, it is desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effect. This can be achieved by using a suspension of poorly water-soluble crystalline or amorphous material. Thus, the rate of drug absorption may depend on its dissolution rate, and subsequently on the crystal size and morphology. Alternatively, delayed absorption of parenterally administered dosage forms can be achieved by dissolving or distributing the drug in an oily medium.

[0126] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral composition may also contain auxiliary agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0127] Solid dosage forms for oral administration include capsules, tablets, pills, powders, cements, putties, and granules. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, for example, sodium citrate or dicalcium phosphate and / or a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) water-retaining agents such as glycerol; d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) dissolution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0128] Furthermore, similar types of solid compositions can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared using coatings and shells, such as enteric coatings and other coatings, which are well known in pharmaceutical formulation technology. They may optionally contain opaque agents, and they may be compositions that optionally release only the active ingredient, or preferentially to a specific part of the intestinal tract, or in a delayed manner. Examples of usable embedding compositions include polymer substances and waxes. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or lactose and high molecular weight polyethylene glycol.

[0129] Furthermore, the active compound may be in the form of microencapsulated tablets having one or more excipients as described above. Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared using coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings, which are well known in pharmaceutical formulation technology. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may, as is common practice, contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffers. These may optionally contain opacifiers, and they may also be compositions that selectively release only the active ingredient, or preferentially, in a delayed manner, to a specific part of the intestinal tract. Examples of usable embedding compositions include polymers and waxes.

[0130] The composition for rectal or vaginal administration is preferably a suppository that can be prepared by mixing the compound of the present invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active compound.

[0131] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and, if required, any necessary preservatives or buffers. Eye drops, ear drops, and eye drops are also intended as they fall within the scope of the present invention. In addition, the present invention intends the use of transdermal patches, which have the further advantage of providing controlled delivery of the compounds to the body. Such dosage forms are prepared by dissolving or distributing the compounds in a suitable culture medium. Absorption enhancers can also be used to increase the fluidity of the compounds through the skin. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compounds in a polymer matrix or gel.

[0132] The compounds described herein may be administered as pharmaceutical compositions comprising the compound of interest in combination with one or more pharmaceutically acceptable carriers. However, it is understood that the total daily dose of the compound and composition may be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose level for any particular patient may depend on a variety of factors, including the disorder and its severity during treatment; the activity of the particular compound used; the particular composition used; the patient's age, weight, general health and prior medical history, sex and diet; the timing, route of administration, and rate of excretion of the particular compound used; the duration of treatment; drugs used in combination with or concurrently with the particular compound used; and well-known analogues in medicine. For example, it is well within the scope of the art to start administration of a compound at a level lower than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The actual dose level of the active ingredient in a pharmaceutical composition may be varied to obtain an amount of the active compound that is effective in achieving the desired therapeutic response for a particular patient and a particular mode of administration. In the treatment of a particular medical condition, repeated or chronic administration of a compound may be necessary to achieve the desired therapeutic response. "Repeated or chronic administration" refers to administering the compound diurnally (i.e., daily) or intermittently (i.e., not daily) over a period of several days, weeks, months, or longer.

[0133] The compositions described herein may be administered together with additional compositions provided to extend the stability, delivery, and / or activity of the compositions, or to be combined with additional therapeutic agents, or to be provided before or after the administration of additional therapeutic agents.

[0134] Combination therapy includes the administration of a single drug formulation containing one or more of the compounds described herein and one or more additional pharmaceuticals, as well as the administration of the compounds and each additional pharmaceutical in their own separate drug formulations. For example, the compounds described herein and one or more additional pharmaceuticals may be administered together to a patient in a single oral composition having each active ingredient in a fixed ratio, such as a tablet or capsule; or each drug may be administered in a separate oral formulation. When separate formulations are used, the compounds and one or more additional pharmaceuticals may be administered essentially at the same time (e.g., simultaneously) or separately at different times (e.g., over time).

[0135] For adults, the dosage is generally about 0.01 to 100 mg / kg, preferably about 0.1 to 1 mg / kg body weight / day by inhalation; about 0.01 to 100 mg / kg, preferably 0.1 to 70 mg / kg, more preferably 0.5 to 10 mg / kg body weight / day by oral administration; and about 0.01 to 50 mg / kg, preferably 0.1 to 1 mg / kg body weight / day by intravenous administration.

[0136] The compositions and methods will be described more fully hereafter with reference to the accompanying drawings, which show embodiments that are part of but not all of the present invention. Indeed, the present invention can be illustrated in many different forms, but should not be construed as being limited to the embodiments described herein.

[0137] Similarly, many modifications and other embodiments of the compositions and methods described herein will remind those skilled in the art that the present invention benefits from the teachings presented in the prior description and the accompanying drawings. It should therefore be understood that the present invention should not be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to fall within the scope of the appended claims. Certain terms are used herein, but they are used in a general and descriptive sense only and not for limitation.

[0138] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as they are commonly understood by those skilled in the art to which the present invention relates. Any methods and materials similar to or corresponding to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0139] 5.Chemical synthesis The compounds of the present invention may be prepared as illustrated in the following scheme and examples.

[0140] Abbreviation: Acetate Ac2O Acetic anhydride aq. Water-based Calcd cat. catalyst c-HCl concentrated HCl DCM Dichloromethane DI deionization DMF (N,N-dimethylformamide) eq. equivalent weight Et ethyl ESI Electrospray Ionization HCl ethyl acetate h or hr hours HRMS high resolution mass spectrometry Me methyl MeOH methanol min. NBS (N-bromosaxinimide) NCI Negative Chemoionization NCS N-chlorosuccinimide NIS N-iodosuccinimide Ph Phenyl ppt precipitate pTsOH / TsOH p-toluenesulfonic acid pyridine rt or rt room temperature sat. saturation Selectfluor® 1-Chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanbis(tetrafluoroborate) TBAF Tetrabutylammonium Fluoride THF (Tetrahydrofuran) TLC (Thin-Layer Chromatography)

[0141] Scheme 1 is R 5 A general method for synthesizing compounds of formula (I) in which is substituted at the para position is shown. 5 Compounds in which the meta-substitution is performed can be synthesized by an analogous process starting with a suitable meta-substituted aniline.

[0142] Scheme 1 [ka]

[0143] Chemical Examples 1) Synthesis of 7-bromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Br,1) 1-1) Synthesis of 7-bromo-2-(4-methoxyphenyl)-2H-indazole-5-ylacetate (5-Ac-Ind-7-Br-4'-OMe,1-1)

[0144] Scheme 2 [ka]

[0145] 3-Bromo-5-(hydroxymethyl)-4-((4-methoxyphenyl)diazenyl)phenol(azo-2-Br-4'-OMe). To a suspension of 4-methoxyaniline (123 mg, 1.00 mmol) in cold DI water (10 mL), c-HCl (0.36 mL) was added, followed by sodium nitrite (71 mg, 1.03 mmol) being gradually added to an ice bath while maintaining the temperature below 5°C. After stirring the reaction mixture below 5°C for 1.5 hours, 3-bromo-5-hydroxymethylphenol (190 mg, 0.94 mmol) in a water-ethanol (1:2, v / v, 5 mL) mixture was added to the reaction mixture in the ice bath, and the pH was then carefully adjusted to a maximum of 7.5 with K2CO3 to precipitate a dark brown solid. During the addition of K2CO3, when the pH reached approximately 7.5, a large amount of brown solid precipitated from the solution. The solid was collected by filtration, washed with DI water, and dried in a drying oven at 60-80°C to obtain a brown solid azo-2-Br-4'-OMe (253 mg). (In this case, it is not necessary to adjust the pH to acidity to collect the solid, however, in some cases, the pH of the solution may need to be acidified to collect more solid.) The collected solid was used without further purification. 1 H NMR(499MHz,CD3OD)δ 7.86(d,J=8.5Hz,2H),7.17(d,J=2.7Hz,1H),7.01(d,J=8.5Hz,2H),6.97( d,J=2.7Hz,1H),4.53(s,2H),3.88(s,3H).ESI(m / z,M+1)337.02,339.02. [ka]

[0146] 5-Ac-Ind-7-Br-4'-OMe(1-1). Pyridine HCl salt (500 mg) was placed in a 50 mL round-bottom flask and heated in an oil bath at 165 °C until completely melted. Dry brown solid azo-2-Br-4'-OMe (152 mg, 0.45 mmol) was added to the melted pyridine HCl solution and heated in the mixture for 5 minutes. Once monitored by SiO2 TLC, all starting materials were converted to the compound indazole, at which point indazole exhibited weak blue fluorescence upon exposure to 254 nm UV. Then, acetic anhydride (300 μL) and pyridine (100 μL) were added to the reaction mixture, and water (50 mL) was added to the reaction solution, resulting in the formation of a gray precipitate. The precipitate was collected by filtration and dried under vacuum to obtain compound 1-1 as a gray solid (140 mg). The collected dried compound (1-1) is pure enough to be used without further purification. 1 H NMR(499MHz,CDCl3)δ 8.38(s,1H),7.81(d,J=10.05Hz,2H),7.40(d,J=2.0Hz,1H),7.32(d,J=2.0Hz,1H),7.03(d,J=10.05Hz,2H),3.88(s,3H),2.32(s,3H). 13 C NMR(126MHz,CDCl3)δ 170.77,159.45,146.63,144.94,135.86,126.34,123.65,122.39,122.2 2,116.23,112.86,110.64,53.72,19.43.ESI(m / z,M+1)361.02,363.02.

[0147] 1-2) Synthesis of 7-bromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Br,1) [ka] 7-Bromo-2-(4-methoxyphenyl)-2H-indazole-5-yl acetate (37.5 mg, 0.10 mmol) was dissolved in dichloromethane (5 mL). Dimethyl boron trifluoride (200 mg, 1.54 mmol) was added to the resulting solution, and the reaction vessel was stirred at rt for 6 hours. Once the starting material had disappeared during silica gel TLC analysis, the solvent and excess dimethyl boron trifluoride evaporated. Potassium carbonate (25 mg) and methanol (500 μL) were added to the resulting product, and the mixture was stirred at rt for 1 hour to evaporate the solvent. The mixture was then treated with 1N HCl water (100 μL) to obtain a precipitate, which was collected by filtration to obtain a light brown solid. This solid was dried to obtain the title compound (Ind-7-Br) (26 mg). 1 HNMR(500MHz,CD3OD-CDCl3)δ 8.26(s,1H),7.64(d,J=8.9Hz,2H),7.23(d,J=2.1Hz,1H),6.93(d,J=8.9Hz,2H),6.92(d,J=2.1Hz,1H). 13 C NMR(126MHz,CD3OD-CDCl3)δ 157.05,153.10,145.93,134.10,124.53,123.57,123.17,121.63,118.02,109.26,102.24.ESI(m / z,M+1)304.99,306.99.

[0148] 2) Synthesis of 7-bromo-3-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndF-7-Br,2) [ka] 5-Ac-IndF-7-Br-4'-OMe was prepared using the fluorination procedure described in International Publication No. 2019226936. 5-Ac-Ind-7-Br-4'-OMe (36.1 mg, 0.10 mmol) was treated with Selectfluor® (1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octanbis(tetrafluoroborate)) (49.28 mg, 0.14 mmol) in acetonitrile (3 mL) at rt for 8 hours until 5-Ac-Ind-7-Br-4'-OMe disappeared by silica gel thin-layer chromatography (in n-hexane, 20% siRNA, Rf=0.5). The reaction mixture was poured into 20 mL of water, extracted three times with 10 mL of siRNA, dried with sodium sulfate, and the siRNA layer was concentrated and loaded onto silica gel for column chromatography. 5-Ac-IndF-7-Br-4'-OMe was obtained in 69% yield by elution with a mixture of ethyl acetate and n-hexane (1:5=v / v). IndF-7-Br(2) was obtained in 73% yield by treatment of 5-Ac-IndF-7-Br-4'-OMe with DCM in MeOH and BF3-SMe2 (20 equivalents) in K2CO3, following the same method as described for the preparation of IndBr-7-Br. 1 H NMR(499MHz,CDCl3)δ 7.53(dd,J=8.8,1.7Hz,2H),7.26(d,J=2.0Hz,1H),6.93(d,J=8.9Hz,2H),6.75(d,J=2.0Hz,1H). 19 F NMR(470MHz,CDCl3)δ -133.53.ESI(m / z,M+1)323.0,325.0.

[0149] 3) Synthesis of 7-bromo-3-chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Br,3) [ka] Using the same method as for producing 5-Ac-IndBr-7-Br-4'-OMe, the reaction of 5-Ac-Ind-7-Br-4'-OMe (36.1 mg, 0.10 mmol) with NCS (20 mg, 0.15 mmol) and a catalytic amount of p-TsOH, followed by treatment with BF3-SMe2 (20 equivalents) in DCM at rt for 6 hours, then with K2CO3, yielded IndCl-7-Br(3) in 77% yield by the same method as for producing IndBr-7-Br. 1 H NMR(499MHz,CD3OD)δ 7.39(d,J=8.9Hz,2H),7.27(d,J=2.1Hz,1H),6.94(d,J=8.9Hz,2H),6.77(d,J=2.1Hz,1H).ESI(m / z,M+1)338.9,340.9,342.9.

[0150] 4) Synthesis of 3,7-dibromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndBr-7-Br,4) [ka] 3,7-Dibromo-2-(4-methoxyphenyl)-2H-Indazole-5-ylacetate (5-Ac-IndBr-7-Br-4'-OMe). Acetyl compound (5-Ac-Ind-7-Br) (75.0 mg, 0.200 mmol) in THF (5 mL) was mixed with NBS (N-bromosuccinimide) (128.0 mg, 0.76 mmol) and a catalytic amount of p-TsOH. The resulting solution was stirred at 55°C for 4 hours while monitoring the reaction with silica gel TLC. The resulting solution was concentrated and loaded onto a silica gel-coated preparative TLC plate (1 mm thick, plate L×W = 20 cm × 20 cm). Developed with a mixture of ethyl acetate and n-hexane (3:7 = v / v), 5-Ac-IndBr-7-Br-4'-OMe (70 mg) was obtained as a light brown solid. 11H NMR (499 MHz, CDCl3) δ 7.59 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 2.0 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.07 (d, J = 8.9 Hz, 2H), 3.91 (s, 3H), 2.35 (s, 3H). ESI (m / z, M+1) 438.9, 440.9, 442.9.

[0151] 3,7-Dibromo-2-(4-hydroxyphenyl)-2H-indazol-5-ol (IndBr-7-Br, 4). Solid 5-Ac-IndBr-7-Br-4’-OMe was dissolved in dichloromethane (5 mL) and treated with boron trifluoride dimethyl sulfide (520 mg, 4.00 mmol) at rt. Once the starting material disappeared by silica gel TLC, the solvent and excess boron trifluoride dimethyl sulfide were evaporated. Then, K2CO3 (50 mg, 0.36 mmol) and MeOH (1.0 mL) were added to the residue, followed by 10 min of sonication, evaporation of the solvent MeOH, acidification with 0.1 N HCl solution, extraction with ethyl acetate (1 mL × 3), and evaporation to give the title IndBr-7-Br (58 mg) (4) as a pale brownish solid. 1 1H NMR (499 MHz, CD3OD) δ 7.37 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 2.1 Hz, 1H), 6.91 (d, J = 8.9 Hz, 2H), 6.73 (d, J = 2.1 Hz, 1H). ESI (m / z, M+1) 382.9 (50%), 384.9 (100%), 386.9 (49%).

[0152] 5). Synthesis of 7-bromo-2-(4-hydroxyphenyl)-3-iodo-2H-indazol-5-ol (IndI-7-Br, 5)

Chemical Structure

[0153] IndI-7-Br(5). The title compound was obtained by reacting 5-Ac-IndI-7-Br-4’-OMe with BF3-SMe2 (20 equivalents) in DCM at rt for 6 h and subsequent treatment with potassium carbonate according to the method for preparing IndBr-7-Br. 1 H NMR(499MHz,CD3OD)δ 7.33(d,J=8.8Hz,2H),7.29(d,J=2.1Hz,1H),6.92(d,J=8.8Hz,2H),6.66(d,J=2.1Hz,1H).ESI(m / z,M+1)430.9,432.9.

[0154] 6). Synthesis of 7-chloro-2-(4-hydroxyphenyl)-2H-indazol-5-ol (Ind-7-Cl, 6) 6-1). chloro-5-(hydroxymethyl)-4-((4-methoxyphenyl)diazenyl)phenol (azo-2-Cl-4’-OMe)

Chemical Structure

[0155] Azo-2-Cl-4'-OMe was obtained as a yellowish solid in 85% yield by reacting p-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-chloro-5-hydroxymethylphenol (145.0 mg, 0.91 mmol) according to the same method described for preparing the azo-2-Br-4'-OMe compound shown in Scheme 2. 1 H NMR(499MHz,CD3OD)δ 7.85(d,J=9.0Hz,2H),7.00(d,J=9.0Hz,2H),6.98(d,J=2.7Hz,1H),6.83(d, J=2.7Hz,1H),4.43(s,2H),3.87(s,3H).ESI(m / z,M+1)293(100%),295(31%).

[0156] 6-2).5-Ac-Ind-7-Cl-4'-OMe(II) [ka] 5-Ac-Ind-7-Cl-4'-OMe(II) was obtained in 78% of azo-2-Cl-4'-OMe (292.0 mg, 1.00 mmol) by treatment with pyridine HCl (700.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.35(s,1H),7.79(d,J=9.0Hz,2H),7.34(d,J=2.0Hz,1H),7.14(d,J=2.0Hz,1H),7.03(d,J=9.0Hz,2H),3.87(s,3H),2.33(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.88,159.98,145.68,145.43,133.92,124.11,123.06,122.81,122.35,1 22.30,114.93,110.20,55.91,21.35.ESI(m / z,M+1)317.0(100%),319(31%).

[0157] 6-3)7-Chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Cl,6) [ka] Ind-7-Cl(6) was prepared in 91% yield by treating 5-Ac-Ind-7-Cl-4'-MeO(II) (31.7 mg, 0.10 mmol) with BF3-SMe2 (20 equivalents) in DCM, followed by K2CO3 in MeOH. 1 H NMR(499MHz,CD3OD)δ 8.16(s,1H),7.60(d,J=8.9Hz,2H),7.03(d,J=2.0Hz,1H),6.91(d,J=8.9Hz,2H),6.85(d,J=2.0Hz,1H). 13 ¹³C NMR (126 MHz, methanol-d4): δ 157.56, 152.13, 143.73, 132.95, 123.96, 123.18, 123.00, 121.42, 120.48, 116.15, 99.42. ESI (m / z, M+1): 261.03 (100%), 263.02 (30%).

[0158] 7) Synthesis of 3,7-dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Cl,7) [ka] 5-Ac-IndCl-7-Cl-4'-OMe was obtained by the reaction of 5-Ac-Ind-7-Cl-4'-OMe(II) (31.7 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol). 1 H NMR(499MHz,CDCl3)δ 7.57(d,J=9.0Hz,2H),7.28(d,J=2.0Hz,1H),7.16(d,J=2.0Hz,1H),7.03(d,J=9.0Hz,2H),3.87(s,3H),2.33(s,3H). 13C NMR(126MHz,CDCl3)δ 169.75,160.66,145.71,144.34,131.27,127.46,124.46,123.39,121.67,119.93,114.59,109.14,55.92,21.30.

[0159] The title compound was obtained by the reaction of 5-Ac-IndCl-7-Cl-4'-OMe with BF3SMe2 (12 equivalents) in DCM and K2CO3 in MeOH, following the same method as described for the preparation of IndBr-7-Br. 1 H NMR(499MHz,CD3OD)δ 7.39(d,J=8.8Hz,2H),7.07(d,J=2.0Hz,1H),6.93(d,J=8.8Hz,2H),6.72(d,J=2.1Hz,1H). 13 C NMR(126MHz,CD3OD)δ 157.83,153.69,143.65,131.19,127.47,123.48,121.69,120.84,119.22,116.88,109.45.ESI(m / z,M+1)295.0(100%),297(61%).

[0160] 8) Synthesis of 7-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-F,8) 3-Fluoro-5-(hydroxymethyl)-4-((4-methoxyphenyl)diazenyl)phenol(azo-2-F-4'-OMe) [ka] Azo-2-Br-4'-OMe was obtained as a yellowish solid in 93% yield from the reaction of p-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-fluoro-5-hydroxymethylphenol (142.0 mg, 1.00 mmol) following the same method described for preparing the azo-2-Br-4'-OMe compound. 11H NMR (499 MHz, CD3OD) δ 7.81 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 2.7 Hz, 1H), 6.62 (dd, J = 12.6, 2.7 Hz, 1H), 4.64 (s, 2H), 3.86 (s, 3H). 19 19F NMR (470 MHz, CD3OD) δ -119.22 (d, J = 12.9 Hz).

[0161] 5-Ac-Ind-7-F-4’-OMe (III)

Chem.

[0162] 7-Fluoro-2-(4-hydroxyphenyl)-2H-indazol-5-ol (Ind-7-F, 8)

Chem.

[0163] 9) Synthesis of 3-chloro-7-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-F,9) [ka] 5-Ac-IndCl-7-F-4'-OMe was obtained by the reaction of 5-Ac-Ind-7-F-4'-OMe(III) (30.0 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol). 1 H NMR(499MHz,CDCl3)δ 7.58(d,J=9.0Hz,1H),7.16(d,J=1.8Hz,1H),7.04(d,J=9.0Hz,1H),6.80(dd,J=10.9,1.9Hz,1H),3.88(s,2H),2.33(s,2H). 19 F NMR(470MHz,CDCl3)δ -126.17(d,J=11.0Hz).

[0164] IndCl-7-F(9) was obtained by reacting 5-Ac-IndCl-7-F-4'-OMe with BF3SMe2 (12 equivalents) and then K2CO3, following the same method described for preparing IndBr-7-Br. 1 H NMR(499MHz,CD3OD)δ 7.41(d,J=8.9Hz,1H),6.95(d,J=8.8Hz,2H),6.72(dd,J=12.1,2.0Hz,1H),6.61(d,J=1.9Hz,1H). 19F NMR(470MHz,CD3OD)δ -129.01(d,J=12.8Hz).ESI(m / z,M+1) 279.0(100%),281.0(31%).

[0165] 10) Synthesis of 7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Me,10) [ka] 3-methyl-5-(hydroxymethyl)-4-((4-methoxyphenyl)diazenyl)phenol (azo-2-Me-4'-OMe). Following the same method as described for the preparation of the azo-2-Br-4'-OMe compound, azo-2-Me-4'-OMe was obtained as a yellowish solid in 78% yield from the reaction of p-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-methyl-5-hydroxymethylphenol (138.0 mg, 1.00 mmol). 1 H NMR(499MHz,CDCl3)δ 7.85(d,J=9.0Hz,2H),7.02(d,J=9.0Hz,2H),6.84(d,J=2.6Hz,1H),6.81(d,J=2.8Hz,1H),4.36(s,2H),3.90(s,3H),2.62(s,3H). 13 C NMR(126MHz,CDCl3)δ 162.14,158.82,146.99,144.38,142.69,132.41,124.56,118.45,116.77,114.71,63.89,55.93,19.47. [ka]

[0166] 5-Ac-Ind-7-Br-4'-OMe(IV) was obtained in 91% yield by treating azo-2-Me-4'-OMe (272.3 mg, 1.00 mmol) with pyridine HCl (800.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe.1 H NMR(499MHz,CDCl3)δ 8.25(s,1H),7.78(d,J=9.1Hz,2H),7.22(d,J=1.7Hz,1H),7.02(d,J=9.2Hz,2H),6.82(d,J=1.7Hz,1H),3.86(s,3H),2.67(s,3H),2.32(s,3H). 13 C NMR(126MHz,CDCl3)δ 170.16,159.44,148.34,145.96,134.34,130.00,122.72,121.85,121.49,121.18,114.76,108.23,55.76,21.31,17.20. [ka]

[0167] 7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Me,10) was prepared in the same manner as described for the preparation of Ind-7-Br(1). Ind-7-Me(10) was prepared in 85% yield by treating 5-Ac-Ind-7-Me-4'-OMe(IV) (29.6 mg, 0.10 mmol) with BF3-SMe2 (20 equivalents) and K2CO3. 1 H NMR(499MHz,CDCl3)δ 7.98(s,1H),7.54(d,J=8.9Hz,1H),6.87(d,J=8.9Hz,2H),6.72(d,J=1.2Hz,1H),6.72(s,1H),2.55(s,1H). 13 C NMR(126MHz,CDCl3)δ 156.98,151.95,146.74,133.55,129.29,123.14,122.93,120.27,120.19,116.23,97.53,17.20.ESI(m / z,M+1)241.1(100%).

[0168] 11) Synthesis of 3-chloro-7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Me,11) [ka] 5-Ac-IndCl-7-Me-4'-OMe was obtained by the reaction of 5-Ac-Ind-7-Me-4'-OMe (29.6 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol). 1 H NMR(499MHz,CDCl3)δ 7.59(d,J=8.9Hz,2H),7.19-7.18(m,1H),7.07(d,J=9.0Hz,2H),6.87(dd,J=2.2,1.2Hz,1H),3.90(s,3H),2.63(s,3H),2.35(s,3H). 13 C NMR(126MHz,CDCl3)δ 170.21,160.39,147.11,146.38,131.74,130.53,127.43,122.73,120.51,119.05,114.56,107.27,55.86,21.38,16.82.

[0169] IndCl-7-Me(11) was obtained by reacting 5-Ac-IndCl-7-Me-4'-OMe with BF3-SMe2 (20 equivalents) and then K2CO3, following the same method described for preparing IndBr-7-Br. 1 H NMR(499MHz,CDCl3)δ 7.33(d,J=8.8Hz,2H),6.87(d,J=8.8Hz,2H),6.73(dd,J=2.3,1.3Hz,1H),6.57-6.57(m,1H),2.46(s,3H). 13 C NMR(126MHz,CDCl3)δ 158.02,152.57,145.21,130.51,129.56,127.51,121.39,119.80,118.78,115.88,95.91,16.59.ESI(m / z,M+1)275.0(100%),277.0(29%).

[0170] 12) Synthesis of 7-bromo-3-chloro-2-(3-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Br-3'-OH,12) [ka] Azo-2-Br-3'-OMe was obtained as a yellowish solid in 91% yield from the reaction of 3-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-bromo-5-hydroxymethylphenol (203.0 mg, 1.00 mmol) following the same method described for preparing the azo-2-Br-4'-OMe compound. 1 H NMR(499MHz,CD3OD)δ 7.52-7.49(m,1H),7.42-7.38(m,2H),7.21(d,J=2.7Hz,1H),7.02(ddt,J=8. 2,2.6,1.3Hz,1H),6.92(d,J=2.6Hz,1H),4.49(d,J=2.3Hz,2H),3.86(s,3H).

[0171] 5-Ac-Ind-7-Br-3'-OMe was obtained in 93% yield by treating azo-2-Br-3'-OMe (361.0 mg, 1.00 mmol) with pyridine HCl (950.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe.

[0172] 5-Ac-IndCl-7-Br-3'-OMe was obtained by the reaction of 5-Ac-Ind-7-Br-3'-OMe (36.1 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol). 1 H NMR(499MHz,CDCl3)δ 7.48(t,J=8.1Hz,1H),7.40(d,J=2.0Hz,1H),7.37(d,J=1.9Hz,1H),7.30-7.27(m,1H ),7.23(t,J=2.3Hz,1H),7.09(ddd,J=8.4,2.5,0.9Hz,1H),3.91(s,3H),2.37(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.75,160.36,145.96,145.55,139.21,130.17,126.93,121.72,119.68,118.42,115.97,112.74,111.89,109.75,55.92,21.30.

[0173] Following the same method described for preparing IndBr-7-Br, IndCl-7-Br-3'-OH(12) was obtained by the reaction of 5-Ac-IndCl-7-Br-3'-OMe with BF3-SMe2 (20 equivalents) and then K2CO3. 1 H NMR(499MHz,CDCl3-CD3OD)δ 7.27(t,J=8.1Hz,1H),7.23(d,J=2.1Hz,1H),7.03(dt,J=7.8,1.0Hz,2H),7.01(t,J=2.1Hz,2H),6.90(d dd,J=8.2,2.4,1.0Hz,2H),6.72(d,J=2.1Hz,1H).ESI(m / z,M+1)338.9(100%),340.9(98%),342.9(29%).

[0174] 13) Synthesis of 7-chloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Cl-3'-F,13) [ka] Ind-7-Cl-3'-F(13) was prepared in 81% yield by treating 5-Ac-Ind-7-Cl-3'-F-4'-OMe(V) (33.5 mg, 0.10 mmol) with BF3-SMe2 (25 equivalents) and K2CO3, following the same method as described for the preparation of Ind-7-Br(1). 1 H NMR(499MHz,CD3OD)δ 8.24(s,1H),7.59(dd,J=11.6,2.6Hz,1H),7.44(dq,J=8.7,2.6,1.4Hz,1H),7.04(d,J=2.0Hz,1H),6.85(d,J=2.0Hz,1H),7.03(t,J=8.8Hz,1H). 19 F NMR(470MHz,CD3OD)δ -135.58-135.97(m).ESI(m / z,M+1)279.03(100%),281.03(31%).

[0175] 14) Synthesis of 3,7-dichloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Cl-3'-F,14) [ka] Following the same method described for preparing the azo-2-Br-4'-OMe compound, azo-2-Cl-3'-F-4'-OMe was obtained as a yellowish solid in 89% yield from the reaction of 3-fluoro-4-methoxyaniline (141.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-chloro-5-hydroxymethylphenol (203.0 mg, 1.00 mmol). 1 H NMR(499MHz,CD3OD)δ 7.74-7.71(m,1H),7.61(dd,J=12.2,2.3Hz,1H),7.17(t,J=8.6Hz,1H),7.01(d,J=2.7Hz,1H),6.96(d,J=2.7Hz,1H),4.62(s,2H),3.97(s,3H). 19 F NMR(470MHz,CD3OD)δ -134.74(dd,J=12.2,8.6Hz).

[0176] 5-Ac-Ind-7-Cl-3'-F-4'-OMe was obtained in 88% yield by treating azo-2-Cl-3'-F-4'-OMe (311.0 mg, 1.00 mmol) with pyridine HCl (910.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.38(s,1H),7.74(dd,J=11.6,2.6Hz,1H),7.67-7.64(m,1H),7.38(d,J=2.0H z,1H),7.17(d,J=1.9Hz,1H),7.11(t,J=8.8Hz,1H),3.99(s,3H),2.36(s,3H). 19 F NMR(470MHz,CDCl3)δ -132.14(t,J=10.1Hz).

[0177] IndCl-7-Cl-3'-F(14) was obtained in 73% yield via two steps: the reaction of 5-Ac-Ind-7-Cl-3'-F-4'-OMe(V) (33.5 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-7-Cl-3'-F-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(499MHz,CD3OD)δ 7.38(dd,J=11.1,2.4Hz,1H),7.28(dq,J=8.9,2.4,1.4Hz,1H),7.08(t,J=8.9Hz,1H),7.08(d,J=2.1Hz),1H 7.01(d,J=2.1Hz,1H). 19 F NMR(470MHz,CD3OD)δ -136.57(t,J=10.25Hz).ESI(m / z,M+1)312.9(100%),314.9(51%).

[0178] 15) Synthesis of 7-chloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol (Ind-7-Cl-2'-F,15) [ka] Ind-7-Cl-2'-F(15) was prepared in 85% yield by treating 5-Ac-Ind-7-Cl-2'-F-4'-OMe(VI) (33.5 mg, 0.10 mmol) with BF3-SMe2 (25 equivalents) and K2CO3, following the same method as described for the preparation of Ind-7-Br(1). 1 H NMR(499MHz,CDCl3)δ 8.13(d,J=2.5Hz,1H),7.67(t,J=9.0Hz,1H),7.06(d,J=2.0Hz,1H),6.85(d,J=2.0Hz,1H),6.74-6.66(m,2H). 19 F NMR(470MHz,CDCl3)δ -123.76(t,J=10.9Hz).ESI(m / z,M+1)297.0(100%),281.0(29%).

[0179] 16) Synthesis of 3,7-dichloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-7-Cl-2'-F,16) [ka] Following the same method described for preparing the azo-2-Br-4'-OMe compound, azo-2-Cl-2'-F-4'-OMe was obtained as a yellowish solid in 87% yield from the reaction of 2-fluoro-4-methoxyaniline (141.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-chloro-5-hydroxymethylphenol (203.0 mg, 1.00 mmol). 1 H NMR(499MHz,CD3OD)δ 7.81(t,J=8.1Hz,1H),7.00(d,J=2.6Hz,1H),6.83(d,J=2.6Hz,1H),6.80-6.75(m,2H),4.41(s,2H),3.87(s,3H). 19 F NMR(470MHz,CD3OD)δ -123.23(dd,J=12.6,8.7Hz).

[0180] 5-Ac-Ind-7-Cl-2'-F-4'-OMe was obtained in 91% yield by treating azo-2-Cl-2'-F-4'-OMe (310.0 mg, 1.00 mmol) with pyridine HCl (900.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(500MHz,CDCl3)δ 8.43(d,J=2.5Hz,1H),7.95(t,J=8.8Hz,1H),7.37(d,J=2.0Hz,1H),7.17(d,J=1.9Hz,1 H),6.86(dd,J=9.2,2.5Hz,1H),6.82(dd,J=12.8,2.6Hz,1H),3.88(s,3H),2.34(s,3H). 19 F NMR(470MHz,CDCl3)δ -122.58(t,J=10.9Hz). 13C NMR(126MHz,CDCl3)δ 169.86,160.85,158.34,152.96,148.52,133.15,126.43,124.03,123.11,122.51,121.73,112.40,110.33,104.12,58.40,20.48.

[0181] IndCl-7-Cl-2'-F(16) was obtained in 71% yield via two steps: the reaction of 5-Ac-Ind-7-Cl-2'-F-4'-OMe(VI) (33.5 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-7-Cl-2'-F-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(400MHz,CDCl3)δ 7.13(t,J=8.8Hz,1H),6.93(d,J=2.0Hz,1H),6.59-6.52(m,3H). 19 F NMR(470MHz,CD3OD)δ -120.40(t,J=9.8Hz).ESI(m / z,M+1)312.9(100%),314.9(60%).

[0182] 17) Synthesis of 3-chloro-6-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-6-F,17) [ka] Azo-3-F-4'-OMe was obtained as a yellowish solid in 89% yield from the reaction of 4-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 2-fluoro-5-hydroxymethylphenol (138.0 mg, 0.97 mmol) using the same method described for preparing the azo-2-Br-4'-OMe compound. 1H NMR(499MHz,CDCl3)δ 7.83(d,J=9.0Hz,2H),7.63(d,J=11.7Hz,1H),7.12(d,J=8.8Hz,1H),7.01(d,J=8.9Hz,2H),5.00(s,2H),3.90(s,2H). 19 F NMR(470MHz,CDCl3)δ -140.33.

[0183] 5-Ac-Ind-6-F-4'-OMe(VII) was obtained in 91% yield by treating azo-3-F-4'-OMe (276.3 mg, 1.00 mmol) with pyridine HCl (800.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.29(s,1H),7.75(d,J=9.1Hz,2H),7.47(d,J=10.6Hz,1H),7.43(d,J=7.4Hz,1H),7.03(d,J=9.0Hz,2H),3.88(s,3H),2.37(s,3H). 19 F NMR(470MHz,CDCl3)δ -127.64(dd,J=10.7,7.6Hz).

[0184] IndCl-6-F(17) was obtained in 76% yield via two steps: the reaction of 5-Ac-Ind-6-F-4'-OMe(VII) (30.0 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-6-F-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(499MHz,CD3OD)δ 7.39(d,J=8.4Hz,21H),7.23(d,J=11.0Hz,1H),6.95(d,J=8.2Hz,2H),6.92(d,J=8.4Hz,1H). 19 F NMR(470MHz,CDCl3)δ -130.34(t,J=8.6Hz).ESI(m / z,M+1)279.0(100%),281.0(28%).

[0185] 18) Synthesis of 3-chloro-6-fluoro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol (IndCl-6-F-2'-Me,18) [ka] Azo-3-F-2'-Me-4'-OMe was obtained as a yellowish solid in 78% yield from the reaction of 2-methyl-4-methoxyaniline (137.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 2-fluoro-5-hydroxymethylphenol (138.0 mg, 0.97 mmol) following the same method described for preparing the azo-2-Br-4'-OMe compound. 1 H NMR(499MHz,CDCl3)δ 7.80(d,J=9.0Hz,1H),7.59(d,J=11.60Hz,1H),7.58(d,J=8.5Hz,1H),7.15(d,J=8 .8Hz,1H),6.84(d,J=8.2Hz,1H),5.06(s,2H),3.90(s,3H),2.65(d,J=0.9Hz,3H). 19 F NMR(470MHz,CDCl3)δ -140.68(t,J=10.4Hz).

[0186] 5-Ac-Ind-6-F-2'-Me-4'-OMe(VIII) was obtained in 88% yield by treating azo-3-F-2'-Me-4'-OMe (290.3 mg, 1.00 mmol) with pyridine HCl (900.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.03(s,1H),7.47(d,J=11.4Hz,1H),7.45(d,J=8.2Hz,1H),7.29(d,J=8.5Hz,1H),6.8 6(d,J=2.7Hz,1H),6.83(dd,J=8.6,2.9Hz,1H),3.85(s,3H),2.37(s,3H),2.17(s,3H). 19F NMR(470MHz,CDCl3)δ -128.17(dd,J=11.1,7.4Hz).

[0187] IndCl-6-F-2'-Me(18) was obtained in 76% yield via two steps: the reaction of 5-Ac-Ind-6-F-2'-Me-4'-OMe(VIII) (30.0 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-6-F-2'-Me-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(499MHz,CD3OD)δ 7.27(d,J=11.1Hz,1H),7.15(d,J=8.5Hz,1H),6.95(d,J=8.4Hz,1H),6.83(d,J=2.7Hz,1H),6.78(dd,J=8.5,2.6Hz,1H). 19 F NMR(470MHz,CDCl3)δ -130.34(dd,J=11.1,7.4Hz).ESI(m / z,M+1)293.0(100%),295.0(27%).

[0188] 19) Synthesis of 3,6-dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol (IndCl-6-Cl,19) [ka] Azo-3-Cl-4'-OMe was obtained as a yellowish solid in 82% yield from the reaction of 4-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 2-chloro-5-hydroxymethylphenol (158.6 mg, 0.97 mmol) using the same method described for preparing the azo-2-Br-4'-OMe compound. 1 H NMR(499MHz,CDCl3)δ 7.89(s,1H),7.85(d,J=9.0Hz,2H),7.15(s,1H),7.02(d,J=9.0Hz,2H),5.01(s,3H),3.91(s,3H).13 C NMR(126MHz,CDCl3)δ 162.63,153.51,146.92,144.67,139.97,125.03,120.65,118.89,116.14,114.70,63.04,55.91.

[0189] 5-Ac-Ind-6-Cl-4'-OMe(IX) was obtained in 90% yield by treating azo-3-Cl-4'-OMe (293.0 mg, 1.00 mmol) with pyridine HCl (900.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.10(s,1H),7.69(d,J=0.8Hz,1H),7.57(d,J=9.0Hz,2H),7.07(d,J=0.9Hz,1H),6.84(d,J=9.0Hz,2H),3.69(s,3H),2.20(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.60,159.88,147.82,142.32,133.95,127.46,122.73,121.24,121.12,119.07,115.01,113.43,55.91,20.92.

[0190] IndCl-6-Cl(19) was obtained in 76% yield via two steps: the reaction of 5-Ac-Ind-6-Cl-4'-OMe(IX) (31.7 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-6-Cl-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(499MHz,CD3OD)δ 7.64(s,1H),7.42(d,J=8.7Hz,2H),6.95(d,J=8.7Hz,2H),6.91(s,1H). 13C NMR(126MHz,CDCl3)δ 162.81,153.20,147.92,133.96,131.67,131.09,122.67,122.34,121.81,119.49,103.14.ESI(m / z,M+1)295.0(100%),297.0(53%).

[0191] 20) Synthesis of 3,6-dichloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol (IndCl-6-Cl-2'-Me,20) [ka] Azo-3-Cl-2'-Me-4'-OMe was obtained as a yellowish solid in 68% yield from the reaction of 2-methyl-4-methoxyaniline (137.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 2-chloro-5-hydroxymethylphenol (159.0 mg, 0.97 mmol) following the same method described for preparing the azo-2-Br-4'-OMe compound. 1 H NMR(499MHz,CDCl3)δ 7.82(s,1H),7.58(d,J=8.9Hz,1H),7.14(s,1H),6.84(d,J=3.0Hz,1H),6.79(dd,J=9.0,2.8Hz,1H),5.03(s,3H),3.87(s,4H),2.73(s,4H).

[0192] Following the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe, 5-Ac-Ind-6-Cl-2'-Me-4'-OMe(X) was obtained in 81% yield by treating azo-3-Cl-2'-Me-4'-OMe (307.0 mg, 1.00 mmol) with pyridine HCl (950.0 mg) and acetic anhydride (1 mL). 1H NMR(499MHz,CDCl3)δ 8.03(d,J=1.0Hz,1H),7.89(d,J=0.8Hz,1H),7.47(s,1H),7.30(d,J=8.6Hz,1H),6.87 (d,J=2.9Hz,1H),6.83(dd,J=8.6,2.8Hz,1H),3.86(s,3H),2.39(s,3H),2.17(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.72,160.37,147.47,142.18,135.72,133.44,127.84,127.27,125.55,120.30,119.16,116.50,113.45,111.98,55.82,20.93,18.23.

[0193] IndCl-6-Cl-2'-Me(20) was obtained in 73% yield via two steps: the reaction of 5-Ac-Ind-6-Cl-2'-Me-4'-OMe(X) (33.0 mg, 0.10 mmol) with NCS (20.0 mg, 0.15 mmol), followed by treatment of the resulting 5-Ac-IndCl-6-Cl-2'-Me-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3. 1 H NMR(499MHz,CD3OD)δ 7.66(s,1H),7.14(d,J=8.5Hz,1H),6.95(s,1H),6.82(d,J=2.8Hz,1H),6.77(dd,J=8.5,2.7Hz,1H). 13 C NMR(126MHz,CD3OD)δ 159.92,149.86,144.68,137.88,129.52,129.30,128.43,120.74,118.52,11 7.59,113.90,99.87,30.03,16.77.ESI(m / z,M+1)309.0(100%),311.0(56%).

[0194] 21) Synthesis of 3-chloro-2-(4-hydroxyphenyl)-2H-indazole-5,7-diol (IndCl-7-OH,21) [ka] Azo-2-OH-4'-OMe was obtained as a yellowish solid in 86% yield from the reaction of 4-methoxyaniline (123.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-hydroxy-5-hydroxymethylphenol (140.1 mg, 0.97 mmol) using the same method described for preparing the azo-2-Br-4'-OMe compound. 1 H NMR(500MHz,CDCl3)δ 7.66(d,J=8.9Hz,2H),6.96-6.93(m,2H),6.52(d,J=2.6Hz,1H),6.24(d,J=2.6Hz,1H),4.99(s,2H),3.84(s,3H). 13 C NMR(126MHz,CD3OD)δ 162.79,161.45,157.06,145.24,144.50,129.08,122.96,114.60,107.54,102.11,60.60,55.33.

[0195] 5-Ac-Ind-7-Br-4'-OMe was obtained in 88% yield by treating azo-2-OH-4'-OMe (274.3 mg, 1.00 mmol) with pyridine HCl (800.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(500MHz,CDCl3)δ 8.32(s,1H),7.75(d,J=9.00,2H),7.40(s,1H),7.02 (d,J=9.00,2H),3.88(s,3H),2.52(s,3H),2.39(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.33,167.98,159.97,142.44,142.21,138.28,133.77,123.01,122.21,121.32,119.71,114.89,111.19,55.89,20.90.

[0196] IndCl-7-OH(21) was obtained in 65% yield via two steps: the reaction of 5-Ac-Ind-7-OAc-4'-OMe (34.0 mg, 0.10 mmol) with NCS (14.6 mg, 0.11 mmol), followed by treatment of the resulting 5-Ac-IndCl-7-OAc-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3.

[0197] 5-Ac-IndCl-7-OAc-4'-OMe: 1 H NMR(500MHz,CDCl3)δ 7.57(d,J=8.9Hz,2H),7.28(d,J=1.9Hz,1H),7.05(d,J=8.9Hz,2H),6.98(d,J=2.0Hz,1H),3.90(s,3H),2.42(s,3H),2.34(s,3H). 13 C NMR(126MHz,CDCl3)δ 169.71,168.85,160.56,145.53,141.15,140.73,131.37,127.46,121.45,120.67,115.63,114.53,108.01,55.89,21.34.

[0198] IndCl-7-OH(21): 1 H NMR(500MHz,CD3OD)δ 7.59(s,1H),7.38(d,J=8.8Hz,2H),6.93(s,1H),6.88(d,J=8.8Hz,2H).ESI(m / z,M+1)277.0.HRMS(ESI,M+1) C 13 H 10 The calculated mass value for ClN2O3 is 277.0380, and the measured value is 277.0370.

[0199] Synthesis of 3-chloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5,7-diol (22) [ka] Following the same method described for preparing the azo-2-Br-4'-OMe compound, azo-2-OH-2'-Me-4'-OMe was obtained as a yellowish solid in 87% yield from the reaction of 2-methyl-4-methoxyaniline (137.0 mg, 1.00 mmol) with NaNO2 (72.0 mg, 1.03 mmol) and 3-hydroxy-5-hydroxymethylphenol (140.1 mg, 0.97 mmol). 1 H NMR(499MHz,CD3OD)δ 7.68(d,J=8.6Hz,1H),6.81(d,J=2.7Hz,1H),6.79(s,1H),6.56(s,1H),6.24(d,J=2.6Hz,1H),5.04-5.01(m,2H),3.82(s,1H),2.50(s,3H). 13 C NMR(126MHz,CD3OD)δ 162.94,161.08,158.14,142.08,136.54,130.03,117.26,115.79,113.23,108.68,108.10,102.54,61.67,55.61,18.26.

[0200] 5-Ac-Ind-7-Br-4'-OMe was obtained in 78% yield by treating azo-2-OH-2'-Me-4'-OMe (288.3 mg, 1.00 mmol) with pyridine HCl (800.0 mg) and acetic anhydride (1 mL) according to the same method described for the preparation of 5-Ac-Ind-7-Br-4'-OMe. 1 H NMR(499MHz,CDCl3)δ 8.09(s,1H),7.37(d,J=1.9Hz,1H),7.34(d,J=8.6Hz,1H),6.98(d,J=1.9Hz,1H),6.88(d,J= 2.8Hz,1H),6.85(dd,J=8.6,2.8Hz,1H),3.87(s,3H),2.43(s,3H),2.35(s,3H),2.20(s,3H). 13C NMR(126MHz,CDCl3)δ 169.95,169.01,160.37,145.10,141.57,141.18,135.98,133.53,128.02,1 26.12,122.88,116.46,114.34,111.90,109.13,55.81,21.41,21.39,18.25.

[0201] IndCl-7-OH-2'-Me(22) was obtained in 63% yield via two steps: the reaction of 5-Ac-Ind-7-OAc-2'-Me-4'-OMe (35.4 mg, 0.10 mmol) with NCS (14.6 mg, 0.11 mmol), followed by treatment of the resulting 5-Ac-IndCl-7-OAc-2'-Me-4'-OMe with BF3-SMe2 (25 equivalents), and then with K2CO3.

[0202] 5-Ac-IndCl-7-OAc-2'-Me-4'-OMe: 1 H NMR(499MHz,CDCl3)δ 7.29(d,J=2.6Hz,1H),7.27(d,J=8.4Hz,1H),7.02(d,J=1.9Hz,1H),6.89(d,J=2.8Hz, 1H),6.86(dd,J=8.5,2.9Hz,1H),3.87(s,3H),2.40(s,3H),2.35(s,3H),2.05(s,3H).

[0203] IndCl-7-OH-2'-Me(22): 1 H NMR(499MHz,CD3OD)δ 7.74(s,1H),7.15(d,J=8.3Hz,3H),6.81(s,3H),6.77(d,J=8.3Hz,4H),6.43(s,1H),2.02(d,J=2.0Hz,3H).ESI(m / z,M+1)291.0.

[0204] 6. Biological Examples Example 1. Relative binding affinity (RBA) assay in ERα and ERβ The methodology for this competitive binding assay of radiometrics, using tritium-labeled estradiol and purified full-length human ERα and ERβ as tracers, has been described (De Angelis et al., 2005). When E2 (estradiol) is 100%, all values ​​are relative binding affinities. This is based on 0.2 nM for ERα and 0.5 nM for ERβ. d Corresponding to this, and then K for each individual compound i The value is calculable.

[0205] [Table 1-1]

[0206] [Table 1-2]

[0207] Many indazoles with substituents at the 7-position exhibit very high binding selectivity for ERβ compared to ERα (Table 1). The RBA for ERβ of compounds 3 (IndCl-7-Br) and 7 (IndCl-7-Cl) was measured to be approximately 300, compared to a value of 100 for estradiol. The RBA for ERβ of compounds 2 (IndF-7-Br), 4 (IndBr-7-Br), and 5 (IndI-7-Br) was measured to be approximately 150. The binding selectivity for ERβ of compounds 6, 12, and 13 compared to ERα was 200-fold, 246-fold, and 469-fold, respectively. The ERβ selectivity of compounds 1, 10, and 15 was 131-fold, 144-fold, and 185-fold, respectively. All other compounds also exhibit good ERβ selectivity. Compounds with a hydroxyl group at position 7 (compound 21, IndCl-7-OH, and compound 22, IndCl-7-OH-2'-Me) showed lower binding affinity to both ERβ and ERα, but the binding specificity of compounds 21 and 22 to ERβ over ERα remains consistent at positions 51 and 12.

[0208] [Table 2]

[0209] Indazoles with substituents at position 6 exhibit good to moderate ERβ selectivity (Table 2).

[0210] Example 2. In vitro cell assay Primary OPC cultures: As previously mentioned, primary OPCs isolated from the cortex of male and female C57BL / 6 mice at postnatal day P1 were treated with 10 nM ligand in differentiation medium for 3 days (Tiwari-Woodruff et al., 2001; Tiwari-Woodruff et al., 2006). The primary OPCs were placed on 8-well chamber slides (3 wells per condition, 2.5 × 10⁶). 5 Cells are cultured in a well for 3 days to allow binding, and then cultured for 5 days in differentiation medium consisting of Sato containing triiodine thyronine and thyroxine, and DMEM-F12 containing penicillin, streptomycin, insulin, N-acetyl-L-cysteine, forskolin, ciliary neurotrophic factor, neurotrophin-3, and platelet-derived growth factor receptor α (Tiwari-Woodruff et al., 2001). A positive control (IndCl), a negative control (a medium consisting of a mixture of medium + EtOH used to dissolve IndCl), and a normal control (differentiation medium alone) are used for comparison. At the end of the treatment period, cells were fixed and simultaneously stained with immunocytochemistry (primary antibody polyclonal chicken myelin basic protein (MBP, Millipore AB9348) and nuclear staining - DAPI), and imaged at 10x magnification using an Olympus BX61 confocal microscope (Olympus America Inc., Center Valley, PA) (3 images / well). Cells were counted using the ImageJ multipoint tool, and the count value was then recorded in the image area (mm²). 2Divide by ). Next, divide the mean cell density for each condition by the cell density under normal conditions. Analysis of OL differentiation consists of counting the number of MBP+ cells and the number of protrusions longer than the respective cell body diameter, and tracking the number of highly branched (having 3 or more protrusions) MBP+ cells (Monnerie et al., 2017). Statistical analysis is performed using GraphPad Prism 6 software (La Jolla, CA). P-values ​​are generated using one-way ANOVA with Tukey's post-hoc test for multiple comparisons, and data are presented as mean ± SEM (for α ≤ 0.05).

[0211] Figures 1A to 1D show the expression of myelin basic protein (MBP) in OL as an indicator of mouse oligodendrocyte precursor cell (OPC) differentiation. + The effects of treatment with 7-substituted indazole compounds on OL (top two panels) and total cell count as an indicator of cell proliferation / survival (DAPI nuclear staining, bottom two panels) are shown. Cells were treated with the listed compounds at 10 nM (Figure 1A, 1B) and 1 nM (Figure 1C, 1D). Compound 7 (IndCl-7-Cl) exhibits a particularly significant increase in MBP expression levels at 1 nM.

[0212] Figures 1A and 1C show bar graphs quantifying the number of MBP+ (oligodendrocyte) mouse OLs for each treatment group in Table 1. The analogues Ind-7-Br (compound 1), IndI-7-Br (compound 5), IndCl-7-Cl (compound 7), and IndCl-7-Cl-3'-F (compound 14) showed a significant increase in the number of MBP+ OLs, with an increased percentage of branched OLs compared to cells treated with the 10 nM medium. In particular, compounds 2 (IndF-7-Br), 3 (IndCl-7-Br), 7 (IndCl-7-Cl), and 9 (IndCl-7-F) were found to have 1.3 to 1.5 times higher MBP expression compared to cells treated with the 1 nM medium. The study used 5 to 6 wells per treatment group. Independent experiments with n=5 to 6 were conducted.

[0213] Figures 1B and 1D show bar graphs quantifying the total number of OLs for each treatment group in Table 1. No significant differences in the total number of cells were observed between groups, except for the decrease in IndF-7-Br (compound 2) and IndCl-7-Br (compound 3) at 10 nM. There were 5-6 wells per treatment group. Independent experiments with n=5-6 were conducted.

[0214] Figures 2A and 2B show MBP as an indicator of OPC differentiation for 6-substituted compounds 17-20 (IndCl-6-F, IndCl-6-F-2'-Me, IndCl-6-Cl, and IndCl-6-Cl-2'-Me). + This study shows the effect of treatment on MBP expression and total cell number (DAPI staining) as an indicator of cell viability or proliferation. Cells were treated with the listed compounds (17-20) at both 1 nM and 10 nM concentrations. Compound 17 (IndCl-6-F) also showed good MBP expression at 1 nM.

[0215] Figure 2A shows bar graphs quantifying the number of MBP+OLs for each treatment group in Table 2. The analogues IndCl-6-F (compound 17) and IndCl-6-F-2'-Me (compound 18) showed a significant increase in the number of MBP+OLs, with an increase in the percentage of branched OLs, compared to medium-treated cells at both 1 nM and 10 nM. 5 wells / treatment group. An independent experiment with n=5 was conducted.

[0216] Figure 2B shows bar graphs quantifying the total number of cells for each treatment group in Table 2. No significant differences in total cell count were observed between groups at both 1 nM and 10 nM levels. 5 wells / treatment group. An independent experiment with n=5 was conducted.

[0217] Example 3.2-hour single-point pharmacokinetic (PK) study The 2-hour single-point pharmacokinetics of IndCl-7-Cl (compound 7), IndCl-7-Br-3'-OH (compound 12), and IndCl-7-Cl-3'-F (compound 14) in 2.5-month-old C57BL / 6 female mice were studied after a single subcutaneous (SC) injection or oral gastric tube feeding (PO) at doses of 5 mg / kg for SC and 25 mg / kg for PO administration in 0.1 mL of each medium. The medium for SC administration was 0.1 mL of 10% ethanol and 90% Miglyol 812N or 10% DMSO saline, and for PO administration, it was 0.1 mL of 2-hydroxypropyl-β-cyclodextrin [HPCD] / 40% aqueous solution (MW approximately 1540). Two hours after injection, blood and brain samples were collected for quantification of the administered compound and processed by liquid chromatography-mass spectrometry (LC-MS / MS) at the University of Illinois Metabolomics Core Facility. The bar graphs show the concentrations analyzed in nM units in blood and brain for each sample and each administration method. The brain uptake ratios of compound 7 (IndCl-7-Cl) in the PO and SC pharmacokinetic studies were approximately 7 and 6, respectively, higher than those in blood.

[0218] Compound 7 (IndCl-7-Cl) exhibits selectively high cerebral uptake in both PO and SC administration. The concentration of compound 7 (IndCl-7-Cl) in the brain after PO administration was significantly higher than after SC injection.

[0219] Figure 4 shows the 2-hour single-point pharmacokinetic study of compound 17. For subcutaneous (SC) injection, the compound was prepared as a homogeneous suspension in 10% ethanol and 90% Miglyol 812N (medium; Sasol), and a dose of 0.1 mL was administered by SC injection at a dose of 5 mg / kg body weight. For PO administration, the compound was dissolved in 40% (2-hydroxypropyl)-β-cyclodextrin (Mw approximately 1540) (HP-CD) and administered via oral gastric tube feeding in a volume of 0.1 mL at a dose of 25 mg / kg body weight.

[0220] After PO administration, the concentration of compound 17 (IndCl-6-F) in the brain was three times higher than the concentration in the blood (n=3).

[0221] Example 4. Evaluation of myelin recovery in a cuprison-induced demyelination mouse model. The demyelination-remyelination cuprizone assay was performed using the previously described method (Moore et al. 2013; Crawford et al. 2009a,b). The animal groups were based on a normal diet (N group). The remaining mice were divided into groups of 5, and all of these mouse groups were fed a cuprizone diet (0.2% cuprizone mixed in solid feed). Six weeks after cuprizone-induced demyelination, the group of 5 mice was perfused (DM group). The other mouse groups were switched to a normal diet for 1.5 weeks to initiate remyelination and treated with either (i) the medium (SC+V) or (ii) the test compound at a dose of 5 mg / kg. For injection, the drug solution was prepared by combining 10% ethanol and 90% Miglyol oil. One and a half weeks after normal diet-induced remyelination, the mice were perfused. Brains were collected, cryopreserved, embedded, dissected, and subjected to intravascular coagulation (IHC) for myelin oligodendrocyte glycoprotein (MOG).

[0222] Figure 5 shows the results of the cuprizon test in female mice (C57BL / 6) after 6 weeks of cuprizon treatment followed by 1.5 weeks of treatment with a specified 7-substituted compound via SC administration. The height of each bar graph represents the level of myelin formation, assessed by MOG expression in the white matter track of the corpus callosum region, quantified by immunohistochemical analysis (IHC) after staining with MOG antibody. N: normal diet, DM: demyelination after 6 weeks of cuprizon treatment, SC-Veh: spontaneous remyelination after injection of matrix in saline only (no compound; recovery is only about 40%). (Mice: approximately 2 months old, SC: subcutaneous injection, n=5).

[0223] Compound 7 promotes remyelination, reaching normal or above-normal myelin levels. Compounds 5 and 14 cause slightly lower levels of remyelination; compound 12 does not promote remyelination.

[0224] Figure 6 shows the results of the cuprizon test in C57BL / 6 female mice for 6 weeks of 0.2% cuprizon treatment followed by 3 weeks of treatment with 6-substituted compound 17 via SC administration. The height of each bar graph represents the level of myelin formation, assessed by MOG expression in the white matter track of the corpus callosum region, quantified by immunohistochemical analysis (IHC) after staining with MOG antibody. N: Normal diet, DM: Demyelination after 6 weeks of cuprizon treatment, SC-Veh: Spontaneous remyelination after injection of matrix in saline only (no compound; recovery is only about 40%). (Mice: approx. 2 months old, SC: subcutaneous injection, n=5-6). 17: MOG staining indicates recovery above normal levels with compound 17. (Mice: approx. 2 months old, sc: subcutaneous administration, n=5).

[0225] Compound 17 was found to promote remyelination, reaching normal or even higher levels.

[0226] Figure 7 shows the uterine hypertrophy effects of compound 5 (IndI-7-Br) and compound 7 (IndCl-7-Cl) in non-ovariectomized mice. Uteruses were collected from mice treated with compound 5 and compound 7 from recovery evaluation studies using a cuprizon-induced demyelinating model. Uterine weight for each compound is expressed as %uterogenetic weight / body weight. Compounds 5 and 7 did not show significantly different uterine hypertrophy effects compared to mice treated with the medium alone.

[0227] Estrogen increases uterine weight primarily by acting through ERα (Hewitt and Korach, 2003). Uterine weight was evaluated to determine whether the tested analogues possessed ERα signaling properties. Compound 5 (IndI-7-Br) and Compound 7 (IndCl-7-Cl) did not significantly stimulate uterine weight after daily SC treatment at 5 mg / kg for 3 weeks.

[0228] Example 5. Experimental autoimmune encephalomyelitis (EAE) EAE induction: Active EAE can be induced in 8-week-old female C57BL / 6 mice, as previously mentioned (Kumar et al., 2013; Hasselmann et al., 2017) (one of three representative EAE experiments). Specifically, the mice receive two subcutaneous injections of MOG35-55 peptide (Mimotopes, Clayton, Victoria, Australia) emulsified with a complete Freund's adjuvant (BD Difco, Franklin Lakes, NJ) containing dead tuberculosis bacteria (M. butyricum) supplemented with Mycobacterium tuberculosis (BD Difco), followed by two intraperitoneal injections of Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA). Mice may be monitored daily according to the standard EAE clinical disease scoring scale revised by Pettinelli and McFarlin (Pettinelli and McFarlin, 1981; Hasselmann et al., 2017). Animals should be maintained in accordance with the guidelines set by the National Institutes of Health and the instructions of the University of California Riverside Office of Research Integrity and the Institutional Animal Care and Use Committee (IACUC), in accordance with the American Association for Laboratory Animal Science (AALAS).

[0229] Treatment: The test compound can be dissolved in 10% ethanol and 90% Miglyol 812N (medium) (Cremer; Sasol, Germany). The positive control group receives a 0.1 mL subcutaneous (sc) injection of 0.05 mg / kg / day E2 on day 0 of EAE (preEAE). Therapeutic treatment (sc) with the medium and various ERβ ligands at 5 mg / kg / day can be initiated on day 8 after EAE induction (postEAE; onset of clinical symptoms) and continued until day 30. Animals are euthanized according to the 2013 AVMA guidelines for euthanasia and can be sacrificed on either day 20-21 for flow cytometry, Luminex analysis and immunohistochemistry or day 30 for electrophysiology after disease induction.

[0230] Rotarod Behavior Assay: Mobilization behavior can be tested up to twice per week for each mouse using a rotorod apparatus (Med Associates, Inc., St. Albans, VT). Specifically, the animal is placed on a rotating horizontal cylinder for up to 200 seconds. The duration for which the mouse maintains walking on the cylinder without falling is recorded. Each mouse is tested at speeds of 3–30 rpm, and three tests are given on any designated day. The three tests are averaged, a single value is reported for each individual mouse, and then the average is calculated for all animals in a given treatment group (Moore et al., 2014). The first two test days before immunization serve as a practical test.

[0231] Histological preparation of tissue: Mice are deeply anesthetized by inhalation of isoflurane (Piramal Healthcare), perfused intracardiacly with phosphate-buffered saline (PBS), and then the tissue is fixed with 10% formalin (Thermo Fisher Scientific). The brain and spinal cord are dissected, post-fixed in 10% formalin (Thermo Fisher Scientific) for 24 hours, then cryoprotected in 30% sucrose (EMD Millipore, Darmstadt, Germany) for 48 hours, embedded in gelatin, and prepared for dissection. Next, the embedded brain and spinal cord are dissected into 40 μm coronal sections using an HM525 NX cryostat (Thermo Fisher Scientific). The sections are collected sequentially and stored at 4°C in PBS with 1% sodium azide until immunohistochemical staining, according to the previously described protocol (Crawford et al., 2010; Moore et al., 2014).

[0232] Immunohistochemistry: Before histological staining, 40 μm floating sections are thoroughly washed with PBS to remove residual sodium azide (Crawford et al., 2010). Sections are permeabilized with 0.3% Triton X100 and 15% normal goat serum (NGS) in 1×PBS. Myelination, gliosis, and immunomarkers may be visualized using the following primary antibodies at a concentration of 1:500 unless otherwise specified: chicken anti-myelin basic protein (MBP; polyclonal, EMD Millipore, Darmstadt, Germany), chicken anti-glial fibrillary acidic protein (GFAP; EMD Millipore, Darmstadt), rat anti-surface antigen classification 45 (CD45; clone 30-F11, BD Biosciences, San Diego, CA), mouse anti-ionized calcium-binding adapter molecule 1 / allogeneic inflammatory factor-1 (Iba1 / AIF1; clone 20A12.1, EMD Millipore, Darmstadt, Germany), goat anti-CXCL1 at 1:250 (R&D systems; Minneapolis, MN), and mouse anti-adenomatous polyposis coli)(CC-1; clone CC-1, Genetex, Irvine, CA). Unless otherwise specified, secondary staining may be performed using ThermoFisher Scientific polyclonal fluorophore conjugate antibodies at a concentration of 1:500: goat anti-chicken Alexa Fluor® 555 (AF555), goat anti-rabbit Alexa Fluor® 647 (AF647), donkey anti-chicken IgY Cy3 (EMD Millipore), goat anti-rat IgG AF647, goat anti-rabbit IgG Cy3 (EMD Millipore), goat anti-mouse IgG2b AF647, and rabbit anti-goat AF647.The nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, 2 ng / ml; Molecular Probes) for 10 minutes after incubation with a secondary antibody. The sections are then placed on a glass slide, dried, and covered with a coverslip in Fluoromount G mounting medium (Thermo Fisher Scientific) for imaging.

[0233] Splenocyte Isolation and Cytokine Analysis: Splenes are collected 20-21 days after EAE induction, before transcardiac perfusion. The spleens are dissected from anesthetized mice and mechanically dissociated into single-cell suspension in chilled RPMI1640 (hereinafter referred to as RPMI) supplemented with pyruvate, L-glutamine, and 10% fetal bovine serum. Red blood cells are lysed by incubation with ACK buffer (VWR), washed, counted, and resuspended in RPMI for cytokine analysis. Splenocytes are then stimulated with 25 μg / ml MOG35-55, and the supernatant is collected after 48 hours (Khalaj et al., 2013; Moore et al., 2013). The levels of anti-inflammatory cytokines (IL-10, IL-13, IL-4, and IL-5), pro-inflammatory cytokines (IFNγ, IL-17, IL-1β, TNFα, IL-6, and IL-2), and chemokines (CXCL1, CXCL10) were measured using the Cytokine Mouse Magnetic Panel for Luminex (Thermo Fisher Scientific; Waltham, MA) and can be performed using the xMAP MAGPIX 100™ instrument (Luminex Corporation, Austin, Tx) according to the manufacturer's instructions for use.

[0234] Transmission electron microscopy: Mice are perfused with PBS, followed by paraformaldehyde / glutaraldehyde, as described above to preserve their ultrastructure, and then embedded in Epon as previously described (Crawford et al., 2010). Serial ultrathin sections of Epon-embedded CCs can be stained with uranyl acetate-lead citrate for electron microscopy analysis. The G ratio may be measured using Fiji v1.0 software (NIH).

[0235] Confocal microscopy: Posterior and anterior column sections of the thoracic spinal cord, as well as CCs, may be imaged using an Olympus BX61 confocal microscope (Olympus America Inc., Center Valley, PA) with 10x and 40x objective lenses. Z-stack projections were compiled using SlideBook 6 software (Intelligent Imaging Innovations, Inc., Denver, CO). Immunostaining may be quantified using unbiased stereochemistry (Crawford et al., 2010). All images (RGB) can be confocal converted to grayscale, split, and separated by color channel using imageJ version 2.2.0-rc-46 / 1.50g (NIH). To avoid experimenter bias, automatic adjustment of brightness, contrast, and staining signal thresholds can be performed by ImageJ. MBP + GFAP + CD45 + , Iba1 + , CC1 + , and CXCL1 + The staining intensity is measured as the percentage area of ​​positive immunoreactivity within the region of interest, and the signal intensity is measured by ImageJ.

[0236] Electrophysiology: To evaluate functional conductivity through CC, electrophysiological recordings of compound action potentials (CAPs) can be measured as described above (Crawford et al., 2009; Crawford et al., 2010). Coronary brain sections are prepared from adult (3-4 month old) C57BL / 6 female mice. That is, the mice are deeply anesthetized under isoflurane and decapitated. The brain is removed and immersed in a partially frozen "slimy" solution of slicing buffer containing (in mM): 87 NaCl, 75 sucrose, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose, 1.3 ascorbic acid, 0.1 kynurenic acid, 2.0 pyruvate, and 3.5 MOPS, aerated with 5% CO2 + 95% O2 (Lauderdale et al., 2015). Coronal sections (350 μm) were prepared using a Leica VT 1000S Vibratome (Bannockburn, IL), and then incubated in slicing buffer at 35°C for 45 minutes. After incubation, the sections were cooled to room temperature over 15 minutes, and then transferred to ACSF (anterior cervical fixation) containing oxygenated 5% CO2 + 95% O2 (in mM): 125 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 26.0 NaHCO3, and 15 glucose. The sections were equilibrated in standard ACSF for at least 15–20 minutes before electrophysiological recording. During electrophysiological recording, the sections were continuously perfused with oxygenated ACSF at a flow rate of 1 mL / min. For CAP recording, the Axon Digidata 1550 may be used with a Multiclamp 700B amplifier and PClamp 10.4 software (Molecular Devices, Sunnyvale, CA). Continuous recording for CC conduction experiments can be low-passed at 10 kHz and digitized at 200 kHz. All experiments should be conducted at room temperature (24-26°C).To stimulate the CC fiber tract, concentric bipolar stimulator electrodes (FHC Neural microtargeting Worldwide, Bowdoin, ME, USA) are placed approximately 1 mm away from a recording electrode (a glass micropipette filled with ACSF) with a resistance of 1–3 MΩ. To induce CAP, an intermittent stimulation protocol is created consisting of eight consecutive sweeps, each 12 ms long, with a 5-second delay between each sweep and the immediate stimulation (0.01 ms duration) following the start of each sweep (Crawford et al., 2009a). Stimulation intensity is manually adjusted using an ISO-Flex stimulator (AMPI). Standardized input-output plots are generated for each section in current-clamp mode by averaging at least four consecutive sweeps together to reduce the signal-to-noise ratio. Brain sections showing near-zero potentials, even when stimulated with maximum current, are not included in the analysis. Electrophysiological data can be analyzed using Clampfit 10.4 software (Molecular Devices, Sunnyvale, CA) and OriginPro 2016 64Bit (OriginLab Corporation).

[0237] Statistical Analysis: All statistics can be performed using Prism 6 software (GraphPad Software, La Jolla, CA). Differences in EAE clinical scores can be determined by two-way disequilibrium ANOVA with Dunnett's multiple comparison test (Hasselmann et al., 2017). Luminex data and immunohistochemical data can be analyzed by either a standard one-way ANOVA with Dunnett's multiple comparison test, or Kruskal-Wallis with Dunn's multiple comparison test, provided that the data satisfy the assumption of normal distribution (D'Agostino-Pearson Omnibus Normality Test) and equal variances across all groups. CAP record analysis can be performed using Clampfit 10.4 software (Molecular Devices, Sunnyvale, CA), OriginPro 2016 64Bit (OriginLab Corporation), and GraphPad Prism 6 (GraphPad software), following previously published studies (Crawford et al., 2009b; Moore et al., 2014). We compare the averaged mean amplitudes using a one-way ANOVA with a post-hoc test employing Tukey's multiple comparison test. All data are presented as mean ± SEM for the two independent experiments. The difference is: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001, **** We consider a value to be statistically significant if p ≤ 0.0001.

[0238] While the present invention is suitable for various modifications and alternative forms, its exemplary embodiments are shown as examples in the drawings and described in detail herein. However, it should be understood that the description of the exemplary embodiments is not intended to limit the invention to any particular form disclosed, but rather to encompass all modifications, equivalents, and alternatives that fall within the spirit and scope of the invention as defined by the above embodiments and the below claims. Therefore, references to the above embodiments and the below claims should be made in order to interpret the scope of the invention.

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Claims

1. Equation (I) 【Chemistry 1】 (In the formula, R １ and R ２ are, independently, hydrogen, halogen, C １～４ alkyl, C １～４ fluoroalkyl, C ２～４ alkenyl, cyano, OH, -OC １～４ alkyl, or -OC １～４ fluoroalkyl, provided that at least one of R １ and R ２ is not hydrogen; R ３ is hydrogen, halogen, or C １～４ It is alkyl; R ４ is hydrogen, halogen, C １～４ Alkyl, C １～４ Fluoroalkyl, or C ２～４ It is an alkenil; R ５ OH and R ５ It is replaced by either a meta or para position; R ６ Each instance of occurrence independently involves halogen and C. １～４ Alkyl, C １～４ Fluoroalkyl, C ２～４ Alkenyl, cyano, OH, -OC １～４ Alkyl, or -OC １～４ It is a fluoroalkyl group; and n is 0, 1, or 2. Compounds thereof, or pharmaceutically acceptable salts thereof.

2. R １ However, halogen, C １～４ Alkyl, C １～４ Fluoroalkyl, C ２～４ Alkenyl, Cyano, -OC １～４ Alkyl, or -OC １～４ A compound according to claim 1, which is a fluoroalkyl compound, or a pharmaceutically acceptable salt thereof.

3. R １ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is hydrogen.

4. R ２ However, halogen, C １～４ Alkyl, C １～４ Fluoroalkyl, C ２～４ Alkenyl, cyano, OH, -OC １～４ Alkyl, or -OC １～４ A compound according to any one of claims 1 to 3, which is a fluoroalkyl compound, or a pharmaceutically acceptable salt thereof.

5. R ２ The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein the compound is hydrogen.

6. R ３ A compound according to any one of claims 1 to 5, wherein the compound is hydrogen, or a pharmaceutically acceptable salt thereof.

7. R ４ A compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, which is a halogen.

8. R ４ A compound according to any one of claims 1 to 6, wherein the compound is hydrogen, or a pharmaceutically acceptable salt thereof.

9. R ５ A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, which is substituted at the meta position.

10. R ５ However, it is substituted at the para position. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

11. A compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein n is 1.

12. Formula (II): 【Chemistry 2】 A compound according to claim 11, or a pharmaceutically acceptable salt thereof, having the above.

13. Formula (III): 【Transformation 3】 A compound according to claim 11, or a pharmaceutically acceptable salt thereof, having the above.

14. R ６ However, at each occurrence, independently, halogen or C １～４ A compound according to any one of claims 1 to 13, which is alkyl, or a pharmaceutically acceptable salt thereof.

15. A compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein n is 0.

16. Formula (IV): 【Chemistry 4】 A compound according to claim 15, or a pharmaceutically acceptable salt thereof, having the above.

17. Formula (V): 【Transformation 5】 A compound according to claim 15, or a pharmaceutically acceptable salt thereof, having the above.

18. 7-bromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-bromo-3-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-bromo-3-chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-dibromo-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-bromo-2-(4-hydroxyphenyl)-3-iodo-2H-indazole-5-ol; 7-chloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-Fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-chloro-7-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-Chloro-7-methyl-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 7-bromo-3-chloro-2-(3-hydroxyphenyl)-2H-indazole-5-ol; 7-Chloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-dichloro-2-(3-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 7-Chloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3,7-dichloro-2-(2-fluoro-4-hydroxyphenyl)-2H-indazole-5-ol; 3-chloro-6-fluoro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3-chloro-6-fluoro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol; 3,6-dichloro-2-(4-hydroxyphenyl)-2H-indazole-5-ol; 3,6-dichloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5-ol; 3-chloro-2-(4-hydroxyphenyl)-2H-indazole-5,7-diol; and 3-chloro-2-(4-hydroxy-2-methylphenyl)-2H-indazole-5,7-diol; A compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of the following.

19. A pharmaceutical composition comprising a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

20. A method for treating a demyelinating disease, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, to a subject in need thereof.

21. The method according to claim 20, wherein the demyelinating disease is multiple sclerosis.

22. The method according to claim 21, wherein the multiple sclerosis is primary progressive multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis.

23. A method for promoting the remyelination of demyelinated axons, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, to a subject in need thereof.

24. A method for differentiating oligodendrocyte progenitor cells, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, to a subject requiring such treatment.

25. A method for treating endometriosis, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, to a subject in need thereof.

26. A compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, for use in the treatment of demyelinating diseases, or in promoting the remyelination of demyelinated axons, or in the differentiation of oligodendrocyte progenitor cells, or in the treatment of endometriosis.

27. Use of a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 19, in the manufacture of a drug for the treatment of demyelinating diseases, or for promoting the remyelination of demyelinated axons, or for the differentiation of oligodendrocyte progenitor cells, or for the treatment of endometriosis.

28. A kit comprising a compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 19, and instructions for use thereof.

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