Carbon-linked BCL inhibitors and senolitic compounds, and their uses

Abstract

The present invention relates to compounds that kill aging cells, namely senolitic compounds, and compounds that inhibit the BCL-2 protein family (BCL-2, BCL-XL, BCL-W, MCL-1, A1, BCL-B). The present invention also provides compounds and methods for treating aging-related diseases or disorders, and compounds and methods for treating diseases or disorders affected by the BCL-2 protein family.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 602,724, filed November 27, 2023, pursuant to 35 USC § 119(e), the entire contents of which are incorporated herein by reference for all purposes.

[0003] The present invention relates to compounds that kill aging cells, namely senolytic compounds, and compounds that inhibit the BCL-2 protein family (BCL-2, BCL-XL, BCL-W, MCL-1, A1, BCL-B). Furthermore, the present invention provides compounds and methods for treating diseases or disorders related to aging cells, and compounds and methods for treating diseases or disorders affected by the BCL-2 protein family. Background Technology

[0004] Senescence is a cellular program that induces a stable state of proliferative arrest in cells to prevent the replication of damaged or old cells. In addition to growth arrest, senescent cells undergo significant phenotypic changes, including chromatin reorganization, increased beta-galactosidase activity (referred to as senescence-associated β-galactosidase or SA-β-Gal), and the secretion of a number of factors that are primarily pro-inflammatory, which are collectively referred to as the senescence-associated secretory phenotype (SASP).

[0005] Replicative senescence is activated during the successive passage of cultured cells (or as cells age within the organism). Senescence is also induced, for example, by oncogene activation, irradiation, and exposure to chemotherapy drugs. Additionally, there are several drugs that induce senescence, with CDK4 / CDK6 inhibitors such as palbociclib being a representative example.

[0006] Stable growth arrest, a characteristic of aging, is mediated by the activation of the p16 / Rb and p53 / p21 pathways. Cyclin-dependent kinase inhibitor p16 INK4a and p21Cip1 inhibits CDK activity, leading to Rb hypophosphorylation and G1 growth arrest (Kuilman et al., Genes Dev 2010 24, 2453-2479). Furthermore, p16 INK4a It is specifically induced during aging and is used to identify senescent cells, either alone or in combination with SA-β-Gal activity, the formation of aging-associated heterochromatin foci (SAHF), and other markers.

[0007] Senescent cells accumulate during aging and are associated with many diseases, including cancer, fibrosis, and numerous aging-related pathologies. Recent evidence suggests that senescent cells are harmful in many pathologies and that their removal offers significant benefits, such as improving these conditions and extending lifespan.

[0008] Senescent cells are present in many precancerous lesions, fibrous tissues (e.g., liver, kidney, heart, pancreas), and old tissues. Senescent cells are also associated with various pathological conditions such as the nervous system (e.g., cerebral aneurysms, Alzheimer's, and Parkinson's), lungs (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and cystic fibrosis), ophthalmology (e.g., cataracts, glaucoma, macular degeneration), musculoskeletal system (e.g., sarcopenia, disc degeneration, osteoarthritis), cardiovascular system (e.g., atherosclerosis, cardiac fibrosis, aortic aneurysm), kidneys (e.g., renal disease, transplant complications), and diabetes, mucositis, hypertension, and osteomyelofibrosis (OMF). Although senescent cells play a protective role against cancer and limit most types of fibrosis, the accumulation of senescent cells during the aging process and many other diseases is considered harmful.

[0009] Evidence regarding the many harmful effects of senescent cells (and the benefits resulting from their selective removal) has been provided by a series of studies in the van Deursen laboratory (Baker et al., Nature 2016 530, 184-189; Baker et al., Nature 2011 479, 232-236; Childs et al., Science 2016 354, 472-477). These studies used transgenic mice (INK4-ATTAC mice) that specifically express inducible fusion proteins in senescent cells (p16). INK4a (Utilizing the promoter). When this fusion protein is activated by the addition of a drug that induces dimerization, selective death of aging cells occurs.

[0010] Using the aforementioned mouse model, the removal of senescent cells extends lifespan by attenuating numerous senescence-related pathologies (Baker et al., Nature 2016 530, 184-189; Baker et al., Nature 2011 479, 232-236; Childs et al., 2016 Science 354, 472-477). The removal of senescent cells delayed tumor formation and attenuated cataract formation, atherosclerosis, and senescence-related deterioration in the kidneys, lipids, and heart, among other organs. The results obtained with INK4-ATTAC mice were partially replicated in a different mouse model (3MR mouse) in which the tk transgenic gene is expressed in senescent cells, thereby enabling the selective removal of senescent cells during ganciclovir treatment (Demaria et al., Dev Cell 2014 31, 722-733). Furthermore, the removal of senescent cells during chemotherapy reduced cancer recurrence and chemotherapy-related side effects. Importantly, the removal of senescent cells has no adverse effects other than delaying wound healing when senescent cells are removed during the healing process (Baker et al., Nature 2016 530, 184-189; Demaria et al., Dev Cell 2014 31, 722-733). However, the dominant integrative hypothesis is that (pro-inflammatory) factors secreted by senescent cells disrupt tissue homeostasis. This suggests that common mechanisms mediated by senescent cells may lead to the effects of many aging-related pathologies.

[0011] Proof-of-concept studies led to the identification of compounds capable of selectively eliminating senescent cells (so-called "senolytics"). Several senolytic compounds have been identified to date, including dasatinib, quercetin, piperlongumine, and BCL-2 protein family inhibitors such as ABT-263 and ABT-737. Currently, BCL-2 protein family inhibitors are the most promising senolytics, having been shown to kill a series of senescent cells in vivo with reproducible effects in transgenic mice. BCL-2 family inhibitors were initially developed as treatments for lymphoma. ABT-737 is a small molecule inhibitor of BCL-2, BCL-XL, and BCL-w, but it has low solubility and oral bioavailability. ABT-263 inhibits the same molecules and is more suitable for in vivo use, but it causes significant thrombocytopenia as a side effect.

[0012] Therefore, it is necessary to identify more compounds and classes of compounds having senolic properties, and / or compounds that inhibit the BCL-2 protein family. The problem to be solved

[0013] In one aspect, a compound of the following structure, or its salt, hydrate, and solvate, is provided to satisfy these requirements and other requirements:

[0014]

[0015] In the above formula, X is a heteroaryl or a substituted heteroaryl; Y is an alkyldiyl, a substituted alkyldiyl, a cycloalkyldiyl, a substituted cycloalkyldiyl, a heteroalkyldiyl or a substituted heteroalkyldiyl; and Z is an alkyl, a substituted alkyl, a cycloalkyl, a substituted cycloalkyl, a cycloalkenyl, a substituted cycloalkenyl, an aryl, a substituted aryl, a cycloheteroalkyl, a substituted cycloheteroalkyl, a heteroalkyl, a substituted heteroalkyl, -OR 16, or -NHR 17 -is; T is -C- or -S- and; V is -C(O)-, -C(N)OR 2 -, -C(N)NR 3 R 4 -, -C(OH)R 5 - or -CHR 6 - and; J is an aryl, a substituted aryl, a heteroaryl or a substituted heteroaryl; K is an aryldiyl, a substituted aryldiyl, a heteroaryldiyl or a substituted heteroaryldiyl; and R 1 is a halo, alkyl, alkenyl, alkynyl, or haloalkyl; R 2 to R 6 , R8, R 11 , R 14 , R 16 , and R 17 is independently -H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl; R 7 -OR 8 or -NHR 9 is; R 9 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl or -SO2R 10 and; R 10is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl or -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 is; R 12 and R 13 is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, or R 12 and R 13 ... forms a cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded; R 15 is an aryl, substituted aryl, heteroaryl or substituted heteroaryl; p is 2-20; n is 0 or 1; and m is 0, 1, 2 or 3.

[0016] In another aspect, a method is provided to treat, prevent, or improve the symptoms of medical disorders, such as, for example, age-related diseases or disorders, and diseases or disorders affected by BCL. Specific details for implementing the invention

[0017] definition

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Where multiple definitions of a term exist in this specification, the definitions in this section shall prevail unless otherwise specified.

[0019] As used in this specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” may include both singular and plural references unless otherwise explicitly stated or clearly indicated by the context. As used in this specification, the term “exemplary” means “serving as an example, case, or illustration.” Any embodiment or feature characterized as “exemplary” in this specification is not to be interpreted as being more desirable or advantageous than other embodiments or features.

[0020] Where components are presented in a list format (e.g., Markush groups), each possible subgroup of the components is also understood to be disclosed, and any one or more components may be removed from the list or group.

[0021] The disclosure of a numerical range is further understood to be a specific disclosure of all possible sub-ranges and all possible individual numerical values ​​(whether integers or fractions) within the range, regardless of the width of the range. Furthermore, unless clearly indicated otherwise, in any method described or claimed herein comprising two or more acts or steps, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are listed, and the disclosure is understood to encompass embodiments in which the order is so limited.

[0022] In general, where an embodiment of the detailed description or claims is referred to as comprising one or more features, the disclosure is further understood to encompass an embodiment that is composed of or essentially constitutes of such feature(s).

[0023] Additionally, any embodiment of the present disclosure, for example, any embodiment or compound found in the prior art, may be explicitly excluded from the claims, regardless of whether specific exclusions are described in the specification.

[0024] Whenever the term "at least" or "greater than" precedes the first number in a series of two or more numbers, the terms "at least" or "greater than" apply to each number in that series.

[0025] Whenever the term "no more than" or "less than" precedes the first number in a series of two or more numbers, the terms "no more than" or "less than" apply to each number in that series.

[0026] As used herein, and unless otherwise specified, when used in relation to a characteristic having a number or a range of numbers, the terms “about” and “approximately” indicate that said number or range of numbers may deviate to a range that would be considered reasonable to a person skilled in the art while still describing a particular characteristic. Specifically, when used in the context herein, the terms “about” and “approximately” indicate that a number or range of numbers may vary by 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the listed number or range of numbers. Additionally, unless the context clearly indicates otherwise, the singular forms “a” and “the” include plural references. Accordingly, for example, a reference to "the compound" includes a plurality of such compounds, and a reference to "the assay" includes a reference to one or more assays and equivalents thereof known to those skilled in the art.

[0027] A dash ("-") not between two letters or symbols is used to indicate the bonding point of a substituent. For example, -C(O)NH2 is bonded through carbon atoms. Dashes at the beginning or end of chemical groups are for convenience; chemical groups may be depicted with or without one or more dashes without losing their conventional meaning. A wavy line drawn through the lines of the structure indicates the bonding point of the group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which chemical groups are written or named.

[0028] The prefix "Cu-v" indicates that the following group has u to v carbon atoms. The u to v carbons include carbons such as u+1 to v, u+2 to v, u+3 to v, u+1 to u+3 to v, u+1 to u+4 to v, u+2 to u+4 to v, etc., and should be understood to encompass all possible permutations of u and v.

[0029] "alkyl" refers to a saturated, branched, or straight-chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane, either by itself or as part of another substituent. Typical alkyl groups include, but are not limited to, methyl; ethyl; propyl, such as propane-1-yl, propane-2-yl, etc.; butyl, such as butane-1-yl, butane-2-yl, 2-methyl-propane-1-yl, 2-methyl-propane-2-yl, etc.; and similar ones. In some embodiments, the alkyl group comprises 1 to 20 carbon atoms (C1-C 20 alkyl). In another embodiment, the alkyl group comprises 1 to 10 carbon atoms (C1-C 10 Alkyl). In another embodiment, the alkyl group comprises 1 to 6 carbon atoms (C1-C6 alkyl).

[0030] "Alkenyl" refers to an unsaturated, branched, straight-chain group having at least one carbon-carbon double bond, derived by removing one hydrogen atom from a single carbon atom of a parent alkane, either by itself or as part of another substituent. The group may be in a cis or trans stereochemical configuration with respect to the double bond(s). Typical alkenyl groups include ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), and prop-2-en-2-yl; Butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, buta-1,3-diene-1-yl, buta-1,3-diene-2-yl, etc.; and similar, but are included but not limited thereto. In some embodiments, the alkenyl group comprises 2 to 20 carbon atoms (C2-C 20 Alkenyl). In another embodiment, the alkenyl group comprises 2 to 10 carbon atoms (C2-C 10 Alkenyl). In another embodiment, the alkenyl group comprises 2 to 6 carbon atoms (C2-C6 alkenyl).

[0031] "Alkynyl" refers to an unsaturated branched or straight-chain type having at least one carbon-carbon triple bond, derived by removing one hydrogen atom from a single carbon atom of a parent alkyne, either by itself or as part of another substituent. Typical alkynyl groups include, but are not limited to, ethinyl; propynyls such as prop-1-in-1-yl, prop-2-in-1-yl, etc.; butynyls such as but-1-in-1-yl, but-1-in-3-yl, but-3-in-1-yl, etc. In some embodiments, the alkynyl group comprises 2 to 20 carbon atoms (C2-C 20 Alkynyl). In another embodiment, the alkynyl group comprises 2 to 10 carbon atoms (C2-C10 Alkynyl). In another embodiment, the alkynyl group comprises 2 to 6 carbon atoms (C2-C6 alkynyl).

[0032] "alkyldiyl" refers to a saturated, branched, or straight-chain hydrocarbon group derived by removing one hydrogen atom from each of two different carbon atoms of a parent alkane, either as itself or as part of another substituent, or by removing two hydrogen atoms from a single carbon atom of a parent alkane. Each of the two monovalent or divalent radical centers can form bonds with the same or different atoms. Typical alkyldiyl groups include methanediyl; ethanediyls such as ethane-1,1-diyl and ethane-1,2-diyl; and propanediyls such as propane-1,1-diyl, propane-1,2-diyl, propane-2,2-diyl and propane-1,3-diyl. Butanediyls such as butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, butane-1,4-diyl, butane-2,2-diyl, 2-methyl-propane-1,1-diyl, 2-methyl-propane-1,2-diyl, etc.; and similar ones, but are not limited thereto. In some embodiments, the alkyldiyl group is (C1-C 20 ) is an alkyldiyl. In another embodiment, the alkyldiyl group is (C1-C 10 ) is an alkyldiyl. In another embodiment, the alkyldiyl group is a (C1-C6) alkyldiyl.

[0033] "Alkenyldiyl" refers to an unsaturated, branched, linear, or cyclic divalent hydrocarbon group derived by removing one hydrogen atom from each of two different carbon atoms of a parent alkane, either as itself or as part of another substituent, or by removing two hydrogen atoms from a single carbon atom of a parent alkane. Each of the two monovalent or divalent radical centers can form bonds with the same or different atoms. Typical alkenyldiyl groups include butyldiyls such as ethene-1,1-diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl, but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl, etc., butyldiyls, but are not limited thereto. In some embodiments, the alkenyldiyl group is (C2-C 20 ) It is an alkenyldiyl. In another embodiment, the alkenyldiyl group is (C2-C 10 ) It is an alkenyldiyl. In another embodiment, the alkenyldiyl group is a (C2-C6) alkenyldiyl.

[0034] "Aryl" refers to a monovalent aromatic hydrocarbon group derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system as defined herein, either as itself or as part of other substituents. Typical aryl groups include, but are not limited to, groups derived from aceanthrilene, acenaphtylene, acephenanthrilene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indan, indene, naphthalene, octacene, octapene, octalene, ovalene, pentacene, pentalene, pentapene, perylene, phenalene, phenanthrene, ficene, pleiaden, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and similar ones. In some embodiments, the aryl group comprises 6 to 30 carbon atoms (C6-C 30 aryl). In another embodiment, the aryl group comprises 6 to 20 carbon atoms (C6-C 20 aryl). In another embodiment, the aryl group comprises 6 to 15 carbon atoms (C6-C 15 aryl). In another embodiment, the aryl group comprises 6 to 10 carbon atoms (C6-C 10 Aril).

[0035] "Arylalkyl" refers to a carbon atom, either itself or as part of another substituent, typically at the terminal or sp² 3 It refers to a non-cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group as defined herein. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethane-1-yl, 1-phenylethene-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 1-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethane-1-yl, and similar ones. In some embodiments, the arylalkyl group is (C7-C 40 ) is an arylalkyl, and for example, the alkyl portion of the arylalkyl group is (C1-C10 ) alkyl and the aryl portion is (C6-C 30 ) is an aryl. In another embodiment, the arylalkyl group is (C7-C 30 ) is an arylalkyl, and for example, the alkyl portion of the arylalkyl group is (C1-C 10 ) alkyl and the aryl portion is (C6-C 20 ) is an aryl. In another embodiment, the arylalkyl group is (C7-C 20 ) is an arylalkyl, for example, the alkyl portion of the arylalkyl group is (C1-C8)alkyl and the aryl portion is (C6-C 12 ) is an aryl. In another embodiment, the arylalkyl group is (C7-C 15 ) is an arylalkyl, for example, the alkyl portion of the arylalkyl group is (C1-C5)alkyl and the aryl portion is (C6-C 10 It is Aril.

[0036] "Arylalkenyl" refers to a non-cyclic alkenyl group in which one of the hydrogen atoms bonded to a carbon atom, either itself or as part of another substituent, is replaced by an aryl group as defined herein. In some embodiments, the arylalkenyl group is (C8-C 40 ) it is an aryl alkenyl, and for example, the alkenyl portion of the aryl alkenyl group is (C2-C 10 ) It is an alkenyl and the aryl part is (C6-C 30 ) It is an aryl. In another embodiment, the aryl alkenyl group is (C8-C 30 ) it is an aryl alkenyl, and for example, the alkenyl portion of the aryl alkenyl group is (C2-C 10 ) It is an alkenyl and the aryl part is (C8-C 20 ) It is an aryl. In another embodiment, the aryl alkenyl group is (C8-C 20 It is an aryl alkenyl, for example, the alkenyl portion of the aryl alkenyl group is a (C2-C8) alkenyl and the aryl portion is (C6-C 12 ) is an aryl. In another embodiment, the aryl alkenyl group is (C8-C 15It is an aryl alkenyl, for example, the alkenyl portion of the aryl alkenyl group is a (C2-C5) alkenyl and the aryl portion is (C6-C 10 It is Aril.

[0037] "Arylalkynyl" refers to a non-cyclic alkynyl group in which one of the hydrogen atoms bonded to a carbon atom, either itself or as part of another substituent, is replaced by an aryl group as defined herein. In some embodiments, the arylalkynyl group is (C8-C 40 ) it is an arylalkynyl, and for example, the alkynyl portion of the arylalkynyl group is (C2-C 10 ) It is an alkynyl and the aryl part is (C6-C 30 ) It is an aryl. In another embodiment, the arylalkynyl group is (C8-C 30 ) it is an arylalkynyl, and for example, the alkynyl portion of the arylalkynyl group is (C2-C 10 ) It is an alkynyl and the aryl part is (C6-C 20 ) It is an aryl. In another embodiment, the arylalkynyl group is (C8-C 20 It is an arylalkynyl, for example, the alkynyl portion of the arylalkynyl group is (C2-C8)alkynyl and the aryl portion is (C6-C 12 ) is an aryl. In another embodiment, the arylalkinyl group is (C8-C 15 It is an arylalkynyl, for example, the alkynyl portion of the arylalkynyl group is (C2-C5)alkynyl and the aryl portion is (C6-C 10 It is Aril.

[0038] A "carbohydrate derivative" is a chemical compound of the general formula C bonded to a group. n H 2n O nIt refers to a carbohydrate. In some embodiments, the carbohydrate derivative typically comprises five or six carbon atoms. In other embodiments, the carbohydrate derivative is a monosaccharide (e.g., glucose, fructose, galactose, ribose). In yet another embodiment, the carbohydrate derivative comprises a disaccharide (e.g., lactose, sucrose, maltose, cellobiose, chitobiose, gentobiose, etc.). In yet another embodiment, the carbohydrate derivative comprises an oligosaccharide (e.g., oligofructose, oligogalactose, raffinose, plantose, veracoose, etc.). In yet another embodiment, the carbohydrate derivative comprises a polysaccharide (e.g., cellulose, amylose, starch, chitin, pectin, galactogen, etc.). In another embodiment, the carbohydrate derivative comprises a protected carbohydrate, such as an ester (e.g., acetate or benzoate, etc.), a silyl derivative, or a carbohydrate protected by any other known alcohol protecting group.

[0039] "Compounds" refers to compounds encompassed by structural formulas disclosed herein, the structures of which include any specific compounds within these formulas disclosed herein. Compounds may be identified by their chemical structures and / or chemical names. The chemical structure determines the uniqueness of the compounds. Compounds described herein may include one or more chiral centers and / or double bonds and thus may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Accordingly, the chemical structures described herein encompass the stereoisomerically pure forms described in the structures (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure forms). The chemical structures described herein also encompass enantiomers and stereoisomer derivatives of the described compounds. Enantiomer and stereoisomer mixtures may be separated using separation techniques or chiral synthesis techniques well known to those skilled in the art to obtain their constituent enantiomers It may be separated into isomers or stereoisomers. The compound may also exist in various tautomer forms, including enol forms, keto forms, and mixtures thereof. Accordingly, the chemical structures described herein encompass all possible tautomer forms of the exemplified compounds. The compound may also be atropisomers. The described compounds also include isotope-labeled compounds in which one or more atoms have atomic masses different from those typically found in nature. Examples of isotopes that may be introduced into the compounds disclosed herein are 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17O, etc., are included but not limited thereto. Compounds may exist in solvated forms as well as in unsolved forms, including hydrated forms. Generally, compounds may be hydrated or solvated. Specific compounds may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the uses considered herein and are intended to fall within the scope of this disclosure. Furthermore, when a partial structure of a compound is illustrated, parentheses should be understood to indicate a bonding point of the partial structure to the rest of the molecule.

[0040] "Cycloalkyl" refers to a saturated cyclic monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent cycloalkane, either by itself or as part of another substituent. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, etc.; and similar ones. In some embodiments, the cycloalkyl group comprises 3 to 20 carbon atoms (C3-C 20 cycloalkyl). In another embodiment, the cycloalkyl group comprises 3 to 10 carbon atoms (C3-C 10 cycloalkyl). In another embodiment, the cycloalkyl group comprises 3 to 8 carbon atoms (C3-C8 cycloalkyl). The term "cyclic monovalent hydrocarbon radical" also includes a single radical and a polycyclic hydrocarbon ring system having 5 to 12 carbon atoms. Exemplary polycyclic cycloalkyl rings include, for example, norbornyl, pinyl, and adamantyl.

[0041] "Cycloalkyldiyl" refers to a cyclic hydrocarbon group derived by removing one hydrogen atom from each of two different carbon atoms of a parent cycloalkane, either as itself or as part of another substituent, or by removing two hydrogen atoms from a single carbon atom of a parent cycloalkane. Each of the two monovalent radical centers or divalent radical centers may form bonds with the same or different atoms. Exemplary polycyclic cycloalkyldiyl rings include, for example, norbornyldiyl, pinyldiyl, and adamantyldiyl.

[0042] "Cycloalkenyl" refers to an unsaturated cyclic monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent cycloalkene, either as itself or as part of another substituent. Typical cycloalkenyl groups include, but are not limited to, cyclopropphenyl, cyclobutenyl, cyclopentenyl, etc.; and similar ones. In some embodiments, the cycloalkenyl group comprises 3 to 20 carbon atoms (C3-C 20 cycloalkenyl). In another embodiment, the cycloalkenyl group comprises 3 to 10 carbon atoms (C3-C 10 Cycloalkenyl). In another embodiment, the cycloalkenyl group comprises 3 to 8 carbon atoms (C3-C8 cycloalkenyl).

[0043] "Cycloheteroalkyl" refers to a cycloalkyl group as defined herein, either as itself or as part of other substituents, wherein one or more carbon atoms (and optionally any associated hydrogen atoms) are each, independently of one another, replaced by a heteroatom or heteroatom group identical or different from that defined in "heteroalkyl" below. In some embodiments, the cycloheteroalkyl group comprises 3 to 20 carbons and heteroatoms (3-20 cycloheteroalkyl). In other embodiments, the cycloheteroalkyl group comprises 3 to 10 carbons and heteroatoms (3-10 cycloheteroalkyl). In yet another embodiment, the cycloheteroalkyl group comprises 3 to 8 carbons and heteroatoms (3-8 cycloheteroalkyl). The term "cyclic monovalent heteroalkyl radical" also includes single radicals and polycyclic heteroalkyl ring systems having 3 to 12 carbons and at least one heteroatom. Exemplary cycloheteroalkyl groups include, for example, azetidine, pyrrolidine, piperazine, piperidine, morpholine, and tetrahydrofuran.

[0044] "Cycloheteroalkenyl" refers to a cycloalkenyl group as defined herein, either as itself or as part of another substituent, wherein one or more carbon atoms (and optionally any associated hydrogen atoms) are each, independently of one another, replaced by a heteroatom or heteroatom group identical or different from that defined in "heteroalkenyl" below. In some embodiments, the cycloheteroalkenyl group comprises 3 to 20 carbons and heteroatoms (3-20 cycloheteroalkenyl). In other embodiments, the cycloheteroalkenyl group comprises 3 to 10 carbons and heteroatoms (3-10 cycloheteroalkenyl). In yet another embodiment, the cycloheteroalkenyl group comprises 3 to 8 carbons and heteroatoms (3-8 cycloheteroalkenyl). The term "cyclic monovalent heteroalkenyl radical" also includes a single radical and a polycyclic heteroalkenyl ring system having 2 to 12 carbons and at least one heteroatom.

[0045] "Halo" refers to the radical -F, -Cl, -Br, or -I, either as itself or as part of another substituent.

[0046] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms (and optionally any associated hydrogen atoms) are each, independently, replaced by the same or different heteroatom or heteroatom group. Typical heteroatom or heteroatom groups that can replace a carbon atom include, but are not limited to, -O-, -S-, -N-, -Si-, -NH-, -S(O)-, -S(O)2-, -S(O)NH-, -S(O)2NH-, etc. The heteroatom or heteroatom group may be located at any internal position of an alkyl, alkenyl, or alkynyl group. Typical heteroatom groups that may be included in these groups are -O-, -S-, -OO-, -SS-, -OS-, -NR501 R 502 , =NN=, -N=N-, -N=N-NR 503 R 504 , -PR 505 -, -P(O)2-, -POR 506 -, -OP(O)2-, -SO-, -SO2-, -SnR 507 R 508 Includes but is not limited to the rest and similars, where R 501 , R 502 , R 503 , R 504 , R 505 , R 506 , R 507 and R 508 is independently hydrogen, alkyl, aryl, substituted aryl, heteroalkyl, heteroaryl, or substituted heteroaryl. In some embodiments, the heteroalkyl group comprises 1 to 20 carbons and heteroatoms (1-20 heteroalkyl). In other embodiments, the heteroalkyl group comprises 1 to 10 carbons and heteroatoms (1-10 heteroalkyl). In yet another embodiment, the heteroalkyl group comprises 1 to 6 carbons and heteroatoms (1-6 heteroalkyl).

[0047] "Heteroalkenyl" refers to an alkenyl group in which one or more carbon atoms (and optionally any associated hydrogen atoms) are each, independently, replaced by the same or different heteroatom or heteroatom group. Typical heteroatom or heteroatom groups that can replace a carbon atom include, but are not limited to, -O-, -S-, -N-, -Si-, -NH-, -S(O)-, -S(O)2-, -S(O)NH-, -S(O)2NH-, etc. The heteroatom or heteroatom group may be located at any internal position of an alkyl, alkenyl, or alkynyl group. Typical heteroatom groups that may be included in these groups are -O-, -S-, -OO-, -SS-, -OS-, -NR501 R 502 , =NN=, -N=N-, -N=N-NR 503 R 504 , -PR 505 -, -P(O)2-, -POR 506 -, -OP(O)2-, -SO-, -SO2-, -SnR 507 R 508 Includes but is not limited to the rest and similars, where R 501 , R 502 , R 503 , R 504 , R 505 , R 506 , R 507 and R 508 is independently hydrogen, alkyl, aryl, substituted aryl, heteroalkyl, heteroaryl, or substituted heteroaryl. In some embodiments, the heteroalkenyl group comprises 1 to 20 carbons and heteroatoms (1-20 heteroalkenyl). In other embodiments, the heteroalkenyl group comprises 1 to 10 carbons and heteroatoms (1-10 heteroalkenyl). In yet another embodiment, the heteroalkenyl group comprises 1 to 6 carbons and heteroatoms (1-6 heteroalkenyl).

[0048] "Heteroalkyldiyl" refers to a saturated, branched, or straight-chain hydrocarbon group derived by removing one hydrogen atom from each of two different carbon atoms of a parent heteroalkane, either as itself or as part of another substituent, or by removing two hydrogen atoms from a single carbon atom of a parent heteroalkane. Each of the two monovalent radical centers or divalent radical centers may form bonds with the same or different atoms. In some embodiments, the heteroalkyldiyl group (C1-C 20 ) is a heteroalkyldiyl. In another embodiment, the heteroalkyldiyl group is (C1-C 10) is a heteroalkyldiyl. In another embodiment, the heteroalkyldiyl group is a (C1-C6) heteroalkyldiyl.

[0049] "Heteroalkenyldiyl" refers to an unsaturated, branched, linear, or cyclic divalent heteroalkenyl group derived by removing one hydrogen atom from each of two different carbon atoms of a parent heteroalkene, either as itself or as part of another substituent, or by removing two hydrogen atoms from a single carbon atom of a parent heteroalkene. Each of the two monovalent radical centers or divalent radical centers may form bonds with the same or different atoms. In some embodiments, the heteroalkenyldiyl group (C2-C 20 ) is a heteroalkenyldiyl. In another embodiment, the heteroalkenyldiyl group is (C2-C 10 ) It is a heteroalkenyl diyl. In another embodiment, the heteroalkenyl diyl group is a (C2-C6) heteroalkenyl diyl.

[0050] "Heteroaryl" refers to a monovalent heteroaromatic radical derived by removing one hydrogen atom from a single atom of a parent heteroaromatic ring system as defined herein, either as itself or as part of another substituent. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, β-carbolin, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indoligin, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazol, isoxazole, naphthiridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyrazine, pyrazol, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidin, quinazolin, quinoline, quinoligin, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthen, and similar substances. In some embodiments, the heteroaryl group comprises 5 to 20 ring atoms (5-20 heteroaryl). In other embodiments, the heteroaryl group comprises 5 to 10 ring atoms (5-10 heteroaryl). Exemplary heteroaryl groups include those derived from furan, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole, indole, pyridine, pyrazol, quinoline, imidazole, oxazole, isoxazole, and pyrazine.

[0051] "Heteroarylalkyl" refers to a carbon atom, either itself or as part of another substituent, typically at the terminal or sp² 3It refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group. In some embodiments, the heteroarylalkyl group is a 6-21-membered heteroarylalkyl, for example, the alkyl portion of the heteroarylalkyl is a (C1-C6) alkyl and the heteroaryl portion is a 5-15-membered heteroaryl. In other embodiments, the heteroarylalkyl is a 6-13-membered heteroarylalkyl, for example, the alkyl portion is a (C1-C3) alkyl and the heteroaryl portion is a 5-10-membered heteroaryl.

[0052] "Heteroarylalkenyl" refers to an acyclic alkenyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group, either as itself or as part of another substituent. In some embodiments, the heteroarylalkenyl group is a 7-21-membered heteroarylalkenyl, for example, the alkenyl portion of the heteroarylalkenyl is a (C2-C6) alkenyl and the heteroaryl portion is a 5-15-membered heteroaryl. In other embodiments, the heteroarylalkenyl is a 7-13-membered heteroarylalkenyl, for example, the alkenyl portion is a (C2-C3) alkenyl and the heteroaryl portion is a 5-10-membered heteroaryl.

[0053] "Heteroarylalkynyl" refers to an acyclic alkynyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by a heteroaryl group, either as itself or as part of another substituent. In some embodiments, the heteroarylalkynyl group is a 7-21-membered heteroarylalkynyl, for example, the alkynyl portion of the heteroarylalkynyl is a (C2-C6) alkynyl and the heteroaryl portion is a 5-15-membered heteroaryl. In other embodiments, the heteroarylalkynyl is a 7-13-membered heteroarylalkynyl, for example, the alkynyl portion is a (C2-C3) alkynyl and the heteroaryl portion is a 5-10-membered heteroaryl.

[0054] "Heteroaryldiyl" refers to a divalent radical derived by removing two hydrogen atoms from two different atoms of a parent heteroaromatic ring system as defined herein, either as itself or as part of another substituent. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, β-carbolin, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indoligin, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazol, isoxazole, naphthiridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyrazine, pyrazol, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidin, quinazolin, quinoline, quinoligin, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthen, and similar substances. In some embodiments, the heteroaryldiyl group comprises 5 to 20 ring atoms (5-20 heteroaryldiyl). In other embodiments, the heteroaryldiyl group comprises 5 to 10 ring atoms (5-10 heteroaryldiyl). Exemplary heteroaryldiyl groups include those derived from furan, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole, indole, pyridine, pyrazol, quinoline, imidazole, oxazole, isoxazole, and pyrazine.

[0055] “Hydrates” refers to the formation of an addduct by introducing water into the form of a compound described herein in stoichiometric proportions of the chemical formula. Methods for preparing hydrates include, but are not limited to, storage in an atmosphere containing water vapor, formulations containing water, or routine pharmaceutical process steps such as, for example, crystallization (i.e., crystallization from water or a mixed aqueous solvent), freeze-drying, wet granulation, aqueous film coating, or spray drying. Hydrates may also be formed from crystalline solvates upon exposure to water vapor under certain circumstances, or upon suspension of an anhydrous material in water. Hydrates may also crystallize into more than one form, resulting in hydrate polymorphism. See examples (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing the hydrate are sufficiently within the scope of those skilled in the art, are entirely ordinary, and require no experiments other than those typical in the art. The hydrate can be characterized and / or analyzed by methods well known to those skilled in the art, such as, for example, single-crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999).In addition, many commercial companies regularly provide services including the manufacture and / or characterization of hydrates, such as HOLODIAG (http: / / www.holodiag.com), located at 27 100 Val de Reuil Bois de Renovation Parmaparque II, France.

[0056] "N-oxide" refers to a compound containing a NO bond in which three additional hydrogens or side chains are bonded to N, or two additional hydrogens or side chains are bonded to N, resulting in a positive charge on the nitrogen. The N-oxide of the present disclosure can be synthesized by oxidation procedures well known to those skilled in the art.

[0057] "Parent aromatic ring system" refers to an unsaturated cyclic or polycyclic ring system having a conjugated pi electron system. Specifically included within the definition of "parent aromatic ring system" are condensed ring systems in which at least one of the rings is aromatic and at least one of the rings is saturated or unsaturated, such as fluorene, indane, indene, phenalene, etc. Typical parent aromatic ring systems include, but are not limited to, acenaphtylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indan, indene, naphthalene, octacene, octaphene, octalene, ovalene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, ficene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and similar ones.

[0058] A "parent heteroaromatic ring system" refers to a parent aromatic ring system in which one or more carbon atoms (and optionally any associated hydrogen atoms) are each independently replaced by the same or different heteroatoms. Typical heteroatoms replacing carbon atoms include, but are not limited to, N, P, O, S, Si, etc. Specifically included within the definition of a "parent heteroaromatic ring system" are condensed ring systems in which at least one of the rings is aromatic and at least one of the rings is saturated or unsaturated, such as, for example, benzodioxane, benzofuran, chromane, chromene, indole, indoline, xanthen, etc. Typical moheoaromatic ring systems include, but are not limited to, asindole, carbazole, β-carbolin, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indoligin, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazol, isoxazole, naphthiridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyrazine, pyrazol, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidin, quinazolin, quinoline, quinoligin, quinoxaline, tetrazole, tydiazol, thiazole, thiophene, triazole, xanthen, and similar ones.

[0059] "Pharmaceutically acceptable salt" refers to a salt of a compound that possesses the desired pharmacological activity of a parent compound. Such salts include: (1) formed from inorganic acids such as hydrochloric acid, hydrobromide, sulfuric acid, nitric acid, phosphoric acid, etc.; Acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvate, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, Acid addition salt formed by organic acids such as lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or (2) a salt formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or a salt formed when coordinately bonded with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucarmine, etc.

[0060] "Preventing" or "prevention" refers to the reduction of the risk of developing a disease or disorder (i.e., preventing at least one of the clinical symptoms of the disease from developing in a patient who may be exposed to or prone to the disease but has not yet experienced or exhibited symptoms of the disease). The application of a therapeutic agent to prevent a disease or disorder is known as "prophylaxis." In some embodiments, the compounds provided herein provide superior prophylaxis due to lower long-term side effects over extended periods.

[0061] "Prodrug" refers to a derivative of a drug molecule that requires modification within the body to release an active drug, as used herein. Prodrugs are frequently pharmacologically inactive until converted into a parent drug, but this is not always the case.

[0062] "Promoiety" refers to a form of a protecting group that converts a drug into a prodrug when used to mask a functional group within a drug molecule, as used herein. Typically, the promoiety will be bound to the drug through a bond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

[0063] A "protecting group" refers to a group of atoms that, when bound to a reactive functional group within a molecule, mask, reduce, or prevent the reactivity of the functional group during chemical synthesis. Examples of protecting groups can be found in Green et al., "Protective Groups in Organic Chemistry," (Wiley, 2nd ed. 1991) and Harrison et al., "Compendium of Synthetic Organic Methods," Vols. 1-8 (John Wiley and Sons, 1971-1996). Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilylethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("FMOC"), nitroveratrilloxycarbonyl ("NVOC"), etc. Representative hydroxy protecting groups include, but are not limited to, benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, triakilcyl ethers and allyl ethers, in which the hydroxyl group is acylated or alkylated.

[0064] "Solvents" refers to the formation of addition products by introducing a solvent into the crystal lattice of the compound described herein in stoichiometric proportions of the chemical formula. Methods for preparing solvates include, but are not limited to, routine pharmaceutical process steps such as storage in an atmosphere containing a solvent, formulations containing a solvent, or, for example, crystallization (i.e., crystallization from a solvent or mixed solvent), vapor diffusion, etc. Solvents may also be formed from other crystalline solvates or hydrates upon exposure to a solvent or suspension of a substance in a solvent under certain circumstances. Solvents may crystallize into two or more forms, resulting in solvate polymorphism. See examples (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing solvates are sufficiently within the scope of those skilled in the art, are entirely ordinary, and require no experimentation other than that typical in the art. Solvents can be characterized and / or analyzed by methods well known to those skilled in the art, such as, for example, single-crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition, many commercial companies, for example, HOLODIAG located at 27-100 Val de l'oeil Bois de Renovation Parc II, France (http: / / www.holodiag.Regularly provides services including the preparation and / or characterization of solvates, such as com).

[0065] "Substituted" means that when used to modify a specific group or radical, one or more hydrogen atoms of that specific group or radical are each, independently, replaced by the same or different substituent(s). Substituent groups useful for substituting saturated carbon atoms in a specific group or radical are R a , halo, -O-, =O, -OR b , -SR b , -S-, =S, -NR c R c , =NR b , =N-OR b , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N-OR b , -N-NR c R c , -NR b S(O)2OR b , =N2, -N3, -S(O)2OR b , -S(O)2NR b OR b , -S(O)2O-, -S(O)2OR b , -OS(O)2R b , -OS(O)2O-, -OS(O)2OR b , -OS(O)2NR c NR c , -P(O)(O-)2, -P(O)(OR b )(O-), -P(O)(OR b )(OR b ), -C(O)R b , -C(O)NR b -OR b , -C(S)OR b , -C(NR b )R b , -C(O)O-, -C(O)OR b , -C(S)OR b , -C(O)NRc R c , -C(NR b )NR c R c , -OC(O)R b , -OC(S)R b , -OC(O)O-, -OC(O)OR b , -OC(O)NR c R c , -OC(NCN)NR c R c , -OC(S)OR b , -NR b C(O)R b , -NR b C(S)R b , -NR b C(O)O-, -NR b C(O)OR b , -NR b C(NCN)OR b , -NR b S(O)2NR c R c , -NR b C(S)OR b , -NR b C(O)NR c R c , -NR b C(S)NR c R c , -NR b C(S)NR b C(O)R a , -NR b S(O)2OR b , -NR b S(O)2R b , -NR b C(NCN)NR c R c , -NR b C(NR b )R b 및 -NR b C(NR b )NR c R c 를 포함하며, 여기서 각 R ais independently a substituted alkyl, substituted alkenyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, heteroaryl, or substituted heteroaryl; and each R b is independently hydrogen, substituted alkyl, substituted alkenyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroalkynyl, substituted heteroalkynyl, arylalkyl, substituted arylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, heteroarylalkyl, substituted heteroarylalkyl, heteroarylalkenyl, substituted heteroarylalkenyl, heteroarylalkynyl, or substituted heteroarylalkynyl; each R c R independently b Or, alternatively, 2 Rs c They form a quaternary, pentylate, sixtylate, or seventylate cycloheteroalkyl, substituted cycloheteroalkyl, cycloheteroalkenyl, substituted cycloheteroalkenyl ring, or a cycloheteroalkyl or cycloheteroalkenyl ring condensed with an aryl group, together with the nitrogen atoms to which they are bonded, and this may optionally include one to four additional identical or different heteroatoms selected from the group consisting of O, N, and S. As a specific example, -NR c R c is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, and N-morphollinyl. In other embodiments, the substituent group useful for substituting a saturated carbon atom in a specific group or radical is R a , Haro, -OR b , -NR c R c , trihalomethyl, -CN, -NR b S(O)2R b , -C(O)R b, -C(O)NR b -OR b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b , -OC(O)OR b , -OS(O)2NR c NR c , -OC(O)NR c R c and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined. In another embodiment, the substituent group useful for substituting a saturated carbon atom in a specific group or radical is R a , Haro, -OR b , -NR c R c , trihalomethyl, -CN, -C(O)R b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b , -OC(O)NR c R c and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined.

[0066] Substituent groups useful for substituting unsaturated carbon atoms in specific groups or radicals are substituted alkyl, -R a , Haro, -O-, -OR b , -SRb, -S-, -NR c R c , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -S(O)2O-, -S(O)2OR b , -OS(O)2R b , -OS(O)2ORb , -OS(O)2O-, -P(O)(O-)2, -P(O)(OR b )(O-), -P(O)(OR b )(OR b ), -C(O)R b , -C(S)R b , -C(NR b )R b , -C(O)O-, -C(O)OR b , -C(S)OR b , -C(O)NR c R c , -C(NR b )NR c R c , -OC(O)R b , -OC(S)R b , -OC(O)O-, -OC(O)OR b , -OC(S)OR b , -OC(O)NR c R c , -OS(O)2NR c NR c , -NR b C(O)R b , -NR b C(S)R b , -NR b C(O)O-, -NR b C(O)OR b , -NR b S(O)2OR a , -NR b S(O)2R a , -NR b C(S)OR b , -NR b C(O)NR c R c , -NR b C(NR b )R b , -NR b C(NR b )NR c R c 및 -C(NR b )NR b C(NR b )NR c R cIncludes, where R a , R b and R c is as previously defined. In other embodiments, the substituent group useful for substituting an unsaturated carbon atom in a specific group or radical is a substituted alkyl, -R a , Haro, -OR b , -SR b , -NR c R c , trihalomethyl, -CN, -S(O)2OR b , -C(O)R b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b , -OC(O)OR b , -OS(O)2NR c NR c , -NR b C(O)R b and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined. In another embodiment, the substituent group useful for substituting an unsaturated carbon atom in a specific group or radical is a substituted alkyl, -R a , Haro, -OR b , -NR c R c , trihalomethyl, -S(O)2OR b , -C(O)R b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b , -NR b C(O)R b and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined.

[0067] Substituents useful for substituting nitrogen atoms in heteroalkyl and cycloheteroalkyl groups are alkyl, -R a , -O-, -OR b , -SR b , -S-, -NR c R c , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R b , -S(O)2O-, -S(O)2OR b , -OS(O)2R b , -OS(O)2O-, -OS(O)2OR b , -P(O)(O-)2, -P(O)(OR b )(O-), -P(O)(OR b )(OR b ), -C(O)R b , -C(S)R b , -C(NR b )R b , -C(O)OR b , -C(S)OR b , -C(O)NR c R c , -C(NR b )NR c R c , -OC(O)R b , -OC(S)R b , -OC(O)OR b , -OC(S)OR b , -NR b C(O)R b , -NR b C(S)R b , -NR b C(O)OR b , -NR b C(S)OR b , -NR b C(O)NR c R c , -NR b C(NR b )R b , -NR b C(NR b )NR c R c and -C(NRb )NR b C(NR b )NR c R c Includes, but is not limited to, where R a , R b and R c is as previously defined. In some embodiments, the substituent group useful for substituting the nitrogen atom in heteroalkyl and cycloheteroalkyl groups is alkyl, R a , Haro, -OR b , -NR c R c , trihalomethyl, -CN, -S(O)2OR b , -OS(O)2R b , -C(O)R b , -C(NR b )R b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b , -OC(O)OR b , -OS(O)2NR c NR c , -NR b C(O)R b and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined. In another embodiment, the substituent group useful for substituting the nitrogen atom in heteroalkyl and cycloheteroalkyl groups is alkyl, R a , Haro, -OR b , -NR c R c , trihalomethyl, -CN, -S(O)2OR b , -C(O)R b , -C(NR b )R b , -C(O)OR b , -C(O)NR c R c , -OC(O)R b, -NR b C(O)R b and -NR b C(O)OR b Includes, where R a , R b and R c is as previously defined.

[0068] Substituent groups from the above list useful for substituting other defined groups or atoms will be obvious to those skilled in the art.

[0069] The substituent used to substitute the defined group may be additionally substituted and typically substituted with one or more identical or different groups selected from the various groups defined above.

[0070] "Subject," "individual," or "patient" are used interchangeably herein and refer to vertebrates, preferably mammals. Mammals include, but are not limited to, rodents, apes, humans, livestock, athletic animals, and companion animals. In some embodiments, the subject, individual, or patient is a member of the species Homo sapiens. In other embodiments, the subject, individual, or patient includes all mammals other than Homo sapiens.

[0071] "Treating" or "treatment" of any disease or disorder, in some embodiments, refers to improving the disease or disorder (i.e., preventing or reducing at least one of the development of the disease or its clinical symptoms). Treatment may also be considered to include preemptive or prophylactic administration to improve, prevent, or prevent at least one of the development of the disease or its clinical symptoms. In additional features, the provided treatment has a lower likelihood of long-term adverse effects over the years. In other embodiments, "treating" or "treatment" refers to improving at least one physical parameter that the patient may not be able to identify. In yet another embodiment, "treating" or "treatment" refers to suppressing the disease or disorder physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

[0072] The "therapeutic effective amount" refers to the amount of a compound sufficient to treat a disease when administered to a patient for the treatment of the disease. The "therapeutic effective amount" will vary depending on the compound, the disease and its severity, as well as the age, body weight, absorption, distribution, metabolism, and excretion of the patient to be treated.

[0073] "Vehicle" refers to a diluent, excipient, or carrier administered together with the compound when it is administered to a subject. In some embodiments, the vehicle is pharmaceutically acceptable.

[0074] compound

[0075] In one aspect, a compound of the following structure, or its salt, hydrate, and solvate, is provided to satisfy these requirements and other requirements:

[0076]

[0077] In the above formula, X is a heteroaryl or a substituted heteroaryl; Y is an alkyldiyl, a substituted alkyldiyl, a cycloalkyldiyl, a substituted cycloalkyldiyl, a heteroalkyldiyl or a substituted heteroalkyldiyl; and Z is an alkyl, a substituted alkyl, a cycloalkyl, a substituted cycloalkyl, a cycloalkenyl, a substituted cycloalkenyl, an aryl, a substituted aryl, a cycloheteroalkyl, a substituted cycloheteroalkyl, a heteroalkyl, a substituted heteroalkyl, -OR 16 , or -NHR 17 -is; T is -C- or -S- and; V is -C(O)-, -C(N)OR 2 -, -C(N)NR 3 R 4 -, -C(OH)R 5 - or -CHR 6 - and; J is an aryl, a substituted aryl, a heteroaryl or a substituted heteroaryl; K is an aryldiyl, a substituted aryldiyl, a heteroaryldiyl or a substituted heteroaryldiyl; and R 1 is a halo, alkyl, alkenyl, alkynyl, or haloalkyl; R 2 to R 6 , R8, R 11 , R 14 , R 16 , and R 17 is independently -H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl; R 7 -OR 8 or -NHR 9 is; R9 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl or -SO2R 10 and; R 10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl or -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 is; R 12 and R 13 is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, or R 12 and R 13 ... forms a cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded; R 15 is an aryl, substituted aryl, heteroaryl or substituted heteroaryl; p is 2-20; n is 0 or 1; and m is 0, 1, 2 or 3.

[0078] In some embodiments, n is 1 and T is -C-. In other embodiments, n is 0 and T is -S-.

[0079] In some embodiments, X is a substituted heteroaryl. In other embodiments, Y is a cycloalkyldiyl or a substituted cycloalkyldiyl.

[0080] In some embodiments, Z is a substituted alkyl or -OR 16 is. In another embodiment, R 7 -OH or NSO2R 10 am.

[0081] In some embodiments, R 10 -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 is. In another embodiment, R 11 is alkyl, and R 12 and R 13 They form a 5- or 6-membered cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded, and R 14 is alkyl, and R 15 is a substituted aryl. In another embodiment, R 11 is an alkyl or substituted alkyl, and R 12 and R 13 They form a 5- or 6-membered cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded, and R 14 is an alkyl or substituted alkyl, and R 15 is an aryl or a substituted aryl.

[0082] In some embodiments, V is -C(O)-.

[0083] In some embodiments, K is a substituted heteroaryldiyl. In other embodiments, J is a heteroaryl. In yet another embodiment, m is 0.

[0084] In some embodiments, n is 1 and T is -C-, X is a substituted heteroaryl, Y is a cycloalkyldiyl, and Z is -OR 16 And, V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, and m is 0. In another embodiment, X is a substituted heteroaryl, Y is a cycloalkyldiyl, and Z is -OR 16 and, V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, m is 0 and; R 7 NSO2R 10 is; R 10 -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 and; R 11 is an alkyl or substituted alkyl, and R 12 and R 13 They form a 5- or 6-membered cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded, and R 14 is an alkyl or substituted alkyl, and R 15 is an aryl or a substituted aryl. In some embodiments, compounds having the following structure are provided:

[0085]

[0086]

[0087]

[0088]

[0089] and

[0090] In some embodiments, n is 0 and T is -S-, X is a substituted heteroaryl, Y is an alkyldiyl, and Z is -OR 16And, V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, and m is 0.

[0091] In some embodiments, a compound having the following structure is provided:

[0092]

[0093] The above compounds can be prepared by well-known procedures, some of which are exemplified in the experimental section.

[0094] Method for Characterizing and Identifying Senolytic Agents

[0095] The characterization of a senolytic agent may be determined using one or more cell-based assays and one or more animal models described herein or described in the art and familiar to those skilled in the art. The senolytic agent may selectively kill one or more types of senescent cells (e.g., senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes). In certain embodiments, the senolytic agent may selectively kill at least senescent fibroblasts.

[0096] Characterizing any substance as a senescent cell-removing substance may be achieved using one or more cell-based assays and one or more animal models as described herein or in the art. Those skilled in the art will readily understand that characterizing a substance as a senescent cell-removing substance and determining the level of cell death caused by said substance may be achieved by comparing the activity of the test substance with an appropriate negative control (e.g., a mediator or diluent alone and / or a composition or compound known in the art as not killing senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing a senescent cell-removing substance also include a control to determine the effect of said substance on non-senescent cells (e.g., quiescent cells or proliferative cells). The senescent cell-removing substance reduces (i.e., degrades) the survival percentage of a plurality of senescent cells compared to one or more negative controls (i.e., reduces the amount of viable senescent cells in animals or in cell-based assays in any way). Conditions for specific in vitro analyses include temperature, buffers (including salts, cations, and media), and other components that maintain the integrity of the test substances and reagents used in the analysis, which are familiar to those skilled in the art and / or can be easily determined through routine experiments.

[0097] Sources of senescent cells for use in analysis may be primary cell cultures or culture-adapted cell lines (including, but not limited to, genetically engineered cell lines that may contain recombinant nucleic acid sequences that are incorporated into chromosomes or are episomes, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiable cell lines, transformed cell lines, etc.). In some embodiments, senescent cells are isolated from biological samples obtained from a host or subject having a senescent cell-related disease or disorder. In other embodiments, non-senescent cells may be obtained from a subject or culture-adapted cell lines, and senescence is induced by methods described herein and in the art, such as irradiation or exposure to a chemotherapy agent (e.g., dozorubicin). Biological samples may be, for example, blood samples, biopsy specimens, body fluids (e.g., lung lavage fluid, ascites fluid, mucosal lavage fluid, synovial fluid, etc.), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation obtained from a subject. Biological samples may be tissue or cell preparations whose morphological integrity or physical state has been destroyed by, for example, incision, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, grinding, freeze-drying, sonication, or any other means for processing a sample derived from a subject or biological source. The subject may be a human or non-human animal.

[0098] Transgenic animal models described herein and in the art can be used to determine the death or removal of senescent cells (see, e.g., Baker et al., Nature, 479 (2011) 232-236; International Application No. WO / 2012 / 177927; International Application No. WO 2013 / 090645). Exemplary transgenic animal models use senescent cells as a positive control (e.g., p16 INK4a It contains a transgenic gene comprising nucleic acids that enable the controlled removal of senescent cells. The presence and level of senescent cells within the transgenic animal can be determined by measuring the levels of detectable markers or markers expressed in the animal's senescent cells. The transgenic gene nucleotide sequence includes, for example, a red fluorescent protein; a green fluorescent protein; and one or more detectable markers such as luciferase to detect the removal of senescent cells.

[0099] Animal models described in this specification or in the art include models recognized in the art to determine the utility of senescent cell removal substances for treating or preventing (i.e., reducing the likelihood of occurrence) specific age-related diseases or disorders, such as atherosclerosis models, osteoarthritis models, COPD models, IPF models, etc. As described in this specification, mouse models of lung diseases, such as bleomycin pulmonary fibrosis models and chronic tobacco smoking models, are applicable to diseases such as COPD and can be routinely practiced by those skilled in the art. Animal models for determining the utility of senescent cell removal substances for treating and / or preventing (i.e., reducing the likelihood of occurrence) chemotherapy and radiation therapy side effects or treating or preventing (i.e., reducing the likelihood of occurrence) metastasis are described in International Applications WO 2013 / 090645 and WO 2014 / 205244. Animal models to determine the utility of substances for treating ocular diseases, particularly age-related macular degeneration, are also routinely used in the field of the art (see, e.g., Pennesi et al.; Mol. Aspects Med. 33 (2012) 487-509; Zeiss et al., Vet. Pathol. 47 (2010) 396-413; Chavala et al., J. Clin. Invest. 123 (2013) 4170-4181).

[0100] Animal models of osteoarthritis have been developed as described herein as a non-limiting example. Osteoarthritis can be induced in animals, for example, by inducing damage to the joint, for example, by surgically cutting the anterior cruciate ligament (ACL) incompletely or completely in the knee. Animal models of osteoarthritis can be used to evaluate the utility of senescent cell removal substances for treating or preventing osteoarthritis (i.e., reducing the likelihood of occurrence), reducing proteoglycan erosion, inducing (i.e., stimulating, enhancing) collagen production (e.g., type 2 collagen), and reducing pain in animals that have undergone ACL surgery. Immunohistology may be performed to investigate the integrity and composition of tissues and cells within the joint. Immunochemical and / or molecular biological techniques, such as analyses to determine levels of inflammatory molecules (e.g., IL-6) and analyses to determine levels of aging indicators as mentioned above, may also be performed using the methods and techniques described herein, which can be routinely practiced by those skilled in the art.

[0101] As another non-limiting example, animal models of atherosclerosis have been developed as described herein. Atherosclerosis can be induced in animals, for example, by feeding them a high-fat diet or by using transgenic animals that are highly susceptible to developing atherosclerosis. Animal models can be used to determine the utility of senescent removal substances to reduce the amount of plaque in atherosclerotic arteries or inhibit plaque formation, to reduce the lipid content of atherosclerotic plaque (i.e., to reduce or lower the amount of lipids within the plaque), and to cause an increase in or reinforce the thickness of the fibrous cap of the plaque. Sudan staining may be used to detect lipid levels within atherosclerotic blood vessels. Immunohistological, immunochemical, and molecular biological analyses (e.g., analyses to determine levels of inflammatory molecules (e.g., IL-6) and levels of aging indicators as mentioned above) may all be performed according to methods described herein and routinely practiced in the art.

[0102] In another non-limiting example, as described herein, mouse models in which animals are administered bleomycin to determine the utility of a substance for treating IPF have been described (e.g., Peng et al., PLoS One 8(4) (2013) e59348. doi: 10.1371 / journal.pone.0059348; see Mouratis et al., Curr. Opin. Pulm. Med. 17 (2011) 355-361). In animal models of lung disease (e.g., bleomycin animal models, smoke exposure animal models, etc.), respiration measurements may be performed to determine elastance, compliance, static compliance, and peripheral arterial oxygen saturation (SpO2). Immunohistological, immunochemical, and molecular biological analyses (e.g., analyses to determine levels of inflammatory molecules (e.g., IL-6) and to determine levels of aging indicators as mentioned above) can all be performed according to methods described herein and routinely practiced in the art.

[0103] As described herein, determining the utility of a senescent cell-removing agent that selectively kills senescent cells in animal models may be performed using one or more statistical analyses familiar to those skilled in the art. For example, statistical analyses such as two-way ANOVA may be used to determine the statistical significance of the difference between a group of animals treated with the agent and a group of animals not treated with the agent (i.e., a negative control group that may include the agent alone and / or a non-senolytic agent). Statistical packages such as SPSS, MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are readily available and routinely used by those skilled in the art of animal models.

[0104] Those skilled in the art will readily understand that characterizing a senescent cell-removing substance and determining the level of death caused by said senescent cell-removing substance can be achieved by comparing the activity of the test substance with an appropriate negative control (e.g., a mediator alone and / or a composition, substance, or compound known in the art as not killing senescent cells) and an appropriate positive control. An in vitro cell-based assay for characterizing said substance also includes a control to determine the effect of said substance on non-senescent cells (e.g., quiescent cells or proliferative cells). A useful senescent cell-removing substance reduces (i.e., degrades) the survival percentage of senescent cells compared to one or more negative controls (i.e., reduces the amount of viable senescent cells in animals or in cell-based assays in any way). Accordingly, the senescent cell-removing substance selectively kills senescent cells compared to the death of non-senescent cells (this may be referred to herein as selectively killing senescent cells compared to non-senescent cells).

[0105] In a specific embodiment (either in vitro analysis or in vivo (in human or non-human animals)), the at least one senescent cell removal substance kills at least 20% of senescent cells and kills 5% or less of non-senescent cells. In another embodiment (either in vitro analysis or in vivo (in human or non-human animals)), the at least one senescent cell removal substance kills at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills about 5% or 10% or less of non-senescent cells. In another embodiment (either in vitro analysis or in vivo (in human or non-human animals)), the at least one senescent cell removal substance kills at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills at least about 5%, 10%, or 15% of non-senescent cells. In another embodiment (either in vitro analysis or in vivo (in human or non-human animals)), the at least one senescent cell removal substance kills at least about 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills at least about 5%, 10%, 15%, 20%, or 25% of non-senescent cells. In another embodiment (either in vitro analysis or in vivo (in human or non-human animals)), the at least one senescent cell removal substance kills at least about 50%, 55%, 60%, or 65% of senescent cells and kills about 5%, 10%, 15%, 20%, 25%, or 30% or less of non-senescent cells. In other words, the senescent cell removal substance has at least 5-25 times, 10-50 times, 10-100 times, or 100-1000 times greater selectivity for killing senescent cells compared to non-senescent cells.

[0106] In relation to specific embodiments of the method for treating age-related diseases or disorders described herein, the percentage of dead senescent cells may refer to the percentage of dead senescent cells within a tissue or organ containing senescent cells that contribute to the onset, progression, and / or worsening of the said disease or disorder. As non-limiting examples, brain tissue, tissues and parts of the eye, lung tissue, heart tissue, arteries, joints, skin, and muscles may contain senescent cells in which the percentage may be reduced as described above by the senescent cell removal material described herein and thereby provide a therapeutic effect. Furthermore, selectively removing at least 20% or at least 25% of senescent cells from the affected tissue or organ may have a clinically significant therapeutic effect.

[0107] In relation to specific embodiments of the method described herein, in treating a cardiovascular disease or disorder associated with arteriosclerosis, e.g., atherosclerosis, by administering a senescent cell-removing substance (i.e., referring to the above in vivo method), the percentage of senescent cells killed may refer to the percentage of senescent cells killed in the affected artery containing the plaque relative to non-senescent cells killed in the arterial plaque. In a specific embodiment, in a method for treating a cardiovascular disease such as atherosclerosis as described herein, the at least one senescent cell-removing substance kills at least 20% of the senescent cells in the artery and kills 5% or less of the non-senescent cells. In another embodiment, the senescent cell-removing substance selectively kills at least 25% of the senescent cells in the arteriosclerotic artery.

[0108] In some embodiments, in relation to the method described herein for treating osteoarthritis by administering a senescent cell-removing substance, the percentage of senescent cells killed may refer to the percentage of senescent cells killed in an osteoarthritis joint relative to non-senescent cells killed in an osteoarthritis joint. In certain embodiments, in the method for treating osteoarthritis as described herein, the at least one senescent cell-removing substance kills at least 20% of the senescent cells in an osteoarthritis joint and kills 5% or less of the non-senescent cells. In other embodiments, the senescent cell-removing substance selectively kills at least 25% of the senescent cells in an osteoarthritis joint.

[0109] In some embodiments, in relation to the method described herein for treating an aging-related lung disease or disorder (e.g., COPD, IPF) by administering at least one senescent cell-removing substance, the percentage of senescent cells killed may refer to the percentage of senescent cells killed in the affected lung tissue relative to non-senescent cells killed in the affected lung tissue. In certain embodiments, in the method for treating an aging-related lung disease and disorder as described herein, the senescent cell-removing substance kills at least 20% of the senescent cells in the affected lung tissue and kills 5% or less of the non-senescent cells. In other embodiments, the senescent cell-removing substance selectively kills at least 25% of the senescent cells in the affected lung tissue.

[0110] In certain embodiments, a method for identifying (i.e., screening) senescent-removing substances useful for treating or preventing (i.e., reducing the likelihood of occurrence) aging-related diseases or disorders is provided. In some embodiments, the method for identifying senescent-removing substances for treating such diseases and disorders comprises inducing senescence in cells to provide established senescent cells. Methods for inducing senescence in cells are described herein and in the art and include, for example, exposure to radiation (e.g., typically 10 Gy is sufficient) or exposure to chemotherapy agents (e.g., dozorubicin or other anthracyclines). After exposure to said substances, cells are cultured for an appropriate amount of time and under appropriate conditions (e.g., medium, temperature, CO2 / O2 levels appropriate for a specific cell type or cell line) so that senescence can be established. As discussed herein, senescence of cells is morphologically altered (e.g., when observed under a microscope); for example, senescence-associated galactosidase (SA-gal), p16 INK4a This can be determined by determining any number of features, such as the production of p21, or any one or more SASP factors (e.g., IL-6, MMP3). Subsequently, a sample of senescent cells is brought into contact with the candidate substance (i.e., mixed, bound, or otherwise allowed to interact with the cells and the substance). Those skilled in the art will understand that the analysis will include appropriate negative and positive controls, whether they are historical controls or concurrently performed controls. For example, a sample of control non-senescent cells, cultured similarly to senescent cells but not exposed to the senescence-inducing substance, is brought into contact with the candidate substance. The viability level of the senescent cells is determined and compared to the viability level of the non-senescent cells. When the viability level of the senescent cells is lower than that of the non-senescent cells, it is identified as a senescent cell removal substance.

[0111] In some embodiments, the method described above for identifying a senescent cell-removing substance may further include steps for identifying whether said senescent cell-removing substance is useful for treating osteoarthritis. The method may further include the step of contacting the identified senescent cell-removing substance with a cell capable of producing collagen; and the step of determining the level of collagen produced by said cell. In some embodiments, said cell is chondrocytes and said collagen is Type 2 collagen. The method may further include the step of determining the therapeutic efficacy of said senescent cell-removing substance by administering a candidate senescent cell-removing substance to a non-human animal having an arthritic lesion in a joint and determining one or more of (a) the level of senescent cells in the joint; (b) the animal's physical function; (c) the level of one or more inflammatory markers; (d) the histology of the joint; and (e) the level of said Type 2 collagen produced, wherein in the animal treated with said senescent cell-removing substance, one or more of the following are observed compared to an animal not treated with said senescent cell-removing substance: (i) a decrease in the level of senescent cells in the joint of the treated animal; (ii) improvement of the body function of the treated animal; (iii) reduction of the level of one or more inflammatory markers in the treated animal; (iv) increase in the histological normality within the joints of the treated animal; and (v) increase in the level of type 2 collagen produced in the treated animal. As described in this specification and in the art, the body function of the animal may be determined by techniques that determine the sensitivity of the leg to an induced or natural osteoarthritis state, for example, by the animal's tolerance to bearing weight on the affected limb or the animal's ability to avoid unpleasant stimuli such as heat or cold.As described herein, determining the usefulness of a substance that kills senescent cells in animal models may be performed using one or more statistical analyses familiar to those skilled in the art. To analyze the data, statistical analyses described herein and routinely performed in the art may be applied.

[0112] In other embodiments, the method described above for identifying a senescent cell-removing substance may further include steps for identifying whether said senescent cell-removing substance is useful for treating cardiovascular diseases induced by or associated with atherosclerosis. Accordingly, the method may further include the step of administering a candidate senescent cell-removing substance to a non-human animal or animal model to determine the usefulness of the substance for reducing the amount of plaque, inhibiting plaque formation in atherosclerotic arteries, reducing the lipid content of atherosclerotic plaque (i.e., reducing or lowering the amount of lipids within the plaque), and / or causing an increase in or reinforcing the thickness of the fibrous cap of the plaque. Sudan staining may be used to detect levels of lipids within atherosclerotic vessels. Immunohistology, analysis to determine levels of inflammatory molecules (e.g., IL-6), and / or analysis to determine levels of aging indicators as mentioned above may all be performed according to methods described herein and routinely practiced in the art.

[0113] In a specific embodiment, the method described herein for identifying a senescent cell-removing substance may further include the step of administering a candidate senescent cell-removing substance to a non-human animal having atherosclerotic plaques and determining the therapeutic efficacy of said senescent cell-removing substance by determining one or more of: (a) the level of senescent cells in the artery; (b) the physical function of the animal; (c) the level of one or more inflammatory markers; and (d) the histology of the affected blood vessel (e.g., artery), wherein in the animal treated with said senescent cell-removing substance, one or more of the following are observed compared to an animal not treated with said senescent cell-removing substance: (i) a decrease in the level of senescent cells in the artery of the treated animal; (ii) an improvement in the physical function of the treated animal; (iii) a decrease in the level of one or more inflammatory markers in the treated animal; and (iv) an increase in the histological normality in the artery of the treated animal. As described herein and in the art, the physical function of the animal may be determined by measuring physical activity. Statistical analysis described herein and routinely performed in the art may be applied to analyze the data.

[0114] In some embodiments, the method described herein for identifying a senescent cell-removing substance may include the step of administering a candidate senescent cell-removing substance to a non-human animal lung disease model, such as a bleomycin model or a smoke-exposed animal model, and determining the therapeutic efficacy of said senescent cell-removing substance by determining one or more of: (a) the level of senescent cells in the lungs; (b) the lung function of the animal; (c) the level of one or more inflammatory markers; and (d) the histology of the lung tissue, wherein one or more of the following are observed in the animal treated with said senescent cell-removing substance compared to an animal not treated with said senescent cell-removing substance: (i) a decrease in the level of senescent cells in the lungs and lung tissue of the treated animal; (ii) an improvement in the lung function of the treated animal; (iii) a decrease in the level of one or more inflammatory markers in the treated animal; and (iv) an increase in histological normality in the lung tissue of the treated animal. Respiratory measurements may be performed to determine elastance, compliance, static compliance, and peripheral arterial oxygen saturation (SpO2). Lung function can be evaluated by determining any one of numerous measurements such as expiratory reserve (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., forced expiratory volume in 1 second, FEV1), FEV1 / FEV ratio, 25% to 75% forced expiratory flow, and maximum spontaneous ventilation (MVV), peak expiratory flow (PEF), and slow vital capacity (SVC). Total lung volume includes total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange across the alveolar capillary membrane can be measured using carbon monoxide diffusing capacity (DLCO). Peripheral arterial oxygen saturation (SpO2) can also be measured. Statistical analysis described herein and routinely performed in the art may be applied to analyze the data.

[0115] Treatment and prevention methods for aging-related diseases and disorders

[0116] The present specification provides a method for treating conditions, diseases, or disorders associated with, linked to, or induced by cellular aging, including age-related diseases and disorders, in subjects requiring such a method. Age-related diseases or disorders may also be referred to herein as aging cell-related diseases or disorders. Age-related diseases and disorders include, for example, cardiovascular diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, lung diseases and disorders, ocular diseases and disorders, metabolic diseases and disorders, neurological diseases and disorders (e.g., neurodegenerative diseases and disorders); age-related diseases and disorders induced by aging; skin conditions; age-related diseases; dermatological diseases and disorders; and transplant-related diseases and disorders. A prominent feature of aging is a progressive loss of function or degeneration occurring at the molecular, cellular, tissue, and organism levels. Age-related degeneration causes well-known pathologies such as sarcopenia, atherosclerosis and heart failure, osteoporosis, pulmonary insufficiency, renal failure, and neurodegeneration (including macular degeneration, Alzheimer's disease, and Parkinson's disease). Although susceptibility to specific age-related diseases varies among different mammalian species, age-related diseases generally begin at the midpoint of species-specific life expectancy (e.g., 50–60 years for humans) and increase with roughly exponential dynamics (see, e.g., Campisi, Annu. Rev. Physiol. 75 (2013) 685–705; Naylor et al., Clin. Pharmacol. Ther. 93 (2013) 105–116).

[0117] Examples of aging-related conditions, disorders, or diseases that can be treated by administering any one of the senescent cell removal substances described herein according to the method described herein include cognitive disorders (e.g., mild cognitive impairment (MCI), Alzheimer's disease and other dementias; Huntington's disease); cardiovascular diseases (e.g., atherosclerosis, diastolic insufficiency, aortic aneurysm, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis); metabolic diseases and disorders (e.g., obesity, diabetes mellitus, metabolic syndrome); motor function disorders and disorders (e.g., Parkinson's disease, motor neuron dysfunction (MND); Huntington's disease); cerebrovascular diseases; emphysema; osteoarthritis; benign prostatic hyperplasia; lung diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiolitis obliterans, asthma); Inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); ophthalmic diseases or disorders (e.g., age-related macular degeneration, cataract, glaucoma, vision loss, presbyopia); diabetic ulcers; metastases; side effects of chemotherapy, side effects of radiation therapy; age-related diseases and disorders (e.g., kyphosis, renal insufficiency, frailty, hair loss, hearing loss, muscle fatigue, skin conditions, sarcopenia, and herniated discs) and other age-related diseases induced by aging (e.g., diseases / disorders caused by radiation exposure, chemotherapy, tobacco smoking, high-fat / high-sugar diet intake, and environmental factors); wound healing; skin nevi; fibrotic diseases and disorders (e.g., cystic fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, oral submucosal fibrosis, cardiac fibrosis, and pancreatic fibrosis). In certain embodiments, any one or more of the diseases or disorders described above or in this specification may be excluded.

[0118] In some embodiments, a method for treating an aging-related disease or disorder is provided by administering a senescent cell removal substance to kill senescent cells (i.e., established senescent cells) associated with the disease or disorder in a subject having the disease or disorder, wherein the disease or disorder is osteoarthritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), or atherosclerosis.

[0119] Cardiovascular disease and disorders

[0120] In another embodiment, the aging-related disease or disorder treated by the method described herein is a cardiovascular disease. The cardiovascular disease may be one or more of angina pectoris, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease (CAD), carotid artery disease, endocarditis, heart attack (coronary artery thrombosis, myocardial infarction [MI]), hypertension, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, diastolic insufficiency, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral artery disease (PAD)), cardiac stress resistance, and stroke.

[0121] In certain embodiments, a method for treating age-related cardiovascular disease associated with or induced by arteriosclerosis (i.e., arteriosclerosis) is provided. The cardiovascular disease may be one or more of atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease); angina pectoris, congestive heart failure, and peripheral vascular disease (e.g., peripheral artery disease (PAD)). The method for treating cardiovascular disease associated with or induced by arteriosclerosis may reduce the likelihood of hypertension, angina pectoris, stroke, and heart attack (i.e., coronary artery thrombosis, myocardial infarction (MI)). In certain embodiments, a method is provided for reducing or delaying the likelihood of thrombotic events, such as stroke or myocardial infarction, by stabilizing atherosclerotic plaque(s) within the blood vessels (e.g., arteries) of a subject. In certain embodiments, this method, which includes the administration of a senescent cell removal substance, reduces (i.e., causes a decrease) the lipid content of atherosclerotic plaques in the blood vessels (e.g., arteries) of the subject and / or increases the thickness of the fibrous cap (i.e. causes an increase, strengthening, or thickening of the fibrous cap).

[0122] Atherosclerosis is characterized by patch-like intima-faults (atheromas) that invade the lumen of medium-sized and large arteries, and these plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect large and medium-sized arteries, including coronary arteries, carotid and cerebral arteries, the aorta and its branches, and the major arteries of the extremities. In some embodiments, a method is provided for inhibiting the formation of atherosclerotic plaques (or causing a reduction, decline, or decrease in the formation of atherosclerotic plaques) by administering a senescent cell removal substance. In other embodiments, a method is provided for reducing (decreasing, declining) the amount (i.e., level) of plaque. A reduction in the amount of plaque within a blood vessel (e.g., artery) may be determined, for example, by a reduction in the plaque surface area, or by a reduction in the extent or degree (e.g., percentage) of occlusion of the blood vessel (e.g., artery), which can be determined by angiography or other visualization methods used in the cardiovascular field. In addition, the present specification provides a method for increasing (or improving, promoting, or enhancing) the stability of atherosclerotic plaques present in one or more blood vessels (e.g., one or more arteries) of a subject, and the method comprises administering any one of the senescent cell removal substances described in the present specification to the subject.

[0123] Subjects suffering from cardiovascular disease may be identified using standard diagnostic methods known in the art for cardiovascular disease. Generally, the diagnosis of atherosclerosis and other cardiovascular diseases is based on symptoms (e.g., chest pain or tightness (angina), numbness or weakness of the arms or legs, difficulty speaking or slurred speech, sagging of facial muscles, leg pain, hypertension, renal failure and / or erectile dysfunction), medical history, and / or physical examination of the patient. The diagnosis may be confirmed by angiography, ultrasonography, or other imaging tests. Subjects at risk of developing cardiovascular disease include subjects who have one or more predisposing factors, such as a family history of cardiovascular disease, and subjects who have other risk factors (i.e., predisposing factors), such as hypertension, dyslipidemia, high cholesterol, diabetes, obesity, tobacco smoking, a sedentary lifestyle, hypertension, etc. In certain embodiments, the cardiovascular disease associated with aging cells is atherosclerosis.

[0124] The efficacy of one or more senescent cell-removing substances in treating or preventing cardiovascular disease (e.g., atherosclerosis) (i.e., reducing or lowering the likelihood of onset or occurrence) can be readily determined by those skilled in the art of medicine and clinical fields. To monitor the health status of a subject, one or any combination of diagnostic methods may be used, including physical examination, evaluation and monitoring of clinical symptoms, and the performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiogram, stress test, non-stress test). The effect of treatment with a senescent cell-removing substance or a pharmaceutical composition containing it may be analyzed using techniques known in the art, for example, by comparing the symptoms of a patient with or at risk of cardiovascular disease who has received treatment with the symptoms of a patient who has not received such treatment or a patient who has received a placebo.

[0125] Inflammatory and autoimmune diseases and disorders

[0126] In certain embodiments, the aging-related disease or disorder is an inflammatory disease or disorder (e.g., osteoarthritis as a non-limiting example) which may be treated or prevented (i.e., the likelihood of occurrence is reduced) according to the method described herein, which includes the administration of a senescent cell removal substance. Other inflammatory or autoimmune diseases or disorders that may be treated by administering a senescent cell removal substance, such as the inhibitors and antagonists described herein, include osteoporosis, psoriasis, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated disc, and lung diseases such as COPD and idiopathic pulmonary fibrosis.

[0127] Osteoarthritis, a degenerative joint disease, is characterized by chondrofibrillation, osteosclerosis, and thickening of the synovium and joint capsule in areas subjected to high mechanical stress. Fibrillation is localized surface disorder involving the cracking of the surface layer of the cartilage. Initial cracking occurs tangentially to the cartilage surface along the axis of the dominant collagen bundle. Collagen within the cartilage becomes disordered, and proteoglycans are lost from the cartilage surface. When the protective and lubricating effects of proteoglycans within the joint are lost, collagen fibers become susceptible to degradation, followed by mechanical destruction. Predisposing risk factors for the development of osteoarthritis include increasing age, obesity, previous joint injury, overuse of the joints, weakness of the thigh muscles, and genetic factors. Symptoms of osteoarthritis include pain or stiffness in the joints (particularly the hips, knees, and lumbar spine) after inactivity or overuse; stiffness that disappears with movement after rest; and pain that worsens after activity or at the end of the day. Osteoarthritis can also affect the neck, small joints of the fingers, the base of the thumb, the ankle, and the big toe. Chronic inflammation is considered a major age-related factor contributing to osteoarthritis. Combined with aging, overuse of the joints and obesity appear to accelerate osteoarthritis.

[0128] A senescent cell removal substance prevents (i.e., reduces the likelihood of) or reduces or inhibits the loss or erosion of the proteoglycan layer within the joint by selectively killing senescent cells, reduces inflammation within the affected joint, and promotes (i.e., stimulates, strengthens, or induces) the production of collagen (e.g., type 2 collagen). The removal of senescent cells reduces the amount (i.e., level) of inflammatory cytokines such as IL-6 produced within the joint and alleviates inflammation. The present specification provides a method for treating osteoarthritis by administering at least one senescent cell removal substance (which may form a pharmaceutical composition by combining with at least one pharmaceutically acceptable excipient) to a subject, and for selectively killing and / or killing senescent cells within the subject's osteoarthritis-related joint, or for inducing the production of collagen (e.g., type 2 collagen) within the subject's joint. The senescent cell-removing substance can also be used to reduce (inhibit, lower) the production of metalloproteinase 13 (MMP-13), which breaks down collagen within the joint, and to repair the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Accordingly, treatment with the senescent cell-removing substance prevents (i.e., reduces the likelihood of occurrence), inhibits, or reduces bone erosion, and slows down (i.e., reduces the rate of) bone erosion. As described in detail herein, in certain embodiments, the senescent cell-removing substance is administered directly to the osteoarthritis-affected joint (e.g., by intra-articular, local, transdermal, intradermal, or subcutaneous delivery). Treatment with the senescent cell-removing substance may also repair or improve joint strength or inhibit the decline of joint strength. Furthermore, the method comprising the administration of the senescent cell-removing substance may reduce joint pain and is therefore useful for pain management in osteoarthritis-affected joints.

[0129] The efficacy of one or more senescent cell removal substances for the treatment or prevention of osteoarthritis in a subject and the monitoring of the subject receiving one or more senescent cell removal substances can be easily determined by a person skilled in the art of medicine and clinical fields. To monitor the health status of the subject, one or any combination of diagnostic methods may be used, including physical examination (determining tenderness, swelling, or redness of the affected joint, etc.), evaluation and monitoring of clinical symptoms (pain, stiffness, mobility, etc.), and the performance of analytical tests and methods described herein and practiced in the art (e.g., determination of levels of inflammatory cytokines or chemokines; X-ray imaging to determine cartilage loss manifested by narrowing of the inter-bone space within the joint; magnetic resonance imaging (MRI) providing detailed images of bone and soft tissues including cartilage). The effect of treatment with one or more senescent cell removal substances can be analyzed by comparing the symptoms of patients who received the treatment with the symptoms of patients who did not receive such treatment or patients who received a placebo, among patients suffering from or at risk of having an inflammatory disease or disorder such as osteoarthritis.

[0130] In certain embodiments, senescent cell removal materials may be used to treat and / or prevent (i.e., reduce or lower the likelihood of occurrence) rheumatoid arthritis (RA). Dysregulation of innate and adaptive immune responses is a characteristic of rheumatoid arthritis (RA), which is an autoimmune disease with an increasing incidence with age. Rheumatoid arthritis is a chronic inflammatory disorder that typically affects the small joints of the hands and feet. While osteoarthritis is caused at least partially by the wear and tear and damage of the joints, rheumatoid arthritis affects the endothelium of the joints, resulting in painful swelling that can cause bone erosion and joint deformation. RA can sometimes affect other organs of the body, such as the skin, eyes, lungs, and blood vessels. RA can occur in subjects of any age, but generally begins to develop after age 40. This disorder is much more common in women. In certain embodiments of the method described herein, RA is excluded.

[0131] Chronic inflammation can also contribute to other age-related or aging-related diseases and disorders, such as kyphosis and osteoporosis. Kyphosis is a severe curvature of the spine and is frequently observed in normal and premature aging (see, e.g., Katzman et al., J. Orthop. Sports Phys. Ther. 40 (2010) 352-360). Age-related kyphosis often develops after osteoporosis has weakened the vertebrae to the point of causing fractures and compression. Some types of kyphosis affect infants, young children, and adolescents. Severe kyphosis can affect the lungs, nerves, and other tissues and organs, causing pain and other problems. Kyphosis has been associated with cellular senescence. The efficacy of senescent-removing substances for the treatment of kyphosis can be evaluated in preclinical animal models used in the relevant technical field. For example, TTD mice exhibit kyphosis (see, e.g., de Boer et al., Science 296 (2002) 1276-1279); another mouse that can be used is the BubR1, which is likewise known to exhibit kyphosis. H / H Mice are included (e.g., see Baker et al., Nature 479 (2011) 232-236). The formation of kyphosis is visually measured over time. The level of senescent cells reduced by treatment with senescent cell removal substances can be determined by detecting the presence of one or more senescent cell-related markers, such as SA-β-Gal staining.

[0132] Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and bone density, which can lead to an increased risk of fracture, and can be treated or prevented by the administration of the senescent cell removal agent described herein. Bone mineral density (BMD) decreases, bone microstructure deteriorates, and the quantity and types of proteins within the bone are altered. Osteoporosis is typically diagnosed and monitored by bone density tests. Postmenopausal women or women with reduced estrogen levels are at the greatest risk. Both men and women over the age of 75 are at risk, but women are twice as likely as men to develop osteoporosis. The level of senescent cells reduced by treatment with the senescent cell removal agent can be determined by detecting the presence of one or more senescent cell-related indicators, such as SA-β-Gal staining.

[0133] In another embodiment, inflammatory / autoimmune disorders that can be treated or prevented (i.e., the likelihood of occurrence reduced) by the senescent cell removal material described herein include irritable bowel syndrome (IBS) and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the digestive tract. In addition to life-threatening complications caused by IBD, the disease can cause pain and debilitation. Ulcerative colitis is an inflammatory bowel disease that causes long-term inflammation in a part of the digestive tract. Symptoms usually develop over time rather than appearing suddenly. Ulcerative colitis generally affects only the innermost lining of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that causes inflammation anywhere along the lining of the digestive tract and often spreads deep into the affected tissue. This can lead to abdominal pain, severe diarrhea, and malnutrition. The inflammation caused by Crohn's disease can affect different areas of the digestive tract. The diagnosis and monitoring of the disease are performed according to methods and diagnostic tests routinely conducted in the relevant technical field, including blood tests, colonoscopy, flexible colonoscopy, barium enema, CT scans, MRI, endoscopy, and imaging of the small intestine.

[0134] Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., reduced likelihood of occurrence) by using senescent cell removal materials include eczema, psoriasis, osteoporosis, and lung diseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis (including, in some cases, radiation-induced oral mucositis). Certain fibrotic or fibrotic conditions of organs, such as renal fibrosis, hepatic fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing, and oral submucosal fibrosis, can be treated with the senescent cell removal materials described herein.

[0135] In certain embodiments, senescent cell-related disorders are inflammatory disorders of the skin, such as psoriasis and eczema, as non-limiting examples, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) according to the method described herein, which includes the administration of a senescent cell-removing substance. Psoriasis is characterized by abnormally excessive and rapid growth of the epidermal layer of the skin. The diagnosis of psoriasis is usually based on the appearance of the skin. Typical skin features of psoriasis are scaly red plaques, papules, or skin patches that can cause pain and itching. In psoriasis, cutaneous and systemic overexpression of various pro-inflammatory cytokines, such as IL-6, a key component of SASP, is observed. Eczema is an inflammation of the skin characterized by redness, skin swelling, itching and dryness, crusting, peeling, blisters, cracking, oozing, or bleeding. The efficacy of senescent cell removal substances for the treatment of psoriasis and eczema, and the monitoring of subjects receiving such senescent cell removal substances, can be easily determined by a person skilled in the art of medicine or clinical fields. Any combination of any one of the diagnostic methods may be used, including physical examination (skin appearance, etc.), evaluation and monitoring of clinical symptoms (itching, swelling, pain, etc.), and the performance of analytical tests and methods described herein and practiced in the art (i.e., determination of levels of pro-inflammatory cytokines).

[0136] Other immune disorders or conditions that can be treated or prevented (i.e., the likelihood of occurrence is reduced) by the senescent cell-removing substances described herein include conditions resulting from the host immune response to organ transplants (e.g., kidney, bone marrow, liver, lung, or heart transplants), such as rejection of the transplanted organ. The senescent cell-removing substances described herein may also be used to treat graft-versus-host disease or reduce the likelihood of its occurrence.

[0137] Lung diseases and disorders

[0138] In some embodiments, a method is provided for treating or preventing (i.e., reducing the likelihood of occurrence) an aging-related disease or disorder by administering a senescent cell removal substance described herein to kill senescent cells (i.e., established senescent cells) associated with the disease or disorder in a subject having the disease or disorder. Aging-related lung diseases and disorders include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

[0139] COPD is a lung disease characterized by a persistent decrease in airflow due to the destruction of lung tissue (emphysema) and the dysfunction of small airways (obstructive bronchiolitis). Major symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excessive sputum production. Elastases derived from neutrophils and macrophages, activated by tobacco smoke, disrupt the extracellular matrix of alveolar structures, expanding air spaces and causing a loss of breathing ability (see, e.g., Shapiro et al., Am. J. Respir. Cell Mol. Biol. 32 (2005) 367-372). COPD is most commonly caused by tobacco smoke (including cigarette smoke, cigar smoke, secondhand smoke, and pipe tobacco smoke), occupational exposure (e.g., exposure to dust, smoke, or fumes), and pollution occurring over decades, which is why aging acts as a risk factor for the development of COPD.

[0140] The processes involved in causing lung damage include, for example, oxidative stress generated by high concentrations of free radicals in cigarette smoke; the release of cytokines resulting from inflammatory responses to irritants in the airways; and damage to anti-proteases caused by cigarette smoke and free radicals, which enables these proteases to damage the lungs. Genetic susceptibility may also contribute to this disease. In about 1% of COPD patients, the disease develops due to a genetic disorder that causes low levels of alpha-1-antitrypsin production in the liver. This enzyme is normally secreted into the bloodstream to help protect the lungs.

[0141] Pulmonary fibrosis is a chronic, progressive lung disease characterized by stiffening and scarring of the lungs, which can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblast activation, increased production of extracellular matrix proteins, and transdifferentiation into contractile myofibroblasts contribute to wound contraction. A provisional matrix fills damaged epithelium and provides a scaffold for epithelial cell migration accompanied by epithelial-mesenchymal transition (EMT). Blood loss associated with epithelial damage induces platelet activation, the production of growth factors, and an acute inflammatory response. Under normal circumstances, the epithelial barrier heals and the inflammatory response resolves. However, in fibrotic diseases, the fibroblast response persists, resulting in unresolved wound healing. The formation of fibroblastic foci is a characteristic of this disease and reflects the locations where persistent fibrosis occurs. As the name implies, the etiology of IPF is unknown. The involvement of cellular senescence in IPF is suggested by the observation that the incidence of the disease increases with age, and by the observation that the lung tissues of IPF patients are rich in SA-β-Gal-positive cells and that levels of the aging marker p21 are elevated (e.g., see Minagawa et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 300 (2011) L391-L401; also, see, e.g., the literature of Minagawa et al.). Short telomeres are a common risk factor for both IPF and cellular senescence (e.g., see Alder et al., Proc. Natl. Acad. Sci. USA 105 (2008) 13051-13056).Without being limited by a specific theory, the contribution of cellular senescence to IPF is suggested by reports that SASP components of senescent cells (e.g., IL-6, IL-8, and IL-1β) promote differentiation from fibroblasts to myofibroblasts and epithelial-mesenchymal transition, leading to extensive remodeling of the extracellular matrix in the alveolar and interstitial spaces (e.g., also see the literature by Naylor et al.).

[0142] Individuals at risk of developing pulmonary fibrosis include people exposed to environmental or occupational contaminants such as asbestosis and silicosis, smokers, people with some typical connective tissue diseases such as rheumatoid arthritis, SLE (systemic lupus erythematosus), and scleroderma, people with other connective tissue diseases such as sarcoidosis and Wegener's granulomatosis, people with infections, people taking certain drugs (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin), people who have received radiation therapy to the chest, and people with a family history of pulmonary fibrosis.

[0143] Symptoms of COPD may include any one of the following: shortness of breath, particularly during physical activity; wheezing; chest tightness; clearing one's throat first thing in the morning due to excessive mucus in the lungs; a chronic cough that produces sputum that may be clear, white, yellow, or green; bluish discoloration of the lips or beds of fingernails (cyanosis); frequent respiratory infections; lack of energy; and unintended weight loss (observed in the later stages of the disease). Patients with COPD may also experience exacerbations, in which symptoms worsen and last for several days or longer. Symptoms of pulmonary fibrosis are known in the art and include, in particular, shortness of breath during exercise; a dry, wheezing cough; rapid, shallow breathing; progressive unintended weight loss; fatigue; joint and muscle pain; and clubbing of the fingers or toes (widening and rounding of the fingertips or toes).

[0144] Subjects suffering from COPD or pulmonary fibrosis may be identified using standard diagnostic methods routinely performed in the relevant technical field. Monitoring the effects of one or more senescent cell removal substances administered to subjects with lung disease or at risk of developing lung disease may be performed using methods typically used for diagnosis. Generally, one or more examinations or tests may be performed, including a physical examination, patient medical history, patient family history, chest X-ray, pulmonary function tests (such as spirometer tests), blood tests (e.g., arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CT scan, and exercise stress test.

[0145] Other lung diseases or disorders that can be treated by using senescent cell removal agents include, for example, emphysema, asthma, bronchiectasis, and cystic fibrosis (see, e.g., Fischer et al., Am J Physiol Lung Cell Mol Physiol. 304(6) (2013) L394-400). These diseases can also be exacerbated by tobacco smoke (including cigarette smoke, cigar smoke, secondhand smoke, and pipe tobacco smoke), occupational exposure (e.g., exposure to dust, smoke, or fumes), infections, and / or contaminants, which induce cellular aging and contribute to inflammation. Emphysema is sometimes considered a subgroup of COPD.

[0146] Bronchiectasis is caused by airway damage that widens, stretches, and scars the airways. Bronchiectasis is usually caused by conditions that damage the airway walls or obstruct the clearing of mucus. Examples of such conditions include cystic fibrosis and primary ciliary dyskinesia (PCD). If only one part of the lung is affected, the disorder may be caused by airway blockage rather than any specific disease.

[0147] The methods described herein for treating or preventing (i.e., reducing the likelihood or occurrence) age-related lung disease or disorder may also be used to treat subjects who are aging and exhibiting loss (or degeneration) of lung function (i.e., reduced or impaired lung function compared to younger subjects) and / or degeneration of lung tissue. The respiratory system undergoes various anatomical, physiological, and immunological changes with age. Structural changes include deformities of the chest wall and thoracic vertebrae that can impair the compliance of the entire respiratory system and increase respiratory effort. The respiratory system undergoes structural, physiological, and immunological changes with age. Compared to young adults, a higher proportion of neutrophils and a lower percentage of macrophages may be found in bronchoalveolar lavage (BAL) of older adults. Persistent low-grade inflammation of the lower respiratory tract can cause proteolytic and oxidant-mediated damage to the lung matrix, leading to the loss of alveolar units and impaired gas exchange across the alveolar membranes observed with aging. Persistent inflammation of the lower respiratory tract can predispose older adults to increased susceptibility to toxic environmental exposures and accelerated decline in lung function (see e.g., Sharma et al., Clinical Interventions in Aging 1 (2006) 253-260). Oxidative stress exacerbates inflammation during the aging process (see e.g., Brod, Inflamm. Res. 49 (2000) 561-570; Hendel et al., Cell Death and Differentiation 17 (2010) 596-606). Changes in redox balance and increased oxidative stress during aging promote the expression of cytokines, chemokines, adhesion molecules, and enzymes (see e.g., Chung et al., Ageing Res. Rev. 8 (2009) 18-30).The constitutive activation and mobilization of macrophages, T cells, and mast cells promote the release of proteolytic enzymes, leading to the degradation of the extracellular matrix, apoptosis, remodeling, and other events that can cause tissue and organ damage during chronic inflammation (see, e.g., Demedts et al., Respir. Res. 7 (2006) 53-63). By administering senescent cell removal substances to aging subjects (including asymptomatic middle-aged adults), senescent cells can be killed and removed from the respiratory tract, thereby slowing down or inhibiting the decline in lung function.

[0148] The efficacy of the senescent cell removal substance can be easily determined by those skilled in the art of medicine and clinical fields. To monitor the health status of the subject, one or any combination of diagnostic methods including physical examination, evaluation and monitoring of clinical symptoms, and the performance of analytical tests and methods described herein may be used. The effect of treatment with the senescent cell removal substance or a pharmaceutical composition containing it may be analyzed using techniques known in the art, for example, by comparing the symptoms of a patient with or at risk of lung disease who has received treatment with the symptoms of a patient who has not received such treatment or a patient who has received a placebo. Additionally, methods and techniques for evaluating the mechanical function of the lungs, such as measuring lung capacity, elastance, and airway hyperresponsiveness, may be performed. To determine lung function and monitor it throughout the treatment period, any one of numerous measurements may be obtained, such as expiratory reserve (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., forced expiratory volume in 1 second, FEV1), FEV1 / FEV ratio, 25% to 75% forced expiratory flow, and maximum spontaneous ventilation (MVV), peak expiratory flow (PEF), and slow vital capacity (SVC). Total lung volume includes total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange across the alveolar capillary membrane can be measured using carbon monoxide diffusing capacity (DLCO). Peripheral arterial oxygen saturation (SpO2) can also be measured, and normal oxygen levels are typically between 95% and 100%. An SpO2 level below 90% suggests hypoxemia in the subject. Values ​​below 80% are considered critical, and intervention is required to maintain brain and heart function and avoid cardiac or respiratory arrest.

[0149] Neurological diseases and disorders

[0150] Age-related diseases or disorders treatable by administering the senescent cell-removing substances described herein include neurological diseases or disorders. These age-related diseases and disorders include Parkinson's disease, Alzheimer's disease (and other forms of dementia), motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, and ocular diseases and disorders such as macular degeneration. Other age-related ocular diseases include glaucoma, vision loss, presbyopia, and cataracts.

[0151] Parkinson's disease (PD) is the second most common neurodegenerative disease. It is a condition that causes brain dysfunction characterized by bradykinesia (slowness of movement), tremors, stiffness, and loss of balance in later stages. Many of these symptoms are caused by the loss of specific neurons within the brain, which results in dopamine deficiency. The disease is characterized by neurodegeneration such as the loss of approximately 50% to 70% of dopaminergic neurons in the substantia nigra pars compacta, severe loss of dopamine in the striatum, and / or the presence of cytoplasmic inclusion bodies (Lewy bodies) composed primarily of alpha-synuclein and ubiquitin. Parkinson's disease is also characterized by motor deficits such as tremors, rigidity, bradykinesia, and / or postural instability. Subjects at risk of developing Parkinson's disease include people with a family history of Parkinson's disease and people exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., Agent Orange), or heavy metals. Aging of dopamine-producing neurons is thought to contribute to the cell death observed in Parkinson's disease through the generation of reactive oxygen species; therefore, the method and senescent cell removal material described herein are useful for the treatment and prevention of Parkinson's disease.

[0152] Methods for detecting, monitoring, or quantifying neurodegenerative and / or motor deficits associated with Parkinson's disease are known in the art, such as histological studies, biochemical studies, and behavioral assessments (see, for example, U.S. Patent Application Publication No. 2012 / 0005765). Symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty initiating or ending voluntary movements, rapid and stiff movements, muscle atrophy, tremor, and changes in heart rate, with normal reflex function and bradykinesia and postural instability. There is growing awareness that patients diagnosed with Parkinson's disease may experience cognitive impairment, including mild cognitive impairment, in addition to physical symptoms.

[0153] Alzheimer's disease (AD) is a neurodegenerative disease characterized by a gradual deterioration of mental function accompanied by memory loss, disorientation, and confusion, leading to severe dementia. Age is the single largest predisposing risk factor for the onset of Alzheimer's disease, and the disease is a major cause of dementia in the elderly (see, e.g., Hebert, et al., Arch. Neurol. 60 (2003) 1119-1122). Early clinical symptoms show a significant resemblance to mild cognitive impairment (see below). As the disease progresses, impaired judgment, confusion, behavioral changes, disorientation, and difficulty walking and swallowing occur.

[0154] Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid plaques in histological specimens. This disease primarily affects the limbic system and cortical regions of the brain. Argyrophilic plaques containing amyloidogenic Aβ fragments of amyloid precursor protein (APP) are scattered throughout the cerebral cortex and hippocampus. Neurofibrillary tangles are found in pyramidal neurons located mainly in the neocortex, hippocampus, and nucleus basalis of Meynert. Other changes are observed, such as granulovacular degeneration of hippocampal pyramidal cells, neuronal loss in the cortex and hippocampus, and gliosis. Individuals at risk of developing Alzheimer's disease include the elderly, people with a family history of Alzheimer's disease, people with genetic risk genes (e.g., ApoE4) or determinant gene mutations (e.g., APP, PS1, or PS2), and people with a history of head trauma or cardiovascular conditions (e.g., hypertension, heart disease, stroke, diabetes, high cholesterol, etc.).

[0155] Numerous behavioral and histopathological assays are known in the art to evaluate Alzheimer's disease phenotypes, characterize therapeutic substances, and evaluate treatments. Histological analysis is typically performed post-mortem. Histological analysis of Aβ levels can be performed by visualizing Aβ deposition on sectioned brain tissue using Thioflavin-S, Congo red, or anti-Aβ staining (e.g., 4G8, 10D5, or 6E10 antibodies) (see, e.g., Holcomb et al., Nat. Med. 4 (1998) 97-100; Borchelt et al., Neuron 19 (1997) 939-945; Dickson et al., Am. J. Path. 132 (1998) 86-101). In vivo methods for visualizing Aβ deposition in transgenic mice are also described. BSB ((trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) and the PET tracer 11C-labeled Pittsburgh compound-B (PIB) bind to amyloid plaques (see, e.g., Skovronsky et al., Proc. Natl. Acad. Sci. USA 97 (2000) 7609-7614; Klunk et al., Ann. Neurol. 55 (2004) 306-319). FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene), an amyloid-affinity Congo Red-type compound containing 19F, enables the visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., Nature Neurosci. 8 (2005) 527-533). Radiolabeled amyloid-beta peptides modified with putrescine label amyloid deposits in vivo in a mouse model of Alzheimer's disease (see, e.g., Wengenack et al., Nat. Biotechnol. 18 (2000) 868-872).

[0156] Increased glial fibrillary acidic protein (GFAP) by astrocytes is a marker of astrocyte activation and gliosis during neurodegeneration. Amyloid plaques are associated with GFAP-positive activated astrocytes and can be visualized through GFAP staining (see, e.g., Nagele et al., Neurobiol. Aging 25 (2004) 663-674; Mandybur et al., Neurology 40 (1990) 635-639; Liang et al., J. Biol. Chem. 285 (2010) 27737-27744). Neurofibrillary tangles can be identified by immunohistochemistry using thioflavin-S fluorescence microscopy and Gallyas silver staining (e.g., Gotz et al., J. Biol. Chem. 276 (2001) 529-534; see U.S. Patent No. 6,664,443). Axonal staining and axonal transport studies using electron microscopy can be used to visualize neuronal degeneration (e.g., see Ishihara et al., Neuron 24 (1999) 751-762).

[0157] Subjects with Alzheimer's disease may be identified using standard diagnostic methods known in the art for Alzheimer's disease. Generally, the diagnosis of Alzheimer's disease is based on symptoms (e.g., progressive decline in memory function, progressive withdrawal from normal activities and frustration with normal activity, apathy, agitation or irritability, aggression, anxiety, sleep disturbances, dysphoria, abnormal motor behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia), medical history, neuropsychological testing, and the patient's neurological and / or physical examination. Cerebrospinal fluid may also be evaluated for various proteins associated with Alzheimer's pathology, including tau, amyloid beta peptide, and AD7C-NTP. Genetic testing for early-onset familial Alzheimer's disease (eFAD), an autosomal dominant genetic disorder, is also available. Clinical genetic testing may be performed on individuals with symptoms of Alzheimer's disease or on family members at risk of patients with early-onset disease. In the United States, mutations for PS2 and APP can be evaluated at clinical or federally approved laboratories under the Clinical Laboratory Improvement Amendments. Commercial testing for PS1 mutations is also available (Elan Pharmaceuticals).

[0158] The efficacy of one or more senescent cell-removing substances described herein and the monitoring of subjects receiving one or more senescent cell-removing substances can be easily determined by those skilled in the art of medicine and clinical fields. To monitor the health status of a subject, one or any combination of diagnostic methods including physical examination, evaluation and monitoring of clinical symptoms, and the performance of analytical tests and methods described herein may be used. The effect of administering one or more senescent cell-removing substances may be analyzed using techniques known in the art, for example, by comparing the symptoms of a patient with or at risk of Alzheimer's disease who has received such treatment with the symptoms of a patient who has not received such treatment or a patient who has received a placebo.

[0159] Mild cognitive impairment (MCI) is a neurological syndrome characterized by the onset and progression of cognitive impairment that falls outside the expected range based on an individual's age and education level, but is not severe enough to interfere with the individual's daily activities. MCI is an aspect of cognitive aging, considered a transitional state between normal aging and dementia, and can progress to dementia (see Pepeu, Dialogues in Clinical Neuroscience 6 (2004) 369-377). MCI that primarily affects memory is known as "amnestic MCI." Individuals with amnestic MCI may begin to forget important information that they previously recalled easily, such as recent events. Amnestic MCI is often considered a prodromal stage of Alzheimer's disease. MCI that affects thinking abilities other than memory is known as "non-amnestic MCI." This type of MCI affects cognitive abilities such as the ability to make correct decisions, judge the timing or sequence of steps required to complete complex tasks, or visual perception. Individuals with non-amnestic MCI are considered to be more likely to progress to other types of dementia (e.g., Lewy body dementia).

[0160] There is a growing awareness in the medical community that patients diagnosed with Parkinson's disease may experience MCI in addition to physical symptoms. Recent studies show that 20 to 30 percent of Parkinson's patients suffer from MCI, and this MCI tends to be non-amnestic. Parkinson's patients with MCI sometimes progress to full-blown dementia (Parkinson's disease with dementia).

[0161] Methods for detecting, monitoring, quantifying, or evaluating neuropathological deficits associated with MCI are known in the art, including astrocyte morphological analysis, acetylcholine release, silver staining for neurodegeneration assessment, and PiB PET imaging for detecting beta-amyloid deposition (e.g., U.S. Patent Application Publication No. 2012 / 0071468; see Pepeu, (2004), cited above). Methods for detecting, monitoring, quantifying, or evaluating behavioral deficits associated with MCI are also known in the art, including the eight-arm radial maze paradigm, the non-matching-to-sample task, the allocentric place determination task in an underwater maze, the Morris maze test, the visuospatial task, the delayed response spatial memory task, and the olfactory novelty test.

[0162] Motor neuron dysfunction (MND) is a group of progressive neurological disorders that destroy motor neurons, the cells responsible for controlling essential muscle activities such as speaking, walking, breathing, and swallowing. It is classified based on whether the degeneration affects upper motor neurons, lower motor neurons, or both. Examples of MND include, but are not limited to, amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease; progressive bulbar palsy; pseudobulbar palsy; primary lateral sclerosis; progressive muscular atrophy; lower motor neuron disease; and spinal muscular atrophy (SMA) (e.g., SMA1, SMA2, also known as Werdnig-Hoffmann disease; SMA3, also known as Kugelberg-Wellander disease; and Kennedy disease), post-polio syndrome, and hereditary spastic paraplegia. The most common MND in adults is amyotrophic lateral sclerosis (ALS), which affects both upper and lower motor neurons. This can affect the muscles of the arms, legs, or face. Primary lateral sclerosis is a disease of upper motor neurons, whereas progressive muscular dystrophy affects only lower motor neurons within the spinal cord. In progressive bulbar palsy, the lowest motor neurons in the brainstem are most severely affected, causing slurred speech and difficulty chewing and swallowing. Slight abnormal signs in the arms and legs are almost always present. Patients with MND exhibit a Parkinson's disease phenotype (e.g., tremor, rigidity, bradykinesia, and / or postural instability). Methods for detecting, monitoring, or quantifying motor and / or other deficits associated with Parkinson's disease, such as MND, are known in the art (see, for example, U.S. Patent Application Publication No. 2012 / 0005765).

[0163] Methods for detecting, monitoring, quantifying, or evaluating motor and histopathological defects associated with MND, including histological, biochemical, and electrophysiological studies and motor activity analysis, are known in the art (see, e.g., Rich et al., J. Neurophysiol. 88 (2002) 3293-3304; Appel et al., Proc. Natl. Acad. Sci. USA 88 (1991) 647-651). Histopathologically, MND is characterized by the death of motor neurons, the progressive accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin within degenerating motor neurons, and the accumulation of abnormal neurofilaments. Additionally, reactive astroglia and microglia are often detected in affected tissues. Patients with MND exhibit one or more motor deficits, including muscle weakness and atrophy, uncontrollable spasms, spasticity, slow and labored movements, and overactive tendon reflexes.

[0164] Ophthalmic diseases and disorders

[0165] In certain embodiments, the age-related disease or disorder is an ophthalmic disease, disorder, or condition, such as presbyopia, macular degeneration, or cataract. In other specific embodiments, the age-related disease or disorder is glaucoma. Macular degeneration is a neurodegenerative disease that causes the loss of photoreceptor cells in the central part of the retina called the macula. Macular degeneration is generally classified into two types: dry and wet. The dry form is more common than the wet form, and about 90% of patients with age-related macular degeneration (ARMD or AMD) are diagnosed with the dry form. The wet form of this disease generally leads to more severe vision loss. Although the exact cause of age-related macular degeneration is not yet known, the number of aged retinal pigment epithelial (RPE) cells increases with age. Age and certain genetic and environmental factors are risk factors for the development of ARMD (see e.g., Lyengar et al., Am. J. Hum. Genet. 74 (2004) 20-39; Kenealy et al., Mol. Vis. 10 (2004) 57-61; Gorin et al., Mol. Vis. 5 (1999) 29). Environmental predisposing factors include omega-3 fatty acid intake (see e.g., Christen et al., Arch. Ophthalmol. 129 (2011) 921-929); estrogen exposure (see e.g., Feshanich et al., Arch. Ophthalmol. 126(4) (2008) 519-524); and increased serum levels of vitamin D (see e.g., Millen et al., Arch. Ophthalmol. 129(4) (2011) 481-89). Genetic predispositional risk factors include reduced levels of Dicer1 (an enzyme involved in the maturation of microRNA) in the eyes of patients with dry AMD, and the reduction in microRNA contributes to the aging cell profile.

[0166] Dry ARMD is associated with atrophy of the RPE layer, which leads to the loss of photoreceptor cells. The dry form of ARMD can result from the aging and thinning of macular tissue and pigmentation within the macula. Cellular senescence appears to inhibit both the replication and migration of RPE, resulting in permanent RPE depletion within the macula of patients with dry AMD (see, e.g., Iriyama et al., J. Biol. Chem. 283 (2008) 11947-11953). In the case of wet ARMD, new blood vessels grow beneath the retina, leaking blood and fluid. This abnormal and leaky choroidal neovascularization causes retinal cell death, leading to blind spots in central vision. Various forms of macular degeneration can occur in younger patients as well. Non-age-related etiologies may be associated with genetics, diabetes, nutritional deficiencies, head trauma, infection, or other factors.

[0167] Vision loss detected by the patient or by an ophthalmologist during a routine eye examination may be the first indicator of macular degeneration. The formation of exudates, or "drusen," beneath Bruch's membrane of the macula is often the first physical sign indicating that macular degeneration may develop. Symptoms include distorted vision of straight lines; in some cases, the center of the visual field appears more distorted than the rest of the scene; dark or blurry areas or "white-out" phenomena appear in the center of the visual field; and / or changes or reductions in color perception. Diagnosis and monitoring of a patient with macular degeneration may be performed by a person skilled in the art of ophthalmology based on routine eye examination procedures recognized in the relevant technical field and the patient's symptom reports.

[0168] Presbyopia is an age-related condition in which the eye's ability to focus on near objects progressively declines as the normal speed and amplitude of accommodation decrease with aging. Loss of elasticity of the lens and loss of contractility of the ciliary muscle have been hypothesized as causes (e.g., Heys et al., Mol. Vis. 10 (2004) 956-963; Petrash, Invest. Ophthalmol. Vis. Sci. 54 (2013) ORSF54-ORSF59). Age-related changes in the mechanical properties of the anterior and posterior lenticular capsules suggest that the mechanical strength of the posterior lenticular capsule significantly decreases with age (see, e.g., Krag et al., Invest. Ophthalmol. Vis. Sci. 44 (2003) 691-696; Krag et al., Invest. Ophthalmol. Vis. Sci. 38 (1997) 357-363).

[0169] The layered structure of the lens capsule also changes, which may be at least partially attributed to changes in tissue composition (e.g., Krag et al., 1997, see ibid. and cited references). The major structural component of the lens capsule is basement membrane type IV collagen organized into a three-dimensional molecular network (e.g., Cummings et al., Connect. Tissue Res. 55 (2014) 8-12; Veis et al., Coll. Relat. Res. 1 (1981) 269-286). Type IV collagen consists of six homologous α chains (α 1-6), which each combine into a heterotrimeric collagen IV protomer containing a specific chain combination of α112, α345, or α556 (see, e.g., Khoshnoodi et al., Microsc. Res. Tech. 71 (2008) 357-370). The protomers share structural similarities with a triple helical collagen domain having a Gly-XY tripeptide sequence (Timpl et al., Eur. J. Biochem. 95 (1979) 255-263) and end in a spherical C-terminal region called the non-collagenous 1 (NC1) domain. The N-terminus consists of a helical domain called the 7S domain (see, e.g., Risteli et al., Eur. J. Biochem. 108 (1980) 239-250), which is also involved in interactions between protomers.

[0170] Studies suggest that type IV collagen influences cellular function, which is inferred from the fact that the basement membrane is located beneath the epithelial layer, and various data support the role of type IV collagen in tissue stabilization (e.g., Cummings et al., cit.). Posterior capsule opacification (PCO) occurs as a complication in approximately 20–40% of patients within a few years following cataract surgery (e.g., Awasthi et al., Arch. Ophthalmol. 127 (2009) 555–562). PCO arises from the proliferation and activation of residual lens epithelial cells along the posterior lens capsule in a response similar to wound healing. Growth factors such as fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, insulin-like growth factor, and interleukins IL-1 and IL-6 can also promote the migration of epithelial cells (e.g., Awasthi et al., cit.; Raj et al., cit.). As discussed herein, the production of these factors and cytokines by senescent cells contributes to SASP. In contrast, in vitro studies have shown that type IV collagen promotes the adhesion of lens epithelial cells (see, e.g., Olivero et al., Invest. Ophthalmol. Vis. Sci. 34 (1993) 2825-2834). The adhesion of type IV collagen, fibronectin, and laminin to intraocular lenses inhibits cell migration and may reduce the risk of secondary cataracts (see, e.g., Raj et al., Int. J. Biomed. Sci. 3 (2007) 237-250).

[0171] Without being limited by any specific theory, the selective killing of senescent cells by the senescent cell-removing substances described herein may slow down or disrupt (delay, inhibit, or prevent) the disordering of type IV collagen networks. Removing senescent cells and thereby eliminating the inflammatory effects of SASP may reduce or inhibit epithelial cell migration, delay (inhibit) the onset of presbyopia, or reduce or mitigate the progressive deterioration of the condition (e.g., mitigating progression from mild to moderate, or from moderate to severe). The senescent cell-removing substances described herein may also be useful after cataract surgery to reduce the likelihood of developing post-cataract (PCO).

[0172] Although direct evidence that cellular senescence is involved in the development of cataracts has not been obtained from human studies, BubR1 hypomorphic mice develop bilateral posterior subcapsular cataracts at a young age, suggesting that cellular senescence may play a role (see, e.g., Baker et al., Nat. Cell Biol. 10 (2008) 825-836). Cataracts cause blurred vision due to clouding of the lens of the eye and can lead to blindness if left untreated. Surgery to remove cataracts is effective and routinely performed. Administering one or more senescent-removing substances described herein may reduce the likelihood of developing cataracts or slow or inhibit their progression. The presence and severity of cataracts can be monitored through ophthalmic examinations using methods routinely performed by those skilled in the art of ophthalmology.

[0173] In certain embodiments, at least one senescent cell removal substance described herein may be administered to a subject at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the senescent cell removal substance may be initiated when the human subject is at least 40 years of age to delay or inhibit the onset or progression of cataracts, presbyopia, and macular degeneration. Since presbyopia occurs in almost all humans, in certain embodiments, the senescent cell removal substance may be administered to the human subject in the manner described herein after the subject has reached the age of 40 to delay or inhibit the onset or progression of presbyopia.

[0174] In certain embodiments, the age-related disease or disorder is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause visual field defects without other dominant symptoms. The absence of symptoms often results in a delayed diagnosis of glaucoma until the disease reaches a terminal stage. Even if subjects with glaucoma do not go blind, their vision is frequently severely impaired. Normally, clear fluid flows into and out of the front part of the eyeball known as the anterior chamber. In individuals with open-angle glaucoma, this fluid drains too slowly, leading to an increase in intraocular pressure (IOP). If left untreated, this high pressure can consequently damage the optic nerve and lead to complete blindness. The loss of peripheral vision is caused by the death of ganglion cells within the retina. Ganglion cells are a type of projection neuron that connects the eyeball to the brain. When SA-β-Gal staining was performed on the cellular network required for fluid outflow, a fourfold increase in aging was observed in glaucoma patients (see, e.g., Liton et al., Exp. Gerontol. 40 (2005) 745-748).

[0175] To monitor the therapeutic effect of inhibiting the progression of glaucoma, the standard automated visual field test is the most widely used technique. In addition, several algorithms for progression detection have been developed (e.g., Wesselink et al., Arch. Ophthalmol. 127(3) (2009) 270-274 and the references cited therein). Additional methods include gonioscopy (examining the angle of drainage of the trabecular meshwork and fluid out of the eye); imaging techniques, e.g., scanning laser ophthalmoscopy (e.g., HRT3), laser polarimetry (e.g., GDX), and optical coherence tomography (OCT); ophthalmoscopy; and a pachymeter to determine the central corneal thickness.

[0176] metabolic diseases or disorders

[0177] Aging-related diseases or disorders treatable by administering the senescent cell removal substance described in this specification include metabolic diseases or disorders. These senescent cell-related diseases and disorders include diabetes, metabolic syndrome, diabetic ulcers, and obesity.

[0178] Diabetes is characterized by high blood sugar levels caused by defects in insulin production, insulin action, or both. The majority (90% to 95%) of all cases of diabetes diagnosed in adults are Type 2 diabetes, characterized by the progressive loss of the pancreas's ability to produce insulin. Diabetes is a leading cause of renal failure, non-traumatic lower limb amputation, and new-onset blindness among U.S. adults. Diabetes is a leading cause of heart disease and stroke and is the seventh leading cause of death in the United States (see, for example, U.S. Centers for Disease Control and Prevention, “National Diabetes Fact Sheet: National Estimates and General Information on Diabetes and Prediabetes in the United States, 2011”). The senescent cell removal material described herein may be used to treat Type 2 diabetes, particularly Type 2 diabetes associated with age, diet, and obesity.

[0179] The involvement of senescent cells in metabolic diseases such as obesity and type 2 diabetes has been suggested as a response to injury or metabolic dysfunction (e.g., see Tchkonia et al., Aging Cell 9 (2010) 667-684). Adipose tissue from obese mice exhibited the induction of aging markers SA-β-Gal, p53, and p21 (e.g., see Tchkonia et al., cit.; see Minamino et al., Nat. Med. 15 (2009) 1082-1087). Concurrent upregulation of pro-inflammatory cytokines such as tumor necrosis factor (TNF) and Ccl2 / MCP1 was observed in the same adipose tissue (e.g., see Minamino et al., cit.). The induction of senescent cells in obesity may have clinical significance, as pro-inflammatory SASP components are also suggested to contribute to type 2 diabetes (e.g., see Tchkonia et al., cit.). Similar upregulation patterns of aging markers and SASP components are associated with diabetes in both mice and humans (e.g., Minamino et al., see cited literature). Therefore, the method described herein, which involves the administration of a senescent cell removal substance, may be useful for the treatment or prevention of type 2 diabetes as well as obesity and metabolic syndrome. Without being limited by any particular theory, inducing the death of aged pre-adipocytes by contacting them with a senescent cell removal substance may provide clinical and health benefits to individuals suffering from any one of diabetes, obesity, or metabolic syndrome.

[0180] Subjects with type 2 diabetes may be identified using standard diagnostic methods known in the art for type 2 diabetes. Generally, the diagnosis of type 2 diabetes is based on symptoms (e.g., increased thirst and frequent urination, increased hunger, weight loss, fatigue, blurred vision, wounds that do not heal well or frequent infections, and / or darkened skin), medical history, and / or physical examination of the patient. Subjects at risk of developing type 2 diabetes include individuals with a family history of type 2 diabetes and those with other risk factors such as overweight, fat distribution, lack of activity, race, age, prediabetes, and / or gestational diabetes.

[0181] The efficacy of the senescent cell removal substance can be readily determined by those skilled in the art of medicine and clinical fields. To monitor the health status of a subject, one or any combination of diagnostic methods may be used, including physical examination, evaluation and monitoring of clinical symptoms, and the performance of analytical tests and methods as described herein. A subject receiving one or more of the senescent cell removal substances described herein for the treatment or prevention of diabetes may be monitored, for example, by analyzing glucose and insulin resistance, energy expenditure, body composition, adipose tissue, skeletal muscle and liver inflammation, and / or lipotoxicity (muscle and liver lipids by in vivo imaging, and lipid accumulation and inflammation of muscle, liver, bone marrow and pancreatic β-cells by histology). Other characteristic features or phenotypes of type 2 diabetes are known and may be analyzed using the manner described herein and other methods and techniques known in the art and routinely practiced.

[0182] Obesity and obesity-related disorders are used to refer to a condition in which a subject's body mass is measurably greater than the ideal level for their height and physique. Body Mass Index (BMI) is a measurement tool used to determine excess weight and is calculated from a subject's height and weight. Humans are considered overweight when their BMI is between 25 and 29, obese when their BMI is between 30 and 39, and morbidly obese when their BMI exceeds 40. Accordingly, the terms obesity and obesity-related refer to human subjects with a BMI value exceeding 30, 35, or 40. The category of obesity not captured by BMI is referred to as "abdominal obesity" in the relevant technical field; this is associated with excessive fat found around the subject's abdomen and is a significant health factor regardless of BMI. The simplest and most frequently used method to measure abdominal obesity is waist circumference. Generally, abdominal obesity is defined as a waist circumference of 35 inches or more for women and 40 inches or more for men. More complex methods for determining obesity require specialized equipment such as magnetic resonance imaging (MRI) or dual-energy X-ray absorptiometry (DEXA).

[0183] A condition or disorder associated with diabetes and cellular senescence is diabetic ulcers (i.e., diabetic wounds). Ulcers refer to the destruction of the skin, which can extend to subcutaneous tissue or even muscle or bone. These lesions occur particularly in the lower extremities. Patients with diabetic venous ulcers show a high presence of cellular senescence in chronic wound sites (e.g., see Stanley et al., J. Vas. Surg. 33 (2001) 1206-1211). Chronic inflammation is also observed in chronic wound sites such as diabetic ulcers (e.g., see Goren et al., Am. J. Pathol. 168 (2006) 65-77), suggesting that the pro-inflammatory cytokine phenotype of senescent cells plays a role in the pathology.

[0184] Subjects who have type 2 diabetes or are at risk of developing type 2 diabetes may exhibit metabolic syndrome. In humans, metabolic syndrome is typically associated with obesity and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes, and insulin resistance. Subjects exhibiting metabolic syndrome may display complex metabolic disorders or abnormalities, which may include, for example, hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis, and one or more of other metabolic disorders.

[0185] kidney failure

[0186] Renal pathologies such as glomerular disease occur in the elderly and can be treated by the administration of senescent-removing compounds (senolytic compounds) described herein. Glomerulonephritis is characterized by inflammation of the kidney and the expression of two proteins, IL-1α and IL-1β (see, e.g., Niemir et al., Kidney Int. 52 (1997) 393-403). IL-1α and IL-1β are considered major regulators of SASP (see, e.g., Coppe et al., PLoS. Biol. 6 (2008) 2853-2868). Glomerular disease is associated with an elevated presence of senescent cells, particularly in fibrotic kidneys (see, e.g., Sis et al., Kidney Int. 71 (2007) 218-226).

[0187] dermatological diseases or disorders

[0188] Diseases or disorders treatable by administering the compounds described herein include dermatological diseases or disorders. These diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and have been discussed in more detail above. Other dermatological diseases and disorders include wrinkles (rhytides, wrinkles due to aging); pruritis (associated with diabetes and aging); dysesthesia (a side effect of chemotherapy associated with diabetes and multiple sclerosis); psoriasis (as described above) and other papulosquamous disorders, e.g., exfoliative dermatitis (erythrodermia), lichen planus, and lichenoid dermatitis; atopic dermatitis (a type of eczema associated with inflammation); and eczematous rash (frequently observed in elderly patients and associated with side effects of certain drugs). Other dermatological diseases and disorders associated with cellular aging include eosinophilic dermatitis (associated with certain types of blood cancer); reactive neutrophilic dermatitis (associated with underlying conditions such as irritable bowel syndrome); Pemphigus (an autoimmune disease in which autoantibodies against desmoglein are formed); pemphigus-like and other immune blistering dermatitis (autoimmune blistering of the skin); fibrohistocytotic proliferative lesions of the skin associated with aging; and cutaneous lymphoma, which is more common in the elderly population. Another dermatological condition that may be treatable according to the method described herein includes cutaneous lupus, which is a symptom of lupus erythema (systemic lupus erythematosus). Late-onset lupus may be associated with reduced function (i.e., decline) of T cells and B cells and reduced cytokines (immune senescence). Other dermatological indications that may be treated using the compounds described herein include, but are not limited to, plaque psoriasis, dermatitis and alopecia induced by cancer treatment (chemotherapy or radiation), vitiligo, alopecia areata, hidradenitis suppurativa, chronic spontaneous urticaria, actinic keratosis, and seborrheic keratosis.

[0189] Metastasis

[0190] In some embodiments, a method is provided for treating or preventing (i.e., reducing the likelihood of occurrence or progression) metastasis that is a disease (or disorder or condition) associated with senescent cells. The senescent cell removal material described herein may also be used according to the method described herein to treat or prevent (i.e., reduce the likelihood of occurrence) metastasis (i.e., the spread and dissemination of cancer or tumor cells) from one organ or tissue to another organ or tissue within the body.

[0191] Diseases or disorders related to senescent cells include metastasis, and subjects with cancer may benefit from administering a senescent cell-removing substance as described herein to inhibit metastasis. Such a senescent cell-removing substance may inhibit tumor proliferation when administered to subjects with cancer according to the method described herein. Cancer metastasis occurs when cancer cells (i.e., tumor cells) spread from the anatomical site of their initial origin and initial settlement to other areas throughout the subject's body. Tumor proliferation can be determined by tumor size, which can be measured in various ways familiar to those skilled in the art, such as PET scans, MRI, CAT scans, and biopsies. The effect of a therapeutic substance on tumor proliferation may also be evaluated by examining the differentiation of tumor cells.

[0192] As used in this specification and in the art, the terms cancer or tumor are clinical descriptors encompassing diseases characterized by cells exhibiting typically abnormal cell proliferation. The term cancer is generally used to describe a malignant tumor or a disease state resulting from such tumor. Alternatively, abnormal growth may be referred to as a neoplasm in the art. The term tumor, as used in relation to tissue, generally refers to any abnormal tissue growth characterized at least partially by excessive and abnormal cell proliferation. A tumor may be metastatic and may spread beyond the anatomical site of its initial origin and settlement to other areas throughout the subject's body. Cancer may include solid tumors or "liquid" tumors (e.g., leukemia and other blood cancers).

[0193] Cells are senescent induced by cancer treatments such as radiation and certain chemotherapy drugs. The presence of senescent cells increases the secretion of inflammatory molecules and promotes tumor progression, which may include the promotion of tumor growth and size increase, the promotion of metastasis, and differentiation changes. When senescent cells are destroyed, tumor progression is significantly inhibited, resulting in tumors that are small in size and show little to no metastatic growth (see, e.g., International Publication WO 2013 / 090645).

[0194] In some embodiments, a method is provided for preventing (i.e., reducing the likelihood of occurrence), suppressing, or inhibiting metastasis of a cancerous subject by administering a senescent cell-removing substance as described herein. In other embodiments, the senescent cell-removing substance is administered for at least one day within a treatment window (i.e., a treatment process) of 7 or 14 days or less. In yet another embodiment, the treatment process is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or less, or 21 days or less. In yet another embodiment, the treatment process is a single day. In yet another embodiment, the senescent cell-removing substance is administered for at least two days within a treatment window of 7 or 14 days or less.

[0195] Since senescence can be induced in cells by cancer treatments such as radiation and certain chemotherapy drugs (e.g., doxorubicin; paclitaxel; gemcitabine; pomalidomide; lenalidomide), the senescence-removing agent described herein may be administered after chemotherapy or radiation therapy to kill (or promote) these senescent cells. As discussed herein and understood in the art, the establishment of senescence, such as that manifested by the presence of an senescence-associated secretory phenotype (SASP), occurs over several days; therefore, the administration of the senescence-removing agent to kill senescent cells and thereby reduce the likelihood of metastasis or the extent of metastasis is initiated when senescence is established. As discussed herein, in the method described herein for treating or preventing (i.e., reducing the likelihood of occurrence or mitigating the severity of) side effects of chemotherapy or radiation therapy, the following treatment steps may be used for the administration of the senescence-removing agent.

[0196] In a specific embodiment, when chemotherapy or radiation therapy is administered in a treatment cycle of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days (or about 2 weeks), 15, 16, 17, 18, 19, 20, or 21 days (or about 3 weeks) or about 4 weeks (about 1 month) of off-therapy (i.e., a break from chemotherapy or radiation therapy), the senescent cell removal substance is administered for at least one day during the off-therapy period, starting on or after the second day of the off-therapy time interval (period) and ending on or before the last day of the off-therapy time interval. As an exemplary example, where n is the number of off-therapy days, the senescent cell-removing material is administered for at least one day and no more than n-1 days during the off-therapy time interval. In some embodiments in which chemotherapy or radiation therapy is administered in a treatment cycle of at least one week of off-therapy following at least one day of on-therapy, the senescent cell-removing material is administered for at least one day during the off-therapy period, starting on or after the second day of the off-therapy time interval and ending on or before the last day of the off-therapy time interval.

[0197] Chemotherapy may be referred to as chemotherapy, chemotherapeutic, or chemotherapeutic drug. Many chemotherapeutic agents are compounds called small organic molecules. Chemotherapy is also a term used to describe a combination of chemotherapy drugs administered to treat a specific cancer. As understood by those skilled in the art, chemotherapy may also refer to a combination of two or more chemotherapy molecules administered in combination, which may be referred to as combination chemotherapy. Numerous chemotherapeutic drugs are used in the field of oncology, including, without limitation, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, and topoisomerase inhibitors.

[0198] Cancers that may metastasize may be solid tumors or liquid tumors (e.g., blood cancers such as leukemia). Cancers that are liquid tumors are classified in the art as cancers occurring in the blood, bone marrow, and lymph nodes, and generally include leukemias (myeloid and lymphoid), lymphomas (e.g., Hodgkin lymphoma), and myelomas (including multiple myeloma). Leukemias include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia (CML), and hair cell leukemia. Solid tumors that occur with higher frequency in humans include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colorectal cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer (including squamous cell carcinoma), kidney cancer, head and neck cancer, throat cancer, squamous cell carcinoma formed on the moist mucosal lining of the nose, mouth, throat, etc.), bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain specific embodiments, the senescent cell-related disease or disorder treated or prevented (i.e., the likelihood of occurrence or progression reduced) by the method described herein is a metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colorectal cancer cells, thyroid cancer cells, gastric cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, renal cancer cells, head and neck cancer cells, throat cancer cells, squamous cell carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

[0199] The method described in this specification is also useful for inhibiting, stopping, or mitigating the progression of metastatic cancer of any type of tumor described in the medical field. Types of cancer (tumors) include the following: adrenocortical carcinoma, pediatric adrenocortical carcinoma, AIDS-associated cancer, anal cancer, appendiceal cancer, basal cell carcinoma, pediatric basal cell carcinoma, bladder cancer, pediatric bladder cancer, bone cancer, brain tumor, pediatric astrocytoma, pediatric brainstem glioma, pediatric central nervous system atypical / teratomyoplastic tumor, pediatric central nervous system embryonic tumor, pediatric central nervous system germ cell tumor, pediatric pharyngeal duct tumor, pediatric ependymoma (ventricular ependymoma) brain tumor, breast cancer, pediatric bronchial tumor, carcinoid tumor, pediatric carcinoid tumor, gastrointestinal carcinoid tumor, primary carcinoma of unknown etiology, pediatric primary carcinoma of unknown etiology, pediatric cardiac tumor, cervical cancer, pediatric cervical cancer, pediatric chordoma, chronic myeloproliferative disorder, colorectal cancer, rectal cancer, pediatric rectal cancer, extrahepatic biliary cancer, intraepithelial ductal carcinoma (DCIS). Endometrial cancer, esophageal cancer, pediatric esophageal cancer, pediatric olfactory neuroblastoma, ocular cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, pediatric gastric cancer, gastrointestinal stromal tumor (GIST), pediatric gastrointestinal stromal tumor (GIST), pediatric extracranial germ cell tumor, extragonadal germ cell tumor, gestational choriocarcinoma, glioma, head and neck cancer, pediatric head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, kidney cancer, renal cell renal carcinoma, Wilms tumor, pediatric renal tumor, Langerhans cell histiocytosis, laryngeal cancer, pediatric laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia (CML), hair cell leukemia, lip cancer, liver cancer (primary), pediatric liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS Related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma (CNS), melanoma, pediatric melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, malignant mesothelioma, pediatric malignant mesothelioma,Metastatic squamous cervical cancer with potential primary site, NUT-controlled midline urinary tract carcinoma, oral cancer, pediatric multiple endocrine neoplasm syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, myeloproliferative neoplasm, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, pediatric nasopharyngeal cancer, neuroblastoma, oral cancer, pediatric oral cancer, oropharyngeal cancer, ovarian cancer, pediatric ovarian cancer, epithelial ovarian cancer, tumor with low malignancy potential ovarian cancer, pancreatic cancer, pediatric pancreatic cancer, pancreatic neuroendocrine tumor (silicate cell tumor), pediatric papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pediatric pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis transitional cell carcinoma, retinoblastoma, salivary gland cancer, pediatric salivary gland cancer, Ewing Sarcomatous tumors, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, pediatric rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, pediatric skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, pediatric squamous cell carcinoma, testicular cancer, pediatric testicular cancer, throat cancer, thymoma and thymic cancer, pediatric thymoma and thymic cancer, thyroid cancer, pediatric thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, endometrial uterine cancer, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

[0200] Side effects of chemotherapy and radiation therapy

[0201] In other embodiments, senescent cell-related disorders or conditions are side effects of chemotherapy or radiation therapy. Examples of chemotherapy agents that induce senescence of non-cancerous cells include anthracyclines (doxorubicin, daunorubicin, etc.); taxols (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more senescent cell-removing substances administered as described herein may be used to treat and / or prevent (i.e., reduce the likelihood or occurrence of) side effects of chemotherapy or radiation therapy. The removal or destruction of senescent cells may improve acute toxicity of chemotherapy or radiation therapy (including acute toxicity including energy imbalance). Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, loss of appetite, diarrhea), peripheral neuropathy, fatigue, malaise, low physical activity, hematological toxicity (e.g., anemia), hepatotoxicity, hair loss (alopecia), pain, infection, mucositis, fluid retention, dermatological toxicity (e.g., rash, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), oral (e.g., stomatitis), gum or throat problems, or any toxic side effects induced by chemotherapy or radiation therapy. For example, toxic side effects induced by radiation therapy or chemotherapy may be improved by the methods described herein. Accordingly, in certain embodiments, a method for improving (reducing, suppressing, or preventing (i.e., reducing the likelihood of occurrence) the acute toxicity of chemotherapy or radiation therapy, or both, in a subject receiving treatment, or for reducing the severity of toxic side effects (i.e., harmful side effects) is provided herein, wherein the method comprises administering to the subject a substance that selectively kills, removes, or destroys senescent cells or promotes their selective destruction.The administration of the senescent cell-removing substance described herein for treating side effects of chemotherapy or radiation therapy, or for reducing the likelihood or severity thereof, may be achieved by the same treatment course described above for the treatment or prevention of metastasis. As described for the treatment or prevention of metastasis (i.e., reduction of the likelihood of occurrence), the senescent cell-removing substance is administered during the chemotherapy rest period or radiation therapy rest period, or after the completion of the chemotherapy or radiation therapy treatment regimen.

[0202] In more specific embodiments, the acute toxicity is an acute toxicity including energy imbalance and may include one or more of weight loss, endocrine changes (e.g., hormonal imbalance, changes in hormonal signaling), and changes in body composition. In certain embodiments, the acute toxicity including energy imbalance is associated with a decrease or decline in the subject's physical activity capacity, such as as manifested by reduced or lowered energy consumption compared to that observed in subjects not receiving medical treatment. As an example, though not intended to be limiting, such acute toxicity effects including energy imbalance include low physical activity. In other embodiments, the energy imbalance includes fatigue or lethargy.

[0203] In some embodiments, the chemotherapy side effect treated or prevented (i.e., the likelihood of occurrence reduced) by the senescent cell-removing agent described herein is cardiotoxicity. Cancer patients being treated with anthracyclines (doxorubicin, daunorubicin, etc.) may be treated with one or more senescent cell-removing agents described herein that reduce, improve, or lower the cardiotoxicity of anthracyclines. As is well understood in the medical field, due to the cardiotoxicity associated with anthracyclines, the maximum lifetime dose that a subject may receive is limited, even if the cancer responds to the drug. Administering one or more senescent cell-removing agents may result in improved prognosis related to the cancer disease by reducing cardiotoxicity, thereby allowing the subject to receive additional amounts of anthracyclines. In some embodiments, cardiotoxicity occurs due to the administration of anthracyclines such as doxorubicin. Doxorubicin is used in patients with ovarian cancer who have failed platinum-based therapy; patients with Kaposi's sarcoma who have failed primary systemic chemotherapy or are intolerant to such therapy; Alternatively, it is an anthracycline topoisomerase inhibitor approved for the treatment of multiple myeloma patients in combination with bortezomib in patients who have never received bortezomib or have received at least one previous treatment. Doxorubicin may cause myocardial damage leading to congestive heart failure if the total lifetime dose to the patient exceeds 550 mg / m². Cardiotoxicity may occur at lower doses if the patient receives mediastinal radiation or other cardiotoxic drugs.

[0204] In other embodiments, the senescent cell removal material described herein may be used in the method provided herein to improve chronic or long-term side effects. Chronic toxic side effects typically occur due to multiple exposures or administrations of chemotherapy or radiation therapy over a longer period. Certain toxic effects appear a long time after treatment (also called delayed toxic effects) and are caused by damage to organs or systems due to treatment. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients who received cancer treatment during childhood (see, e.g., Hudson et al., JAMA 309 (2013) 2371-2381). Without being limited by any particular theory, by destroying senescent cells, particularly certain normal cells whose senescence has been induced by chemotherapy or radiation therapy, the likelihood of chronic side effects occurring may be reduced, the severity of chronic side effects may be reduced or alleviated, or the onset of chronic side effects may be delayed. Chronic and / or delayed toxic side effects occurring in subjects who have received chemotherapy or radiation therapy include, but are not intended to be limited to, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, cognitive impairment, peripheral neuropathy, secondary cancer, cataracts and other vision problems, hearing loss, chronic fatigue, reduced lung capacity, and lung disease.

[0205] Furthermore, by administering a senescent cell removal substance to subjects with cancer to kill or eliminate senescent cells, sensitivity to chemotherapy or radiation therapy can be improved in a clinically or statistically significant way compared to cases where the substance is not administered. In other words, when a senescent cell removal substance is administered to subjects undergoing chemotherapy or radiation therapy, the development of resistance to chemotherapy or radiation therapy can be suppressed.

[0206] Age-related diseases and disorders

[0207] The senescent cell removal substance described herein selectively kills senescent cells. In this way, targeting senescent cells during the aging process can serve as a preventive strategy. Therefore, administering the senescent cell removal substance described herein to a subject can prevent comorbidities in elderly subjects and delay the time of death. Furthermore, the selective killing of senescent cells can strengthen the subject's immune system, extend their health span, and improve their quality of life.

[0208] Senescent cell removal substances may also be useful for treating or preventing (i.e., reducing the likelihood of occurrence) age-related diseases or disorders that occur as part of the natural aging process or when a subject is exposed to aging-inducing substances or factors (e.g., radiation, chemotherapy, tobacco smoking, a high-fat / high-sugar diet, or other environmental factors). Age-related disorders or diseases or age-sensitive traits may be associated with aging-inducing stimuli. The efficacy of the treatment methods described herein may be manifested by reducing the number of symptoms of age-related disorders or age-sensitive traits associated with aging-inducing stimuli, reducing the severity of one or more symptoms, or delaying the progression of age-related disorders or age-sensitive traits associated with aging-inducing stimuli. In other embodiments, preventing age-related disorders or age-sensitive traits associated with aging-inducing stimuli means preventing (i.e., reducing the likelihood of occurrence) or delaying the onset of age-related disorders or age-sensitive traits associated with aging-inducing stimuli, or preventing or delaying the recurrence of one or more age-related disorders or age-sensitive traits associated with aging-inducing stimuli. Age-related diseases or conditions include, for example, renal failure, kyphosis (curvature of the spine), herniated disc, frailty, hair loss, hearing loss, vision loss (blindness or visual impairment), muscle fatigue, skin conditions, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Vision loss refers to a state in which a subject previously had vision but currently lacks it. Various scales have been developed to describe the range of vision and vision loss based on visual acuity.Age-related diseases and conditions also include dermatological conditions, and include, for example, without limitation, the treatment of one or more of the following conditions: wrinkles including superficial fine wrinkles, hyperpigmentation, scars, keloids, dermatitis, psoriasis, eczema (including seborrheic eczema), rosacea, vitiligo, ichthyosis vulgaris, dermatomyositis, and actinic keratosis. Frailty has been defined as a clinically recognizable state of increased vulnerability resulting from age-related reserve and functional decline across multiple physiological systems, which impairs a subject's ability to cope with routine or acute stressors. Frailty may be characterized by reduced energetic features such as low grip strength, low energy, slowed walking speed, low physical activity, and / or unintended weight loss. Studies have suggested that a diagnosis of senility may be made to a patient when three of the five above characteristics are observed (see, e.g., Fried et al., J. Gerontol. A Biol. Sci. Med, Sci. 56(3) (2001) M146-M156; Xue, Clin. Geriatr. Med. 27(1) (2001) 1-15). In certain embodiments, aging and aging-related diseases and disorders may be treated or prevented (i.e., the likelihood of occurrence is reduced) by administering a senescent removal substance. Senescent removal substances may inhibit the aging of adult stem cells, inhibit the accumulation of aged adult stem cells, cause them to die, or promote their removal. The importance of preventing stem cell aging to maintain tissue regenerative capacity is, for example, Park et al., J. Clin. Invest. 113 (2004) 175-179; and discussed in Sousa-Victor, Nature 506 (2014) 316-321, etc.

[0209] Methods for measuring aging are known in the art. For example, aging in bone tissue can be measured by the occurrence of non-vertebral fractures, hip fractures, total fractures, vertebral fractures, recurrent fractures, functional recovery after fracture, decreased bone density in the lumbar spine and hips, frequency of knee buckling, use of non-steroidal anti-inflammatory drugs (NSAIDs), number of painful joints, and osteoarthritis. Additionally, aging in muscles can be measured by functional decline, frequency of falls, reaction time and grip strength, decrease in muscle mass of the upper and lower extremities, and 10-meter walking speed during double task performance. Furthermore, aging in the cardiovascular system can be measured by changes in systolic and diastolic blood pressure, occurrence of hypertension, major cardiovascular events such as myocardial infarction, stroke, and congestive heart disease, and cardiovascular disease mortality. Additionally, aging in the brain can be measured by cognitive decline, occurrence of depression, and occurrence of dementia. Additionally, aging in the immune system can be measured by infection rates, upper respiratory infection rates, flu-like disease rates, occurrence of severe infections leading to hospitalization, cancer incidence, implant infection rates, and gastrointestinal infection rates. Other indicators of aging may include, but are not limited to, deterioration of oral health, tooth loss, frequency of gastrointestinal symptoms, changes in fasting blood glucose and / or insulin levels, body composition, decline in renal function, quality of life, occurrence of impairments related to activities of daily living, and occurrence of nursing home admissions. Methods for measuring skin aging are known in the art and may include transepidermal water loss (TEWL), skin moisture content, skin elasticity, analysis of the area ratio of crow's feet, sensitivity, radiance, roughness, freckles / blemishes, sagging, skin tone uniformity, smoothness, and curvature (changes in depth).

[0210] The administration of the senescent cell removal substance described herein may extend survival compared to the expected survival time of the subject without treatment. Subjects requiring treatment include not only subjects who already suffer from a disease or disability, but also subjects who are prone to or at risk of developing a disease or disability, and subjects who require preventive treatment for a disease, condition, or disability. Subjects may have a genetic predisposition to develop a disease or disability from which benefits can be derived from the removal of senescent cells, or may be of a specific age at which receiving the senescent cell removal substance to delay the progression or reduce the severity of a disease, including age-related diseases or disabilities, may provide clinical benefits.

[0211] In another embodiment, a method for treating an aging-related disease or disorder is provided, further comprising the step of identifying a subject who may benefit from treatment with a senescent cell removal substance described herein (i.e., phenotypic analysis; individualized treatment). The method first comprises the step of detecting the level of senescent cells in a subject, for example, in a specific organ or tissue of the subject. Biological samples, for example, blood samples, serum or plasma samples, biopsy specimens, body fluids (e.g., lung lavage fluid, ascites fluid, mucosal lavage fluid, synovial fluid, vitreous fluid, cerebrospinal fluid), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation from the subject may be obtained from the subject. The level of senescent cells may be determined according to any in vitro analysis method or technique described herein. For example, senescent cells are morphological (e.g., as observed under a microscope); aging-related β-galactosidase (SA-β-Gal), p16 INK4aIt may be detected by the generation of aging-related indicators such as p21, PAI-1, or any one or more SASP factors (e.g., IL-6, MMP3). To determine the ability of a senescent cell removal substance to kill senescent cells in a subject without unwanted toxicity to non-senescent cells, the senescent and non-senescent cells of the biological sample may be used in an in vitro cell analysis in which the cells are exposed to any senescent cell removal substance described herein. Additionally, these methods may be used to monitor the levels of senescent cells in the subject before, during, and after treatment with the senescent cell removal substance. In certain embodiments, the presence of senescent cells may be detected (e.g., by determining the level of senescent cell indicator mRNA expression), and accordingly, the treatment process and / or non-treatment interval (resting period) may be adjusted.

[0212] GPX4 Treatment and Prevention Methods for Diseases and Disorders

[0213] A method for treating, preventing, or improving the symptoms of a glutathione peroxidase 4 (GPX4)-related disease in a subject is provided, comprising administering an effective amount of one or more compounds disclosed herein or a composition disclosed herein. In some embodiments, the GPX4-related disease is cancer, neurotic disorder, neurodegenerative disorder, spondylometaphyseal dysplasia, mixed cerebral palsy, pontocerebellar hypoplasia, or male infertility.

[0214] In some embodiments, the GPX4-related disease is cancer. Non-limiting examples of cancer include hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colorectal cancer, diffuse large B-cell lymphoma, leukemia, lung cancer, clear cell carcinoma, or non-small cell lung carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In other embodiments, the cancer is metastatic. In yet another embodiment, the cancer is hypersensitive to ferroptosis. In yet another embodiment, the cancer is refractory to standard cancer treatment. In yet another embodiment, the cancer has mesenchymal characteristics. In yet another embodiment, the cancer is multitherapy-resistant cancer.

[0215] The present specification also provides a method for controlling the activity of GPX4 in a subject, comprising administering an effective amount of one or more compounds disclosed herein or a composition disclosed herein. In some embodiments, the control comprises inhibiting the activity of GPX4.

[0216] The present specification also provides a method for increasing the level of peroxide in a subject, comprising administering an effective amount of one or more compounds disclosed in the present specification or a composition disclosed in the present specification. Non-limiting examples of peroxides include hydrogen peroxide, organic hydroperoxides, lipid peroxides, and combinations thereof.

[0217] The present specification also provides a method for inducing ferroptosis in a cell, comprising contacting the cell with an effective amount of one or more compounds disclosed herein or a composition disclosed herein. The cell may have abnormal lipid accumulation. In some embodiments, the cell is a cancer cell, which includes, but is not limited to, hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colorectal cancer, diffuse large B-cell lymphoma, leukemia, lung cancer, clear cell carcinoma, or non-small cell lung carcinoma cells. In other embodiments, the cancer cell is a hepatocellular carcinoma cell.

[0218] In some embodiments, the cancer cells are metastatic. In other embodiments, the cancer cells are hypersensitive to perophtosis. In yet another embodiment, the cancer has mesenchymal characteristics. In yet another embodiment, the cancer is a multitherapy-resistant cancer.

[0219] Hypersensitivity to the above-mentioned perophtosis can be identified by NADPH levels (NADPH abundance), GCH1 expression, NF2-YAP activity, EMT signature, and GPX4 expression. In some embodiments, the cancer cells are selected from the group consisting of hepatocellular carcinoma, sarcoma, glioma, renal cell carcinoma, ovarian cancer, prostate cancer, breast cancer, pancreatic cancer, melanoma, colorectal cancer, diffuse large B-cell lymphoma, leukemia, lung cancer, clear cell carcinoma, or non-small cell lung carcinoma cells. In some embodiments, the cancer is hepatocellular carcinoma cells.

[0220] Pharmaceutical composition and method of administration

[0221] The present specification also provides a pharmaceutical composition comprising a senescent cell-removing substance as described herein and at least one pharmaceutically acceptable excipient, which may be referred to as a pharmaceutically suitable excipient or carrier (i.e., a non-toxic substance that does not interfere with the activity of the active ingredient). The pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension, or emulsion (e.g., a microemulsion). The excipients described herein are examples and are by no means limiting. An effective dose or therapeutically effective dose refers to an amount of one or more senescent cell-removing substances that is effective to produce a desired therapeutic effect when administered to a subject, whether as a single dose or as part of a series of doses.

[0222] When two or more senescent cell-removing substances are administered to a subject for the treatment of a disease or disorder described herein, each senescent cell-removing substance may be formulated into a separate pharmaceutical composition. A pharmaceutical compound comprising each separate pharmaceutical composition may be prepared (for convenience, this may be referred to, for example, as a first pharmaceutical composition and a second pharmaceutical composition comprising a first and a second senescent cell-removing substance, respectively). Each pharmaceutical composition in the compound may be administered simultaneously (i.e., in combination) and via the same route of administration, or administered at different times via the same or different routes of administration. Alternatively, two or more senescent cell-removing substances may be formulated together in a single pharmaceutical composition.

[0223] In another embodiment, at least one senescent cell removal substance and mTOR, NF- K A combination of at least one inhibitor of the B or PI3K pathway may be administered to a subject requiring this. In order to selectively kill senescent cells, at least one senescent cell removal agent and mTOR, NF- KWhen one or more inhibitors of the B or PI3K pathways are used together, each substance may be formulated into the same pharmaceutical composition or into separate pharmaceutical compositions. Pharmaceutical formulations containing each separate pharmaceutical composition may be prepared, for convenience, for example, a senescent cell removal substance and mTOR, NF- K A first pharmaceutical composition and a second pharmaceutical composition may be referred to as comprising one or more inhibitors of the B or PI3K pathways. Each pharmaceutical composition in the formulation may be administered simultaneously and through the same route of administration, or administered at different times by the same or different routes of administration.

[0224] The pharmacokinetics of a senescent cell-removing substance (or one or more metabolites thereof) administered to a subject may be monitored by determining the levels of the senescent cell-removing substance in biological fluids, e.g., blood, blood fractions (e.g., serum), and / or urine, and / or other biological samples or biological tissues from the subject. Any method practiced in the art and described herein for detecting such substances may be used to measure the levels of the senescent cell-removing substance during the treatment process.

[0225] The dosage of the senescent-removing substance described herein for treating senescent cell-related diseases or disorders may vary depending on the subject's condition, namely the stage of the disease, the severity of symptoms caused by the disease, and the overall health status, as well as age, gender, weight, and other factors apparent to those skilled in the art of medicine. The pharmaceutical composition may be administered in a manner appropriate for the disease to be treated, as determined by those skilled in the art of medicine. In addition to the factors described herein and above regarding the use of the senescent-removing substance for treating senescent-related diseases or disorders, the appropriate duration and frequency of administration of the senescent-removing substance may also be determined or controlled by factors such as the patient's condition, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. The optimal dosage of the substance may generally be determined using experimental models and / or clinical trials. The optimal dosage may vary depending on the subject's body mass, weight, or blood volume. It is generally preferred to use the minimum dosage sufficient to provide effective treatment. The design and conduct of preclinical and clinical studies for the senescent-removing substances described herein (including cases where they are administered for preventive benefit) are fully within the scope of the art of those skilled in the relevant field. When two or more senescent-removing substances are administered to treat aging-related diseases or disorders, the optimal dosage of each senescent-removing substance may differ from when any one substance is administered alone as monotherapy, for example, and may be lower. In certain embodiments, the two combined senescent-removing substances exhibit a synergistic or additive effect, and either substance may be used in a smaller amount than when administered alone. The amount of senescent-removing substance that may be administered per day is, for example, between about 0.01 mg and 100 mg per kg of body weight (e.g., about 0.It may be between 1 and 1 mg / kg, between about 1 and 10 mg / kg, between about 10 and 50 mg / kg, or between about 50 and 100 mg / kg. In other embodiments, the amount of senescent cell removal substance that may be administered per day is between about 0.01 mg and 1000 mg per kg of body weight, between about 100 and 500 mg / kg, or between about 500 and 1000 mg / kg. The optimal dosage (per day or per course of treatment) may vary depending on the aging-related disease or disorder to be treated, and may also vary depending on the route of administration and the treatment regimen.

[0226] A pharmaceutical composition comprising a senescent cell-removing substance may be formulated in a manner suitable for a delivery method using techniques routinely practiced in the art. The composition may be in the form of a solid (e.g., tablet, capsule), a semi-solid (e.g., gel), a liquid, or a gas (aerosol). In other specific embodiments, the senescent cell-removing substance (or a pharmaceutical composition comprising it) is administered by bolus infusion. In specific embodiments in which the senescent cell-removing substance is delivered by injection, the senescent cell-removing substance is delivered via blood vessels to an organ or tissue containing senescent cells to be killed, according to techniques routinely practiced by those skilled in the art of medicine.

[0227] Pharmaceutically acceptable excipients are well known in the pharmaceutical field, for example, Rowe et al., Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 th Ed., 2006, and Remington: The Science and Practice of Pharmacy (Gennaro, 21 stIt is described in Ed. Mack Pub. Co., Easton, Pa. (2005). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate-buffered saline at physiological pH. Pharmaceutical compositions may be provided with preservatives, stabilizers, dyes, buffers, etc. Additionally, antioxidants and suspending agents may also be used. Generally, the type of excipient is selected based on the chemical composition of the active ingredient(s) as well as the mode of administration. Alternatively, the compositions described herein may be formulated as lyophilizates. The compositions described herein may be lyophilized using one or more suitable excipient solutions to solubilize and / or dilute the material(s) of the composition upon administration, or formulated into other types of lyophilized products. In other embodiments, the material may be encapsulated in liposomes using techniques known and practiced in the art. Pharmaceutical compositions may be formulated for any suitable mode of administration described herein and in the art.

[0228] The pharmaceutical composition may be delivered to a subject in need of it by any of several routes known to those skilled in the art. By example, though not intended to be limiting, the composition may be delivered orally, intravenously, intraperitoneally, by injection (e.g., bolus injection), subcutaneously, enterally, rectalally, intranasally, by inhalation, orally, sublingually, intramuscularly, transdermally, intradermally, topically, intraocularly, vaginally, rectally, or intracranially, or by any combination thereof. In certain embodiments, administration of the dose described above is made intravenously, intraperitoneally, by direct administration to a target tissue or organ, or via a subcutaneous route. In certain embodiments, the delivery method comprises a drug-coated or drug-impregnated stent in which the drug is a senescent cell-removing material. Formulations suitable for such delivery methods are described in more detail herein.

[0229] In a specific embodiment, a senescent cell-removing substance (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) is administered directly to a target tissue or organ containing senescent cells that contribute to the manifestation of a disease or disorder. In a specific embodiment for treating osteoarthritis, the at least one senescent cell-removing substance is administered directly (i.e., into the joint cavity) to the osteoarthritis joint of a subject requiring it. In another specific embodiment, the senescent cell-removing substance(s) may be administered to the joint via a local, transdermal, intradermal, or subcutaneous route. In another specific embodiment, the present specification provides a method for treating a cardiovascular disease or disorder associated with arteriosclerosis, such as atherosclerosis, by administering directly into an artery. In other embodiments, a senescent cell-removing substance for treating age-related lung disease or disorder (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) may be administered, for example, by inhalation, intranasal, endotracheal intubation, or subarachnoid space to deliver the senescent cell-removing substance more directly to the affected lung tissue. As other examples, but not intended to be limiting, the senescent cell-removing substance (or a pharmaceutical composition containing a senescent cell-removing substance) may be delivered directly to the eye by injection (e.g., intraocular or vitreous) or by conjunctival application of a cream, ointment, gel, or eye drop to the suborbital area. In more specific embodiments, the senescent cell-removing substance or a pharmaceutical composition containing a senescent cell-removing substance may be formulated as a time-controlled release (also referred to as sustained release or controlled release) composition or administered by a bolus injection.

[0230] The pharmaceutical composition (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery, or other methods) may be in liquid form. The liquid pharmaceutical composition may comprise, for example, one or more of the following: water, saline solution, preferably physiological saline, Ringer's solution, sterile diluents such as isotonic sodium chloride, fixed oils that may act as solvents or suspension media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antimicrobial agents; antioxidants; chelating agents; buffers; and tonicity modifiers such as sodium chloride or dextrose (glucose). The supplemental (parenteral) composition may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The use of physiological saline is preferred, and the injectable pharmaceutical composition is preferably sterile. In another embodiment, for the treatment of an ophthalmic condition or disease, the liquid pharmaceutical composition may be applied to the eye in the form of eye drops. The liquid pharmaceutical composition may be delivered orally.

[0231] For oral formulations, at least one of the senescent cell-removing substances described herein may be used alone or in combination with suitable additives to form tablets, powders, granules, or capsules, and may be combined with diluents, buffers, humectants, preservatives, coloring agents, and flavorings if desired. The compounds may be formulated with buffers and / or an enteric coating to protect the compounds from the low pH of the gastric environment. The senescent cell-removing substance included in the pharmaceutical composition may be formulated for oral delivery in liquid, solid, or semi-solid formulations with flavorings and / or an enteric coating.

[0232] Pharmaceutical compositions comprising any senescent cell-removing material described herein may be formulated for sustained release or sustained release (also referred to as time-controlled release or controlled release). Such compositions may be prepared using generally well-known techniques and administered, for example, orally, rectally, by intradermal or subcutaneous implantation, or by implantation at a desired target site. Sustained-release formulations may contain a compound dispersed within a carrier matrix and / or a compound contained within a reservoir enclosed by a membrane that controls the release rate. Excipients for use in such formulations may be biocompatible and biodegradable; preferably, said formulations provide a relatively constant level of active ingredient release. The amount of active substance contained in sustained-release formulations depends on the implantation site, the release rate and the expected release duration, and the characteristics of the condition, disease, or disorder to be treated or prevented.

[0233] In certain embodiments, a pharmaceutical composition comprising a senescent cell removal substance is formulated for transdermal, intradermal, or topical administration. The composition may be administered in the form of powder / talc or other solids, liquids, sprays, aerosols, ointments, foams, creams, gels, or pastes using a syringe, bandage, transdermal patch, insert, or syringe-shaped applicator. This is preferably in the form of a controlled-release or sustained-release formulation that is administered topically or injected directly (intradermal or subcutaneously) into the skin near or within the site to be treated. The active composition may also be delivered via iontophoresis. Preservatives may be used to prevent the growth of fungi and other microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate (sodium benzoate), sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

[0234] A pharmaceutical composition comprising a senescent cell removal substance may be formulated as an emulsion for topical application. The emulsion contains one liquid dispersed throughout the body of a second liquid. The emulsion may be an oil-in-water emulsion or a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. The oil phase may contain other pharmaceutically approved oily excipients. Suitable surfactants include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. The composition for topical application may also include at least one suitable suspending agent, antioxidant, chelating agent, emollient, or moisturizer.

[0235] Ointments and creams may be formulated on an aqueous or oily base, for example, with the addition of a suitable thickener and / or gelling agent. Lotions may be formulated on an aqueous or oily base and generally contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners, or coloring agents. Liquid sprays may be delivered from a pressurized pack, for example, through a specially molded closure. Water-in-oil type emulsions may also be used in compositions, patches, bandages, and articles. Such systems are semi-solid emulsions, microemulsions, or foam emulsion systems.

[0236] Transdermal or topical formulations for controlled or sustained release can be achieved by adding time-controlled release additives, such as polymeric structures or matrices available in the art. For example, the composition may be administered via the use of a hot-melt extruded article, such as a bioadhesive hot-melt extruded film. The formulation may comprise a cross-linked polycarboxylic acid polymer formulation. The cross-linking agent may be present in an amount that provides appropriate adhesion to ensure the system remains attached to the surface of target epithelial or endothelial cells for a sufficient time to achieve the desired release of the compound.

[0237] An insert, transdermal patch, bandage, or article may comprise a mixture or coating of a polymer that provides the release of an active substance at a constant rate over a long period of time. In some embodiments, the article, transdermal patch, or insert comprises a water-soluble pore-forming agent, such as polyethylene glycol (PEG), which can be mixed with a water-insoluble polymer to increase the durability of the insert and extend the release of the active ingredient.

[0238] Polymer formulations may be utilized to provide controlled or sustained release. Bioadhesive polymers described in the art may be used. As an example, sustained-release gels and compounds may be contained within a polymer matrix, such as a hydrophobic polymer matrix. Examples of polymer matrices include microparticles. The microparticles may be microspheres, and the core may be a material different from the polymer shell. Alternatively, the polymer may be cast into a thin slab or film, a powder prepared by grinding or other standard techniques, or a gel such as a hydrogel. The polymer may be in the form of a coating to facilitate the delivery of senescent cell removal materials, or may be part of a bandage, stent, catheter, artificial blood vessel, or other device. The matrix may be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art.

[0239] A kit is provided comprising a unit dose (unit formulation) of one or more substances described herein, which is typically an oral or injectable dose. Such a kit may comprise a container holding the unit dose, an information package insert (guide) describing the method of use of the drug and the associated benefits in treating aging cell-related diseases, and optionally a device or apparatus for delivering the composition.

[0240] Combination therapy

[0241] The compounds and compositions disclosed herein may also be used in combination with one or more other active ingredients. In certain aspects, said compounds may be administered in combination with or sequentially with other therapeutic agents. Such other therapeutic agents include those known for the treatment, prevention, or improvement of one or more of the symptoms disclosed herein. Many such therapeutic agents are known in the art.

[0242] Any suitable combination of the compounds and compositions provided herein with one or more of the therapeutic agents and optionally one or more additional pharmacologically active substances is to be understood to be within the scope of this disclosure. In some aspects, the compounds and compositions provided herein are administered before or after one or more additional active ingredients.

[0243] Examples of compounds that may be administered together with the compounds disclosed herein include dasatinib, quercetin, fisetin, luteolin (leeutolin), curcumin, curcumin analog EF24, navitoclax (ABT263), A1331852, A1155463, geldanamycin, tanespimycin, albepimycin, piperolongumein, FOXO-4 peptide, Nutlin3a, cardiac glycosides (e.g., auabain, proscilaridin A, digoxin, etc.), HSP-90 inhibitors, triptolide, EF-24, procyanidin C1, azithromycin, rocithromycin, 25-hydroxycholesterol, SSK1, BIRC5 knockout, BCL-2 inhibitors, Src inhibitors. PD-1, CTLA-4 ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, ofatumumab, blinatumumab, daratumumab, elotuzumab, obinutuzumab, talimozin laherparebec, necitumumab, lenalidomide, dinutusimab and combinations thereof, including but not limited to these.

[0244] Finally, it should be noted that alternative methods of implementing the present invention exist. Accordingly, these aspects should be considered illustrative and not restrictive, and the present invention is not limited to the details provided herein but may be modified within the scope of the appended claims and equivalents.

[0245] All publications and patents cited in this specification are incorporated herein by reference in their entirety.

[0246] The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0247] Examples

[0248] Scheme 1 shows the preparation of the key intermediate 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (109).

[0249]

[0250] (1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-carboxylic acid (101)

[0251]

[0252] A mixture of bromine (12.28 mL) and iron (2.68 g, 48.01 mmol) was stirred at 0 °C for 1 hour. Then, (1r, 3R, 5S, 7r)-3,5-dimethyladamantan-1-carboxylic acid (10.00 g, 48.01 mmol) (100) was added. The reaction mixture was stirred at room temperature for 2 days. After the addition of ethyl acetate, the resulting mixture was carefully treated with a saturated solution of sodium thiosulfate at 0 °C and stirred for 15 minutes. After filtering through a Celite pad and rinsing with ethyl acetate, the organic phase was separated, washed with a saturated sodium thiosulfate solution and brine (salt water), dried over a sodium sulfate layer, and concentrated under reduced pressure to obtain (1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-carboxylic acid (14.3 g, unrefined) (101) as a pale yellow oil. ESI-MS [MH] - Calculated value (C 13 H 19BrO2): 285.06, 287.06; Measured: 285.00, 287.00.

[0253] ((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methanol (102)

[0254]

[0255] BH3-THF (1 M in THF, 149 mL, 150.00 mmol) was slowly added dropwise at 0 °C to a stirred solution of (1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-carboxylic acid (14.3 g, 50.00 mmol) (101) in THF (35 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was quenched with methanol (180 mL). After stirring at room temperature for 30 minutes, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 8%) in petroleum ether to obtain ((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methanol (6.8 g, 52% yield over two steps) (102) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 4.51 (t, J = 5.6 Hz, 1H), 3.05 (d, J = 5.6 Hz, 2H), 1.91 (d, J = 2.3 Hz, 6H), 1.23-1.02 (m, 6H), 0.85 (s, 6H).

[0256] 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-1H-pyrazole (103)

[0257]

[0258] CMBP (7.10 g, 29.43 mmol) was added to a stirred mixture of ((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methanol (6.70 g, 24.52 mmol) (102) and 1H-pyrazole (2.01 g, 29.43 mmol) in toluene (80 mL) under a nitrogen atmosphere. The reaction mixture was stirred at 90 °C for 5 hours. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 10%) in dichloromethane to obtain 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-1H-pyrazole (7.4 g, 93%) (103) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.62 (d, J = 2.1 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 6.23 (t, J = 2.0 Hz, 1H), 3.90 (s, 2H), 1.97-1.79 (m, 6H), 1.22-0.96 (m, 6H), 0.83 (s, 6H). ESI-MS [M+H] + Calculated value (C 16 H 23 BrN2): 323.10, 325.10; Measured: 323.10, 325.10.

[0259] 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole (104)

[0260]

[0261] n-butyllithium (2.5 M in THF, 40.21 mL, 100.54 mmol) was added dropwise at -78 °C to a stirred solution of 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-1H-pyrazole (6.50 g, 20.11 mmol) (103) in THF (120 mL) under a nitrogen atmosphere. After the addition was finished, the reaction mixture was stirred at -50 °C for 1.5 hours. Then, CH3I (17.12 g, 120.64 mmol) was added dropwise. After stirring the reaction mixture at this temperature for 1.5 hours, it was quenched with a saturated solution of NH4Cl (200 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic layer was washed with water (2 x 300 mL) and brine (salt water) (2 x 300 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 60%) in petroleum ether to obtain 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole (6.6 g, 97%) (104) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.31 (d, J = 1.6 Hz, 1H), 6.03-5.99 (m, 1H), 3.79 (s, 2H), 2.23 (s, 3H), 2.01 (s, 2H), 1.90-1.85 (m, 4H), 1.27-1.12 (m, 6H), 0.83 (s, 6H). ESI-MS [M+H] + Calculated value (C 17 H 25 BrN2): 337.12, 339.12; Measured: 337.10, 339.10.

[0262] 2-(((1s,3R,5S,7r)-3,5-dimethyl-7-((5-methyl-1H-pyrazole-1-yl)methyl)adamantan-1-yl)oxy)ethanol-1-ol (105)

[0263]

[0264] A mixture of 1-(((1r,3s,5R,7S)-3-bromo-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole (6.60 g, 19.57 mmol) (104) and DIEA (17.04 mL, 97.84 mmol) in ethane-1,2-diol (44 mL) was stirred at 120 °C for 6 hours. The cooled reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 x 80 mL). The combined organic layer was washed with water (2 x 150 mL) and brine (salt water) (2 x 150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2-(((1s,3R,5S,7r)-3,5-dimethyl-7-((5-methyl-1H-pyrazol-1-yl)methyl)adamantan-1-yl)oxy)ethanol-1-ol (6.5 g, unrefined) (105) as a yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.29 (d, J = 1.6 Hz, 1H), 6.02-5.96 (m, 1H), 4.44 (t, J = 5.6 Hz, 1H), 3.78 (s, 2H), 3.43-3.36 (m, 2H), 3.35-3.31 (m, 2H), 2.23 (s, 3H), 1.34 (s, 2H), 1.28-1.21 (m, 4H), 1.13-1.04 (m, 4H), 1.04-0.97 (m, 2H), 0.84 (s, 6H). ESI-MS [M+H] + Calculated value (C 19 H 30 N2O2): 319.23; Measured: 319.20.

[0265] 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol (106)

[0266]

[0267] TBSCl (3.69 g, 24.49 mmol) was added to a stirred mixture of 2-(((1s,3R,5S,7r)-3,5-dimethyl-7-((5-methyl-1H-pyrazole-1-yl)methyl)adamantan-1-yl)oxy)ethanol-1-ol (6.5 g, 20.41 mmol) (105) and imidazole (1.81 g, 26.53 mmol) in DCM (120 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water (150 mL) and extracted with dichloromethane (3 x 100 mL). The combined organic layer was washed with water (2 x 150 mL) and brine (salt water) (150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 32%) in petroleum ether to obtain 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole (6.3 g, 71%) (106) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.28 (d, J = 1.8 Hz, 1H), 5.98 (d, J = 1.6 Hz, 1H), 3.78 (s, 2H), 3.59 (t, J = 5.3 Hz, 2H), 3.35 (t, J = 5.3 Hz, 2H), 2.22 (s, 3H), 1.33 (s, 2H), 1.27-1.20 (m, 4H), 1.13-1.05 (m, 4H), 1.02-0.94 (m, 2H), 0.86 (s, 9H), 0.83 (m, 6H), 0.03 (s, 6H). ESI-MS [M+H] + Calculated value (C 25 H 44 N2O2Si): ​​433.32; Measured: 433.30.

[0268] 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-4-iodo-5-methyl-1H-pyrazol (107)

[0269]

[0270] NIS (9.20 g, 40.91 mmol) was added to a stirred solution of 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole (5.90 g, 10.56 mmol) (106) in DMF (100 mL). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was quenched with water (150 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was washed with water (200 mL) and brine (salt water) (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 25%) in petroleum ether to obtain 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-4-iodo-5-methyl-1H-pyrazole (7.5 g, 98%) (107) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) δ 7.44 (s, 1H), 3.89 (s, 2H), 3.59 (t, J = 5.2 Hz, 2H), 3.35 (t, J = 5.2 Hz, 2H), 2.24 (s, 3H), 1.31 (s, 2H), 1.29-1.20 (m, 4H), 1.15-1.05 (m, 4H), 1.04-0.94 (m, 2H), 0.86 (s, 9H), 0.83 (s, 6H), 0.02 (s, 6H). ESI-MS [M+H] + Calculated value (C 25 H 43 IN2O2Si): ​​559.21; Measured: 559.25.

[0271] 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol (109)

[0272]

[0273] iPrMgCl-LiCl (1.3 M in THF, 15.49 mL, 20.14 mmol) was added dropwise at 0 °C to a stirred solution of 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-4-iodo-5-methyl-1H-pyrazole (7.50 g, 13.43 mmol) (107) in THF (90 mL) under a nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 1 hour. Then, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (12.49 g, 67.13 mmol) (108) was added. The reaction mixture was stirred at 0°C for 2 hours, quenched with an aqueous solution of saturated ammonium chloride (200 mL), and then extracted with ethyl acetate (3 x 150 mL). The combined organic layer was washed with water (200 mL) and brine (salt water) (2 x 200 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 32%) in petroleum ether to obtain 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (6.4 g, 85%) (109) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.46 (s, 1H), 3.80 (s, 2H), 3.59 (t, J = 5.1 Hz, 2H), 3.35 (t, J = 5.1 Hz, 2H), 2.35 (s, 3H), 1.33 (s, 2H), 1.28-1.22 (m, 14H), 1.21-1.01 (m, 8H), 0.84 (s, 9H), 0.83 (s, 6H), 0.01 (s, 6H). ESI-MS [M+H] + Calculated value (C 31 H 55BN2O4Si): 559.40; Measured: 559.45.

[0274] Scheme 2 is compound 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (1) and the core intermediate tert-butyl This describes the preparation of 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (116).

[0275]

[0276] Tert-butyl 3-bromo-6-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)picolinate (112)

[0277]

[0278] NaH (724 mg, 18.11 mmol, 60%) was added at 0°C to a stirred mixture of tert-butyl 3-bromo-6-fluoropicolinate (2.00 g, 7.24 mmol) (110) and 2-(6-chloro-5-methylpyridazine-3-yl)acetonitrile (1.46 g, 8.69 mmol) (111) in DMF (60 mL) under a nitrogen atmosphere. After the addition, the reaction mixture was stirred at room temperature for 16 hours, quenched with water (100 mL), and extracted with ethyl acetate (3 x 80 mL). The combined organic layer was washed with water (150 mL) and brine (salt water) (150 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain tert-butyl 3-bromo-6-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)picolinate (3.2 g, unpurified) (112) as an orange solid. ESI-MS [M+H] + Calculated value (C 17 H 16BrClN4O2): 423.01, 425.01; Measured: 423.00, 425.00.

[0279] Tert-butyl 3-bromo-6-(6-bromo-5-methylpyridazine-3-carbonyl)picolinate (113)

[0280]

[0281] Potassium 2-methylpropane-2-oleate (740 mg, 6.61 mmol) was added to a solution of tert-butyl 3-bromo-6-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)picolinate (3.20 g, unpurified) (112) in acetonitrile (80 mL). The reaction mixture was stirred at room temperature for 30 minutes. Then, H2O2 (2.82 mL, 36.34 mmol, 30%) was added dropwise at 0 °C. After the addition was finished, the reaction mixture was heated to room temperature and stirred for 3.5 hours, then quenched with water (120 mL) and extracted with ethyl acetate (3 x 80 mL). The combined organic layer was washed with water (150 mL) and brine (salt water) (150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 30%) in petroleum ether to obtain tert-butyl 3-bromo-6-(6-bromo-5-methylpyridazine-3-carbonyl)picolinate (1.8 g, 58% yield over two steps) (113) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.3 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 8.05 (s, 1H), 2.55 (s, 3H), 1.61 (s, 9H). ESI-MS [M+H] + Calculated value (C 16 H 15 BrClN3O3): 412.00, 414.00; Measured: 412.05, 414.05.

[0282] Tert-butyl 3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)picolinate (114)

[0283]

[0284] A mixture of tert-butyl 3-bromo-6-(6-bromo-5-methylpyridazine-3-carbonyl)picolinate (500 mg, 1.21 mmol) (113), 1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (677 mg, 1.21 mmol) (109), PdCl2(amphos)2 (257 mg, 0.36 mmol), and Cs2CO3 (1.18 g, 3.64 mmol) in dioxane (16 mL) and water (1.6 mL) five times The mixture was degassed and refilled with nitrogen. The reaction mixture was heated at 90 °C for 2 hours. The cooled reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layer was washed with brine (salt water) (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 34%) in petroleum ether to obtain tert-butyl 3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazol-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)picolinate (340 mg, 37%) (114) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.32 (s, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.45 (s, 1H), 3.92 (s, 2H), 3.60 (t, J = 5.4 Hz, 2H), 3.36 (t, J = 5.4 Hz, 2H), 2.25 (s, 3H), 1.43 (s, 2H), 1.34 (s, 9H), 1.29-1.23 (m, 4H), 1.22-1.10 (m, 5H), 1.10-1.03 (m, 3H), 1.02-0.95 (m, 1H), 0.88-0.82 (m, 15H), 0.03 (s, 6H). ESI-MS [M+H] + 계산치 (C 41 H 58 ClN5O5Si): 764.39, 766.39; 실측치: 764.55, 766.55.

[0285] Tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (116)

[0286]

[0287] A mixture of tert-butyl 3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)picolinate (320 mg, 0.41 mmol) (114), benzo[d]thiazole-2-amine (76 mg, 0.50 mmol) (115), XantPhos (48 mg, 0.08 mmol), Pd2(dba)3 (38 mg, 0.04 mmol) and Cs2CO3 (409 mg, 1.26 mmol) in dioxane (16 mL) was degassed five times and refilled with nitrogen. The reaction mixture was heated at 110 °C for 2 hours. The cooled reaction mixture was diluted with water (40 mL) and extracted with ethyl acetate (3 x 40 mL). The combined organic layer was washed with brine (salt water) (60 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 36%) in petroleum ether to obtain tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (280 mg, 76%) (116) as a yellow solid. ESI-MS [M+H] + Calculated value (C 48 H 63 N7O5SSi): 878.44; Measured: 878.65.

[0288] Example 1: 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (1)

[0289]

[0290] A mixture of tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (90 mg, 0.10 mmol) (116) and 4 M HCl in dioxane (10 mL) was stirred at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by Prep-HPLC under the following conditions: Column: XBridge Prep OBD C18 column, 30 x 150 mm, 5 μm; Mobile phase A: water (10 nmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient elution: from 21% B to 51% B for 10 min; wavelength: 254 nm / 220 nm; RT (min): 8.08; thereby 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (38.2 mg, 51%) (1) was obtained as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.06-7.96 (m, 2H), 7.95-7.84 (m, 2H), 7.66 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 3.88 (s, 2H), 3.42-3.38 (m, 2H), 3.35-3.33 (m, 2H), 2.46 (s, 3H), 2.29 (s, 3H), 1.40 (s, 2H), 1.33-1.10 (m, 8H), 1.07 (q, J = 12.6 Hz, 2H), 0.87 (s, 6H). ESI-MS [M+H] + Calculated value (C 38 H 41N7O5S): 708.29; Measured: 708.45.

[0291] Scheme 3 represents the preparation of 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (2).

[0292]

[0293] Tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (117)

[0294]

[0295] TBAF (1 M in THF, 0.39 mL, 0.39 mmol) was added to a stirred solution of tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (170 mg, 0.19 mmol) (116) in THF (6 mL) at 0 °C. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was washed with water (20 mL) and brine (salt solution) (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by Prep-TLC (ethyl acetate) to obtain tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (120 mg, 81%) (117) as a yellow solid. ESI-MS [M+H] + Calculated value (C 42 H 49N7O5S): 764.35; Measured: 764.55.

[0296] Tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (118) and tert-butyl 3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)-6-(5-methyl-6-(((Z)-3-tosylbenzo[d]thiazole-2(3H)-ylidene)amino)pyridazine-3-carbonyl)picolinate (119)

[0297]

[0298] TsCl (75 mg, 0.39 mmol) was added at 0 °C to a mixture of tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3s,5R,7S)-3-(2-hydroxyethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (100 mg, 0.13 mmol), DMAP (8 mg, 0.07 mmol), and TEA (66 mg, 0.65 mmol) in DCM (8 mL) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was washed with brine (salt water) (2 x 50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to produce tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (118) and tert-butyl A mixture of 3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)-6-(5-methyl-6-(((Z)-3-tosylbenzo[d]thiazole-2(3H)-ylidene)amino)pyridazine-3-carbonyl)picolinate (140 mg, unpurified state) (119) was obtained as a yellow solid. ESI-MS [M+H] + Calculated value (C 49 H 55 N7O7S2): 918.36; Measured: 918.35. ESI-MS [M+H] + Calculated value (C 56 H61 N7O9S3): 1072.37; Measured: 1072.40.

[0299] Tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (120)

[0300]

[0301] Tert-butyl 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinate (118) and tert-butyl A mixture of 3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-(tosyloxy)ethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)-6-(5-methyl-6-(((Z)-3-tosylbenzo[d]thiazole-2(3H)-ylidene)amino)pyridazine-3-carbonyl)picolinate (140 mg, unpurified) (119) was dissolved in DMF (6 mL). Then, KI (18 mg, 0.11 mmol) and morpholine (228 mg, 2.62 mmol) were added. The reaction mixture was stirred at 50 °C for 16 hours. The cooled reaction mixture was quenched with water (15 mL) and extracted with ethyl acetate (3 x 15 mL). The combined organic layer was washed with brine (salt solution) (2 x 50 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by Prep-TLC (methanol / dichloromethane = 1:10) to obtain tert-butyl 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-[1-({3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methanol)methyl]-5-methylpyrazole-4-yl]pyridine-2-carboxylate (75 mg, 69% yield over two steps) (120) as a yellow solid. ESI-MS [M+H] + Calculated value (C 46 H 56 N8O5S): 833.41; Measured: 833.60.

[0302] Example 2: 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (2)

[0303]

[0304] A mixture of tert-butyl 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-[1-({3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-carboxylate (120) (65 mg, 0.08 mmol) in TFA (6 mL) was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by Prep-HPLC under the following conditions: Column: XBridge Prep OBD C18 column, 30 x 150 mm, 5 μm; Mobile phase A: water (10 mmol / L NH4HCO3), Mobile phase B: acetonitrile; Flow rate: 60 mL / min; Gradient elution: from 23% B to 53% B for 10 minutes; wavelength: 254 nm / 220 nm; RT (min): 8.5; thereby 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (31.9 mg, 53%) (2) was obtained as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 3.89 (s, 2H), 3.72-3.61 (m, 4H), 3.58 (t, J = 5.2 Hz, 2H), 2.82-2.63 (m, 6H), 2.48 (s, 3H), 2.25 (s, 3H), 1.38-1.18 (m, 8H), 1.19-1.11 (m, 2H), 1.11-0.99 (m, 2H), 0.87 (s, 6H). ESI-MS [M+H] + Calculated value (C 42 H 48 N8O5S): 777.35; Measured: 777.50.

[0305] Scheme 4 represents the preparation of 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylic acid (3).

[0306]

[0307]

[0308] Tert-butyl 2-(6-chloro-5-methylpyridazine-3-yl)-2-cyanoacetate (122)

[0309]

[0310] A mixture of 3,6-dichloro-4-methylpyridazine (10.0 g, 61.35 mmol) (121), tert-butyl 2-cyanoacetate (9.53 g, 67.48 mmol) and potassium carbonate (17.0 g, 123.1 mmol) in DMF (120 mL) was stirred at 80 °C for 1 day. The cooled reaction mixture was poured into ice water (200 mL) and acidified with concentrated hydrochloric acid until pH = 2. The resulting mixture was extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with brine (salt water) (2 x 300 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain tert-butyl 2-(6-chloro-5-methylpyridazine-3-yl)-2-cyanoacetate (29.0 g, unpurified) (122) as a yellow solid. ESI-MS [MH] - Calculated value (C 12 H 14 ClN3O2): 266.08, 268.08; Measured: 265.95, 267.95.

[0311] 2-(6-chloro-5-methylpyridazine-3-yl)acetonitrile (123)

[0312]

[0313] A mixture of tert-butyl 2-(6-chloro-5-methylpyridazine-3-yl)-2-cyanoacetate (122) (29.0 g, unpurified) in TFA (50 mL) and DCM (200 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water (200 mL) and extracted with ethyl acetate (3 x 150 mL). The combined organic layer was washed with brine (salt water) (200 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 50%) in petroleum ether to obtain 2-(6-chloro-5-methylpyridazine-3-yl)acetonitrile (6.5 g, unpurified) (123) as a yellow solid.1 H NMR (400 MHz, CDCl3) δ 7.54 (s, 1H), 4.13 (s, 2H), 2.47 (s, 3H). ESI-MS [M+H] + Calculated values ​​(C7H6ClN3): 168.03, 170.03; Measured values: 168.05, 170.05.

[0314] Ethyl 5-bromo-2-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)thiazole-4-carboxylate (125)

[0315]

[0316] NaHMDS (2 M in THF, 13.9 mL, 27.72 mmol) was added dropwise at -10 °C to a stirred mixture of ethyl 5-bromo-2-chlorothiazole-4-carboxylate (5.0 g, 18.48 mmol) (123) and 2-(6-chloro-5-methylpyridazine-3-yl)acetonitrile (4.03 g, 24.04 mmol) (124) in THF (100 mL) under a nitrogen atmosphere. After stirring at -10 °C for 2 hours, the reaction mixture was quenched with water (200 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with brine (salt water) (300 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain ethyl 5-bromo-2-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)thiazole-4-carboxylate (10.0 g, unpurified) (125) as a brown solid. ESI-MS [M+H] + Calculated value (C 13 H 10 BrClN4O2S): 400.94, 402.94; Measured: 401.00, 403.00.

[0317] Ethyl 5-bromo-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (126)

[0318]

[0319] KOtBu (2.08 g, 18.57 mmol) was added to a solution of ethyl 5-bromo-2-((6-chloro-5-methylpyridazine-3-yl)(cyano)methyl)thiazole-4-carboxylate (10.0 g, unpurified) (125) in acetonitrile (200 mL). The reaction mixture was stirred at room temperature for 30 minutes. Then, the reaction mixture was cooled to 0 °C and H2O2 (14.6 g, 128.86 mmol, 30%) was added. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with water (300 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with brine (salt water) (300 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 5%) in dichloromethane to obtain ethyl 5-bromo-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (4.2 g, 58% yield over two steps) (126) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 1.0 Hz, 1H), 4.48 (q, J = 7.2 Hz, 2H), 2.57 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H). ESI-MS [M+H] + Calculated value (C 12 H9BrClN3O3S): 389.92, 391.92; Measured: 389.95, 391.95.

[0320] Ethyl 5-(3-((tert-butyldimethylsilyl)oxy)prop-1-in-1-yl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (127)

[0321]

[0322] Triethylamine (3.03 g, 30.0 mmol), Pd(PPh3)2Cl2 (351 mg, 0.5 mmol), and CuI (190 mg, 1.0 mmol) were added at 0 °C to a stirred solution of ethyl 5-bromo-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (3.9 g, 10.0 mmol) (126) and tert-butyldimethyl(prop-2-in-1-yloxy)silane (2.55 g, 15.0 mmol) in THF (150 mL) under a nitrogen atmosphere. After stirring at 0 °C for 3 hours, the reaction mixture was quenched with water (200 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic layer was washed with brine (salt water) (200 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 10%) in dichloromethane to obtain ethyl 5-(3-((tert-butyldimethylsilyl)oxy)prop-1-in-1-yl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (2.3 g, 46%) (127) as a black solid. 1 H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 4.66 (s, 2H), 4.46 (q, J = 7.2 Hz, 2H), 2.56 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H), 0.94 (s, 9H), 0.17 (s, 6H). ESI-MS [M+H] + Calculated value (C 21 H 26 ClN3O4SSi): 480.11, 482.11; Measured: 480.10, 482.10.

[0323] Ethyl 5-(3-((tert-butyldimethylsilyl)oxy)propyl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (128)

[0324]

[0325] A mixture of ethyl 5-(3-((tert-butyldimethylsilyl)oxy)prop-1-in-1-yl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (2.3 g, 4.79 mmol) (127) and Pd / C (3.0 g) in EtOH (120 mL) was stirred at room temperature under a hydrogen (H2, 2 atm) atmosphere for 8 hours. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was dissolved in EtOH (30 mL) and stirred at room temperature under an oxygen (O2) atmosphere for 2 days. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 40%) in petroleum ether to obtain ethyl 5-(3-((tert-butyldimethylsilyl)oxy)propyl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (1.0 g, 43%) (128) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 4.44 (q, J = 6.8 Hz, 2H), 3.72 (t, J = 6.0 Hz, 2H), 3.41 (t, J = 7.2 Hz, 2H), 2.55 (s, 3H), 2.09-1.94 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H), 0.90 (s, 9H), 0.06 (s, 6H). ESI-MS [M+H] + Calculated value (C 21 H 30 ClN3O4SSi): 484.10, 486.14; Measured: 484.15, 486.15.

[0326] Ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-((tert-butyldimethylsilyl)oxy)propyl)thiazole-4-carboxylate (129)

[0327]

[0328] A mixture of ethyl 5-(3-((tert-butyldimethylsilyl)oxy)propyl)-2-(6-chloro-5-methylpyridazine-3-carbonyl)thiazole-4-carboxylate (900 mg, 1.86 mmol) (128), benzo[d]thiazole-2-amine (280 mg, 1.86 mmol) (115), Pd2(dba)3 (170 mg, 0.19 mmol), XantPhos (215 mg, 0.37 mmol), and Cs2CO3 (1.81 g, 5.58 mmol) in dioxane (30 mL) was degassed five times and refilled with nitrogen. The reaction mixture was heated at 110 °C for 2 hours. The cooled reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3 x 100 mL). The combined organic layer was washed with brine (salt water) (150 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with ethyl acetate (0 to 50%) in dichloromethane to obtain ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-((tert-butyldimethylsilyl)oxy)propyl)thiazole-4-carboxylate (890 mg, 80%) (129) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.63-7.50 (m, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 4.35 (q, J = 6.8 Hz, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.35 (t, J = 7.2 Hz, 2H), 2.47 (s, 3H), 1.97-1.87 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H), 0.90 (s, 9H), 0.06 (s, 6H). ESI-MS [M+H] + Calculated value (C 28 H 35 N5O4S2Si): ​​598.19; Measured: 598.25.

[0329] Ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-hydroxypropyl)thiazole-4-carboxylate (130)

[0330]

[0331] 4 M HCl (5 mL, 20.07 mmol) in dioxane was added to a solution of ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-((tert-butyldimethylsilyl)oxy)propyl)thiazole-4-carboxylate (400 mg, 0.67 mmol) (129) in dioxane (8 mL). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water (50 mL) and based with a saturated aqueous solution of Na2CO3 until pH = 8. The resulting mixture was extracted with methanol (10%) (4 x 100 mL) in dichloromethane. The combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by Prep-TLC (methanol / dichloromethane = 1:10) to obtain ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-hydroxypropyl)thiazole-4-carboxylate (290 mg, 89%) (130) as a yellow solid. ESI-MS [M+H] + Calculated value (C 22 H 21 N5O4S2): 484.10; Measured: 484.05.

[0332] Ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylate (132)

[0333]

[0334] DIAD (182 mg, 0.90 mmol) was added dropwise at 0 °C to a mixture of ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-hydroxypropyl)thiazole-4-carboxylate (290 mg, 0.60 mmol) (130), 4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenol (127 mg, 0.66 mmol) (131) and PPh3 (236 mg, 0.90 mmol) in THF (15 mL) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was quenched with water (50 mL) and extracted with methanol (10%) (4 x 100 mL) in dichloromethane. The combined organic layer was washed with brine (salt solution) (100 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by Prep-TLC (methanol / dichloromethane = 1:10) to obtain ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylate (100 mg, 25%) (132) as a yellow solid. ESI-MS [M+H] + Calculated value (C 33 H 31 FN6O4S2): 659.18; Measured: 659.15.

[0335] Example 3: 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylic acid (3)

[0336]

[0337] A 2 M aqueous solution of LiOH (1.5 mL, 3.04 mmol) was added to a solution of ethyl 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylate (100 mg, 0.15 mmol) (132) in THF (10 mL). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was quenched with water (20 mL) and acidified with 1 M hydrochloric acid until pH = 2. The resulting mixture was extracted with methanol (10%) (4 x 100 mL) in dichloromethane. The combined organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by Prep-HPLC under the following conditions: column: XBridge Prep OBD C18 column, 30 x 150 mm, 5 μm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient elution: from 22% B to 52% B for 10 min; wavelength: 254 nm / 220 nm; RT (min): 8.5; thereby, 2-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-5-(3-(4-(3-(dimethylamino)prop-1-in-1-yl)-2-fluorophenoxy)propyl)thiazole-4-carboxylic acid (8.5 mg, 8.6%) (3) was obtained as a brown solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.34-6.99 (m, 4H), 4.13 (s, 2H), 3.47 (t, J = 7.4 Hz, 2H), 3.41 (s, 2H), 2.42 (s, 3H), 2.31-2.05 (m, 8H). ESI-MS [M+H] + Calculated value (C 31 H 27FN6O4S2): 631.15; Measured: 631.15.

[0338] Scheme 5 shows the preparation of the intermediate 6-sulfamoylhexanoate (136).

[0339]

[0340] Ethyl 6-(sodiooxysulfonyl)hexanoate (134)

[0341]

[0342] Sodium sulfite (36.72 g, 291.33 mmol, 1.30 equivalents) was added in portions at room temperature to a stirred solution of ethyl 6-bromohexanoate (133) (50.00 g, 224.10 mmol, 1.00 equivalents) in 250.0 mL of water. The resulting mixture was stirred overnight under reflux, cooled to room temperature, and concentrated under reduced pressure to obtain ethyl 6-(sodioxysulfonyl)hexanoate (134) (55 g, unpurified) as a white solid. The unpurified product was used directly in the next step without purification. LCMS (ESI) [M+H] + : 247.1

[0343] Ethyl 6-(chlorosulfonyl)hexanoate (135)

[0344]

[0345] Sulfonyl dichloride (228.50 g, 1920.81 mmol, 8.6 equivalents) was added dropwise at 0 °C to a stirred solution of ethyl 6-(sodioxsulfonyl)hexanoate (134) (55.00 g, 223.35 mmol, 1.00 equivalents) and N,N-dimethylformamide (27.5 mL, 355.34 mmol, 1.5 equivalents) in 550.0 mL of tetrahydrofuran. The resulting mixture was stirred at 70 °C for 2 hours and concentrated under reduced pressure to obtain ethyl 6-(chlorosulfonyl)hexanoate (135) (50.0 g, unpurified) as a yellow oil. The unpurified product was used directly in the next step without purification. LCMS (ESI) [M+H] + : 243.0

[0346] Ethyl 6-sulfamoylhexanoate (136)

[0347]

[0348] NH3·H2O (72.20 g, 2060.07 mmol, 10 equivalents) was dropwise added at 0 °C to a stirred solution of ethyl 6-(chlorosulfonyl)hexanoate (135) (50.0 g, 206.00 mmol, 1.0 equivalent) in 500 mL of acetonitrile. The resulting mixture was stirred at 0 °C for 20 minutes and concentrated under reduced pressure. The residue was then purified by silica gel column chromatography (50% ethyl acetate in petroleum ether) to obtain ethyl 6-sulfamoylhexanoate (136) (12.1 g, 26.3% yield) as a yellow oil. LCMS (ESI) [M+H] + : 224.1.

[0349] Scheme 6 represents the preparation of the compound (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl]methyl}-5-methylpiazole-4-yl)pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (4).

[0350]

[0351] Ethyl 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoate (137)

[0352]

[0353] In a stirred solution of 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl)pyridine-2-carboxylic acid (2) (3.00 g, 3.86 mmol, 1.0 equivalent) and ethyl 6-sulfamoylhexanoate (136) (17.24 g, 77.22 mmol, 20.0 equivalent) in 30 mL of dichloromethane, (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (1.38 g, 8.88 (mmol, 2.3 equivalents) and N,N-dimethylpyridine-4-amine (1.08 g, 8.88 mmol, 2.3 equivalents) were added in portions. The resulting mixture was stirred overnight at room temperature and concentrated under reduced pressure, after which the residue was purified by the reverse phase method under the following conditions: column, C18 column; mobile phase, acetonitrile in water (10 mmol / L NH4HCO3), 1% to 70% gradient elution for 15 minutes; detector, UV 254 nm; Thus, ethyl 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoate (137) (1.58 g, 41.6% yield) was obtained as a yellow solid. LCMS (ESI) [M+H] + : 982.4.

[0354] 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoic acid (138)

[0355]

[0356] Lithium hydroxide (0.20 g, 8.20 mmol, 5.1 equivalents) was added at room temperature to a stirred solution of ethyl 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoate (137) (1.58 g, 1.60 mmol, 1.00 equivalents) in 8 mL of water and 8 mL of tetrahydrofuran. The mixture was stirred overnight at room temperature and the pH was adjusted to 5 with an aqueous hydrochloric acid solution. The mixture was then concentrated under reduced pressure and purified by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% FA) in water, 10% to 80% gradient elution for 15 minutes; detector, UV 254 nm; thereby, 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoic acid (138) (1.24 g, 80.7% yield) was obtained as a yellow solid. LCMS (ESI) [M+H] + : 954.4

[0357] Example 4: (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (4)

[0358]

[0359] 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoic acid (138) (300.0 mg, 0.31 mmol, 1.0 equivalent) in 3.0 mL of N,N-dimethylformamide (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidin-2-carboxamide (139) (279.5 mg, 0.62 mmol, 2.0 equivalents), (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (244.0 mg, 1.57 mmol, 5.0 equivalents) and 1H-1,2,3-benzotriazole-1-ol (212.4 mg, 1.57 mmol, 5.0 equivalents) were mixed with N-ethyl-N-isopropylpropane-2-amine (406.3 mg, 3.14 mmol, 10.0) at room temperature in a stirred solution of (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidin-2-carboxamide (139) (279.5 mg, 0.62 mmol, 2.0 equivalents), (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (244.0 mg, 1.57 mmol, 5.0 equivalents), and 1H-1,2,3-benzotriazole-1-ol (212.4 mg, 1.57 mmol, 5.0 equivalents). Equivalents were added dropwise. The resulting mixture was stirred overnight at room temperature and quenched with water; the precipitated solid was collected by filtration and dried under reduced pressure. The unpurified product (200 mg) was purified by Prep-HPLC under the following conditions (Column: Xselect CSH Prep C18, 30*150 mm 5 μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient elution (B%): from 30% B to 45% B over 10 min; Wavelength: 254 / 220 nm; RT1 (min): 11.05 min).Thus, (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (4) (TFA salt, 51.3 mg, yield 11.8%, purity 99.2%) was obtained as a yellow solid. LCMS (ESI) [M+H]. + : 1380.6. 1H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 9.67 (s, 1H), 8.99 (s, 1H), 8.37 - 8.35 (d, J = 7.8 Hz, 1H), 8.23 - 8.18 (m, 2H), 8.10 - 8.08 (d, J = 8.1 Hz, 1H), 7.89 - 7.78 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 9.2 Hz, 1H), 7.52 (s, 1H), 7.52 (s, 1H), 7.47 - 7.40 (m, 3H), 7.38 - 8.36 (d, J = 8.3 Hz, 2H), 7.27 (t, J = 7.7 Hz, 1H), 5.23-4.81 (m, 1H), 4.51 - 4.49 (d, J = 9.3 Hz, 1H), 4.42 - 4.40 (d, J = 8.0 Hz, 1H), 4.27 (s, 1H), 3.98 (s, 3H), 3.75 - 3.67 (m, 4H), 3.52-3.37 (m, 4H), 3.28 (s, 2H), 3.12 (s, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.28 (s, 3H), 2.24 - 2.17 (m, 1H), 2.10 - 1.98 (m, 2H), 1.79 (td, J = 8.3, 4.2 Hz, 1H), 1.61 (d, J = 8.7 Hz, 2H), 1.46 (d, J = 15.3 Hz, 4H), 1.39 -1.30 (m, 9H), 1.25 (d, J = 8.5 Hz, 1H), 1.19 (d, J = 3.1 Hz, 4H), 1.07 (d, J = 6.5 Hz, 2H), 0.90 (d, J = 8.2 Hz, 15H).

[0360] Scheme 7 represents the preparation of (2S,4S)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (5).

[0361]

[0362] Example 5: (2S,4S)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (5)

[0363]

[0364] 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoic acid (138) (400.0 mg, 0.422 mmol, 1.0 equivalent) in 4 mL of N,N-dimethylformamide N-ethyl-N-isopropylpropane-2-amine (541.8 mg, 4.19 mmol, 10.0 equivalents) at room temperature in a mixture of (2S,4S)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidin-2-carboxamide (140) (372.7 mg, 0.83 mmol, 2.0 equivalents), (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (325.40 mg, 2.095 mmol, 5 equivalents), and 1H-1,2,3-benzotriazole-1-ol (283.2 mg, 2.09 mmol, 5.0 equivalents) The resulting mixture was stirred overnight at room temperature and quenched with water; the precipitated solid was collected by filtration and dried under reduced pressure. The unpurified product (260 mg) was purified by Prep-HPLC under the following conditions (Column: Xselect CSH Prep C18, 30*150 mm 5 μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient elution (B%): Isocratic from 30% to 47% B over 14 min; Wavelength: 254 nm / 220 nm; RT1 (min): 13.87 min).Thus, (2S,4S)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (5) (TFA salt, 52.3 mg, yield 12.0%, purity 97.5%) was obtained as a yellow solid. LCMS (ESI) [M+H]. + : 1380.6. 1 H NMR (400 MHz, DMSO-d6) δ 12.16 (s, 1H), 9.70 (s, 1H), 8.99 (s, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.23 ​​- 8.15 (m, 2H), 8.09 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.59 (s, 1H), 7.52 (s, 1H), 7.46 - 7.41 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 7.7 Hz, 1H), 4.92 (t, J = 7.2 Hz, 1H), 4.43 (d, J = 8.8 Hz, 1H), 4.34 - 4.30 (m, 1H), 4.19 (t, J = 5.6 Hz, 1H), 3.96 (d, J = 15.5 Hz, 4H), 3.90-3.85.

[0365] Scheme 8 shows the preparation of the intermediate (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidine-2-carboxamide (144).

[0366]

[0367] Tert-butyl N-[(2S)-1-[(2S,4R)-2-{[(4-ethynylphenyl)methyl]carbamoyl}-4-hydroxypyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamate (143)

[0368]

[0369] In a stirred solution of 1-(4-ethynylphenyl)methaneamine (141) (700.0 mg, 5.33 mmol, 1.0 equivalent), (2S,4R)-1-[(2S)-2-[(tert-butoxycarbonyl)amino]-3,3-dimethylbutanoyl]-4-hydroxypyrrolidin-2-carboxylic acid (142) (918.9 mg, 2.66 mmol, 0.5 equivalent), (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (662.7 mg, 4.26 mmol, 0.8 equivalent), and 1H-1,2,3-benzotriazole-1-ol (576.8 mg, 4.26 mmol, 0.8 equivalent) in 8 mL of N,N-dimethylformamide at room temperature N-ethyl-N-isopropylpropane-2-amine (1.86 g, 14.40 mmol, 2.7 equivalents) was added. The resulting mixture was stirred overnight at room temperature, quenched with 20 mL of water, and extracted with ethyl acetate (3 x 20 mL). The combined organic layer was washed with brine (salt water) (2 x 20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (10% ethyl acetate in petroleum ether) to obtain tert-butyl N-[(2S)-1-[(2S,4R)-2-{[(4-ethynylphenyl)methyl]carbamoyl}-4-hydroxypyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamate (143) (650.0 mg, yield 26.6%) as a yellow oil. LCMS (ESI) [M+H] + : 458.3.

[0370] (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidine-2-carboxamide (144)

[0371]

[0372] 2 mL of HCl (gas) in 1,4-dioxane was added dropwise at room temperature to 5.0 mL of N-[(2S)-1-[(2S,4R)-2-{[(4-ethynylphenyl)methyl]carbamoyl}-4-hydroxypyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamate (143) (650.0 mg, 1.42 mmol, 1.0 equivalent) in 1,4-dioxane. The resulting mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure, after which the residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, CH3CN (0.1% FA) in water, 10% to 80% gradient elution for 15 minutes; detector, UV 254 nm; Thus, (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidine-2-carboxamide (144) (200.0 mg, yield 39.3%) was obtained as a yellow oil. LCMS (ESI) [M+H] + : 358.2.

[0373] Scheme 9 represents the preparation of (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidin-2-carboxamide (6).

[0374]

[0375] Example 6: (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidin-2-carboxamide (6)

[0376]

[0377] 6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanoic acid (138) (400.0 mg, 0.41 mmol, 1.0 equivalent), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl]-N-(4-ethinylbenzyl)-4-hydroxypyrrolidin-2-carboxamide (224.7 mg, 0.63 N-ethyl-N-isopropylpropane-2-amine (433.45 mg, 3.352 mmol, 8 equivalents) was added dropwise at room temperature to a stirred solution of (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (260.3 mg, 1.67 mmol, 4.0 equivalents) and 1H-1,2,3-benzotriazole-1-ol (226.5 mg, 1.67 mmol, 4.0 equivalents). The resulting mixture was stirred overnight at room temperature, quenched with 30 mL of water, and the precipitated solid was collected by filtration to obtain 260 mg of unpurified product. The unpurified product was purified by Prep-HPLC under the following conditions (Column: Xselect CSH Prep C18, 30*150 mm 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: ACN; Flow rate: 60 mL / min; Gradient elution (B%): Isocratic from 29% to 48% B over 14 min; Wavelength: 254 nm / 220 nm; RT1 (min): 13.18 min).Thus, (2S,4R)-1-[(2S)-2-[6-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)hexanamido]-3,3-dimethylbutanoyl]-N-[(4-ethynylphenyl)methyl]-4-hydroxypyrrolidine-2-carboxamide (TFA salt, 51.0 mg, yield 8.6%, purity 97.7%) was obtained as a yellow solid. LCMS (ESI) [M+H]. + : 1293.6. 1 H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 9.73 (s, 1H), 8.53 (s, 1H), 8.19 (d, J = 13.8 Hz, 2H), 8.09 (s, 1H), 7.94 - 7.78 (m, 2H), 7.55 (d, J = 26.7 Hz, 2H), 7.46 -7.24 (m, 6H), 4.52 (d, J = 8.9 Hz, 1H), 4.39 (s, 3H), 4.15 (d, J = 22.7 Hz, 3H), 3.97 (d, J = 19.8 Hz, 4H), 3.66 (d, J = 25.0 Hz, 6H), 3.39 (s, 4H), 3.12 (s, 2H), 2.28 (s, 4H), 2.03 (d, J = 17.9 Hz, 2H), 1.88 (s, 1H), 1.61 (s, 2H), 1.48 (s, 5H), 1.34 (s, 6H), 1.19 (s, 5H), 1.07 (s, 2H), 0.90 (d, J = 7.3 Hz, 15H).

[0378] Reaction Scheme 10 is 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl)-N-[4-(3-{[(2S)-1-[(2S,4R)-4-hydroxy-2-{[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]carbamoyl}pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl}azetidine-1-yl)benzenesulfonyl]pyridine-2-carboxamide (7) shows the manufacture of.

[0379]

[0380] Ethyl 1-(4-sulfamoylphenyl)azetidine-3-carboxylate (147)

[0381]

[0382] Cs2CO3 (13.81 g, 42.39 mmol, 4.0 equivalents) and 3-chloropyridine, 3-bis[2,6-bis(heptan-4-yl)phenyl]-4,5-dichloro-2,3-dihydro-1H-imidazole-2-yl}dichloropalladium (0.52 g, 0.53 mmol, 0.05 equivalents) were added to a stirred solution of 4-iodobenzenesulfonamide (145) (3.00 g, 10.59 mmol, 1.0 equivalent) and ethylazetidine-3-carboxylate (146) (2.74 g, 21.19 mmol, 2.0 equivalents) in 30 mL of 1,4-dioxane at room temperature under a nitrogen atmosphere. The obtained mixture was further stirred at 100 °C for 1 hour, quenched with 300 ml of water, extracted with dichloromethane (3 x 100 ml), and dried over anhydrous Na₂SO₄. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (16% ethyl acetate in petroleum ether) to obtain ethyl 1-(4-sulfamoylphenyl)azetidine-3-carboxylate (147) (1.5 g, yield 49.7%) as a white solid. LCMS (ESI) [M+H] + : 285.

[0383] ethyl 1-[4-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)phenyl]azetidine-3-carboxylate (148)

[0384]

[0385] In a stirred solution of ethyl 1-(4-sulfamoylphenyl)azetidine-3-carboxylate (147) (1.00 g, 3.51 mmol, 1.0 equivalent) and 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-carboxylic acid (2) (1.37 g, 1.75 mmol, 0.5 equivalent) in 20 mL of dichloromethane, at room temperature, (3-{[(ethylimino)methylidene]amino}propyl)dimethylamine hydrochloride (0.82 g, 5.27 mmol, 1.5 equivalents) and N,N-dimethylpyridine-4-amine (0.86 g, 7.03 mmol, 2.0 equivalents) were added in portions. The obtained mixture was stirred overnight at room temperature and concentrated under reduced pressure, after which the residue was purified by silica gel column chromatography (16% methanol in dichloromethane) to obtain ethyl 1-[4-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)phenyl]azetidine-3-carboxylate (148) (550.0 mg, yield 14.9%) as a brownish-yellow solid. LCMS (ESI) [M+H] + : 1043.

[0386] 1-(4-(N-(6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picoline oil)sulfamoyl)phenyl)azetidine-3-carboxylic acid (149)

[0387]

[0388] Lithium hydroxide (63.0 mg, 2.63 mmol, 5.0 equivalents) was added in portions at room temperature to a stirred solution of ethyl 1-[4-({6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl]pyridine-2-yl}formamidosulfonyl)phenyl]azetidine-3-carboxylate (550.0 mg, 0.52 mmol, 1.0 equivalent) in 5 mL of tetrahydrofuran and 3 mL of water. The resulting mixture was stirred overnight at room temperature. The pH of the reaction mixture was adjusted to 6 with concentrated hydrochloric acid. The obtained mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% FA) in water, 10% to 90% gradient elution for 15 minutes; detector, UV 254 nm; thereby, 1-(4-(N-(6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolineol)sulfamoyl)phenyl)azetidine-3-carboxylic acid (400.0 mg, yield 74.8%) was obtained as a yellow solid. LCMS (ESI) [M+H] + : 1015.4.

[0389] Example 7: 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl)-N-[4-(3-{[(2S)-1-[(2S,4R)-4-hydroxy-2-{[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]carbamoyl}pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl}azetidine-1-yl)benzenesulfonyl]pyridine-2-carboxamide (7)

[0390]

[0391] 1-(4-(N-(6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1r,3R,5S,7s)-3,5-dimethyl-7-(2-morpholinoethoxy)adamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picoline oil)sulfamoyl)phenyl)azetidine-3-carboxylic acid (149) (200.0 mg, 0.19 mmol, 1.0 equivalent) and in 5.0 ml of N,N-dimethylformamide (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (139) (175.3 mg, 0.39 mmol, 2.0 equivalents) at room temperature [(dimethylamino)({3H-[1,2,3]triazoro[4,5-b]pyridine-3-yloxy})methylidene]dimethylazanium; Hexafluoro (374.9 mg, 0.98 mmol, 5.0 equivalents), 1H-1,2,3-benzotriazole-1-ol (133.0 mg, 0.98 mmol, 5.0 equivalents), and N-ethyl-N-isopropylpropane-2-amine (254.0 mg, 1.97 mmol, 10.0 equivalents) were added in portions. The resulting mixture was stirred overnight at room temperature, and the residue was purified by silica gel column chromatography (16% methanol in dichloromethane) to obtain 120 mg of the target product (60% purity) as a yellow solid.The unpurified product was further purified by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile in water (10 mmol / L NH4HCO3), 30% to 60% gradient elution for 15 minutes; detector, UV 254 nm; Thus, 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-(1-{[(1S,3R,5S,7R)-3,5-dimethyl-7-[2-(morpholine-4-yl)ethoxy]adamantan-1-yl}methyl)-5-methylpyrazole-4-yl)-N-[4-(3-{[(2S)-1-[(2S,4R)-4-hydroxy-2-{[(1S)-1-[4-(4-methyl-1,3-thiazole-5-yl)phenyl]ethyl]carbamoyl}pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl}azetidine-1-yl)benzenesulfonyl]pyridine-2-carboxamide (7) (41.3 mg (yield 14.5%, purity 93.6%) was obtained as a yellow solid. LCMS (ESI) [M+H]. + : 1441.7. 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.39 - 8.37 (d, J = 7.8 Hz, 1H), 8.17 - 8.06 (m, 3H), 7.98 -7.96 (d, J = 8.1 Hz, 1H), 7.92 - 7.83 (m, 1H), 7.65 - 7.54 (m, 3H), 7.47 - 7.08 (m, 8H), 6.46 - 6.34 (m, 2H), 5.11 - 5.10 (d, J = 3.5 Hz, 1H), 4.98 - 4.88 (m, 1H), 4.57 - 4.55 (d, J = 9.2 Hz, 1H), 4.44 (t, J = 8.1 Hz, 1H), 4.28 (s, 1H), 4.05 (t, J = 8.1 Hz, 1H), 3.98 (t, J = 7.9 Hz, 1H), 3.90 (t, J = 6.7 Hz, 1H), 3.86 - 3.84 (d, J = 7.9 Hz, 3H), 3.73 - 3.69 (m, 1H), 3.67 - 3.59 (m, 5H), 3.54 (t, J = 5.9 Hz, 3H), 2.62 (s, 6H), 2.46 (s, 3H), 2.14 (s, 3H), 2.07 - 1.97 (m, 1H), 1.86 -1.66 (m, 1H), 1.38 (q, J = 6.7, 5.8 Hz, 5H), 1.30 (s, 4H), 1.25 - 1.23 (d, J = 12.2 Hz, 3H), 1.18 - 1.15 (d, J = 12.3 Hz, 2H), 1.10 - 1.00 (m, 3H), 0.95 - 0.93 (d, J = 8.7 Hz, 9H), 0.89 (s, 7H).

[0392] Reaction Scheme 11 represents the preparation of 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-(((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid; trifluoroacetic acid (8).

[0393]

[0394] (S)-2-(2,2-dimethyl-1,3-dioxolane-4-yl)acetaldehyde (151)

[0395]

[0396] Pyridinium chlorochromate (17.7 g, 82.0 mmol, 2.0 equivalents) was added at 0 °C to a stirred solution of (S)-2-(2,2-dimethyl-1,3-dioxolane-4-yl)ethanol-1-ol (150) (6.0 g, 41.0 mmol, 1.0 equivalent) and 3 Å molecular sieve in 100 mL of dichloromethane. The resulting mixture was stirred at room temperature for 3 hours and filtered, after which the filter cake was washed with dichloromethane (2 x 100 mL). The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluting with 50% ethyl acetate in petroleum ether) to obtain (S)-2-(2,2-dimethyl-1,3-dioxolane-4-yl)acetaldehyde (151) (1.00 g, yield 16.9%) as a pale yellow oil.

[0397] 6-(6-chloro-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (153)

[0398]

[0399] 3-(1-(((1r,3s,5R,7S)-3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)picolinic acid (152) (1.0 g, 1.65 mmol, 1.0 equivalent) was added at room temperature to a stirred solution of (S)-2-(2,2-dimethyl-1,3-dioxolane-4-yl)acetaldehyde (1.0 g, unpurified, 30% purity) in 25 mL of dichloromethane. The obtained mixture was stirred at room temperature for 30 minutes, sodium triacetoxyborohydride (0.7 g, 3.4 mmol, 0.5 equivalents) was added dropwise over 10 minutes at 0 °C, and the mixture was stirred at room temperature for 1 day. Subsequently, the reaction mixture was filtered, the filter cake was washed with ethyl acetate (3 x 100 mL), and the filtrate was concentrated under reduced pressure. The residue was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% formic acid) in water, 10% to 80% gradient elution for 30 minutes; detector, UV 254 nm; Thus, 6-(6-chloro-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (153) (320.0 mg, yield 6.4%) was obtained as a pale yellow oil. LCMS (ESI) [M+H] + : 722.

[0400] 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (154)

[0401]

[0402] In 10 mL of 1,4-dioxane, 6-(6-chloro-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (153) (320.0 mg, 0.44 mmol, 1.0 equivalent), benzo[d]thiazole-2-amine (115) (66.0 mg, 0.44 mmol, 1.0 equivalent) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthen (77.0 mg, 0.13 mmol, 0.3 Tris(dibenzylideneacetone)dipalladium-chloroform adduct (121.0 mg, 0.13 mmol, 0.3 equivalents) and cesium carbonate (289.0 mg, 0.88 mmol, 2.0 equivalents) were added dropwise to a stirred solution of equivalents at 25 °C. The resulting mixture was stirred at 90 °C for 2 hours under a nitrogen atmosphere and filtered; afterward, the filter cake was washed with ethyl acetate (3 x 50 mL). The filtrate was concentrated under reduced pressure, and the residue was purified by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% formic acid) in water, 10% to 80% gradient elution for 30 minutes; detector, UV 254 nm; Thus, 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (154) (150.0 mg, yield 40.4%) was obtained as an orange oil. LCMS (ESI) [M+H] + : 835.

[0403] Example 8: 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-(((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (8)

[0404]

[0405] 2 mL of trifluoroacetic acid was dropwise added at 0 °C to a stirred solution of 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-((2-((S)-2,2-dimethyl-1,3-dioxolane-4-yl)ethyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (150.0 mg, 0.18 mmol, 1.0 equivalent) in 4 mL of dichloromethane. The resulting mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure, after which the residue was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; Mobile phase, acetonitrile (0.1% formic acid) in water, 10% to 90% gradient elution for 30 minutes; detector, UV 254 nm; thereby 6-(6-(benzo[d]thiazole-2-ylamino)-5-methylpyridazine-3-carbonyl)-3-(1-(((1S,3s,5R,7S)-3-(2-(((S)-3,4-dihydroxybutyl)amino)ethoxy)-5,7-dimethyladamantan-1-yl)methyl)-5-methyl-1H-pyrazole-4-yl)picolinic acid (8); trifluoroacetic acid (82.5 mg as trifluoroacetate, yield 50.5%, purity 96.8%) was obtained as an orange solid. LCMS (ESI) [M+H] + : 795.50. 1H NMR (400 MHz, DMSO-d6) δ 13.44 (s, 1H), 8.32 - 8.25 (m, 2H), 8.17 - 8.10 (m, 2H), 8.06 (d, J = 8.1 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.54 - 7.45 (m, 2H), 7.44 (m, 1H), 7.31 - 7.23 (m, 1H), 5.13 - 4.79 (m, 2H), 3.93 (s, 2H), 3.62 - 3.48 (m, 3H), 3.36 (m, 1H), 3.24 (m, 1H), 3.10 - 3.03 (m, 4H), 2.52 (s, 3H), 2.29 (s, 3H), 1.87 - 1.77 (m, 1H), 1.59 (m, 1H), 1.48 (s, 2H), 1.39 - 1.27 (m, 4H), 1.19 (s, 4H), 1.18 - 1.15 (m, 2H), 0.89 (s, 6H).

[0406] Reaction Scheme 12 shows the preparation of 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}adamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (9).

[0407]

[0408] Tert-butyl 3-{1-[(3-{2-[(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylate (156)

[0409]

[0410] Cesium carbonate (47.95 g, 147.17 mmol, 2.0 equivalents) at 0 °C in a stirred solution of tert-butyl N-{2-[(3,5-dimethyl-7-{[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole-1-yl]methyl}adamantan-1-yl)oxy]ethyl}carbamantane (155) (40.00 g, 73.58 mmol, 1.0 equivalent) and tert-butyl 3-bromo-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylate (113) (91.10 g, 220.76 mmol, 3.0 equivalents) in 1500 mL of 1,4-dioxane and 500 mL of water and bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) (5.21 g, 7.35 mmol, 0.1 equivalent) were added. The resulting mixture was stirred at 75 °C for 2 hours and filtered, after which the filter cake was washed with ethyl acetate (3 x 200 mL). The filtrate was concentrated under reduced pressure, diluted with 2000 mL of water, and extracted with ethyl acetate (3 x 1500 mL). The combined organic layer was washed with water (3 x 500 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50% ethyl acetate in petroleum ether) to obtain tert-butyl 3-{1-[(3-{2-[(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylate (156) (47.00 g, yield 85.2%) as a pale yellow oil. LCMS (ESI) [M+H] + : 749.

[0411] 3-(1-{[3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl]methyl}-5-methylpyrazole-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (152)

[0412]

[0413] 20 mL of trifluoroacetic acid was added dropwise at 0 °C to a stirred solution of tert-butyl 3-{1-[(3-{2-[(tert-butoxycarbonyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylate (156) (5.00 g, 6.67 mmol, 1.0 equivalent) in 60 mL of dichloromethane. The resulting mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure, after which the residue was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; Mobile phase, acetonitrile in water (0.1% ammonium hydroxide), 10% to 80% gradient elution for 30 minutes; detector, UV 254 nm; thereby 3-(1-{[3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl]methyl}-5-methylpyrazole-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (152) (2.30 g, yield 58.1%) was obtained as a pale yellow oil. LCMS (ESI) [M+H] + : 593.

[0414] 3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (158)

[0415]

[0416] Triethylamine (3.92 g, 38.78 mmol, 10.0 equivalents) was dropwise added at 0 °C to a stirred solution of 3-(1-{[3-(2-aminoethoxy)-5,7-dimethyladamantan-1-yl]methyl}-5-methylpyrazole-4-yl)-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (152) (2.30 g, 3.87 mmol, 1.0 equivalent) and 4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutyl ethanesulfonate (157) (2.60 g, 5.81 mmol, 1.5 equivalents) in 50 mL of N,N-dimethylformamide. The obtained mixture was stirred at room temperature for 2 days, diluted with 200 mL of water, and extracted with ethyl acetate (3 x 150 mL). The combined organic layer was washed with water (3 x 100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% ammonium hydroxide) in water, 10% to 90% gradient elution for 30 minutes; detector, UV 220 nm; Thus, 3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (158) (2.00 g, yield 49.6%) was obtained as a pale yellow oil. LCMS (ESI) [M+H] + : 1040.

[0417] 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (159)

[0418]

[0419] In a stirred solution of 3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}-6-(6-chloro-5-methylpyridazine-3-carbonyl)pyridine-2-carboxylic acid (2.00 g, 1.92 mmol, 1.0 equivalent), benzo[d]thiazole-2-amine (115) (1.0 equivalent) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthen (0.11 g, 0.19 mmol, 0.1 equivalent) in 30 mL of 1,4-dioxane, cesium carbonate (1.25 g, 3.84 mmol, 2.0 equivalents) and tris(dibenzylideneacetone)dipalladium-chloroform adduct (0.18 g, 0.19 mmol, 0.1 equivalents) were added dropwise. The resulting mixture was stirred at 90 °C for 2 hours under a nitrogen atmosphere. The resulting mixture was filtered, and the filter cake was washed with ethyl acetate (3 x 30 mL). The filtrate was concentrated under reduced pressure. The residual product was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; mobile phase, acetonitrile (0.1% formic acid) in water, 10% to 80% gradient elution for 30 minutes; detector, UV 254 nm; Thus, 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (159) (910.0 mg, yield 41.0%) was obtained as a dark green solid. LCMS (ESI) [M+H] + : 1154.

[0420] Example 9: 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}adamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (9)

[0421]

[0422] 3 mL of trifluoroacetic acid was dropwise added at 0 °C to a stirred solution of 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3-{2-[(2-{4-[(tert-butyldiphenylsilyl)oxy]-2,2-dimethylbutoxysulfonyl}ethyl)amino]ethoxy}-5,7-dimethyladamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (159) (600.0 mg, 0.52 mmol, 1.0 equivalent) in 6 mL of dichloromethane. The resulting mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure, after which the residue was purified twice by reverse-phase flash chromatography under the following conditions: column, C18 column; Mobile phase, acetonitrile (0.1% formic acid) in water, 10% to 90% gradient elution for 30 minutes; detector, UV 254 nm; thereby 6-[6-(1,3-benzothiazole-2-ylamino)-5-methylpyridazine-3-carbonyl]-3-{1-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}adamantan-1-yl)methyl]-5-methylpyrazole-4-yl}pyridine-2-carboxylic acid (9) (258.2 mg, yield 60.9%, purity 92.6%, free basic state) was obtained as a yellow solid. LCMS (ESI) [M+H] + : 815.45. 1H NMR (400 MHz, DMSO-d6) δ 13.13(s, 1H), 9.15 (s, 1H), 8.14 - 8.12 (d, J = 17.7 Hz, 2H), 8.02 (s, 1H), 7.91 - 7.90 (d, J = 7.6 Hz, 1H), 7.53 (m, 2H), 7.47 - 7.38 (m, 1H), 7.27 (t, J = 7.7 Hz, 1H), 3.90 (m, 2H), 3.56 (m, 2H), 3.21 (m, 2H), 3.03 (m, 2H), 2.85 (t, J = 6.3 Hz, 2H), 2.54 (m, 3H), 2.25 (m, 3H), 1.35 - 1.30 (m, 8H), 1.23-1.18 (m, 2H), 1.08 (d, J = 4.5 Hz, 2H), 0.89 (s, 6H).

[0423] Example 10: Cell Aging Analysis Procedure and Results

[0424] Cells were maintained in DMEM medium (high glucose, 4 mM L-glutamine, no sodium pyruvate) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37°C containing 5% carbon dioxide. Similar to previous studies in the field (e.g., Aoshiba et al., 2003 Eur Respir J 22:436-443), cellular senescence was induced after treatment with sublethal concentrations of chemotherapy agents (bleomycin, gemcitabine, doxorubicin, 5-fluorouridine, mitomycin C) followed by a resting period without chemotherapy agents. Cellular senescence was confirmed by lower EdU (5-ethinyl-2'-deoxyuridine) incorporation and higher SA-β-Gal (senescence-associated beta-galactosidase) staining compared to untreated cells during proliferation. Senescent cells were seeded into 96-well plates at cell densities of 5,000 to 15,000 cells per well. The following day, the compound was added in two replicates across 10 different concentration ranges covering approximately 4 Log10 units. After incubating cells with the compound for 24 to 72 hours, cell viability was quantified using CellTiter-Glo (Promega) or XTT (R&D Systems). The viability of treated cells was determined relative to untreated control cells. A graph of mean viability normalized against the logarithm of the compound concentration was fitted to a 4-parameter Hill function with a variable slope. The bottom was restricted to be greater than 0. The absolute IC was calculated by interpolating the mean X value at Y=50. 50 ...was determined. The results in Table 1 show that many compounds exhibit activity within cells and can induce a significant concentration-dependent decrease in the viability of aged A549 and IMR90 cells.

[0425] The results are presented in Table 1 below.

[0426] Table 1

[0427]

[0428] *IE-05 to 1E-06 M, ** 1E-07 to 1E-06 M, *** 1E-08 to 1E-07 M, **** Less than 1E-08 M, nt. Not evaluated.

Claims

Claim 1 Compounds with the following structure: or as a salt, hydrate, and solvate thereof, wherein: X is a heteroaryl or a substituted heteroaryl; Y is an alkyldiyl, a substituted alkyldiyl, a cycloalkyldiyl, a substituted cycloalkyldiyl, a heteroalkyldiyl or a substituted heteroalkyldiyl; Z is an alkyl, a substituted alkyl, a cycloalkyl, a substituted cycloalkyl, a cycloalkenyl, a substituted cycloalkenyl, an aryl, a substituted aryl, a cycloheteroalkyl, a substituted cycloheteroalkyl, a heteroalkyl, a substituted heteroalkyl, -OR 16 , or -NHR 17 -is;T is -C- or -S- and;V is -C(O)-, -C(N)OR 2 -, -C(N)NR 3 R 4 -, -C(OH)R 5 - or -CHR 6 - and; J is an aryl, a substituted aryl, a heteroaryl or a substituted heteroaryl; K is an aryldiyl, a substituted aryldiyl, a heteroaryldiyl or a substituted heteroaryldiyl; and R 1 is a halo, alkyl, alkenyl, alkynyl, or haloalkyl; R 2 -R 6 , R8, R 11 , R 14 , R 16 , and R 17 is independently -H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl; R 7 -OR 8 or -NHR 9 is;R 9 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl or -SO2R 10 Igo;R 10 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl; or -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 is;R 12 and R 13 is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroalkenyl, substituted heteroalkenyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, or R 12 and R 13 They form a cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded; R 15 is an aryl, substituted aryl, heteroaryl or substituted heteroaryl; p is 2-20; n is 0 or 1; and m is 0, 1, 2 or 3. Claim 2 A compound of claim 1, wherein n is 1 and T is -C-. Claim 3 A compound according to claim 1, wherein n is 0 and T is -S-. Claim 4 In paragraph 1, X is a substituted heteroaryl compound. Claim 5 In claim 1, Y is a compound that is a cycloalkyldiyl or a substituted cycloalkyldiyl. Claim 6 In claim 1, Z is a substituted alkyl or -OR 16 Phosphorus compound. Claim 7 In paragraph 1, R 7 -OH or NSO2R 10 Phosphorus compound. Claim 8 In Paragraph 7, R 10 -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 Phosphorus compound. Claim 9 In paragraph 8, R 11 is an alkyl or substituted alkyl, and R 12 and R 13 They form a 5- or 6-membered cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded, and R 14 is an alkyl or substituted alkyl, and R 15 is a compound that is an aryl or a substituted aryl. Claim 10 In paragraph 1, V is a compound that is -C(O)-. Claim 11 In paragraph 1, K is a substituted heteroaryldiyl compound. Claim 12 In paragraph 1, J is a heteroaryl compound. Claim 13 A compound in which m is 0 in paragraph 1. Claim 14 In paragraph 2, X is a substituted heteroaryl, Y is a cycloalkyldiyl, and Z is -OR 16 A compound in which V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, and m is 0. Claim 15 In paragraph 2, X is a substituted heteroaryl, Y is a cycloalkyldiyl, and Z is -OR 16 and, V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, m is 0 and; R 7 NSO2R 10 is; R 10 -(CH2) p CONHCHR 11 NHR 12 CHR 13 CONHCR 14 R 15 and; R 11 is an alkyl or substituted alkyl, and R 12 and R 13 They form a 5- or 6-membered cycloheteroalkyl or substituted cycloheteroalkyl ring together with the atoms to which they are bonded, and R 14 is an alkyl or substituted alkyl, and R 15 is a compound that is an aryl or a substituted aryl. Claim 16 In claim 1, a compound having the following structure: . Claim 17 In paragraph 3, X is a substituted heteroaryl, Y is an alkyldiyl, and Z is -OR 16 A compound in which V is -C(O)-, K is a substituted heteroaryldiyl, J is a heteroaryl, and m is 0. Claim 18 In paragraph 17, a compound having the following structure: . Claim 19 The compound of paragraph 1 and a pharmaceutically acceptable medium. Claim 20 A method for treating an aging-related disease or disorder, comprising the step of administering a therapeutically effective amount of a compound according to claim 1 to a subject requiring treatment for an aging-related disease or disorder.

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