Drug conjugates of sugar derivatives and their use as senolytic agents

Abstract

Provided herein are agents and methods for selectively killing senescent cells associated with a number of conditions and diseases, including age-related conditions and diseases. As disclosed herein, diseases and disorders associated with senescent cells can be treated or prevented by administering at least one senolytic agent or pharmaceutical composition thereof. Diseases or disorders associated with senescent cells that may be treated or prevented by the agents and methods described herein include, but are not limited to, cardiovascular diseases or disorders, arteriosclerosis, e.g., cardiovascular diseases and disorders associated with atherosclerosis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory or autoimmune diseases or disorders, pulmonary diseases or disorders, nervous system diseases or disorders, skin diseases or disorders, chemotherapy side effects, radiation therapy side effects, metastasis, and metabolic diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 243,539, filed September 13, 2021 under U.S. Patent Act 119(e), which is incorporated by reference in its entirety.

[0002] Provided herein are senolytic agents for selectively killing senescent cells associated with a number of conditions and diseases, including age-related conditions and diseases. As disclosed herein, senescent cell-associated diseases and disorders can be treated or prevented by administering at least one senolytic agent or pharmaceutical composition thereof. Senescent cell-associated diseases or disorders that can be treated or prevented by the methods described herein include, but are not limited to, cardiovascular diseases or disorders, arteriosclerosis, e.g., cardiovascular diseases and disorders associated with atherosclerosis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory or autoimmune diseases or disorders, pulmonary diseases or disorders, nervous system diseases or disorders, skin diseases or disorders, chemotherapy side effects, radiation therapy side effects, metastasis, and metabolic diseases. [Background technology]

[0003] Aging is a risk factor for most chronic diseases, disabilities, and declining health. Senescent cells, which are cells that have ceased replicating, accumulate in older individuals and may contribute in part or in part to the deterioration of cells and tissues, which underlie aging and age-related diseases (see, e.g., Childs et al., Nat. Rev. Drug Discov. 16 (2017) 718-735). Cells can also senesce after exposure to environmental, chemical, or biological insults, or as a result of disease (see, e.g., Demaria et al., Cancer Discovery 7 (2017) 165-176; Schafer et al., Nat. Commun. 8 (2017) doi: 10.1038 / ncommsl4532).

[0004] Senocyte-eliminating agents with a variety of pharmacological mechanisms have been previously described in the art. Senocyte-eliminating agents can be specific inhibitors of one or more Bcl-2 anti-apoptotic protein family members, where the inhibitors include at least Bcl-xL (e.g., Bcl-2 / Bcl-xL / Bcl-w inhibitors; selective Bcl-xL inhibitors; Bcl-xL / Bcl-w inhibitors (e.g., navitoclax, ABT-737, A1331852, A1155463); see, e.g., Childs et al., supra; Zhu et al., Aging 9 (2017) 955-965; Yosef et al., Nature Commun. (2016) doi:10.1038); Akt kinase-specific inhibitors (e.g., MK-2206); receptor tyrosine kinase inhibitors (e.g., dasatinib, Zhu et al., Aging Cell 2010 : 1111-1122); 14(2015)654-658); CDK4 / 6 inhibitors (e.g., palbociclib, see Whittaker et al., Pharmacol. Ther. 173(2017)83-105); mTOR inhibitors (e.g., rapamycin, see Laberge et al., Nat. Cell Biol. 17(2015)1049-1061); MDM2 inhibitors (e.g., nutrin-3, RG-7112, see U.S. Patent Application Publication No. 2016 / 0339019); Hsp90 inhibitors (e.g., 17-DMAG, ganetespib, Fuhrmann-Stroissnigg et al., Pharmacol. Ther. 173(2017)83-105); al., Nat. Commun. 8 (2017) doi:10.1038 / s41467-017-00314-z); flavones (e.g., quercetin, fisetin, see Zhu et al., Aging Cell 14 (2015) 654-658; Zhu et al., Aging 9 (2017) 955-965); or histone deacetylase inhibitors (e.g., panobinostat, see Samaraweera et al., Sci. Rep. 7 (2017) 1900. doi:10.1038 / s41598-017-01964-l). Summary of the Invention [Problem to be solved by the invention]

[0005] A considerable challenge has been the identification of senolytic agents that selectively kill senescent cells while sparing non-senescent cells. Moreover, many known senolytic agents were initially developed as cytotoxic anti-cancer agents and subsequently repurposed for the "selective" elimination of senescent cell populations. Dose-limiting toxicity in hematopoietic cells is a frequently observed side effect that limits the clinical utility of anti-senescence therapies, as proliferating cells are often more sensitive to the cytotoxic or cytostatic effects of anti-tumor agents (e.g., neutropenia is a well-characterized toxicity associated with the use of anti-apoptotic Bcl-2 family protein inhibitors, see Leverson et al., Sci. Transl. Med. (2015) 7:279ra40. doi:10.1126 / scitranslmed.aaa4642). Pulsed administration of such senolytic agents has been proposed as a mechanism to minimize the exposure of non-senescent cells to these molecules, potentially limiting off-target effects. Therefore, what is needed are senolytic agents with improved selectivity for killing senescent cells with minimal toxicity to non-senescent cells. [Means for solving the problem]

[0006] These and other needs are met by providing non-toxic prodrugs of senolytic agents that are activated by hydrolase enzymes that preferentially accumulate in senescent cells. In one embodiment, the hydrolase enzymes are glycosidases, and these senescence-associated elevated intracellular glycosidase activities are exploited to convert non-toxic prodrug derivatives of pro-apoptotic agents (I) into the toxic pro-apoptotic parent compound (II), resulting in the specific killing of senescent cells. [ka]

[0007] In some embodiments, Compound (II) can promote apoptosis in non-proliferating cells.

[0008] In one aspect, non-toxic prodrugs of toxic senolytic agents are provided that specifically result in senescent cell death when attached to an active senolytic agent in senescent cells. In some embodiments, prodrugs of histone deacetylase inhibitors are provided. In other embodiments, prodrugs of Hsp90 inhibitors are provided. In still other embodiments, prodrugs of topoisomerase 1 inhibitors are provided. In still other embodiments, prodrugs of DNA alkylating agents are provided. In still other embodiments, prodrugs of Akt1 inhibitors are provided. In still other embodiments, prodrugs of proteasome inhibitors are provided. In still other embodiments, prodrugs of Bcl2 inhibitors are provided. Derivatives, including salts, solvates, hydrates, and metabolites, of the prodrugs described herein are also provided. Further provided are pharmaceutical compositions comprising the prodrugs provided herein and a vehicle.

[0009] In another aspect, also provided herein is a method of treating, preventing, or ameliorating the symptoms of a medical disorder in a subject, such as a cardiovascular disease or disorder, arteriosclerosis, such as cardiovascular diseases and disorders associated with atherosclerosis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory or autoimmune diseases or disorders, pulmonary diseases or disorders, nervous system diseases or disorders, skin diseases or disorders, chemotherapy side effects, radiation therapy side effects, metastasis, and metabolic diseases. In carrying out the method, a therapeutically effective amount of a compound or a pharmaceutical composition thereof is administered to the subject.

[0010] In yet another aspect, a method of treating an age-related disease or condition is provided, the method comprising administering to a subject a composition comprising a therapeutically effective amount of one or more senolytic agents provided herein.

[0011] In yet another aspect, there is provided a method for delaying at least one feature of aging in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of one or more senolytic agents provided herein.

[0012] In yet another aspect, a method of killing treatment-induced senescent cells is provided, the method comprising administering to a subject who has undergone a DNA damaging treatment a composition comprising a therapeutically effective amount of one or more senolytic agents provided herein. [Brief description of the drawings]

[0013] [Figure 1] Representative IMR90 images are illustrated (from left to right: SA-β-Gal, SA-α-Fuc, EdU incorporation assay [EdU fluorophore visualized in FITC channel, counterstained with DAPI]). [Diagram 2] A representative A549 image incorporation assay is illustrated [EdU fluorophore visualized in the FITC channel, counterstained with DAPI]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event that there are a plurality of definitions for a term herein, the definition in this section prevails unless stated otherwise.

[0015] As used herein, and unless otherwise specified, the terms "about" and "approximately," when used in connection with a property with a numerical value or a range of values, indicate that the value or range of values ​​may deviate to an extent that would be considered reasonable by one of ordinary skill in the art, while still describing the particular property. Specifically, the terms "about" and "approximately," when used in this context, indicate that the numerical value or range of values ​​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 recited value or range of values. Also, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "compound" includes a plurality of such compounds, and a reference to an "assay" includes a reference to one or more assays and equivalents thereof known to those of ordinary skill in the art.

[0016] A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -C(O)NH2 is attached through a carbon atom. Dashes before or after chemical groups are for convenience; chemical groups may be shown with or without one or more dashes without losing their ordinary meaning. A wavy line drawn through a line in a structure indicates the point of attachment of the group. Unless chemically or structurally required, no directionality is indicated or implied by the order in which chemical groups are described or named.

[0017] Prefix “C” u~v indicates that the group below has u through v carbon atoms. It should be understood that u through v carbons includes carbons u+1 to v, u+2 to v, u+3+v, etc., u+1 to u+3 to v, u+1 to u+4 to v, u+2 to u+4 to v, etc., covering all possible permutations of u and v.

[0018] "Hallmarks of aging" as used herein include, but are not limited to, a generalized decline in the immune system, muscle atrophy and loss of muscle strength, decreased skin elasticity, delayed wound healing, retinal atrophy, decreased lens transparency, decreased hearing, osteoporosis, sarcopenia, graying hair, wrinkling of the skin, decreased vision, frailty, and declining cognitive function.

[0019] "Age-related diseases or conditions", as used herein, include, but are not limited to, degenerative diseases or functional impairment disorders, such as Alzheimer's disease, Parkinson's disease, cataracts, macular degeneration, glaucoma, frailty, muscle weakness, cognitive decline, atherosclerosis, acute coronary syndromes, myocardial infarction, stroke, hypertension, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, type 2 diabetes, obesity, adipose dysfunction, coronary artery disease, cerebrovascular disease, periodontal disease, disability associated with cancer treatment, such as atrophy and fibrosis in various tissues. , brain and heart injuries, treatment-related myelodysplastic syndromes, and diseases associated with accelerated aging and / or defects in DNA damage repair and telomere maintenance, such as progeroid syndromes (i.e., Hutchinson-Gilford progeria syndrome, Werner syndrome, Bloom syndrome, Rothmund-Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum, sulfur deficiency hair growth dysgenesis, xeroderma pigmentosum-Cockayne syndrome complex, restrictive dermopathy), ataxia-telangiectasia, Fanconi anemia, Friedreich's ataxia, dyskeratosis congenita, aplastic anemia, and the like.

[0020] "Alkyl" by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkyl groups include, but are not limited to, methyl; ethyl; propyl, e.g., propan-1-yl, propan-2-yl, etc.; butyl, e.g., butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, etc. In some embodiments, an alkyl group contains 1 to 20 carbon atoms (C1-C 20In other embodiments, the alkyl group contains 1 to 10 carbon atoms (C1 to C 10 In yet other embodiments, an alkyl group contains 1 to 6 carbon atoms (C1-C6 alkyl).

[0021] "Alkenyl" by itself or as part of another substituent refers to an unsaturated branched, straight chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of a hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond. Typical alkenyl groups include, but are not limited to, ethenyl; propenyl, e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyl, e.g., 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, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and the like. In some embodiments, the alkenyl group contains 1 to 20 carbon atoms (C 20 In another embodiment, the alkenyl group contains 1 to 10 carbon atoms (C 10 In yet other embodiments, an alkenyl group contains 1 to 6 carbon atoms (C1-C6 alkenyl).

[0022] "Alkynyl" by itself or as part of another substituent refers to an unsaturated branched, straight chain, or cyclic alkyl radical having at least one carbon-carbon triple bond derived by removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, e.g., prop-1-yn-1-yl, prop-2-yn-1-yl, and the like; butynyl, e.g., but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In some embodiments, an alkynyl group contains 1 to 20 carbon atoms (C1-C2). 20 In another embodiment, the alkynyl group contains 1 to 10 carbon atoms (C 10 In yet other embodiments, the alkynyl group contains 1 to 6 carbon atoms (C1-C6 alkynyl).

[0023] "Aryl" by itself or as part of another substituent 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. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In some embodiments, an aryl group contains 6 to 20 carbon atoms (C6-C8). 20 In another embodiment, the aryl group contains 6 to 15 carbon atoms (C 15 In yet another embodiment, the aryl group contains 6 to 10 carbon atoms (C 10 aryl).

[0024] "Arylalkyl", by itself or as part of another substituent, refers to an alkyl group consisting of a carbon atom, typically a terminal or sp 3 It refers to a non-cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group as defined herein. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. In some embodiments, an arylalkyl group is any of the groups (C6-C 30 ) arylalkyl, for example, the alkyl portion of the arylalkyl group is (C1-C 10 ) alkyl, and the aryl portion is (C6-C 20 In another embodiment, the arylalkyl group is (C6-C 20 )arylalkyl, for example, the alkyl portion of the arylalkyl group is (C1-C8)alkyl and the aryl portion is (C6-C 12 In yet another embodiment, the arylalkyl group is (C6-C 15 ) arylalkyl, for example, the alkyl portion of the arylalkyl group is (C1-C5) alkyl and the aryl portion is (C6-C 10 ) aryl.

[0025] "Arylalkenyl," by itself or as part of another substituent, refers to a non-cyclic alkenyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group, as defined herein. In some embodiments, an arylalkenyl group is an alkyl group such as (C6-C 30 ) arylalkenyl, for example, the alkenyl portion of the arylalkenyl group is (C1-C 10 ) alkenyl, and the aryl portion is (C6-C 20 In another embodiment, the arylalkenyl group is (C6-C 20)arylalkenyl, for example, the alkenyl portion of the arylalkenyl group is (C1-C8)alkenyl and the aryl portion is (C6-C 12 In yet another embodiment, the arylalkenyl group is (C6-C 15 )arylalkenyl, for example, the alkenyl portion of the arylalkenyl group is (C1-C5)alkenyl and the aryl portion is (C6-C 10 ) aryl.

[0026] "Arylalkynyl," by itself or as part of another substituent, refers to a non-cyclic alkynyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with an aryl group, as defined herein. In some embodiments, an arylalkynyl group is an aryl group selected from the group consisting of (C6-C 30 ) arylalkynyl, for example, the alkynyl portion of the arylalkynyl group is (C1-C 10 ) alkynyl, and the aryl portion is (C6-C 20 In another embodiment, the arylalkynyl group is (C6-C 20 )arylalkynyl, for example, the alkynyl portion of the arylalkenyl group is (C1-C8)alkynyl and the aryl portion is (C6-C 12 In yet another embodiment, the arylalkynyl group is (C6-C 15 )arylalkynyl, for example, the alkynyl portion of the arylalkynyl group is (C1-C5)alkynyl and the aryl portion is (C6-C 10 ) aryl.

[0027] A "carbohydrate derivative" is a compound having the general formula C n H 2n O nIn some embodiments, the carbohydrate derivative typically contains 5 or 6 carbon atoms. In other embodiments, the carbohydrate derivative is a monosaccharide (e.g., glucose, fructose, galactose, ribose). In still other embodiments, the carbohydrate derivative comprises a disaccharide (e.g., lactose, sucrose, maltose, cellobiose, chitobiose, gentobiose, etc.). In still other embodiments, the carbohydrate derivative comprises an oligosaccharide (e.g., oligofructose, oligogalactose, raffinose, plantose, veracose, etc.). In still other embodiments, the carbohydrate derivative comprises a polysaccharide (e.g., cellulose, amylose, starch, chitin, pectin, galactogen, etc.).

[0028] "Cycloalkyl," by itself or as part of another substituent, refers to a saturated cyclic monovalent hydrocarbon radical derived by removal of one hydrogen atom from a single carbon atom of a parent cycloalkane. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, and the like. In some embodiments, cycloalkyl groups contain 3 to 20 carbon atoms (C1-C 15 In another embodiment, the cycloalkyl group contains 3 to 10 carbon atoms (C1 to C 10 In yet other embodiments, the cycloalkyl group contains 3 to 8 carbon atoms (C1-C8 cycloalkyl). The term "cyclic monovalent hydrocarbon radical" also includes polycyclic hydrocarbon ring systems having single radicals and 3 to 12 carbon atoms. Exemplary polycyclic cycloalkyl rings include, for example, norbornyl, vinyl, and adamantyl.

[0029] "Cycloalkenyl," by itself or as part of another substituent, refers to an unsaturated cyclic monovalent hydrocarbon radical derived by removal of one hydrogen atom from a single carbon atom of a parent cycloalkene. Typical cycloalkenyl groups include, but are not limited to, cyclopropene, cyclobutene, cyclopentene, and the like. In some embodiments, cycloalkenyl groups contain 3 to 20 carbon atoms (C1-C 20 In other embodiments, the cycloalkenyl group contains 3 to 10 carbon atoms (C 10 In yet another embodiment, the cycloalkenyl group contains 3 to 8 carbon atoms (C1-C8 cycloalkenyl). The term "cyclic monovalent hydrocarbon radical" also includes single radicals and polycyclic hydrocarbon ring systems having 3 to 12 carbon atoms.

[0030] "Cycloheteroalkyl," by itself or as part of another substituent, refers to a cycloalkyl group, as defined herein, in which one or more of the carbon atoms (and optionally any associated hydrogen atoms) are each, independently of the other, replaced with the same or different heteroatoms or heteroatom groups, as defined below under "heteroalkyl." In some embodiments, a cycloheteroalkyl group contains 3 to 20 carbon and heteroatoms ( 1~20 In other embodiments, the cycloheteroalkyl group contains 3 to 10 carbon and heteroatoms (cycloheteroalkyl). 1~10 In yet other embodiments, the cycloheteroalkyl group contains 3 to 8 carbon and heteroatoms (cycloheteroalkyl). 1~8 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.

[0031] "Cycloheteroalkenyl," by itself or as part of another substituent, refers to a cycloalkenyl group, as defined herein, in which one or more of the carbon atoms (and optionally any associated hydrogen atoms) are each, independently of the other, replaced with the same or different heteroatoms or heteroatom groups, as defined below under "heteroalkenyl." In some embodiments, a cycloheteroalkenyl group contains 3 to 20 carbon and heteroatoms ( 1~20 In other embodiments, the cycloheteroalkenyl group contains 3 to 10 carbon and heteroatoms ( 1~10 In yet other embodiments, the cycloheteroalkenyl group contains 3 to 8 carbon and heteroatoms (e.g., cycloheteroalkenyl). 1~8 The term "cyclic monovalent heteroalkenyl radical" also includes single radical and polycyclic heteroalkenyl ring systems having 3 to 12 carbons and at least one heteroatom.

[0032] "Compound" refers to a compound encompassed by the structural formulas disclosed herein, including any specific compound within these formulas whose structures are disclosed herein. Compounds may be identified by their chemical structure and / or chemical name. The chemical structure is determinative of the identity of the compound. Compounds described herein may contain one or more chiral centers and / or double bonds, and therefore may exist as stereoisomers, e.g., double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Thus, chemical structures depicted herein encompass stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) depicted in the structure. Chemical structures depicted herein also encompass enantiomeric and stereoisomeric derivatives of the depicted compounds. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. Compounds may also exist in several tautomeric forms, including enol, keto and mixtures thereof. Thus, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds. The compounds described also include isotopically labeled compounds in which one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds disclosed herein include, but are not limited to, 2 H, 3 H, 11 C. 13 C. 14 C. 15 N, 18 O. 17O and the like. Compounds can exist in unsolvated and solvated forms, including hydrated forms. In general, compounds can be hydrated or solvated. Certain compounds can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the intended uses herein and are intended to be within the scope of this disclosure. Furthermore, when a partial structure of a compound is illustrated, it should be understood that parentheses indicate the attachment point of the partial structure to the remainder of the molecule.

[0033] "DNA damaging therapy" as used herein includes, but is not limited to, g-irradiation, alkylating agents such as nitrogen mustards (e.g., chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (streptozocin, carmustine, lomustine), alkyl sulfonates (e.g., busulfan), triazines (dacarbazine, temozolomide) and ethylenimines (e.g., thiotepa, altretamine), platinum drugs such as cisplatin, carboplatin, oxalaplatin, antimetabolites such as 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxylase inhibitors (HLA, H ... These include thiourea, methotrexate, pemetrexed, pentostatin, thioguanine, anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, antitumor antibiotics such as actinomycin-D, bleomycin, mitomycin-C, mitoxantrone, topoisomerase inhibitors such as topoisomerase I inhibitors (e.g., topotecan, irinotecan) and topoisomerase II inhibitors (e.g., etoposide, teniposide, mitoxantrone), mitotic inhibitors such as taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine) and estramustine.

[0034] "Halo," by itself or as part of another substituent, refers to the radicals -F, -Cl, -Br, or -I.

[0035] "Heteroalkyl" refers to an alkyl group in which one or more of the carbon atoms (and optionally any associated hydrogen atoms) are replaced, each independently of the other, with the same or different heteroatoms or heteroatomic groups. Exemplary heteroatoms or heteroatomic groups that can replace the carbon atoms include, but are not limited to, -O-, -S-, -N-, -Si-, -NH-, -S(O)-, -S(O)2-, -S(O)NH-, -S(O)2NH-, and the like, and combinations thereof. The heteroatoms or heteroatomic groups can be located at any interior position of the alkyl, alkenyl, or alkynyl group. Exemplary heteroatomic groups that can be included in these groups include, but are not limited to, -O-, -S-, -OO-, -SS-, -OS-, -NR 501 R 502 , =NN=, -N=N-, -N=N-NR 503 R 404 , -PR 505 -, -P(O)2-, -POR 506 -, -OP(O)2-, -SO-, -SO2-, -SnR 507 R 508 etc., 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 contains 1 to 20 carbon and heteroatoms ( 1~20 In other embodiments, the heteroalkyl group contains 1 to 10 carbon and heteroatoms (heteroalkyl). 1~10 In yet other embodiments, the heteroalkyl group contains 1 to 6 carbon and heteroatoms (heteroalkyl). 1~6 Heteroalkyl).

[0036] "Heteroalkenyl" refers to an alkenyl group in which one or more of the carbon atoms (and optionally any associated hydrogen atoms) are replaced, each independently of the other, with the same or different heteroatoms or heteroatomic groups. Exemplary heteroatoms or heteroatomic groups that can replace the carbon atoms include, but are not limited to, -O-, -S-, -N-, -Si-, -NH-, -S(O)-, -S(O)2-, -S(O)NH-, -S(O)2NH-, and the like, and combinations thereof. The heteroatoms or heteroatomic groups can be located at any interior position of the alkyl, alkenyl, or alkynyl group. Exemplary heteroatomic groups that can be included in these groups include, but are not limited to, -O-, -S-, -OO-, -SS-, -OS-, -NR 501 R 502 , =NN=, -N=N-, -N=N-NR 503 R 404 , -PR 505 -, -P(O)2-, -POR 506 -, -OP(O)2-, -SO-, -SO2-, -SnR 507 R 508 etc., 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, heteroalkenyl groups contain 1 to 20 carbon and heteroatoms (e.g., 1~20 In other embodiments, the heteroalkenyl group contains 1 to 10 carbon and heteroatoms ( 1~10 In yet other embodiments, the heteroalkenyl group contains 1 to 6 carbon and heteroatoms (e.g., 1~6 heteroalkenyl).

[0037] "Heteroaryl" by itself or as part of another substituent 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. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. In some embodiments, heteroaryl groups contain 5 to 20 ring atoms (5-20 membered heteroaryls). In other embodiments, heteroaryl groups contain 5 to 10 ring atoms (5-10 membered heteroaryls). Exemplary heteroaryl groups include those derived from furan, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole, indole, pyridine, pyrazole, quinoline, imidazole, oxazole, isoxazole, and pyrazine.

[0038] "Heteroarylalkyl" by itself or as part of another substituent means an alkyl group having a carbon atom, typically a terminal or sp 3It refers to a non-cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with a heteroaryl group. In some embodiments, the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., 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 group is a 6-13 membered heteroarylalkyl, e.g., the alkyl portion is a (C1-C3)alkyl and the heteroaryl portion is a 5-10 membered heteroaryl.

[0039] "Heteroarylalkenyl," by itself or as part of another substituent, refers to an acyclic alkenyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with a heteroaryl group. In some embodiments, a heteroarylalkenyl group is a 6- to 21-membered heteroarylalkyl, e.g., the alkenyl portion of the heteroarylalkenyl is a (C1-C6)alkenyl and the heteroaryl portion is a 5- to 15-membered heteroaryl. In other embodiments, a heteroarylalkenyl is a 6- to 13-membered heteroarylalkenyl, e.g., the alkenyl portion is a (C1-C3)alkyl and the heteroaryl portion is a 5- to 10-membered heteroaryl.

[0040] "Heteroarylalkynyl," by itself or as part of another substituent, refers to an acyclic alkenyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced with a heteroaryl group. In some embodiments, a heteroarylalkynyl group is a 6- to 21-membered heteroarylalkyl, e.g., the alkynyl portion of the heteroarylalkynyl is a (C1-C6)alkynyl and the heteroaryl portion is a 5- to 15-membered heteroaryl. In other embodiments, a heteroarylalkynyl is a 6- to 13-membered heteroarylalkynyl, e.g., the alkynyl portion is a (C1-C3)alkyl and the heteroaryl portion is a 5- to 10-membered heteroaryl.

[0041] "Hydrate" refers to the incorporation of water in stoichiometric proportions into the crystal lattice of a compound described herein, resulting in the formation of an adduct. Methods for making hydrates include, but are not limited to, storage in an atmosphere containing water vapor, a dosage form containing water, or a conventional pharmaceutical processing step, such as crystallization (i.e., from water or mixed aqueous solvents), lyophilization, wet granulation, aqueous film coating, or spray drying. Hydrates may also form from crystalline solvates under certain circumstances by exposure to water vapor or by suspension of anhydrous materials in water. Hydrates may also crystallize in multiple forms, resulting in hydrate polymorphs. See, for example, (Guillory, K., Chapter 5, pp. 202205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing hydrates are well within the realm of one skilled in the art, are completely routine, and do not require experimentation beyond that which is typical in the art. Hydrates may be characterized and / or analyzed by methods well known to those skilled in the art, such as single crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition, many commercial companies, such as HOLODIAG, Pharmaparc II, Voie de 1'Innovation, 27100 Val de Reuil, France (http: / / www.holodiag.com), routinely provide services that include the preparation and / or characterization of hydrates.

[0042] "Parent Aromatic Ring System" refers to an unsaturated cyclic or polycyclic ring system having a conjugated p-electron system. Specifically included within the definition of "Parent Aromatic Ring System" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as fluorene, indane, indene, phenalene, and the like. Exemplary parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like.

[0043] "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 with 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 "parent heteroaromatic ring system" are fused ring systems in which one or more of the rings is aromatic and one or more of the rings is saturated or unsaturated, such as benzodioxanes, benzofurans, chromanes, chromenes, indoles, indolines, xanthenes, etc. Exemplary parent heteroaromatic ring systems include, but are not limited to, arsindole, carbazole, b-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.

[0044] "Pharmaceutically acceptable salt" refers to a salt of a compound that possesses the desired pharmacological activity of the parent compound. Such salts can be formed with: (1) inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids, such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, 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-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, benzoic ... or (2) salts 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 ion, or an aluminum ion; or coordinates with an organic base, e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.

[0045] "Preventing" or "prevention" refers to a reduction in the risk of acquiring a disease or disorder (i.e., resulting in the non-occurrence of at least one clinical symptom of a disease in a patient who may be exposed to or susceptible to the disease, but who has not yet experienced or exhibited symptoms of the disease). The application of a treatment to prevent or prevent a disease or disorder is known as "prophylaxis." In some embodiments, the compounds provided herein provide superior prophylaxis with lower side effects over the long term.

[0046] "Prodrug," as used herein, refers to a derivative of a drug molecule that requires a transformation within the body to release the active drug. Prodrugs are frequently, but not necessarily, pharmacologically inactive until converted to the parent drug.

[0047] "Promoiety," as used herein, refers to a form of protecting group that, when used to mask a functional group within a drug molecule, converts the drug into a prodrug. Typically, a promoiety is attached to the drug via a bond that is cleaved in vivo by enzymatic or nonenzymatic means.

[0048] "Protecting group" refers to a class of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group during chemical synthesis. Examples of protecting groups are described in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2003). nd 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-trimethylsilyl-ethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitroveratryloxycarbonyl ("NVOC"), and the like. Representative hydroxy protecting groups include, but are not limited to, those in which the hydroxy group is acylated or alkylated, such as benzyl, and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, and allyl ethers.

[0049] "Senescence" or "senescent cells", as used herein, refers to a state in which cells acquire one or more markers of senescence in response to some cellular stress. Such markers can typically include permanent withdrawal from cell cycle, expression of bioactive secretome of inflammatory factors, altered methylation, senescence-associated heterochromatin structures (SAHF), expression markers for oxidative stress, expression of markers for DNA damage, protein and lipid modifications, morphological hallmarks of senescence, altered lysosomes / vacuoles, and expression of senescence-associated b-galactosidase (see Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI10.1007 / 978-1-62703-239-l_4, (Copyright) Springer Science+Business Media, LLC 2013).

[0050] "Senolytic agents," as used herein, refer to agents that "selectively" (preferentially or to a greater extent) destroy, kill, or remove senescent cells, or promote the selective destruction of senescent cells. In other words, a senolytic agent destroys or kills senescent cells in a biologically, clinically, and / or statistically significant manner, as compared to its ability to destroy or kill non-senescent cells. A senolytic agent is used in an amount and for a time sufficient to selectively kill established senescent cells, but insufficient to kill non-senescent cells in a clinically or biologically meaningful manner. In certain embodiments, the senolytic agents described herein alter at least one signaling pathway in a manner that results in inducing (i.e., initiating, stimulating, triggering, activating, promoting) the death of senescent cells.

[0051] "Solvate" refers to the incorporation of a solvent in a stoichiometric ratio into the crystal lattice of a compound described herein, resulting in the formation of an adduct. Methods for making solvates include, but are not limited to, storage in an atmosphere containing a solvent, a dosage form containing a solvent, or a routine pharmaceutical processing step, such as crystallization (i.e., from a solvent or a mixture of solvents), vapor diffusion, and the like. Solvates may also form from other crystalline solvates or hydrates under certain circumstances upon exposure to a solvent or upon suspension of a material in a solvent. Solvates may crystallize in multiple forms, resulting in solvate polymorphs. See, for example, (Guillory, K., Chapter 5, pp. 205208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing solvates are well within the realm of one skilled in the art, are completely routine, and do not require experimentation beyond that which is typical in the art. Solvates may be characterized and / or analyzed by methods well known to those skilled in the art, such as single crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). In addition, many commercial companies, such as HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27100 Val de Reuil, France (http: / / www.holodiag.com), routinely provide services that include the preparation and / or characterization of solvates.

[0052] "Substituted," when used to modify a particular group or radical, means that one or more hydrogen atoms of the particular group or radical are each replaced, independently of one another, with the same or different substituents. Substituents useful for replacing saturated carbon atoms in a particular group or radical include 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)2R b , =N2, -N3, -S(O)2R b , -S(O)NR b R 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)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)Rb , -OC(S)R b , -O-C(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 and -NR b C(NR b )NR c R c where each R a is independently aryl, substituted aryl, heteroalkyl, substituted heteroalkyl, heteroaryl, or substituted heteroaryl; each R bis independently hydrogen, alkyl, heteroalkyl, substituted heteroalkyl, arylalkyl, substituted arylalkyl, heteroarylalkyl, or substituted heteroarylalkyl; each R c are independently b or alternatively, two R c together with the nitrogen atom to which they are attached form a 4-, 5-, 6- or 7-membered cycloheteroalkyl, substituted cycloheteroalkyl or cycloheteroalkyl fused to an aryl group which may optionally contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S. Specific examples include -NR c R c is meant to include -NH, -NH-alkyl, N-pyrrolidinyl, and N-morpholinyl. In other embodiments, substituents useful for replacing saturated carbon atoms in particular groups or radicals are R a , Halo, -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 R c , -OC(O)NR c R c , and -NR b C(O)OR b where each R a is independently alkyl, aryl, or heteroaryl; each R b are independently hydrogen, R a , heteroalkyl, arylalkyl, and heteroarylalkyl; each R c are independently b or alternatively, two R ctogether with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered cycloheteroalkyl ring.

[0053] Substituents useful for replacing unsaturated carbon atoms in certain groups or radicals include -R a , halo, -O - , -OR b , -SR b , -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)2OR b , -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 , -O-C(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 and -NR b C(NR b )NR c R c where R a , R b and R c is as previously defined. In other embodiments, substituents useful for replacing unsaturated carbon atoms in particular groups or radicals are -R a , Halo, -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 where R a , R b and R c is as previously defined.

[0054] Useful substituents for replacing a nitrogen atom in heteroalkyl and cycloheteroalkyl groups include, but are not limited to, -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 and -NR b C(NR b )NR c R c where R a , R b and R cis as previously defined. In some embodiments, substituents useful for substituting a nitrogen atom in heteroalkyl and cycloheteroalkyl groups are R a , Halo, -OR b , -NR c R c , trihalomethyl, -CN, -S(O)2OR b , -OS(O)2R b , -OS(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 , -OC(O)OR b , -OS(O)2NR c NR c , -NR b C(O)R b and -NR b C(O)OR b where R a , R b and R c is as previously defined.

[0055] Substituents from the above list that are useful to replace other particular groups or atoms will be apparent to those of skill in the art.

[0056] The substituents used to substituted a particular group can typically be further substituted with one or more of the same or different groups selected from the various groups identified above.

[0057] The terms "subject," "individual," or "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, including, but not limited to, murines, rodents, simians, humans, farm animals, sport animals, and pets.

[0058] "Treating" or "treatment" of any disease or disorder, in some embodiments, refers to reversing the disease or disorder (i.e., arresting or reducing the occurrence of the disease or at least one of its clinical symptoms). Treatment may also be considered to include preemptive or prophylactic administration to reverse, arrest or prevent the occurrence of the disease or at least one of its clinical symptoms. In a further feature, a given treatment has a lower chance of long-term side effects over multiple years. In other embodiments, "treating" or "treatment" refers to reversing at least one physical parameter that may not be discernible by the patient. In yet other embodiments, "treating" or "treatment" refers to inhibiting the disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In yet other embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

[0059] "Therapeutically effective amount" means the amount of a compound that, when administered to a patient for treating a disease, is sufficient to treat the disease. The "therapeutically effective amount" varies depending on the compound, the disease and its severity, and the age, weight, absorption, distribution, metabolism and excretion, etc., of the patient being treated.

[0060] "Vehicle" refers to an excipient, additive, or carrier with which a compound is administered to a subject. In some embodiments, the vehicle is pharma- ceutically acceptable.

[0061] Reference will now be made in detail to certain embodiments of the compounds and methods. The disclosed embodiments are not intended to limit the scope of the appended claims. On the contrary, the appended claims are intended to cover all alternatives, modifications, and equivalents.

[0062] Senolytic agent Provided herein are non-toxic prodrugs of senolytic agents that are activated by hydrolase enzymes that preferentially accumulate in senescent cells. In some embodiments, the hydrolase enzymes are glycosidases, and senescence-associated elevated intracellular glycosidase activity is exploited to convert non-toxic prodrug derivatives of pro-apoptotic agents into the toxic pro-apoptotic parent compound, which results in the specific killing of senescent cells.

[0063] In some embodiments, there is provided a compound of formula (III) or (IV), or pharma-ceutically acceptable salts, hydrates, and solvates thereof, wherein: [ka] R1 is R 18 C(O)NH—, where R 18 is a residue of a histone deacetylase inhibitor, a residue of an Hsp90 inhibitor, a residue of a topoisomerase inhibitor, a residue of an Akt1 inhibitor, a residue of a DNA alkylating agent, a residue of a proteosome inhibitor, or a residue of a Bcl2 inhibitor; L is a linker; n is 0 or 1; R2 is -H, -F, -OH, -OC(O)R9, or -OC(O)OR 10 and R3 is -H, -F, -OH, -OC(O)R 11 OR-OC(O)OR 12 and R4 is -H, -F, -OH, -OC(O)R 13 OR-OC(O)OR 14 Alternatively, both R and R together with the atom to which they are attached form R at the acetal carbon atom. 17 Alternatively, R3 and R4 together with the atoms to which they are attached form a five-membered cyclic carbonate; R5 is -CH3, -CH2F, -CHF2, -CF3, -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 R6 is -H or -F; R7 is -H or -F; R8 is -H or -F; R9 to R 17are independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, or substituted heteroaryl; with the proviso that when R5 is -CH2F, -CHF2, or -CF3, then one of R2, R3, or R4 is -H or -F; with the proviso that when R5 is -CH3, -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 when R7 is -F or -H, then R8 is -F only when R6 is -F or -H; when R7 is -F or -H, then R8 is -F only when R6 is -F or -H; when R7 is -F or -H, then R3 is -F only when R8 is -F or -H, then R2 is -F.

[0064] In some embodiments, R6 is -F if and only if R4 is -F; R7 is -F if and only if R3 is -F; and R8 is -F if and only if R2 is -F.

[0065] Linker L is a moiety that optionally connects a sugar group to R1, as defined herein. The linker can be a chemically cleavable linker, a photolabile linker, or an enzymatically cleavable linker (see, for example, U.S. Pat. Nos. 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). The linker can vary in structure and length. The linker can be hydrophobic or hydrophilic, long or short, rigid, semi-rigid or flexible, etc.; the only requirement is that the linker be cleaved from the residue of the senolytic agent following hydrolysis of the sugar moiety to liberate the free senolytic agent.

[0066] Linkers L include, but are not limited to, the structures exemplified below. In some embodiments, L is: [ka] wherein X is -O- or -NH- and o is 1 to 20. In another embodiment, L is [ka] It is.

[0067] In yet other embodiments, L is [ka] It is.

[0068] In some embodiments, L is [ka] where X and Y are independently O, S or NR 20 and R 20 In other embodiments, X and Y are independently O or NR 20 It is.

[0069] In some embodiments, L is [ka] where X, Y and Z are independently O, S or NR 21 and R 21 In other embodiments, X, Y and Z are independently O or NR 21 It is.

[0070] In some embodiments, R5 is -CH3 and R2 is -H or -F. In other embodiments, R5 is -CH3 and R3 is -H or -F. In still other embodiments, R5 is -CH3 and R4 is -H or -F. In still other embodiments, R5 is -CH3, R2 is -F and R8 is -F. In still other embodiments, R5 is -CH3, R3 is -F and R7 is -F. In still other embodiments, R5 is -CH3, R4 is -F and R6 is -F.

[0071] In some embodiments, R5 is -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 and R2 is -H or -F. In other embodiments, R5 is -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 and R3 is -H or -F. In yet another embodiment, R5 is -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 and R4 is -H or -F. In yet another embodiment, R5 is -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 and R2 is -F and R8 is -F. In yet other embodiments, R5 is -CH2OH, -CH2OC(O)R 15 Or -CH2OC(O)OR 16 and R is -F. In yet another embodiment, R is -CHOH, -CHOC(O)R 15 Or -CH2OC(O)OR 16 where R4 is -F and R6 is -F.

[0072] In some embodiments, R5 is -CH2F, -CHF2, or -CF3, and R2 is -H or -F. In other embodiments, R5 is -CH2F, -CHF2, or -CF3, and R3 is -H or -F. In still other embodiments, R5 is -CH2F, -CHF2, or -CF3, and R4 is -H or -F. In still other embodiments, R5 is -CH2F, -CHF5, or -CF3, R2 is -F, and R8 is -F. In still other embodiments, R5 is -CH2F, -CHF2, or -CF3, R3 is -F, and R7 is -F. In still other embodiments, R5 is -CH2F, -CHF2, or -CF3, R4 is -F, and R6 is -F.

[0073] In some embodiments, R2 is -H or -F and R3 is -H or -F. In other embodiments, R2 is -H or -F and R4 is -H or -F. In other embodiments, R3 is -H or -F and R4 is -H or -F. In other embodiments, R2 is -H or -F, R3 is -F and R7 is -F. In other embodiments, R2 is -H or -F, R4 is -F and R6 is -F. In other embodiments, R3 is -H or -F, R4 is -F and R6 is -F. In other embodiments, R2 is -F, R8 is -F and R3 is -H or -F. In other embodiments, R2 is -F, R8 is -F and R4 is -H or -F.

[0074] In some embodiments, R2 is -F and R8 is -F. In other embodiments, R3 is -F and R7 is -F. In yet other embodiments, R4 is -F and R6 is -F.

[0075] In some embodiments, R2 is -H or -F. In some other embodiments, R3 is -H or -F.

[0076] In yet other embodiments, R4 is -H or -F.

[0077] In some of the above embodiments, R9 to R 17 is independently alkyl, alkenyl, alkynyl, aryl, substituted aryl, cycloalkyl, cycloheteroalkyl, or heteroaryl. In other embodiments, R 17 is independently alkyl, alkenyl, aryl, substituted aryl, or cycloheteroalkyl. In still other of the above embodiments, R 17 is independently (C1-C4) alkyl, (C1-C4) alkenyl, phenyl, substituted phenyl, or (C5-C7) cycloheteroalkyl.

[0078] In some of the above embodiments, the anomeric carbon is the S stereoisomer. In others of the above embodiments, the anomeric carbon is the R stereoisomer.

[0079] In some of the above embodiments, R is R 18 C(O)NH—, where R 18 is a residue of a hydroxamic acid inhibitor. In other embodiments, R 18 is a residue of dacinostat, panobinostat, xinostat, or CUDC-907. In still other of the above embodiments, R1 is a residue of an HSP inhibitor. In still other of the above embodiments, R1 is a residue of a topoisomerase inhibitor. In still other of the above embodiments, R1 is a residue of an Akt1 inhibitor. In still other of the above embodiments, R1 is a residue of a DNA alkylating agent. In still other of the above embodiments, R1 is a residue of a Bcl2 inhibitor.

[0080] Hydroxamic acid derivative HDAC inhibitors include, but are not limited to, vorinostat (suberoylanilide hydroxamic acid or SAHA (1)), belinostat (2) and panobinostat (3). Several other hydroxamic acid derivative HDAC inhibitors (e.g., compounds (4)-(13)) have been investigated for the treatment of both hematological and solid tumors as single agents or in combination therapy with other oncolytic compounds. In addition to inhibiting various enzymes within HDAC classes I, II and IV, hydroxamic acid derivatives have been designed to concurrently inhibit other therapeutic targets, such as CUDC-101 (12) (which potently inhibits EGFR and HER-2 kinases) and CUDC-907 (13) (which further inhibits various PI3K isoforms). Many other hydroxamic acid derivative HDAC inhibitors have been disclosed, including the natural product trichostatin A (14), isolated from Streptomyces, and a number of synthetically derived compounds, representative examples of which include compounds (15)-(21) and others disclosed in Roche and Bertrand (supra); or any of the hydroxamic acids disclosed in U.S. Pat. Nos. 5,369,108, 5,932,616, 6,087,367, and 6,511,990. [ka] [ka] [ka]

[0081] Senescent cell ablative activity has been previously reported for the pan-HDAC inhibitor panobinostat (3) (Samaraweera et al., supra), and senescence has been shown to be associated with a decrease in global histone acetylation (Li et al., Proteomics 13 (2013) 2585-2596). Several reports have documented the reduction in Bcl-xL expression mediated by HDAC inhibitors (see, e.g., Cao et al., Am. J. Respir. Cell Mol. Biol. 25 (2001) 562-568; Rada-Iglesias et al., Genome Res. 17 (2007) 708-719; Frys et al., Br. J. Haematol. 169 (2015) 506-519). Without wishing to be bound by theory, one pharmacological basis for the senolytic activity of HDAC inhibitors may be mediated by a reduction in the levels of anti-apoptotic Bcl-xL protein.

[0082] Compounds of formula (III) or (IV) (wherein R1 is R 18 The hydroxamic acid HDAC inhibitors RCOH (V) and the sugar oxime compounds (VI) and (VII), which are C(O)NH-, are conveniently synthesized by coupling the carboxylic acid precursors of the hydroxamic acid HDAC inhibitors RCOH (V) with the sugar oxime compounds (VI) and (VII), respectively, in the presence of an acyl coupling reagent, such as a carbodiimide (e.g., EDC) or, alternatively, after prior activation as an acyl chloride or mixed acylating anhydride. [ka] Sugar alkoxyamines (VI) and (VII) can be prepared by conventional methods, for example from halo compounds (VIII) and (IX), respectively (X = Cl, Br or F) (Thomas et al. Bioorg. Med. Chem. Lett. 17 (2007) 983-986). [ka]

[0083] The HDAC inhibitory activity of hydroxamic acid derivative compounds is usually determined by the zinc chelating activity of the free hydroxamic acid moiety (see, e.g., Roche and Bertrand, supra). Thus, masking the hydroxamic acid functionality as a glycoside derivative in compounds of formula (III) or (IV) ensures that these prodrugs are inactive as HDAC inhibitors but are activated by hydrolysis in the lysosomes of senescent cells.

[0084] Hsp90 inhibitors include, but are not limited to, the resorcinol compounds AT13387 (onarespib, (22)), NYP-AUY922 (luminespib, (23)), ganetespib (24), VER-50589 (25), VER-49009 (26), CCT018159 (27) and KW-2478 (28), 2-(4-aminocyclohexanol)-benzamide derivatives exemplified by SNX-2112 (29) and (SNX-7081) (30). One of skill in the art will recognize that glycoconjugates of Hsp90 inhibitors exemplified by compounds of formulae (III) and (IV) are senolytic compounds. [ka]

[0085] Compounds of formula (III) and (IV), where R1 is an Hsp90 inhibitor, can be prepared by reaction of compounds of formula (X) with protected donor moieties (R2-R8 are not -OH) under classical BF3-mediated glycosylation or Koenigs-Knorr coupling conditions (Shie et al., Carbohydrate Res. 341 (2006) 443-456; Brough et al., J. Med. Chem. 51 (2008) 196-218), and the regioisomers thus obtained are separated by chromatographic means. Alternatively, the phenolic hydroxyl of the resorcinol compound (X) can be first selectively protected to allow regioselective glycosylation. [ka] [ka]

[0086] Topoisomerase I (TOPI) inhibitory compounds include, but are not limited to, camptothecin (31), SN-38 (32), topotecan (33) (see, e.g., Jain et al., Current Genomics 18 (2017) 75-92; Liu et al., Med. Res. Rev. 35 (2015) 753-789), indenoisoquinolines (exemplified by compounds (34)-(39) (Cineili et al., J. Med. Chem. 55 (2012) 10844-10862; Lv et al., J. Med. Chem. 59 (2016) 4890-4899)) and dibenzonaphthyridones (exemplified by compounds (40)-(42) (see, e.g., Sooryakumar et al., Mol. Cancer 2016: 111-113). Ther. 10(2011)1490-1499). [ka]

[0087] Compounds of formula (III) and formula (IV) (wherein R1 is a topoisomerase I (TOPI) inhibitor) may be prepared by the methods previously disclosed herein.

[0088] DNA alkylating agents include compounds such as DNA-reactive spirocyclopropylcyclohexadienones (46) derived from duocarmycin SA (43). Compounds (44) of formula (III) (see, for example, Tietze et al., Angew. Chem. Int. Ed. 45 (2006) 6574-6577; Tietze et al., J. Med. Chem. 52 (2009) 537-543) may be significantly less cytotoxic than the hydrolyzed seco product (45), which undergoes in situ so-called Winstein cyclization to give DNA-reactive spirocyclopropylcyclohexadienones (46) (those skilled in the art will recognize that compounds of formula (IV) can also be used in place of compounds of formula (III)). [ka]

[0089] The compound of formula (III), compound (44), is synthesized from compound (47) (prepared by the method of Tietze et al., op. cit.). [ka]

[0090] Other DNA alkylating agents include, for example, conjugates of cytotoxic pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) as senolytic agents. PBDs are a family of antitumor antibiotics, including the natural product anthramycin (53), that exert their cytotoxic effects by covalently binding to the exocyclic NH2 groups of guanine residues in the minor groove of DNA via their N10-C11 imine functional groups (see, for example, Antonow and Thurston, Chem. Rev. 111 (2011) 2815-2864; Mantaj et al., Angew. Chem. Int. Ed. 56 (2017) 462-488). PBD monomers exhibit considerable cytotoxicity, and by linking two PBD monomers via a linker, they give rise to PBD dimers that are capable of interstrand DNA cross-linking. SJG-136 (54) is one such dimer with high cytotoxic potency that has been used to construct antibody-drug conjugates with clinical utility. [ka]

[0091] The compound of formula (III), compound (55), is prepared from compound (56) (prepared by the method of Kamal et al., ChemMedChem 3 (2008) 794-802): [ka]

[0092] One skilled in the art will appreciate that a compound of formula (XII) can also be used in place of a compound of formula (XI) to provide a compound of formula (IV).

[0093] Compound (58), a compound of formula (III), may be prepared using a similar approach. Those skilled in the art will recognize that compounds of formula (IV) may be prepared in a similar manner. [ka]

[0094] Akt inhibitors include, but are not limited to, ipatasertib (or GDC-0068) (59), AZD5363 (60) and triciribine (61). [ka]

[0095] Compounds of formula (III) and formula (IV) (wherein R1 is an Akt inhibitor) may be prepared by the methods previously disclosed herein.

[0096] Proteasome inhibitors include, but are not limited to, delanzomib (62). [ka]

[0097] Bcl2 inhibitors include, but are not limited to, the compounds exemplified below. [ka]

[0098] Senocyte ablative compounds include those exemplified in Table 1 below. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0099] The senolytic compounds can be made by the methods illustrated below in Schemes 1 to 7. Other procedures for making senolytic compounds are within the realm of one of ordinary skill in the art.

[0100] Methods for characterizing and identifying senolytic agents Characterizing the senolytic agent can be determined using one or more cell-based assays and one or more animal models described herein or in the art and familiar to those skilled in the art. The senolytic agent can 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 can selectively kill at least senescent fibroblasts.

[0101] Characterizing an agent as a senolytic agent can be accomplished using one or more cell-based assays and one or more animal models described herein or in the art. Those skilled in the art will readily recognize that characterizing an agent as a senolytic agent and determining the level of killing by the agent can be accomplished by comparing the activity of the test agent to an appropriate negative control (e.g., vehicle or excipient only, and / or a composition or compound known in the art that does not kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing senolytic agents also include controls for determining the effect of the agent on non-senescent cells (e.g., quiescent or proliferating cells). Senescent cell-based agents reduce (i.e., decrease) the percent survival of a plurality of senescent cells (i.e., in some modes, reduce the amount of viable senescent cells in an animal or cell-based assay) compared to one or more negative controls. Conditions for a particular in vitro assay include temperature, buffers (including salts, cations, media), and other components that maintain the integrity of the test agents and reagents used in the assay, and are familiar to those of skill in the art and / or can be readily determined by routine experimentation.

[0102] The source of senescent cells for use in the assay can be a primary cell culture or a culture-adapted cell line, including, but not limited to, a genetically engineered cell line, immortalized or immortalizable cell line, somatic cell hybrid cell line, differentiated or differentiable cell line, transformed cell line, etc., that may contain a chromosomally integrated or episomal recombinant nucleic acid sequence. In some embodiments, the senescent cells are isolated from a biological sample obtained from a host or subject having a disease or disorder associated with senescent cells. In other embodiments, non-senescent cells may be obtained from a subject or may be a culture-adapted line, and senescence is induced by methods described herein and in the art, for example, by exposure to irradiation or chemotherapeutic agents (e.g., doxorubicin). The biological sample can be, for example, a blood sample, a biopsy specimen, a bodily fluid (e.g., lung lavage, ascites, mucosal lavage, synovial fluid, etc.), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation obtained from a subject. A biological sample may be a tissue or cell preparation whose morphological integrity or physical state has been disturbed by, for example, dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, micronization, lyophilization, 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.

[0103] Transgenic animal models as described herein and in the art may be used to determine the death or elimination of senescent cells (see, e.g., Baker et al., supra; Nature, 479 (2011) 232-236; International Application WO 2012 / 177927; International Application WO 2013 / 090645). An exemplary transgenic animal model contains a transgene comprising a nucleic acid that allows for controlled elimination of senescent cells (e.g., p16INK4a positive senescent cells) as a positive control. The presence and level of senescent cells in the transgenic animal can be determined by measuring a detectable label or the level of a label expressed in the senescent cells of the animal. The transgene nucleotide sequence comprises a detectable label, e.g., one or more of red fluorescent protein; green fluorescent protein; and one or more luciferases to detect the elimination of senescent cells.

[0104] The animal models described herein or in the art include art-recognized models for determining the efficacy of senolytic agents to treat or prevent (i.e., reduce the likelihood of occurrence) a particular aging-associated disease or disorder, such as an atherosclerosis model, an osteoarthritis model, a COPD model, an IPF model, etc. As described herein, lung disease mouse models, such as the bleomycin pulmonary fibrosis model, and the chronic cigarette smoking model, are applicable to diseases, such as COPD, and may be routinely performed by one of skill in the art. Animal models for determining the efficacy of senolytic agents to treat and / or prevent (i.e., reduce the likelihood of occurrence) chemotherapy and radiotherapy side effects models, or to treat or prevent metastasis (i.e., reduce the likelihood of occurrence) are described in International Patent Publication Nos. WO 2013 / 090645 and WO 2014 / 205244. Animal models for determining the efficacy of drugs for treating ocular diseases, particularly age-related macular degeneration, are also routinely used in 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).

[0105] As a non-limiting example and as described herein, osteoarthritis animal models have been developed. For example, osteoarthritis can be induced in animals by inducing injury to a joint, e.g., the knee, by surgical transection (incomplete or complete) of the anterior cruciate ligament. The osteoarthritis animal model can be used to assess the effectiveness of senolytic agents that treat or prevent osteoarthritis (i.e., reduce the likelihood of its occurrence), cause a reduction in proteoglycan erosion, induce (i.e., stimulate, enhance) collagen (e.g., type 2 collagen) production, and reduce pain in animals undergoing ACL surgery. Immunohistology can be performed to examine the integrity and composition of tissues and cells in the joint. Immunochemistry and / or molecular biology techniques can also be performed, such as assays to determine the levels of inflammatory molecules (e.g., IL-6), and assays to determine the levels of senescence markers as described above, using the methods and techniques described herein that can be routinely performed by those skilled in the art.

[0106] As another non-limiting example and as described herein, atherosclerosis animal models have been developed. Atherosclerosis can be induced in animals, for example, by feeding the animals a high-fat diet or by using transgenic animals that are highly susceptible to the development of atherosclerosis. The animal models can be used to determine the effectiveness of senolytic agents that reduce the amount of plaque in atherosclerotic arteries or inhibit the formation of plaque, reduce the lipid content of atherosclerotic plaques (i.e., reduce, decrease the amount of lipids in the plaque), and cause or promote an increase in the fibrous cap thickness of the plaque. Sudan staining can be used to detect the level of lipids in atherosclerotic blood vessels. Immunohistology and immunochemistry and molecular biology assays (e.g., for determining the levels of inflammatory molecules (e.g., IL-6) and for determining the levels of senescence markers as mentioned above) can all be performed by methods routinely performed in the art and described herein.

[0107] In yet another non-limiting example, a mouse model in which animals are treated with bleomycin as described herein has been described to determine the efficacy of drugs to treat IPF (see, e.g., Peng et al., PLoS One 8(4)(2013)e59348.doi:10.1371 / journal.pone.0059348; Mouratis et al., Curr.Opin.Pulm.Med.17(2011)355-361). In pulmonary disease animal models (e.g., bleomycin animal models, smoke exposure animal models, etc.), respiration measurements can be performed to determine elastance, compliance, static compliance, and peripheral capillary oxygen saturation (SpO2). Immunohistology and immunochemistry and molecular biology assays (e.g., for determining levels of inflammatory molecules (e.g., IL-6) and for determining levels of senescence markers as described above) can all be performed by methods described herein that are routinely performed in the art.

[0108] Determining the effectiveness of the senolytic agent in selectively killing senescent cells as described herein in an animal model may be performed using one or more statistical analyses familiar to those skilled in the art. For example, statistical analyses, such as two-way analysis of variance (ANOVA), may be used to determine the statistical significance of differences between animal groups treated with the agent and groups not treated with the agent (i.e., a negative control group that may contain only vehicle 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.

[0109] Those skilled in the art will readily recognize that characterizing a senolytic agent and determining the level of killing by the senolytic agent can be accomplished by comparing the activity of the test agent with an appropriate negative control (e.g., vehicle only and / or a composition, agent, or compound known in the art not to kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing agents also include controls to determine the effect of the agent on non-senescent cells (e.g., quiescent or proliferating cells). Useful senolytic agents reduce (i.e., decrease) the percent survival of senescent cells (i.e., in some manner, reduce the amount of viable senescent cells in an animal or cell-based assay) compared to one or more negative controls. Thus, senolytic agents selectively kill senescent cells compared to the killing of non-senescent cells (which may be referred to herein as selectively killing senescent cells over non-senescent cells).

[0110] In certain embodiments (in in vitro assays or in vivo (in a human or non-human animal)), the at least one senolytic agent kills at least 20% of senescent cells and kills 5% or less of non-senescent cells. In other embodiments (in in vitro assays or in vivo (in a human or non-human animal)), the at least one senolytic agent kills at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills about 5% or less or 10% or less of non-senescent cells. In yet other embodiments (in in vitro assays or in vivo (in a human or non-human animal)), the at least one senolytic agent kills at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills about 5%, 10%, or 15% or less of non-senescent cells. In yet other embodiments (in in vitro assays or in vivo (in a human or non-human animal)), the at least one senolytic agent kills at least about 40%, 45%, 50%, 55%, 60%, or 65% of senescent cells and kills about 5%, 10%, 15%, 20%, or 25% or less of non-senescent cells. In yet other embodiments (in in vitro assays or in vivo (in a human or non-human animal)), at least one senolytic agent kills at least about 50%, 55%, 60%, or 65% of senescent cells and kills about 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, or 30% or less of non-senescent cells. Stated another way, the senolytic agent has at least 5-25, 10-50, 10-100, or 100-1000 times greater selectivity for killing senescent cells over non-senescent cells.

[0111] With respect to certain embodiments of the methods described herein for treating a disease or disorder associated with aging, the percentage of dead senescent cells may refer to the percentage of dead senescent cells in a tissue or organ that contains senescent cells that contribute to the initiation, progression, and / or exacerbation of the disease or disorder. As non-limiting examples, brain tissue, eye tissue and parts, lung tissue, heart tissue, arteries, joints, skin, and muscles may contain senescent cells that can be reduced in such percentages by the senolytic agents described herein, thereby achieving a therapeutic effect. Furthermore, selectively removing at least 20% or at least 25% of senescent cells from an affected tissue or organ can have a clinically meaningful therapeutic effect.

[0112] In certain embodiments of the methods described herein, e.g., treating a cardiovascular disease or disorder associated with arteriosclerosis, e.g., atherosclerosis, by administering a senolytic agent (i.e., in connection with the in vivo methods described above), the percentage of killed senescent cells may refer to the percentage of killed senescent cells in the affected artery containing the plaque, relative to the non-senescent cells killed in the arterial plaque. In certain embodiments, in the methods for treating a cardiovascular disease, e.g., atherosclerosis, as described herein, at least one senolytic agent kills at least 20% of senescent cells and kills no more than 5% of non-senescent cells in the artery. In other embodiments, the senolytic agent selectively kills at least 25% of senescent cells in the arteriosclerotic artery.

[0113] In some embodiments, with respect to the methods described herein for treating osteoarthritis by administering a senolytic agent, the percentage of senescent cells killed may refer to the percentage of senescent cells killed in the osteoarthritic joint relative to the non-senescent cells killed in the osteoarthritic joint. In certain embodiments, in the methods for treating osteoarthritis as described herein, at least one senolytic agent kills at least 20% of senescent cells and kills 5% or less of non-senescent cells in the osteoarthritic joint. In other embodiments, the senolytic agent selectively kills at least 25% of senescent cells in the osteoarthritic joint.

[0114] In some embodiments, with respect to the methods described herein for treating a senescence-associated lung disease or disorder (e.g., COPD, IPF) by administering at least one senolytic agent, the percentage of killed senescent cells may refer to the percentage of killed senescent cells in the affected lung tissue relative to the percentage of killed non-senescent cells in the affected lung tissue. In certain embodiments, in the methods for treating a senescence-associated lung disease or disorder as described herein, the senolytic agent kills at least 20% of senescent cells and kills 5% or less of non-senescent cells in the affected lung tissue. In other embodiments, the senolytic agent selectively kills at least 25% of senescent cells in the affected lung tissue.

[0115] In certain embodiments, methods are provided for identifying (i.e., screening) agents that are useful senolytic agents for treating or preventing (i.e., reducing the likelihood of occurrence of) diseases or disorders associated with senescence. In some embodiments, methods for identifying senolytic agents for treating such diseases and disorders include inducing cells to undergo senescence and providing established senescent cells. Methods for inducing cells to undergo senescence are described herein and in the art and include, for example, exposure to radiation (e.g., 10 Gy is typically sufficient) or chemotherapeutic agents (e.g., doxorubicin or other anthracyclines). Following exposure to the agent, the cells are cultured for an appropriate time and under appropriate conditions (e.g., medium, temperature, CO2 / O2 levels appropriate for a given cell type or cell line) to allow senescence to become established. As discussed herein, cellular senescence can be determined by any number of characteristics, such as changes in morphology (e.g., as viewed by a microscope); for example, by determining the production of galactosidase (SA-gal), p16INK4a, p21, or any one or more SASP factors (e.g., IL-6, MMP3) associated with senescence. The sample of senescent cells is then contacted with a candidate agent (i.e., mixed, combined, or in some manner allowing the cells and agent to interact). Those skilled in the art will recognize that the assay includes appropriate controls (negative and positive) performed historically or in parallel. For example, a sample of control non-senescent cells that have been cultured similarly to the senescent cells but have not been exposed to a senescence-inducing agent is contacted with the candidate agent. The level of survival of the senescent cells is determined and compared to the level of survival of the non-senescent cells. When the level of survival of the senescent cells is less than the level of survival of the non-senescent cells, a senolytic agent is identified.

[0116] In some embodiments, the above method of identifying a senolytic agent may further include a step of identifying whether the senolytic agent is useful for treating osteoarthritis. The method may further include contacting the identified senolytic agent with a cell capable of producing collagen; and determining the level of collagen produced by the cell. In some embodiments, the cell is a chondrocyte, and the collagen is type 2 collagen. The method may further include administering a candidate senolytic agent 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 physical function of the animal; (c) the level of one or more markers of inflammation; (d) joint histology; and (e) the level of type 2 collagen produced, thereby determining the therapeutic efficacy of the senolytic agent, wherein one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) a decrease in the level of senescent cells in the joints of the treated animal; (ii) improved physical function of the treated animal; (iii) a decrease in the level of one or more markers of inflammation in the treated animal; (iv) an increase in histological normality in the joints of the treated animal; and (v) an increase in the level of type 2 collagen produced in the treated animal. As described herein and in the art, the physical function of an animal can be determined by techniques that determine the susceptibility of the foot to induced or natural osteoarthritis conditions, for example, by the animal's resistance to bearing weight on the affected limb, or the animal's ability to move away from unpleasant stimuli, such as heat or cold. Determining the effectiveness of an agent that kills senescent cells as described herein in an animal model can be performed using one or more statistical analyses that are familiar to those skilled in the art. Data can be analyzed by applying statistical analyses as described herein and routinely performed in the art.

[0117] In other embodiments, the above method of identifying a senolytic agent may further include a step for identifying whether the senolytic agent is useful for treating a cardiovascular disease caused by or associated with arteriosclerosis. Thus, the method may further include administering the senolytic candidate agent in a non-human animal or animal model to determine the effectiveness of the agent in reducing the amount of plaque, inhibiting the formation of plaque in an atherosclerotic artery, reducing the lipid content of atherosclerotic plaque (i.e., reducing, decreasing the amount of lipid in the plaque), and / or causing or promoting an increase in the fibrous cap thickness of the plaque. Sudan staining may be used to detect the level of lipids in atherosclerotic blood vessels. Immunohistochemical assays for determining the level of inflammatory molecules (e.g., IL-6) and / or assays for determining the level of senescence markers as described above may all be performed by methods described herein and routinely performed in the art.

[0118] In certain embodiments, the methods described herein for identifying senolytic agents may further include administering a candidate senolytic agent to a non-human animal having an atherosclerotic plaque and determining one or more of: (a) the level of senescent cells in an artery; (b) the animal's physical function; (c) the level of one or more markers of inflammation; (d) the histology of the affected blood vessel (e.g., artery); thereby determining the therapeutic efficacy of the senolytic agent, where one or more of the following are observed in the treated animal compared to an animal not treated with the senolytic agent: (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 markers of inflammation in the treated animal; (iv) an increase in 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 may be applied to analyze the data as described herein and as routinely performed in the art.

[0119] In some embodiments, the methods described herein for identifying senolytic agents may include administering a candidate senolytic agent to a non-human animal lung disease model, such as a bleomycin model or a smoke-exposed animal model, and 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 markers of inflammation; (d) the histology of the lung tissue, thereby determining the therapeutic efficacy of the senolytic agent, where one or more of the following is observed in the treated animal compared to an animal not treated with the senolytic agent: (i) a decrease in the level of senescent cells in the lungs and lung tissue of the treated animal; (ii) improved lung function of the treated animal; (iii) a decrease in the level of one or more markers of inflammation in the treated animal; and (iv) an increase in histological normality in the lung tissue of the treated animal. Respiratory measurements may be taken to determine elastance, compliance, static compliance, and peripheral capillary oxygen saturation (SpO2). Pulmonary function may be assessed by determining any one of a number of measurements, such as expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in 1 second, FEV1), FEV1 / FEV ratio, forced expiratory flow rate of 25% to 75%, and maximum voluntary ventilation (MVV), peak expiratory flow rate (PEF), slow exhaust vital capacity (SVC). Total lung capacity includes total lung capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange across the alveolar-capillary membrane may be measured using the diffusion capacity for carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO2) may also be measured. Data may be analyzed by applying statistical analysis as described herein and routinely performed in the art.

[0120] Methods for treating and preventing age-related diseases and disorders Provided herein is a method for treating conditions, diseases, or disorders associated with, related to, or caused by cellular senescence, including age-related diseases and disorders, in a subject in need thereof. Aging-related diseases or disorders may also be referred to herein as senescent cell-related diseases or disorders. Aging-related diseases and disorders include, for example, cardiovascular diseases and disorders, inflammatory diseases and disorders, autoimmune diseases and disorders, pulmonary diseases and disorders, eye diseases and disorders, metabolic diseases and disorders, nervous system diseases and disorders (e.g., neurodegenerative diseases and disorders); age-related diseases and disorders induced by senescence; skin conditions; age-related diseases; skin diseases and disorders; and transplantation-related diseases and disorders. A hallmark of aging is the gradual loss of function or degeneration that occurs at the molecular, cellular, tissue, and organism levels. Age-related degeneration gives rise to well-recognized pathologies, such as sarcopenia, atherosclerosis and heart failure, osteoporosis, pulmonary insufficiency, renal failure, neurodegeneration (including macular degeneration, Alzheimer's disease, and Parkinson's disease), and many others. Although different mammalian species vary in their susceptibility to certain age-related pathologies, collectively, age-related pathologies generally occur with roughly exponential kinetics beginning at approximately the midpoint of the species-specific life span (e.g., 50-60 years for humans) (see, e.g., Campisi, Annu. Rev. Physiol. 75 (2013) 685-705; Naylor et al., Clin. Pharmacol. Ther. 93 (2013) 105-116).

[0121] Examples of conditions, disorders, or diseases associated with aging that may be treated by administering any one of the senolytic agents described herein according to the methods 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 dysfunction, aortic aneurysm, angina, arrhythmias, 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, metabolic syndrome); motor function diseases and disorders (e.g., Parkinson's disease, motor neuron dysfunction (MND); Huntington's disease); cerebrovascular diseases; emphysema; osteoarthritis; benign prostatic hyperplasia; pulmonary diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD)); D), emphysema, bronchiolitis obliterans, asthma); inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); eye diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, blindness, presbyopia); diabetic ulcers; metastases; chemotherapy side effects, radiation therapy side effects; age-related diseases and disorders (e.g., kyphosis, renal dysfunction, frailty, hair loss, hearing loss, muscle wasting, skin conditions, sarcopenia, and herniated discs) and other age-related diseases induced by aging (e.g., diseases / disorders due to irradiation, chemotherapy, tobacco smoking, eating a fatty / sugar diet, and environmental factors); wound healing; skin nevi; fibrotic diseases and disorders (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucous fibrosis, cardiac fibrosis, and pancreatic fibrosis). In certain embodiments, any one or more of the diseases or disorders listed above or described herein may be excluded.

[0122] In some embodiments, a method is provided for treating a disease or disorder associated with senescence by killing senescent cells (i.e., established senescent cells) associated with the disease or disorder in a subject having the disease or disorder by administering a senolytic agent, where the disease or disorder is osteoarthritis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease (COPD); or atherosclerosis.

[0123] Cardiovascular Diseases and Disorders In another embodiment, the aging-related disease or disorder treated by the methods described herein is a cardiovascular disease. The cardiovascular disease can be any one or more of angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease (CAD), carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction [MI]), high blood pressure / hypertension, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, cardiac diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral arterial disease (PAD)), cardiac stress resistance, and stroke.

[0124] In certain embodiments, a method is provided for treating aging-related cardiovascular disease associated with or caused by arteriosclerosis (i.e., hardening of arteries). The cardiovascular disease may be any 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 arterial disease (PAD)). The method for treating cardiovascular disease associated with or caused by arteriosclerosis may reduce the likelihood of occurrence of high blood pressure / hypertension, angina pectoris, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In certain embodiments, a method is provided for stabilizing atherosclerotic plaques in a subject's blood vessel (e.g., artery), thereby reducing the likelihood of occurrence or delaying the occurrence of thrombotic events, such as stroke or myocardial infarction. In certain embodiments, these methods involving administration of a senolytic agent reduce (i.e., result in a reduction in) the lipid content of atherosclerotic plaque and / or increase fibrous cap thickness (i.e., increase, enhance or promote fibrous cap thickening) in a blood vessel (e.g., an artery) of a subject.

[0125] Atherosclerosis is characterized by patchy intimal plaques (atheromas) that encroach on the lumen of medium and large arteries; the plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect large and medium arteries, including coronary, carotid, and cerebral arteries, the aorta and its branches, and major arteries of the limbs. In some embodiments, methods are provided for inhibiting the formation of atherosclerotic plaques (or reducing, lowering, causing a decrease in the formation of atherosclerotic plaques) by administering a senolytic agent. In other embodiments, methods are provided for reducing (reducing, lowering) the amount (i.e., level) of plaque. A reduction in the amount of plaque in a blood vessel (e.g., an artery) can be determined, for example, by a reduction in the surface area of ​​plaque or by a reduction in the extent or degree (e.g., percent) of obstruction of the blood vessel (e.g., an artery), which can be determined by angiography or other visualization methods used in the cardiovascular artery. Also provided herein is a method of increasing the stability (or improving, promoting, enhancing the stability) of atherosclerotic plaque present in one or more blood vessels (e.g., one or more arteries) in a subject, the method comprising administering to the subject any one of the senolytic agents described herein.

[0126] Subjects suffering from cardiovascular disease can be identified using standard diagnostic methods known in the art for cardiovascular disease. In general, the diagnosis of atherosclerosis and other cardiovascular diseases is based on the patient's symptoms (e.g., chest pain or pressure (angina), numbness or weakness in the arms or legs, difficulty speaking or slurred speech, drooping facial muscles, leg pain, high blood pressure, renal failure and / or erectile dysfunction), medical history, and / or physical examination. Diagnosis may be confirmed by angiography, ultrasound, or other imaging tests. Subjects at risk of developing cardiovascular disease include those with any one or more predisposing factors, such as a family history of cardiovascular disease, as well as those with other risk factors (i.e., predisposing factors), such as high blood pressure, dyslipidemia, high cholesterol, diabetes, obesity and cigarette smoking, sedentary lifestyle, and high blood pressure. In certain embodiments, the cardiovascular disease that is a disease / disorder associated with senescent cells is atherosclerosis.

[0127] The efficacy of one or more senolytic agents for treating or preventing (i.e., reducing or decreasing the likelihood of the occurrence or occurrence of) cardiovascular disease (e.g., atherosclerosis) can be readily determined by one skilled in the medical and clinical arts. The health status of a subject can be monitored using one or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performing analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress test, non-stress test). The effect of treatment with a senolytic agent, or a pharmaceutical composition comprising the same, can be analyzed using techniques known in the art, such as comparing the symptoms of patients suffering from or at risk for cardiovascular disease who have been treated with those of patients who have not been treated with such treatment or who have been treated with a placebo.

[0128] Inflammatory and autoimmune diseases and disorders In certain embodiments, the disease or disorder associated with senescence is an inflammatory disease or disorder, such as, by way of non-limiting example, osteoarthritis, which may be treated or prevented (i.e., reduce the likelihood of occurrence) by the methods described herein involving administration of a senolytic agent. Other inflammatory or autoimmune diseases or disorders that may be treated by administering a senolytic agent, such as the inhibitors and antagonists described herein, include osteoporosis, psoriasis, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated disc, and the pulmonary diseases COPD and idiopathic pulmonary fibrosis.

[0129] Osteoarthritis, a degenerative joint disease, is characterized by fibrillation of cartilage tissue at sites of high mechanical stress, bone sclerosis, and thickening of the synovium and joint capsule. Fibrillation is a localized superficial disorganization involving splitting of the superficial layers of cartilage tissue. Early splitting is loosely associated with the cartilage surface and follows the axis of the predominant collagen bundle. Collagen within the cartilage disorganizes and proteoglycans are lost from the cartilage surface. In the absence of the protective and lubricating effects of proteoglycans in the joint, collagen fibers become susceptible to degradation and mechanical destruction ensues. Predisposing risk factors for the development of osteoarthritis include aging, obesity, previous joint injury, joint overuse, weak thigh muscles, and genetics. Symptoms of osteoarthritis include sore or stiff joints, especially in the hips, knees, and lower back after inactivity or overuse; stiffness after rest that subsides after movement; and pain that worsens after activity or toward the end of the day. Osteoarthritis can also affect the neck, pinky knuckles, base of the thumb, ankle, and big toe. Chronic inflammation is thought to be the major age-related factor contributing to osteoarthritis. Overuse of the joints and obesity, combined with aging, appear to promote osteoarthritis.

[0130] By selectively killing senescent cells, the senolytic agent prevents (i.e., reduces the likelihood of occurrence), reduces or inhibits loss or erosion of the proteoglycan layer in the joint, reduces inflammation in the affected joint, and promotes (i.e., stimulates, enhances, induces) collagen (e.g., type 2 collagen) production. Removal of senescent cells results in a reduction in the amount (i.e., level) of inflammatory cytokines, such as IL-6, produced in the joint, reducing inflammation. Provided herein are methods for selectively killing senescent cells in osteoarthritic joints of a subject and / or inducing collagen (e.g., type 2 collagen) production in a subject's joints for treating osteoarthritis by administering to the subject at least one senolytic agent (which may be combined with at least one pharma- ceutically acceptable excipient to form a pharmaceutical composition). The senolytic agent may also be used to decrease (inhibit, reduce) the production of metalloproteinase 13 (MMP-13), which breaks down collagen, in joints, and to repair the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Treatment with the senolytic agent may also thereby prevent (i.e., reduce the likelihood of its occurrence), inhibit, or reduce erosion, or slow down (i.e., reduce the rate of) bone erosion. As described in detail herein, in certain embodiments, the senolytic agent is administered directly to the osteoarthritic joint (e.g., by intra-articular, topical, transcutaneous, intradermal, or subcutaneous delivery). Treatment with the senolytic agent may also repair, improve, or inhibit the deterioration of joint strength. Furthermore, methods including administering the senolytic agent may reduce joint pain, and are therefore useful for pain management of osteoarthritic joints.

[0131] The effectiveness of one or more senolytic agents for the treatment or prevention of osteoarthritis in a subject, and the monitoring of a subject receiving one or more senolytic agents, can be readily determined by one skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination (e.g., determining tenderness, swelling, or redness of the affected joint), assessment and monitoring of clinical symptoms (e.g., pain, stiffness, mobility), and performance of analytical tests and methods described herein and practiced in the art (e.g., determining levels of inflammatory cytokines or chemokines; X-rays to determine loss of cartilage tissue as indicated by narrowing of the space between bones in a joint; magnetic resonance imaging (MRI), providing detailed images of bones and soft tissues, including cartilage tissue), may be used to monitor the health status of a subject. The effect of treatment with one or more senolytic agents can be analyzed by comparing symptoms of patients suffering from or at risk for an inflammatory disease or disorder, e.g., osteoarthritis, who have received the treatment with symptoms of patients who have not received such treatment or who have received a placebo treatment.

[0132] In certain embodiments, senolytic agents may be used to treat and / or prevent (i.e., reduce or decrease the likelihood of occurrence) rheumatoid arthritis (RA). Dysregulation of innate and adaptive immune responses characterizes rheumatoid arthritis (RA), an autoimmune disease whose incidence increases with age. Rheumatoid arthritis is a chronic inflammatory disorder that typically affects small joints in the hands and feet. While osteoarthritis results at least in part from wear and tear on the joints, rheumatoid arthritis affects the lining of the joints, causing painful swelling that can lead to bone erosion and joint deformation. RA may also occasionally affect other organs of the body, such as the skin, eyes, lungs, and blood vessels. RA may occur in subjects of any age; however, RA usually begins to occur after age 40. The disorder is much more common in women. In certain embodiments of the methods described herein, RA is excluded.

[0133] Chronic inflammation may also contribute to other age-related or aging-related diseases and disorders, such as kyphosis and osteoporosis. Kyphosis is a severe curvature in the spine that is often seen with normal and premature aging (see, for example, Katzman et al., J. Orthop. Sports Phys. Ther. 40 (2010) 352-360). Age-related kyphosis often occurs after osteoporosis weakens the spinal bones enough to crack and compress. Some types of kyphosis target infants or teenagers. Some kyphosis can affect the lungs, nerves, and other tissues and organs, resulting in pain and other problems. Kyphosis has been associated with cellular senescence. Characterizing the performance of senolytic agents to treat kyphosis can be determined in preclinical animal models used in the art. As an example, TTD mice develop kyphosis (see, e.g., de Boer et al., Science 296 (2002) 1276-1279); other mice that can be used include BubR1 mice, which are also known to develop kyphosis (see, e.g., Baker et al., Nature 479 (2011) 232-236). Kyphosis formation is measured visually over time. The level of senescent cells reduced by treatment with a senolytic agent can be determined by detecting the presence of one or more markers associated with senescent cells, for example, by SA-□-Gal staining.

[0134] Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density that can lead to an increased risk of fracture, which can be treated or prevented by administration of the senolytic agents described herein. Bone mineral density (BMD) decreases, bone microarchitecture deteriorates, and the amount and type of protein in bone changes. Osteoporosis is typically diagnosed and monitored by bone mineral density testing. Postmenopausal women or women with reduced estrogen are at greatest risk. While both men and women over 75 years of age are at risk, women are twice as likely as men to develop osteoporosis. The level of senescent cells reduced by treatment with a senolytic agent can be determined by detecting the presence of one or more markers associated with senescent cells, for example, by SA-□-Gal staining.

[0135] In yet other embodiments, inflammatory / autoimmune disorders that may be treated or prevented (i.e., the likelihood of occurrence is reduced) with the senolytic agents described herein include irritable bowel syndrome (IBS) and inflammatory bowel disease, such as ulcerative colitis and Crohn's disease. Inflammatory bowel disease (IBD) involves chronic inflammation of all or parts of the digestive tract. In addition to the life-threatening complications that result from IBD, the disease can be painful and debilitating. Ulcerative colitis is an inflammatory bowel disease that results in long-lasting inflammation in parts of the digestive tract. Symptoms usually occur over time rather than suddenly. Ulcerative colitis usually affects only the innermost lining of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that results in inflammation anywhere along the lining of the digestive tract, often spreading deep into the affected tissues. This can cause abdominal pain, severe diarrhea, and malnutrition. The inflammation caused by Crohn's disease can involve different areas of the digestive tract. Diagnosis and monitoring of disease is performed by methods and diagnostic tests routinely performed in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small bowel imaging.

[0136] Other inflammatory or autoimmune diseases that may be treated or prevented (i.e., the likelihood of occurrence is reduced) by using senolytic agents include eczema, psoriasis, osteoporosis, and pulmonary 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 submucous fibrosis, may be treated with the senolytic agents described herein.

[0137] In certain embodiments, the disorder associated with senescent cells is an inflammatory disorder of the skin that can be treated or prevented (i.e., reduce the likelihood of occurrence) by the methods described herein, including administration of a senolytic agent, such as, for example and without limitation, psoriasis and eczema. Psoriasis is characterized by abnormally excessive and rapid growth of the epidermal layer of the skin. Diagnosis of psoriasis is usually based on the appearance of the skin. Typical skin features of psoriasis are scaly erythema, papules, or patches of skin that can be painful and itchy. In psoriasis, cutaneous and systemic overexpression of various pro-inflammatory cytokines, such as IL-6, an important component of the SASP, is observed. Eczema is an inflammation of the skin characterized by redness, skin swelling, itching, and dryness, crusting, peeling, blistering, cracking, weeping, or bleeding. The effectiveness of senolytic agents for the treatment of psoriasis and eczema, as well as the monitoring of subjects receiving such senolytic agents, can be readily determined by those skilled in the art of medicine or clinical sciences. One or any combination of diagnostic methods including physical examination (e.g., appearance of the skin), assessment or monitoring of clinical symptoms (e.g., itching, swelling, and pain), and performing analytical tests and methods described herein and practiced in the art (i.e., determining levels of pro-inflammatory cytokines).

[0138] Other immune disorders or conditions that may be treated or prevented (i.e., reduce the likelihood of occurrence) with the senolytic agents described herein include conditions resulting from a host immune response to an organ transplant (e.g., kidney, bone marrow, liver, lung, or heart transplant), e.g., rejection of the transplanted organ. The senolytic agents described herein may also be used to treat or reduce the likelihood of occurrence of graft-versus-host disease.

[0139] Lung Diseases and Disorders In some embodiments, methods are provided for treating or preventing (i.e., reducing the likelihood of occurrence of) a senescence-associated disease or disorder that is a pulmonary disease or disorder by killing senescent cells (i.e., established senescent cells) associated with the disease or disorder in a subject having the disease or disorder by administering a senolytic agent described herein. Senescence-associated pulmonary diseases and disorders include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

[0140] COPD is a lung disease defined by persistent poor airflow resulting from the breakdown of lung tissue (emphysema) and dysfunction of the small airways (obstructive bronchiolitis). Primary symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excess sputum production. Elastase from neutrophils and macrophages activated by cigarette smoke destroys the extracellular matrix of alveolar structures, resulting in enlarged air spaces and loss of respiratory capacity (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 smoking, second-hand smoke, and pipe smoking), occupational exposure (e.g., exposure to dust, smoke, or fumes), and pollution, which occurs over decades, thereby implicating aging as a risk factor for developing COPD.

[0141] Processes involved in causing lung damage include, for example, oxidative stress produced by high concentrations of free radicals in cigarette smoke; cytokine release due to inflammatory responses to irritants in the airways; and impaired function of antiprotease enzymes by cigarette smoke and free radicals, allowing proteases to damage the lungs. Genetic susceptibility may also contribute to the disease. In about 1% of people with COPD, the disease is caused by a genetic disorder that results in low levels of alpha-1-antitrypsin production in the liver. This enzyme is normally secreted into the bloodstream to help protect the lungs.

[0142] Pulmonary fibrosis is a chronic and progressive lung disease characterized by lung stiffening and scarring, which can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblasts are activated, production of extracellular matrix proteins increases, and transdifferentiation into contractile myofibroblasts contributes to wound contraction. A temporary matrix seals the injured epithelium and provides a scaffold for epithelial cell migration involving epithelial-mesenchymal transition (EMT). Blood loss associated with epithelial injury induces platelet activation, production of growth factors, and an acute inflammatory response. Usually, the epithelial barrier heals and the inflammatory response resolves. However, in fibrotic diseases, the fibroblast response continues and wound healing remains unresolved. The formation of fibroblastic nests is a hallmark of the disease and reflects the location of ongoing fibrosis. As the name implies, the pathogenesis of IPF is unknown. The involvement of cellular senescence in IPF has been suggested by the observation that the incidence of the disease increases with age and that lung tissue in IPF patients is enriched for SA-□-Gal-positive cells and contains elevated levels of the senescence marker p21 (see, e.g., Minami et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 300 (2011) L391-L401; see also, e.g., Naylor et al., supra). Short telomers are a common risk factor for both IPF and cellular senescence (see, e.g., Alder et al., Proc. Natl. Acad. Sci. USA 105 (2008) 13051-13056). Without wishing to be bound by theory, the contribution of cellular senescence to IPF has been suggested by reports that SASP components of senescent cells, e.g., IL-6, IL-8, and IL-1□, promote fibroblast-to-myofibroblast differentiation and epithelial-mesenchymal transition, leading to extensive remodeling of the extracellular matrix of the alveoli and interstitial spaces (see, e.g., Minagawa et al., supra).

[0143] Subjects at risk of developing pulmonary fibrosis include those exposed to environmental or occupational pollutants, such as asbestosis and silicosis; those who smoke cigarettes; those with some typical connective tissue diseases, such as rheumatoid arthritis, SLE, and scleroderma; those with other diseases involving connective tissue, such as sarcoidosis and Wegener's granulomatosis; those with infections; those taking certain medications (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin); those who have been subjected to radiation therapy to the chest; and those with a family history of pulmonary fibrosis.

[0144] Symptoms of COPD may include any one of the following: shortness of breath, especially during physical activity; wheezing; chest tightness; excess mucus in the lungs, which requires clearing the throat first thing in the morning; chronic coughing that produces phlegm, which may be clear, white, yellowish, or greenish in color; blue lips or nail beds (cyanosis); frequent airway infections; lack of energy; unintentional weight loss (observed in later stages of the disease). Subjects with COPD may also experience exacerbations, during which symptoms worsen and last for several days or longer. Symptoms of pulmonary fibrosis are known in the art and include shortness of breath, especially during exercise; dry, harsh cough; rapid, shallow breathing; gradual unintentional weight loss; fatigue; painful joints and muscles; and clubbing (the tips of the fingers or toes become enlarged and rounded).

[0145] Subjects suffering from COPD or pulmonary fibrosis can be identified using standard diagnostic methods routinely practiced in the art. Monitoring the effect of one or more senolytic agents administered to subjects with or at risk of developing pulmonary disease can be performed using methods typically used for diagnosis. In general, one or more of the following examinations or tests can be performed: physical examination, patient medical history, patient family medical history, chest x-ray, pulmonary function tests (e.g., spirometry), blood tests (e.g., arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CT scan, and exercise testing.

[0146] Other lung diseases or disorders that may be treated by using senolytic 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 may also be exacerbated by tobacco smoke (including cigarette smoke, cigar smoking, second-hand smoke, and pipe smoking), occupational exposures (e.g., exposure to dust, smoke, or fumes), infections, and / or pollutants, which induce cells into senescence, thereby contributing to inflammation. Emphysema is sometimes considered to be a subset of COPD.

[0147] Bronchiectasis results from damage to the airways, causing them to become enlarged, sagging, and compromised. Bronchiectasis is usually caused by a medical condition that damages the airway walls or prevents the airways from clearing mucus. Examples of such conditions include cystic fibrosis and primary ciliary dyskinesia (PCD). When only a portion of the lung is affected, the damage may be caused by a blockage rather than a medical condition.

[0148] The methods described herein for treating or preventing (i.e., reducing the likelihood or occurrence of) aging-related lung diseases or disorders may also be used to treat subjects who are elderly and have 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 chest wall and thoracic vertebrae deformations that can impair total respiratory system compliance causing additional effort to breathe. The respiratory system undergoes structural, physiological, and immunological changes with age. An increased proportion of neutrophils and a lower percentage of macrophages can be found in bronchoalveolar lavage fluid (BAL) of elderly subjects compared to younger adults. Persistent low-grade inflammation in the lower airways can result in proteolytic and oxidant-mediated damage to the lung matrix, resulting in the loss of alveolar units and impaired gas exchange across the alveolar membrane seen with aging. Persistent inflammation of the lower airways may predispose older adults to increased susceptibility to toxic environmental exposures and accelerated lung function decline. (See, e.g., Sharma et al., Clinical Interventions in Aging 1 (2006) 253-260). Oxidative stress exacerbates inflammation during aging (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 in aging promote the expression of cytokines, chemokines, and adhesion molecules and enzymes (See, e.g., Chung et al., Ageing Res. Rev. 8 (2009) 18-30). Homeostatic activation and recruitment of macrophages, T cells, and mast cells promotes the release of proteases, triggering extracellular matrix degradation, cell death, remodeling, and other events that can lead to tissue and organ damage during chronic inflammation (see, e.g., Demedts et al., Respir. Res. 7 (2006) 53-63).By administering senolytic agents to elderly subjects (including asymptomatic middle-aged adults), the decline in lung function may be slowed or inhibited by killing and removing senescent cells from the airways.

[0149] The effectiveness of the senolytic agent can be easily determined by those skilled in the art of medicine and clinical sciences. The health status of the subject can be monitored using one or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performing analytical tests and methods described herein. The effect of treatment with the senolytic agent, or pharmaceutical composition comprising the agent, can be analyzed using techniques known in the art, for example, by comparing the symptoms of patients suffering from or at risk of lung disease who have been treated with those who have not been treated or who have been treated with a placebo. In addition, methods and techniques for evaluating lung mechanical function can be performed, for example, techniques for measuring lung volume, elastance, and airway hyperresponsiveness. To determine and monitor pulmonary function throughout the procedure, any one of a number of measurements may be obtained, including expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in 1 second, FEV1), FEV1 / FEV ratio, forced expiratory flow rate of 25% to 75%, and maximum voluntary ventilation (MVV), peak expiratory flow rate (PEF), slow exhaust vital capacity (SVC). Total lung capacity 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 the diffusion capacity for carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO2) can also be measured; normal oxygen levels are typically 95% to 100%. An SpO2 level below 90% suggests that the subject has hypoxemia. Values ​​below 80% are considered serious and require interventional therapy to preserve brain and heart function and prevent cardiac or respiratory arrest.

[0150] Nervous System Diseases and Disorders Age-related diseases or disorders treatable by administering the senolytic agents described herein include nervous system diseases or disorders. Such age-related diseases and disorders include Parkinson's disease, Alzheimer's disease (and other dementias), motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, and eye diseases and disorders, such as age-related macular degeneration. Other age-related eye diseases are glaucoma, blindness, presbyopia, and cataracts.

[0151] Parkinson's disease (PD) is the second most common neurodegenerative disease. It is a condition that causes brain damage characterized by slowness of movement (bradykinesia), shaking, stiffness, and in later stages, impaired balance. Many of these symptoms are due to the loss of certain neurons in the brain, which results in a lack of dopamine. The disease is characterized by neurodegeneration, such as the loss of about 50% to 70% of dopamine neurons in the substantia nigra pars compacta, profound loss of dopamine in the striatum, and / or the presence of intracytoplasmic inclusions (Lewy bodies) composed primarily of alpha-synuclein and ubiquitin. Parkinson's disease is also characterized by spontaneous movement disorders, such as tremor, rigidity, bradykinesia, and / or postural instability. Subjects at risk of developing Parkinson's disease include those with a family history of Parkinson's disease and those exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., Agent Orange), or heavy metals. Senescence of dopamine-producing neurons is thought to contribute to the observed cell death in PD through the production of reactive oxygen species (see, e.g., Cohen et al., J. Neural Transm. Suppl. 19 (1983) 89-103); therefore, the methods and senolytic agents described herein are useful for the treatment and prevention of Parkinson's disease.

[0152] Methods for detecting, monitoring or quantifying neurodegenerative deficits and / or locomotor impairments associated with Parkinson's disease, such as histological studies, biochemical tests, and behavioral assessments, are known in the art (see, for example, US Patent Application Publication No. 2012 / 0005765).Symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty in starting or ending voluntary movements, jerky and stiff movements, muscle atrophy, shaking (tremor), and changes in heart rate, normal reflexes, slow movement, and postural instability.In addition to their physical symptoms, it is becoming increasingly recognized that people diagnosed with Parkinson's disease may have cognitive decline, including mild cognitive decline.

[0153] Alzheimer's disease (AD) is a neurodegenerative disease that presents with a slowly progressive mental decline accompanied by memory loss, disorientation, and confusion, resulting in severe dementia. Age is the single greatest predisposing risk factor for developing AD, which is the leading cause of dementia in the elderly (see, e.g., Hebert, et al., Arch.Neural.60(2003)1119-1122). Early clinical symptoms show striking similarities 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 (senile) plaques in histological specimens. The disease primarily involves limbic and cortical regions of the brain. Argyrophilic plaques containing amyloidogenic AD fragments of amyloid precursor protein (APP) are scattered throughout the cerebral cortex and hippocampus. Neurofibrillary tangles are found in pyramidal neurons located primarily in the neocortex, hippocampus, and nucleus basalis of Meynert. Other changes are observed, such as granulovacuolar degeneration in pyramidal cells of the hippocampus, as well as neuronal loss and gliosis in the cortex and hippocampus. Subjects at risk of developing Alzheimer's disease include those who are older, those with a family history of Alzheimer's disease, those with genetic risk genes (e.g., ApoE4) or critical gene mutations (e.g., APP, PSI, or PS2), and those with a history of head trauma or heart / vascular conditions (e.g., hypertension, heart disease, stroke, diabetes, high cholesterol, etc.).

[0155] Several behavioral and histopathological assays are known in the art to evaluate Alzheimer's disease phenotypes, characterize therapeutic agents, and assess treatments. Histological analysis is typically performed postmortem. Tissue analysis of A□ levels can be performed using thioflavin-S, Congo red, or anti-A□ staining (e.g., 4G8, 10D5, or 6E10 antibodies) to visualize A□ deposits on dissected brain tissue (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 of visualizing A□ deposits in transgenic mice have also been described. BSB ((trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) and PET tracer 11C-labeled Pittsburgh compound-B (PIB) binds to AP 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). 19 The F-containing amyloid-affinity Congo Red-type compound, FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene), allows visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., Nature Neurosci. 8 (2005) 527-533). Radiolabeled putrescine-modified amyloid-beta peptides label amyloid deposits in vivo in mouse models 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 for astroglial activation and gliosis during neurodegeneration. AP plaques are associated with GFAP-positive activated astrocytes and can be visualized by 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 Gallias silver staining (see, e.g., Gotz et al., J. Biol. Chem. 276 (2001) 529-534; U.S. Patent No. 6,664,443). Axonal staining and axonal transport studies by electron microscopy can be used to visualize neuronal degeneration (see, for example, Ishihara et al., Neuron 24 (1999) 751-762).

[0157] Subjects suffering from Alzheimer's disease can be identified using standard diagnostic methods known in the art for Alzheimer's disease. In general, the diagnosis of Alzheimer's disease is based on the patient's symptoms (e.g., progressive decline in memory function, gradual retreat from and irritability to normal activities, apathy, agitation or irritability, aggression, anxiety, sleep disorder, dysphoria, abnormal motor behavior, disinhibition, social withdrawal, loss of appetite, hallucinations, dementia), medical history, neuropsychological tests, neurological tests, and / or physical examinations. Cerebrospinal fluid can also be tested for various proteins that have been linked to Alzheimer's pathology, including tau, amyloid beta peptide, and AD7C-NTP. Genetic tests are also available for early-onset familial Alzheimer's disease (eFAD), an autosomal dominant inherited disease. Clinical genetic tests are available for individuals with AD symptoms or potentially at-risk family members of patients with early-onset disease. In the United States, mutations for PS2 and APP can be tested in clinical laboratories or laboratories federally certified under the Clinical Laboratory Improvement Amendments. A commercial test for PS1 mutations is also available (Elan Pharmaceuticals).

[0158] The effectiveness of one or more senolytic agents described herein and the monitoring of subjects receiving one or more senolytic agents can be easily determined by those skilled in the art of medicine and clinical practice. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performing analytical tests and methods described herein, can be used to monitor the health status of a subject. The effect of administering one or more senolytic agents can be analyzed using techniques known in the art, for example, by comparing the symptoms of patients suffering from or at risk of Alzheimer's disease who have been treated with those of patients who have not been treated or who have been treated with a placebo.

[0159] Mild cognitive decline (MCI) is a brain function syndrome involving the onset and evolution of cognitive decline beyond what would be expected based on an individual's age and education, but not enough to impede an individual's daily activities. MCI is an aspect of cognitive aging that is considered to be a transition state between normal aging and dementia to which it can be converted (see Pepeu, Dialogues in Clinical Neuroscience 6 (2004) 369-377). MCI that primarily affects memory is known as "amnestic MCI". People with amnestic MCI may begin to forget important information that they previously easily recalled, such as recent events. Amnestic MCI is often seen as 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 thinking abilities, such as the ability to make correct decisions and determine the time or sequence of steps required to complete a complex task, or visual perception. Individuals with non-amnestic MCI are thought to be more likely to convert to other types of dementia (eg, dementia with Lewy bodies).

[0160] Those in the medical arts have an increasing awareness that individuals diagnosed with Parkinson's disease may have MCI in addition to their physical symptoms. Recent studies indicate that 20-30% of people with Parkinson's disease have MCI, and their MCI tends to be non-amnestic. Parkinson's disease patients with MCI may progress to developing full-blown dementia (Parkinson's disease with dementia).

[0161] Methods for detecting, monitoring, quantifying or assessing neuropathological deficits associated with MCI are known in the art, including morphological analysis of astrocytes, release of acetylcholine, silver staining to assess neurodegeneration, and PiB PET imaging to detect beta amyloid deposits (see, e.g., U.S. Patent Application Publication No. 2012 / 0071468; Pepeu, (2004), supra). Methods for detecting, monitoring, quantifying or assessing 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 the water 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 that control important voluntary muscle activities, such as speaking, walking, breathing, and swallowing. It is classified according to 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, also known as Werdnig-Hoffmann disease, SMA2, also known as Kugelberg-Welander disease, SMA3, also known as Kennedy disease), post-polio syndrome, and hereditary spastic paraplegia. In adults, the most common MND is amyotrophic lateral sclerosis (ALS), which affects both upper and lower motor neurons. It can affect muscles of the arms, legs, or face. Primary lateral sclerosis is a disease of the upper motor neurons, while progressive muscular atrophy only affects the lower motor neurons in the spinal cord. In progressive bulbar palsy, the lowest motor neurons in the brainstem are most affected, resulting in slurred speech and difficulty chewing and swallowing. Mildly abnormal signs in the arms and legs are most often present. Patients with MND exhibit Parkinson's phenotypes (e.g., have tremor, rigidity, bradykinesia, and / or postural instability). Methods for detecting, monitoring, or quantifying locomotor activity and / or other defects associated with Parkinson's disease, such as MND, are known in the art (see, e.g., U.S. Patent Application Publication No. 2012 / 0005765).

[0163] Methods for detecting, monitoring, quantifying or assessing motor disorders and histopathological deficits associated with MND are known in the art, including histopathological, biochemical, and electrophysiological studies, as well as motor activity analysis (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 death of motor neurons, progressive accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin, and abnormal neurofilament accumulation in degenerating motor neurons. In addition, reactive astroglia and microglia are often detected in affected tissues. Patients with MND exhibit one or more motor disorders, including muscle weakness and wasting, uncontrollable twitching, spasticity, slow and effortful movements, and exaggerated tendon reflexes.

[0164] Eye Diseases and Disorders In certain embodiments, the aging-related disease or disorder is an eye disease, disorder, or condition, such as presbyopia, macular degeneration, or cataract. In other certain embodiments, the aging-related disease or disorder is glaucoma. Macular degeneration is a neurodegenerative disease that results in 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 the disease usually results in more significant vision loss. While the exact cause of age-related macular degeneration is still unknown, the number of senescent retinal pigment epithelium (RPE) cells increases with age. Age and certain genetic and environmental factors are risk factors for developing 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 predisposing risk factors include reduced levels of Dicer1 (an enzyme involved in the maturation of microRNAs) in the eyes of patients with dry AMD, with reduced microRNAs contributing to a senescent cell profile.

[0165] Dry ARMD is associated with atrophy of the RPE layer, which results in the loss of photoreceptor cells. Dry forms of ARMD can be attributed to aging and thinning of macular tissue, as well as to pigment deposition in the macula. Aging appears to inhibit both RPE replication and migration, resulting in permanent RPE loss in the macula of dry AMD patients (see, for example, Iriyama et al., J.Biol.Chem.283(2008)11947-11953). In wet ARMD, new blood vessels grow under the retina, leaking blood and fluid. This abnormal leaky choroidal neovascularization leads to the death of retinal cells, resulting in blind spots in central vision. Different forms of macular degeneration can also occur in younger patients. Non-age-related etiologies can be linked to genetics, diabetes, nutritional deficiencies, head injuries, infections, or other factors.

[0166] The first indication of macular degeneration may be the decline in vision noticed by the patient or by the ophthalmologist during a routine eye examination. The formation of exudates or "drusen" under the Bruch's membrane of the retina macula is often the first physical sign that macular degeneration may occur. Symptoms include the perception of straight lines being distorted, and in some cases, the central vision appears more distorted than the rest of the scene; dark, blurred areas or "whiteouts" appear in the central vision; and / or color perception is altered or reduced. The diagnosis and monitoring of subjects with macular degeneration can be achieved by those skilled in the art of eye care by regular eye examination procedures generally accepted in the art and by the subject reporting symptoms.

[0167] Presbyopia is an age-related condition that refers to the progressively diminished ability of the eye to focus on nearby objects, as the speed and power of accommodation of normal eyes decreases with age. Loss of elasticity of the crystalline lens and loss of contractile power of the ciliary muscle have been hypothesized as its causes (see, 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 lens capsule suggest that the mechanical strength of the posterior lens capsule decreases significantly 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).

[0168] The laminar structure of the capsule also changes, which may be due, at least in part, to changes in the composition of the tissue (see, e.g., Krag et al., 1997, supra, and references cited therein). The major structural component of the lens capsule is basement membrane type IV collagen, which is organized into a three-dimensional molecular network (see, 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 is composed of six homologous □ chains (□1-6) that assemble into heterotrimeric collagen IV protomers, each of which contains a specific combination of □112, □345, or □556 chains (see, e.g., Khoshnoodi et al., Microsc. Res. Tech. 71 (2008) 357-370). The protomers share the structural similarity of a triple-helical collagenous domain with a triplet peptide sequence of Gly-XY, terminating in a globular C-terminal region termed the non-collagenous 1 (NC1) domain (Timpl et al., Eur. J. Biochem. 95 (1979) 255-263). The N-terminus is composed of a helical domain termed the 7S domain (see, e.g., Risteli et al., Eur. J. Biochem. 108 (1980) 239-250), which is also involved in protomer-protomer interactions.

[0169] Studies have suggested that collagen IV influences cellular functions inferred from the positioning of the basement membrane beneath the epithelial layer, and data support a role for collagen IV in tissue stabilization (see, e.g., Cummings et al., supra). Posterior capsule opacification (PCO) occurs as a complication in approximately 20-40% of patients later in life after cataract surgery (see, e.g., Awasthi et al., Arch. Ophthalmol. 127 (2009) 555-562). PCO results from the proliferation and activity of residual lens epithelial cells along the posterior capsule in a response akin 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 epithelial cell migration (see, e.g., Awasthi et al., supra; Raj et al., supra). As discussed herein, these factors, as well as the production of cytokines by senescent cells, contribute to the SASP. In contrast, in vitro studies show that collagen IV promotes the adhesion of lens epithelial cells (see, e.g., Olivero et al., Invest. Ophthalmol. Vis. Sci. 34 (1993) 2825-2834). The adhesion of collagen IV, fibronectin, and laminin to the intraocular lens may inhibit cell migration and reduce the risk of PCO (see, e.g., Raj et al., Int. J. Biomed. Sci. 3 (2007) 237-250).

[0170] Without being bound to any particular theory, selective killing of senescent cells by the senolytic agents described herein may slow down or prevent (delay, inhibit, delay) the disorganization of the type IV collagen network. Removal of senescent cells, and thus removal of the inflammatory effects of SASP, may reduce or inhibit epithelial cell migration and may delay (suppress) the onset of presbyopia or reduce or slow down the progressive severity of the condition (e.g., slow down the progression from mild to moderate or from moderate to severe). The senolytic agents described herein may also be useful for post-cataract surgery to reduce the likelihood of the appearance of PCO.

[0171] While direct evidence for the involvement of cellular senescence in the development of cataracts has not been obtained from human studies, BubR1 dwarf mice develop posterior subcapsular cataracts bilaterally at an early age, suggesting that aging may play a role (see, e.g., Baker et al., Nat. Cell Biol. 10 (2008) 825-836). Cataracts are opacifications of the eye's lens that result in blurred vision and, if left untreated, may result in blindness. Surgery is effective and is routinely performed to remove cataracts. Administration of one or more of the senolytic agents described herein may result in a reduced likelihood of cataract appearance or may slow or inhibit the progression of cataracts. The presence and severity of cataracts can be monitored by eye examination using methods routinely performed by those skilled in the art of ophthalmology.

[0172] In certain embodiments, at least one senolytic agent described herein may be administered to a subject at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the senolytic agent may be initiated when the human subject is at least 40 years old to delay or inhibit the onset or development of cataracts, presbyopia, and macular degeneration. Because almost all humans develop presbyopia, in certain embodiments, the senolytic agent may be administered to a human subject in a manner as described herein after the subject reaches the age of 40 to delay or inhibit the onset or development of presbyopia.

[0173] In certain embodiments, the aging-related disease or disorder is glaucoma. Glaucoma is a broad term used to describe a group of diseases that often result in visual field loss without other predominant symptoms. The absence of symptoms often results in a delay in the diagnosis of glaucoma until the late stages of the disease. Even if subjects with glaucoma do not become blind, their vision is often severely impaired. Normally, a clear fluid flows in and out of the front part of the eye, known as the anterior chamber. In individuals with wide-angle glaucoma, this fluid drains slowly, resulting in an increase in pressure within the eye. If left untreated, this high pressure can subsequently damage the optic nerve and result in complete blindness. The loss of peripheral vision is due to the death of ganglion cells in the retina. Ganglion cells are a specific type of projection neuron that connects the eye to the brain. When the cellular network required for fluid drainage was subjected to SA-□-Gal staining, a four-fold increase in senescence was observed in glaucoma patients (see, e.g., Liton et al., Exp. Gerontol. 40 (2005) 745-748).

[0174] To monitor the effect of treatment on inhibiting the progression of glaucoma, standard automated perimetry (visual field test) is the most widely used technique. In addition, several algorithms for progression detection have been developed (see, for example, Wesselink et al., Arch. Ophthalmol. 127(3)(2009)270-274, and references therein). Further methods include gonioscopy (examining the trabecular meshwork and the angle at which fluid drains from the eye); imaging techniques such as scanning laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and ocular coherence tomography); ophthalmoscopy; and pachymetry to determine central corneal thickness.

[0175] Metabolic disease or disorder Senescence-associated diseases or disorders treatable by administering senolytic agents include metabolic diseases or disorders. Such senescent cell-associated diseases and disorders include diabetes, metabolic syndrome, diabetic ulcers, and obesity.

[0176] Diabetes is characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. The vast majority (90-95%) of all diagnosed cases of diabetes in adults are type 2 diabetes, characterized by the gradual loss of insulin production by the pancreas. Diabetes is the leading cause of kidney failure, non-traumatic lower limb amputations, and new cases of blindness in adults in the United States. Diabetes is the leading cause of heart disease and stroke, and the seventh leading cause of death in the United States (see, e.g., Centers for Disease Control and Prevention, National diabetes fact sheet: national estimates and general information on diabetes and pre-diabetes in the United States, 2011 ("Diabetes Fact Sheet"). The senolytic agents described herein may be used to treat type 2 diabetes, particularly type 2 diabetes associated with age, diet, and obesity.

[0177] 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 (see, e.g., Tchkonia et al., Aging Cell 9 (2010) 667-684). Adipose tissue from obese mice showed induction of senescence markers SA-□-Gal, p53, and p21 (see, e.g., Tchkonia et al., supra; Minamino et al., Nat. Med. 15 (2009) 1082-1087). Concomitant upregulation of proinflammatory cytokines, such as tumor necrosis factor-□□ and Ccl2 / MCP1, was observed in the same adipose tissue (see, e.g., Minamino et al., supra). The induction of senescent cells in obesity has potential clinical implications, as proinflammatory SASP components are also suggested to contribute to type 2 diabetes (see, e.g., Tchkonia et al., supra). Similar patterns of upregulation of senescence markers and SASP components are associated with diabetes in both mice and humans (see, e.g., Minamino et al., supra). Thus, the methods described herein, which include administering a senolytic agent, may be useful for treating or preventing type 2 diabetes, as well as obesity and metabolic syndrome. Without wishing to be bound by theory, contacting senescent preadipocytes with a senolytic agent, thereby killing the senescent preadipocytes, may provide clinical and health benefits to individuals with any one of diabetes, obesity, or metabolic syndrome.

[0178] Subjects suffering from type 2 diabetes can be identified using standard diagnostic methods known in the art for type 2 diabetes. Generally, diagnosis of type 2 diabetes is based on the patient's symptoms (e.g., increased thirst and frequent urination, increased hunger, weight loss, fatigue, blurred vision, slow-healing sores or frequent infections, and / or darkened areas of skin), medical history, and / or physical examination. Subjects at risk of developing type 2 diabetes include those with a family history of type 2 diabetes, as well as those with other risk factors, such as excess weight, fat distribution, inactivity, race, age, prediabetes, and / or gestational diabetes.

[0179] The effectiveness of the senolytic agent can be easily determined by those skilled in the art of medicine and clinical sciences. Physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods, such as one or any combination of diagnostic methods including those described herein, can be used to monitor the health status of a subject. Subjects receiving one or more senolytic agents described herein for the treatment or prevention of diabetes can be monitored, for example, by assaying glucose and insulin resistance, energy expenditure, body composition, adipose tissue, skeletal muscle, hepatitis, and / or lipotoxicity (muscle and liver lipids by in vivo imaging, and lipid accumulation and inflammation in muscle, liver, bone marrow, and pancreatic □-cells by histology). Other characteristic features or phenotypes of type 2 diabetes are known and can be assayed by using other methods and techniques known and routinely performed in the art, as described herein.

[0180] Obesity and obesity-related disorders are used to refer to the condition of a subject having a body mass that is measurably greater than that which is ideal for their height and build. Body mass index (BMI) is a measurement tool used to determine excess body weight and is calculated from the subject's height and weight. A person is considered overweight when they have a BMI between 25 and 29; a person is considered obese when they have a BMI between 30 and 39, and a person is considered severely obese when they have a BMI >40. Thus, the words obesity and obesity-related refer to human subjects with a body mass index value greater than 30, greater than 35, or greater than 40. The category of obesity not captured by BMI is referred to in the art as "abdominal obesity," which is related to excess fat around the waist of a subject, which is an important factor in health, independent of BMI. The simplest and most frequently used measure of abdominal obesity is waist size. Generally, abdominal obesity in women is defined as a waist size of 35 inches or more, and in men as a waist size of 40 inches or more. More sophisticated methods for determining obesity require specialized equipment, such as magnetic resonance imaging or dual energy X-ray absorptiometry machines.

[0181] A condition or disorder associated with diabetes and aging is diabetic ulcers (i.e., diabetic wounds). Ulcers are breakdowns of the skin that can extend to involve subcutaneous tissue or even muscle or bone. These lesions occur especially in the lower extremities. Patients with diabetic venous ulcers show an increased presence of cellular senescence at the site of chronic wounds (see, e.g., Stanley et al., J. Vas. Surg. 33 (2001) 1206-1211). Chronic inflammation is also observed at the site of chronic wounds, such as diabetic ulcers (see, e.g., Goren et al., Am. J. Pathol. 168 (2006) 65-77), suggesting that the proinflammatory cytokine phenotype of senescent cells plays a role in pathology.

[0182] The subject who has type 2 diabetes or is at risk of developing type 2 diabetes may have metabolic syndrome.Metabolic syndrome in humans is typically associated with obesity and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes, and insulin resistance.The subject who has metabolic syndrome may show a cluster of metabolic disorders or abnormalities, which may include, for example, one or more of hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis, and other metabolic disorders.

[0183] Renal dysfunction Renal pathologies, such as glomerular disease, occur in elderly people and can be treated by administering the senolytic compounds described herein. Glomerulonephritis is characterized by renal inflammation and the expression of two proteins, IL1□ and IL1□ (see, e.g., Niemir et al., Kidney Int. 52 (1997) 393-403). IL1□ and IL1□ are considered to be master regulators of the SASP (see, e.g., Coppe et al., PLoS. Biol. 6 (2008) 2853-2868). Glomerular disease is associated with an increased presence of senescent cells, especially in fibrotic kidneys (see, e.g., Sis et al., Kidney Int. 71 (2007) 218-226).

[0184] Skin disease or disorder Age-associated diseases or disorders treatable by administering the senolytic agents described herein include skin diseases or disorders. Such age-associated diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and are discussed in more detail above. Other age-associated skin diseases and disorders include wrinkles (age wrinkles); pruritus (linked to diabetes and aging); paresthesias (a chemotherapy side effect linked to diabetes and multiple sclerosis); psoriasis (as described) and other papular-squamous disorders such as erythroderma, lichen planus, and lichenoid skin diseases; atopic dermatitis (a form of eczema, associated with inflammation); eczematous rash (often observed in older patients and linked to side effects of certain drugs). Other skin diseases and disorders associated with aging include eosinophilic dermatoses (linked to certain types of blood cancers); reactive neutrophilic dermatoses (associated with underlying diseases, e.g., inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies are formed against desmoglein); pemphigoid and other immune blistering dermatoses (autoimmune blistering of the skin); fibrohistiocytic proliferation of the skin (linked to aging); and cutaneous lymphomas, which are more common in older populations. Another skin disease that may be treatable by the methods described herein includes cutaneous lupus, a symptom of lupus erythematosus. Late-onset lupus may be linked to decreased (i.e., reduced) function of T and B cells and cytokines (immunosenescence) associated with aging.

[0185] Metastasis In some embodiments, methods are provided for treating or preventing (i.e., reducing the likelihood of the occurrence or development of) a disease (or disorder or condition) associated with senescent cells, which is metastasis. The senolytic agents described herein may also be used in accordance with the methods described herein for treating or preventing (i.e., reducing the likelihood of the occurrence of) metastasis (i.e., the spread and metastasis of cancer or tumor cells) from one organ or tissue to another organ or tissue in the body.

[0186] Senescent cell-associated diseases or disorders include metastasis, and subjects with cancer may benefit from administration of a senolytic agent as described herein to inhibit metastasis. Such senolytic agents, when administered to a subject with cancer by the methods described herein, may inhibit tumor growth. Cancer metastasis occurs when cancer cells (i.e., tumor cells) spread beyond their anatomical location of origin and initial colonization to other regions throughout the subject's body. Tumor growth may be determined by tumor size, which may be measured by a variety of methods familiar to those skilled in the art, for example, by PET scanning, MRI, CAT scan, biopsy. The effect of a therapeutic agent on tumor growth may also be evaluated by examining tumor cell differentiation.

[0187] As used herein and in the art, the term cancer or tumor is a clinically descriptive term encompassing diseases that are typically characterized by cells exhibiting abnormal cell proliferation. The term cancer is generally used to describe a malignant tumor, or a pathology resulting from a tumor. Alternatively, the abnormal growth may be referred to in the art as a neoplasm. For example, with respect to tissue, the term tumor generally refers to any abnormal tissue growth characterized, at least in part, by excessive and abnormal cell proliferation. A tumor may be metastatic, spreading beyond its anatomical location of occurrence and its initial colonization to other areas throughout the subject's body. Cancer may include solid tumors or may include "liquid" tumors (e.g., leukemia and other blood cancers).

[0188] Cells are induced to senesce by cancer therapy, e.g., radiation and certain chemotherapy drugs. The presence of senescent cells increases the secretion of inflammatory molecules and promotes tumor progression, which may include promoting tumor growth and tumor size, promoting metastasis, and altering differentiation. When senescent cells are destroyed, tumor progression is significantly inhibited, resulting in tumors with small size and little or no metastatic growth observed (see, e.g., International Application WO 2013 / 090645).

[0189] In some embodiments, methods are provided for preventing (i.e., reducing the likelihood of occurrence), inhibiting, or delaying metastasis in a subject with cancer by administering a senolytic agent as described herein. In other embodiments, the senolytic agent is administered on one or more days within a therapeutic time (i.e., treatment course) of 7 days or less or 14 days or less. In still other embodiments, the treatment course is 2 days or less, 3 days or less, 4 days or less, 5 days or less, 6 days or less, 7 days or less, 8 days or less, 9 days or less, 10 days or less, 11 days or less, 12 days or less, 13 days or less, 14 days or less, 15 days or less, 16 days or less, 17 days or less, 18 days or less, 19 days or less, 20 days or less, or 21 days or less. In still other embodiments, the treatment course is a single day. In still other embodiments, the senolytic agent is administered on two or more days within a therapeutic time of 7 days or less or 14 days or less.

[0190] Because cells can be induced to senescent by cancer therapy, e.g., radiation and certain chemotherapy drugs (e.g., doxorubicin; paclitaxel; gemcitabine; pomalidomide; lenalidomide), the senolytic agents described herein can be administered after chemotherapy or radiation therapy to kill (or promote the death of) these senescent cells. As discussed herein and understood in the art, the establishment of senescence, indicated, for example, by the presence of a senescence-associated secretory phenotype (SASP), occurs over a period of days; therefore, administration of a senolytic agent to kill senescent cells and thereby reduce the likelihood of occurrence or the extent of metastasis begins when senescence is established. As discussed herein, the following treatment courses for administration of a senolytic agent can be used in the methods described herein for treating or preventing (i.e., reducing the likelihood of occurrence or reducing the severity of) side effects of chemotherapy or radiation therapy:

[0191] In certain embodiments, when chemotherapy or radiotherapy is administered in a treatment cycle of at least one day of treatment onset (i.e., chemotherapy or radiotherapy) followed by 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, 21 days (or about 3 weeks), or about 4 weeks (about 1 month) of treatment rest (i.e., chemotherapy or radiotherapy rest), the senolytic agent is administered on one or more days during the treatment rest time interval (period) beginning on or after the second day of the treatment rest time interval and ending on or before the last day of the treatment rest time interval. As an illustrative example, the senolytic agent is administered on at least one day and no more than n-1 days of the treatment rest time interval, where n is the number of days in the treatment rest. In some embodiments, when chemotherapy or radiation therapy is administered in a treatment cycle of at least one day of treatment onset (i.e., chemotherapy or radiation therapy), followed by at least one week of treatment rest, the senolytic agent is administered on one or more days during the treatment rest time interval starting on or after day 2 of the treatment rest time interval and ending on or before the last day of the treatment rest time interval.

[0192] Chemotherapy can be referred to as chemotherapy, chemotherapy agents, or chemotherapy drugs. Many chemotherapy agents are compounds that are referred to as small organic molecules. Chemotherapy is also a term used to describe the combination of chemotherapy drugs administered to treat a particular cancer. As those skilled in the art will understand, chemotherapy can also refer to the combination of two or more chemotherapy molecules that are administered in a coordinated manner, which can be referred to as polychemotherapy. Numerous chemotherapy drugs are used in the field of oncology, including, but not limited to, alkylating agents; antimetabolites; anthracyclines, plant alkaloids; and topoisomerase inhibitors.

[0193] Cancers that can metastasize can be solid tumors or liquid tumors (e.g., blood cancers, e.g., leukemias). Liquid tumor cancers are classified in the art as occurring in the blood, bone marrow, and lymph nodes, and generally include leukemias (myeloid and lymphocytic), lymphomas (e.g., Hodgkin's lymphoma), and melanomas (including multiple myeloma). Leukemias include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia. Cancers that are solid tumors and occur more frequently in humans include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer (including squamous cell skin cancer), kidney cancer, head and neck cancer, throat cancer, squamous cell carcinoma that forms on the moist mucosal linings of the nose, mouth, throat, etc., bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain embodiments, the disease or disorder associated with senescent cells that is treated or prevented (i.e., the occurrence or likelihood of occurrence is reduced) by the methods described herein is metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, gastric cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, kidney 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.

[0194] The methods described herein are also useful for inhibiting, delaying or slowing the progression of metastatic cancer of any one of the types of tumors described in the medical arts, including the following types of cancer (tumor): adrenocortical carcinoma, pediatric adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, basal cell carcinoma, pediatric basal cell carcinoma, bladder cancer, pediatric bladder cancer, bone cancer, brain tumor, pediatric astrocytoma, pediatric brain stem glioma, pediatric central nervous system atypical teratoma / rhabdoid tumor, pediatric central nervous system embryonal tumor, pediatric central nervous system germ cell tumor, pediatric craniopharyngioma brain tumor, pediatric ependymoma brain tumor, breast cancer, pediatric bronchial tumor, carcinoid tumor, pediatric carcinoid tumor, gastrointestinal carcinoid tumor, carcinoma of unknown primary, pediatric carcinoma of unknown primary, pediatric cardiac (cardiac) tumor, pediatric bronchial tumor, pediatric endothelial ... pancreatic (hepatic) tumors, cervical cancer, childhood cervical cancer, childhood chordoma, chronic myeloproliferative disorders, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood esthesioneuroblastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric (stomach) cancer, childhood gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), childhood gastrointestinal stromal tumor (GIST), childhood extracranial germ cell tumor, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell renal cancer, Wilms' tumor, childhood kidney tumor, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous cell neck cancer of unknown primary, midline duct carcinoma involving the NUT gene, oral cavity cancer, childhood multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, myeloproliferative neoplasm, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, pediatric nasopharyngeal cancer, neuroblastoma, oral cavity cancer, pediatric oral cavity cancer, oropharyngeal cancer, ovarian cancer, pediatric ovarian cancer,Epithelial ovarian cancer, low-grade malignant potential ovarian cancer, pancreatic cancer, childhood pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), childhood papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, childhood pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis transitional cell carcinoma, retinoblastoma, salivary gland cancer, childhood salivary gland cancer, Ewing sarcoma family tumors, Kaposi's sarcoma, osteosarcoma, striated muscle Includes sarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, childhood skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, throat cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, endometrial uterine cancer, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0195] Side effects of chemotherapy and radiation therapy In other embodiments, the disorder or condition associated with senescent cells is a side effect of chemotherapy or a side effect of radiation therapy. Examples of chemotherapeutic agents that induce non-cancer cells to senesce include anthracyclines (e.g., doxorubicin, daunorubicin); taxol (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more of the senolytic agents administered as described herein may be used to treat and / or prevent (i.e., reduce the likelihood or occurrence of) a side effect of chemotherapy or a side effect of radiation therapy. Removal or destruction of senescent cells may ameliorate acute toxicity, including acute toxicity involving energy imbalance of chemotherapy or radiation therapy. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, lethargy, low physical activity, hematologic toxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, skin toxicity (e.g., rash, dermatitis, hyperpigmentation, hives, photosensitivity, nail changes), mouth (e.g., oral mucositis), gum or throat problems, or any toxic side effects caused by chemotherapy or radiation therapy. For example, toxic side effects caused by radiation therapy or chemotherapy may be ameliorated by the methods described herein. Thus, in certain embodiments, provided herein are methods for ameliorating (reducing, inhibiting, or preventing (i.e., reducing the likelihood of occurrence)) acute toxicity or reducing the severity of toxic side effects (i.e., adverse side effects) of chemotherapy or radiotherapy or both in a subject undergoing treatment, wherein the method comprises administering to the subject an agent that selectively kills, removes, or destroys, or promotes the selective destruction of, senescent cells. Administration of the senolytic agents described herein to treat or reduce the likelihood of occurrence or reduce the severity of chemotherapy or radiotherapy side effects may be accomplished by the same courses of treatment described above for the treatment / prevention of metastasis.As described for treating or preventing metastasis (i.e., reducing the likelihood of its occurrence), the senolytic agent is administered during chemotherapy- or radiotherapy-off time intervals or after a chemotherapy or radiotherapy treatment regimen has been completed.

[0196] In more particular embodiments, acute toxicity is acute toxicity involving 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, acute toxicity involving energy imbalance refers to a decrease or reduction in a subject's ability to be physically active as indicated by a decrease or decline in expenditure of energy, as compared to that observed in subjects not receiving the drug therapy. As a non-limiting example, such acute toxic effects involving energy imbalance include reduced physical activity. In other embodiments, energy imbalance includes fatigue or lethargy.

[0197] In some embodiments, the side effect of chemotherapy that is treated or prevented (i.e., the likelihood of occurrence is reduced) by the senolytic agents described herein is cardiotoxicity. A subject with cancer that is being treated with an anthracycline (e.g., doxorubicin, daunorubicin) may be treated with one or more senolytic agents described herein that reduce, ameliorate, or reduce the cardiotoxicity of the anthracycline. As is well understood in the medical arts, the cardiotoxicity associated with anthracyclines limits the maximum lifetime dose that a subject can receive, even if the cancer is responsive to the drug. Administration of one or more senolytic agents may reduce cardiotoxicity, allowing additional amounts of anthracycline to be administered to the subject, resulting in an improved prognosis associated with the cancer disease. In some embodiments, the cardiotoxicity results from administration of an anthracycline, e.g., doxorubicin. Doxorubicin is an anthracycline topoisomerase inhibitor approved for treating patients with ovarian cancer after failure of platinum-based therapy; Kaposi's sarcoma after failure of or intolerance to first-line systemic chemotherapy; or multiple myeloma in combination with bortezomib in patients who have not previously received bortezomib or who have received at least one prior therapy. The total lifetime dose for patients is 550 mg / m 2 Above this dose, doxorubicin can result in myocardial damage, which can lead to congestive heart failure. Cardiotoxicity can occur even at lower doses if the patient is also receiving mediastinal radiation or another cardiotoxic drug.

[0198] In other embodiments, the senolytic agents described herein may be used in the methods as provided herein to reverse chronic or long-term side effects. Chronic toxic side effects typically result from multiple exposures or administrations of chemotherapy or radiation therapy over a longer period of time. Certain toxic effects appear long after treatment (also referred to as delayed toxic effects) and result from damage to organs or systems by the treatment. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients treated for cancer during childhood (see, e.g., Hudson et al., JAMA 309 92013) 2371-2381). Without being bound to any particular theory, by destroying senescent cells, particularly normal cells that have been induced into senescence by chemotherapy or radiation therapy, the likelihood of the appearance of chronic side effects may be reduced, or the severity of chronic side effects may be reduced or attenuated, or the time of onset of chronic side effects may be delayed. Chronic and / or delayed toxic side effects occurring in subjects undergoing chemotherapy or radiation therapy include, by way of non-limiting example, cardiomyopathy, congestive heart disease, inflammation, early menopause, osteoporosis, infertility, cognitive impairment, peripheral neuropathy, secondary cancers, cataracts and other vision problems, hearing loss, chronic fatigue, reduced lung capacity, and pulmonary disease.

[0199] Furthermore, by administering a senolytic agent to kill or remove senescent cells in a subject with cancer, sensitivity to chemotherapy or radiotherapy may be enhanced in a clinically or statistically significant manner than if the senolytic agent is not administered, In other words, when a senolytic agent is administered to a subject treated with chemotherapy or radiotherapy, respectively, the development of chemotherapy or radiotherapy resistance may be inhibited.

[0200] Age-Related Diseases and Disorders The senolytic agents described herein selectively kill senescent cells. In this way, targeting senescent cells in the aging process can be a preventative strategy. Thus, administration of the senolytic agents described herein to a subject can prevent co-morbid diseases and delay death in elderly subjects. Furthermore, selective killing of senescent cells can boost the immune system, extend health span, and improve quality of life in a subject.

[0201] Senocyte-eliminating agents 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 that occur when a subject is exposed to a senescence-inducing agent or factor (e.g., irradiation, chemotherapy, tobacco smoking, a fatty / sugar diet, other environmental factors). Age-related disorders or diseases, as well as age-sensitive traits, may be associated with a senescence-inducing stimulus. The efficacy of the methods of treatment described herein may be manifested by reducing the number of symptoms, reducing the severity of one or more symptoms, or slowing the progression of an age-related disorder or age-sensitive trait associated with a senescence-inducing stimulus. In other embodiments, preventing an age-related disorder or age-sensitive trait associated with an aging-inducing stimulus refers to preventing (i.e., reducing the likelihood of occurrence) or delaying the onset of an age-related disorder or age-sensitive trait associated with an aging-inducing stimulus, or the reappearance of an age-related disorder or age-sensitive trait associated with one or more aging-inducing stimuli. Age-related diseases or conditions include, for example, renal dysfunction, kyphosis, herniated disc, frailty, hair loss, hearing loss, blindness (blindness or visual impairment), muscle wasting, skin conditions, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Blindness refers to the absence of vision when a subject previously had vision. Various scales have been developed to describe the degree of vision and blindness based on visual acuity. Age-related diseases and conditions also include treating dermatological conditions, such as, but not limited to, one or more of the following conditions: wrinkles, including superficial crepe 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 declines in reserve and function across multiple physiological systems, which impairs the subject's ability to cope with daily or acute stressors.Frailty may be characterized by impaired energy characteristics, such as low grip strength, low energy, slow walking speed, low physical activity, and / or unintentional weight loss. Studies have suggested that when three of the five above characteristics are observed, a patient may be diagnosed with frailty (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 diseases and disorders associated with aging may be treated or prevented (i.e., reduce the likelihood of their occurrence) by administering a senolytic agent. The senolytic agent may inhibit senescence or inhibit the accumulation of adult stem cells, kill senescent adult stem cells, or promote their removal. The importance of preventing senescence in stem cells to maintain the regenerative potential of tissues is discussed, for example, in Park et al., J. Clin. Invest. 113 (2004) 175-179; and Sousa-Victor, Nature 506 (2014) 316-321.

[0202] Methods for measuring aging are known in the art.For example, aging can be measured in bone by accidental non-vertebral fracture, accidental hip fracture, accidental total fracture, accidental vertebral fracture, accidental repeated fracture, functional recovery after fracture, loss of bone mineral density in lumbar spine and hip, rate of knee fracture, use of NSAIDs, number of painful joints, and osteoarthritis.Aging can also be measured in muscle by functional decline, rate of falls, reaction time and grip strength, loss of muscle mass in upper and lower limbs, and dual task 10 meter walking speed.Furthermore, aging can be measured in cardiovascular system by changes in systolic and diastolic blood pressure, incident hypertension, major cardiovascular events such as myocardial infarction, stroke, congestive heart disease, and cardiovascular mortality.Furthermore, aging can be measured in brain by cognitive decline, incident depression, and incident dementia. Aging can also be measured in the immune system by infection rates, upper respiratory infection rates, influenza-like illness rates, incident severe infections resulting in hospitalization, incident cancers, implant infection rates, and gastrointestinal infection rates. Other signs of aging can include, but are not limited to, poor oral health, tooth loss, GI symptoms rates, changes in fasting glucose and / or insulin levels, body composition, poor kidney function, quality of life, incident disability in activities of daily living, and incident nursing home admission. Methods for measuring skin aging are known in the art and can include transepidermal water loss (TEWL), skin moisturization, skin elasticity, crow's feet wrinkle area ratio analysis, sensitivity, radiance, roughness, blotches, laxity, skin tone uniformity, softness, and relief (depth variation).

[0203] Administration of the senolytic agents described herein can prolong survival when compared to the expected survival if the subject does not receive treatment. Subjects in need of treatment include those who already have a disease or disorder, as well as those prone to having or at risk of developing a disease or disorder, and those in which the disease, condition, or disorder is to be treated prophylactically. Subjects may have a genetic predisposition to developing a disease or disorder that would benefit from the elimination of senescent cells, or may be of a certain age, where receiving the senolytic agent provides a clinical benefit of delaying the onset of or reducing the severity of a disease, including an age-related disease or disorder.

[0204] In other embodiments, a method for treating a disease or disorder associated with senescence is provided, further comprising identifying a subject that would benefit from treatment with a senolytic agent described herein (i.e., examining expression patterns; individualized treatment). The method comprises first detecting the level of senescent cells in a subject, for example, in a particular organ or tissue of the subject. A biological sample may be obtained from a subject, for example, a blood sample, serum or plasma sample, a biopsy specimen, a bodily fluid (e.g., lung lavage, peritoneal fluid, mucosal lavage, synovial fluid, vitreous fluid, spinal fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation from the subject. The level of senescent cells may be determined by any of the assays or techniques described herein in vitro. For example, senescent cells may be detected by morphology (e.g., as viewed by a microscope); production of senescence-associated markers, such as senescence-associated □-galactosidase (SA-□-gal), pl6INK4a, p21, PAI-1, or any one or more SASP factors (e.g., IL-6, MMP3). Senescent and non-senescent cells of a biological sample may also be used in in vitro cell assays, where the cells are exposed to any one of the senolytic agents described herein to determine the ability of the senolytic agent to kill the subject's senescent cells without undesirable toxicity to non-senescent cells. Additionally, these methods may be used to monitor the level of senescent cells in a subject before, during, and after treatment with a senolytic agent. In certain embodiments, the presence of senescent cells may be detected (e.g., by determining the level of mRNA senescent cell marker expression), and the treatment course and / or non-treatment interval may be adjusted accordingly.

[0205] Combination therapy The senolytic agents and compositions disclosed herein may also be used in combination with one or more other active ingredients. In certain embodiments, the compounds may be administered in combination or sequentially with another therapeutic agent. Such other therapeutic agents include those known for the treatment, prevention, or amelioration of one or more of the symptoms or disorders described herein.

[0206] It should be understood that any suitable combination of the compounds and pharmaceutical compositions provided herein with one or more of the above therapeutic agents, and optionally one or more additional pharmacologically active substances, is considered to be within the scope of the present disclosure. In some embodiments, the compounds and pharmaceutical compositions provided herein are administered prior to or subsequent to the one or more additional active ingredients.

[0207] Pharmaceutical Compositions and Methods of Administration Also provided herein is a pharmaceutical composition comprising a senolytic agent as described herein and at least one pharma- ceutically acceptable additive, which may also be referred to as a pharma- ceutically suitable additive or carrier (i.e., a non-toxic material 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 additives described herein are examples and are in no way limiting. An effective amount or therapeutically effective amount refers to an amount of one or more senolytic agents administered to a subject, either as a single dose or as part of a series of doses, effective to produce a desired therapeutic effect.

[0208] When two or more senolytic agents are administered to a subject for the treatment of a disease or disorder described herein, each of the senolytic agents may be formulated into a separate pharmaceutical composition. A pharmaceutical preparation may be prepared that includes each of the separate pharmaceutical compositions (which may be conveniently referred to as, for example, a first pharmaceutical composition and a second pharmaceutical composition that include, respectively, a first and a second senolytic agent). Each of the pharmaceutical compositions in the preparation may be administered simultaneously (i.e., concurrently) and via the same route of administration, or may be administered at different times by the same or different routes of administration. Alternatively, two or more senolytic agents may be formulated together in a single pharmaceutical composition.

[0209] In other embodiments, a combination of at least one senolytic agent and at least one inhibitor of the mTOR, NF-□B, or PI3K pathway may be administered to a subject in need thereof. When at least one senolytic agent and one or more inhibitors of the mTOR, NF-□B, or PI3K pathway are both used together in the methods described herein for selectively killing senescent cells, each of the agents may be formulated into the same pharmaceutical composition or may be formulated in separate pharmaceutical compositions. A pharmaceutical preparation may be prepared that includes each of the separate pharmaceutical compositions (which may be conveniently referred to as, for example, a first pharmaceutical composition and a second pharmaceutical composition, each of which includes a senolytic agent and one or more inhibitors of the mTOR, NF-□B, or PI3K pathway, respectively). Each of the pharmaceutical compositions in the preparation may be administered simultaneously and via the same route of administration, or may be administered at different times by the same or different routes of administration.

[0210] The pharmacokinetics of a senolytic agent (or one or more of its metabolites) administered to a subject may be monitored by determining the level of the senolytic agent in a biological fluid, such as blood, a blood fraction (e.g., serum), and / or urine, and / or other biological sample or biological tissue from the subject. Any method practiced in the art and described herein for detecting agents may be used to measure the level of the senolytic agent during the course of treatment.

[0211] The dose of the senolytic agent described herein for treating a disease or disorder associated with senescent cells may depend on the condition of the subject, i.e., the stage of the disease, the severity of the symptoms caused by the disease, the overall physical health, as well as the age, sex, and weight, and other factors that will be apparent to one skilled in the art of medicine. The pharmaceutical composition may be administered in a manner that is appropriate for the disease being treated, as determined by one skilled in the art of medicine. In addition to the factors described herein and above that relate to the use of the senolytic agent for treating a disease or disorder associated with senescence, the appropriate duration and frequency of administration of the senolytic agent may also be determined or adjusted by factors such as the condition of the patient, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. The optimal dose of the agent may generally be determined using experimental models and / or clinical trials. The optimal dose may depend on the subject's body mass, weight, or blood volume. The use of the minimum dose sufficient to achieve effective treatment is usually preferred. The design and execution of preclinical and clinical studies for the senolytic agents described herein (including when administered for prophylactic benefit) is well within the skill of one of ordinary skill in the relevant art. When two or more senolytic agents are administered to treat a disease or disorder associated with aging, the optimal dose of each senolytic agent may be different, e.g., lower, than when either agent is administered alone as a single drug treatment. In certain embodiments, two senolytic agents in combination act synergistically or additively, and either agent may be used in a lower amount than when administered alone. The amount of senolytic agent that may be administered per day may be, for example, about 0.01 mg / kg to 100 mg / kg (e.g., about 0.1 to 1 mg / kg, about 1 to 10 mg / kg, about 10 to 50 mg / kg, about 50 to 100 mg / kg body weight). In other embodiments, the amount of senolytic agent that may be administered per day is about 0.01 mg / kg to 1000 mg / kg, about 100 to 500 mg / kg, or about 500 to 1000 mg / kg of body weight.The optimal dosage (per day or per treatment course) may differ for the age-related disease or disorder being treated and may vary with the route of administration and treatment regimen.

[0212] A pharmaceutical composition comprising a senolytic agent can be formulated in a manner suitable for the delivery method by using techniques routinely practiced in the art. The composition can be in the form of a solid (e.g., tablet, capsule), semi-solid (e.g., gel), liquid, or gas (aerosol). In other specific embodiments, the senolytic agent (or pharmaceutical composition comprising it) is administered as a bolus injection. In certain embodiments, when the senolytic agent is delivered by injection, the senolytic agent is delivered to the organ or tissue containing the senescent cells to be killed via the blood vessels by a person skilled in the art of medicine by techniques routinely practiced.

[0213] Pharmaceutically acceptable excipients are well known in the pharmaceutical art and are described, for example, in 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 stEd. Mack Pub. Co., Easton, Pa. (2005). Exemplary pharma- ceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes, buffers, and the like may be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents may also be used. In general, the type of excipient is selected based on the mode of administration and the chemical composition of the active ingredient. Alternatively, the compositions described herein may be formulated as a lyophilizate. The compositions described herein may be lyophilized or otherwise formulated as a lyophilized product using one or more suitable excipient solutions to solubilize and / or dilute the agent of the composition upon administration. In other embodiments, the agent may be encapsulated in liposomes using techniques known and practiced in the art. The pharmaceutical composition may be formulated for any suitable mode of administration as described herein and in the art.

[0214] The pharmaceutical composition may be delivered to a subject in need thereof by any one of several routes known to those skilled in the art. As non-limiting examples, the composition may be delivered orally, intravenously, intraperitoneally, by injection (e.g., bolus injection), subcutaneously, intestinal, rectally, intranasally, by inhalation, buccal, sublingual, intramuscular, transdermal, intradermal, topically, intraocular, vaginal, rectal, or intracranial injection, or by any combination thereof. In certain embodiments, the administration of such doses is intravenous, intraperitoneal, directly into the target tissue or organ, or by subcutaneous route. In certain embodiments, the delivery method includes a stent coated or impregnated with a drug, and the drug is a senolytic agent. Formulations suitable for such delivery methods are described in more detail herein.

[0215] In certain embodiments, the senolytic agent (which may be combined with at least one pharma- ceutically acceptable excipient to form a pharmaceutical composition) is administered directly to a target tissue or organ that contains senescent cells that contribute to the manifestation of a disease or disorder. In certain embodiments, when treating osteoarthritis, at least one senolytic agent is administered directly to the osteoarthritic joint (i.e., intra-articularly) of a subject in need thereof. In other certain embodiments, the senolytic agent may be administered to the joint by topical, transdermal, intradermal, or subcutaneous routes. In other certain embodiments, provided herein are methods for treating cardiovascular diseases or disorders associated with arteriosclerosis, e.g., atherosclerosis, by administering directly to an artery. In other embodiments, the senolytic agent (which may be combined with at least one pharma-ceutically acceptable excipient to form a pharmaceutical composition) for treating pulmonary diseases or disorders associated with senescence may be administered by inhalation, intranasally, by intubation, or intrathecally, e.g., to provide the senolytic agent more directly to the affected lung tissue. As another non-limiting example, the senolytic agent (or a pharmaceutical composition comprising the senolytic agent) may be delivered directly to the eye by injection (e.g., intraocular or intravitreal) or by conjunctival application of a cream, ointment, gel, or eye drops under the eyelid. In more specific embodiments, the senolytic agent or a pharmaceutical composition comprising the senolytic agent may be formulated as a sustained release (also called extended release, controlled release) composition or may be administered as a bolus injection.

[0216] The pharmaceutical composition (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery, or other methods) may be in the form of a liquid. The liquid pharmaceutical composition may, for example, contain one or more of the following: a sterile excipient, such as water, saline, preferably saline, Ringer's solution, isotonic sodium chloride, a fixed oil that may act as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol, or other solvent; an antibacterial agent; an antioxidant; a chelating agent; a buffer and an agent for adjusting osmotic pressure, such as sodium chloride or dextrose. The parenteral composition may be enclosed in an ampoule, a disposable syringe, or a multiple dose vial made of glass or plastic. The use of saline is preferred, and the injectable pharmaceutical composition is preferably sterile. In other embodiments, for the treatment of ophthalmic conditions or diseases, the liquid pharmaceutical composition may be applied to the eye in the form of eye drops. The liquid pharmaceutical composition may be delivered orally.

[0217] For oral formulations, at least one of the senolytic agents described herein can be used alone or in combination with suitable additives to produce tablets, powders, granules, or capsules, along with excipients, buffers, wetting agents, preservatives, colorants, and flavoring agents, as needed. The compounds may be formulated with buffers and / or enteric coatings that provide protection of the compounds from the low pH of the stomach environment. The senolytic agents included in the pharmaceutical composition may be formulated for oral delivery, for example, with flavoring agents in liquid, solid, or semi-solid formulations, and / or with enteric coatings.

[0218] Pharmaceutical compositions comprising any one of the senolytic agents described herein may be formulated for sustained or delayed release (also referred to as sustained or controlled release). Such compositions may generally be prepared using well-known techniques and administered, for example, by oral, rectal, intradermal, or subcutaneous implantation, or by implantation at the desired target site. Sustained release formulations may contain the compound dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate-limiting membrane. Additives for use within such formulations may be biocompatible and biodegradable; preferably, the formulation provides a relatively constant level of release of the active component. The amount of active agent contained within a sustained release formulation will depend on the site of implantation, the rate and expected duration of release, and the nature of the condition, disease, or disorder to be treated or prevented.

[0219] In certain embodiments, the pharmaceutical composition comprising the senolytic agent is formulated for transdermal, intradermal, or topical administration. The composition can be administered as a powder / talc or other solid, liquid, spray, aerosol, ointment, foam, cream, gel, paste, using a syringe, bandage, transdermal patch, insert, or syringe-like applicator. It is preferably in the form of a controlled or sustained release formulation that is administered topically or injected directly into the skin (intradermal or subcutaneous) adjacent to or within the area to be treated. The active composition can also be delivered via iontophoresis. Preservatives can 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 propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

[0220] The pharmaceutical composition comprising the senolytic agent can be formulated as an emulsion for topical application. The emulsion contains one liquid distributed in a second liquid. The emulsion can be an oil-in-water emulsion or a water-in-oil emulsion. Either or both of the oil and water phases can contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other additives. The oil phase can contain other oil-based pharma- ceutical approved additives. Suitable surfactants include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. The composition for topical application can also contain at least one suitable suspending agent, antioxidant, chelating agent, emollient, or moisturizing agent.

[0221] Ointments and creams can be formulated with an aqueous or oily base, for example, by adding suitable thickening and / or gelling agents.Lotions can be formulated with an aqueous or oily base, and generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents, or colorants.Liquid sprays can be delivered, for example, from pressurized packs via specially shaped closures.Oil-in-water emulsions can also be used in compositions, patches, dressings, and articles.These systems are semisolid emulsions, microemulsions, or foam emulsion systems.

[0222] Controlled or sustained release transdermal or topical formulations can be achieved by the addition of time-release additives available in the art, such as polymeric structures, matrices. For example, the composition can be administered by the use of hot-melt extrusion articles, such as bioadhesive hot-melt extruded films. The formulation can include cross-linked polycarboxylic acid polymer formulations. The cross-linking agent can be present in an amount that provides adequate adhesion to allow the system to remain attached to the target epithelial or endothelial cell surface for a sufficient period of time to allow the desired release of the compound.

[0223] The insert, transdermal patch, dressing or article may include a polymer blend or coating that provides for release of the active agent at a constant rate over an extended period of time. In some embodiments, the article, transdermal patch or insert includes a water-soluble pore former, such as polyethylene glycol (PEG), that may be blended with a water-insoluble polymer to increase the durability of the insert and extend the release of the active ingredient.

[0224] Polymer formulations can also be utilized to achieve controlled or sustained release. Bioadhesive polymers described in the art can be used. As an example, sustained release gels and compounds can be incorporated into polymer matrices, e.g., hydrophobic polymer matrices. Examples of polymer matrices include microparticles. The microparticles can be microspheres, where the core can be of a different material than the polymer shell. Alternatively, the polymer can be cast as a thin plate or film, a powder produced by milling or other standard techniques, or a gel, e.g., a hydrogel. The polymer can also be in the form of a coating or part of a bandage, stent, catheter, vascular graft, or other device that facilitates delivery of the senolytic agent. Matrices can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art.

[0225] Kits are provided having unit doses of one or more of the agents described herein, typically in oral or injectable doses. Such kits may include a container containing the unit doses, an informational insert describing the use and associated benefits of the agent in treating diseases associated with senescent cells, and optionally an instrument or device for delivery of the composition.

[0226] All references and patents are incorporated herein in their entirety for all purposes.

[0227] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. EXAMPLES

[0228] [ka] Scheme 1 illustrates the preparation of key intermediate 203.

[0229] (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (202) [ka] To a mixture of 2-methylindole-3-ethylamine (200) (15.0 g, 86.1 mmol) and 4-formylcinnamic acid (201) (15.2 g, 86.1 mmol) in THF:DCM:MeOH (200 mL:200 mL:25 mL) was added acetic acid (1.0 mL). Sodium triacetoxyborohydride (43.8 g, 206.6 mmol) was then added portionwise at ambient temperature. The reaction mixture was stirred overnight at ambient temperature and filtered through a sintered glass funnel. The precipitate was washed with ethyl acetate (300 mL), water (300 mL) and saturated NaHCO3 solution (150 mL) followed by drying under high vacuum to give the desired product (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (202) (26 g, 70% yield). LC / MS (Method A): RT=2.41 min; m / z=334.41, found=335.4 [M+H].

[0230] (E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (203) [ka] (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (202) (260 mg, 0.77 mmol, 1.0 equiv) and sodium bicarbonate (245 mg, 2.92 mmol, 3.8 equiv) were suspended in dioxane-water (3:1) (5.1 mL, 0.15 M). Fmoc chloride was added portionwise at 0° C. (230 mg, 0.89 mmol, 1.15 equiv). The reaction mixture was allowed to warm to room temperature. Analysis by LCMS showed the desired product. Dilute HCl was added until the pH was about pH 2. The aqueous solution was extracted twice with ethyl acetate and the combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give an orange solid. The crude product was subjected to normal phase purification (eluting with 10-100% ethyl acetate in hexanes). The product fractions were combined and concentrated to dryness to give (E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (203) as an off-white solid (240 mg, 56% yield). LC / MS (Method A): RT=3.91 min; m / z=556.6, found=557.6 [M + H], total time = 6 min.

[0231] [ka] Scheme 2 illustrates the preparation of key intermediate 211.

[0232] 1,2,3,4-Tetra-O-acetyl-L-fucose (205) [ka] L-Fucose (204) (50 g, 0.3 mol) was dissolved in a solution of acetic anhydride (400 mL, 4.23 mol) and pyridine (800 mL, 9.9 mol). The reaction mixture was stirred at room temperature overnight, concentrated under reduced pressure, and the residue was diluted with EtOAc (2000 mL), washed with water (1000 mL), 10% aqueous citric acid (3×700 mL), water (1000 mL), and brine (1000 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was azeotroped with toluene (200 mL) and dried under high vacuum to give the crude product 1,2,3,4-tetra-O-acetyl-L-fucose (205) (100 g, quantitative), which was used in the next step without further purification.

[0233] (2S,3R,4R,5S)-4,5-Bis(acetyloxy)-6-bromo-2-methyloxan-3-yl acetate (206) [ka] 1,2,3,4-Tetra-O-acetyl-L-fucose (205) (101 g, 0.3 mol) was dissolved in anhydrous dichloromethane (500 mL) and cooled to 0° C. Then HBr (33% in AcOH, 135 mL) was added and the reaction mixture was allowed to warm to room temperature with stirring for 2 h. The reaction mixture was poured into an ice / water mixture and the organic layer was separated. The aqueous phase was extracted with CHCl (200 mL). The organic layer was washed with saturated NaHCO (100 mL), brine (150 mL), dried over anhydrous NaSO and concentrated under reduced pressure to give (2S,3R,4R,5S)-4,5-bis(acetyloxy)-6-bromo-2-methyloxan-3-yl acetate, compound (206) (115 g, quantitative) as a yellow oil. The crude material was used in the next step without further purification.

[0234] (2S,3R,4S)-4-(Acetyloxy)-2-methyl-3,4-dihydro-2H-pyran-3-yl acetate (207) [ka] To a stirred refluxing solution of Zn (111 g, 1.7 mol) and 1-methyl-imidazole (25 mL, 0.31 mol) in anhydrous ethyl acetate (1200 mL) was added dropwise (2S,3R,4R,5S)-4,5-bis(acetyloxy)-6-bromo-2-methyloxan-3-yl acetate (206) (100 g, 0.28 mol) in anhydrous ethyl acetate (200 mL) over 40 min. The reaction mixture was heated to reflux for 3 h until TLC analysis indicated the reaction was complete. The reaction mixture was cooled to room temperature and stirred for an additional 30 min, then filtered through a pad of celite. Concentration under reduced pressure gave the crude product, which was purified by silica gel flash chromatography (0-10% EtOAc in hexanes) to give the desired product (2S,3R,4S)-4-(acetyloxy)-2-methyl-3,4-dihydro-2H-pyran-3-yl acetate, compound (207) (38 g, 63% yield).

[0235] (2S,3R,4S)-4,6-Bis(acetyloxy)-2-methyloxan-3-yl acetate (208) [ka] To a cold solution of (2S,3R,4S)-4-(acetyloxy)-2-methyl-3,4-dihydro-2H-pyran-3-yl acetate, compound (207) (75 g, 0.35 mol) in anhydrous dichloromethane (500 mL) (ice / water bath) was added acetic acid (190 mL, 3.3 mol) and acetic anhydride (290 mL, 3 mol). The reaction mixture was stirred for 15 min and a 33% HBr solution in AcOH (19 mL) was added. The reaction mixture was stirred for an additional 30 min at which point the solution turned pale yellow. TLC analysis indicated complete consumption of starting material (lower spot, 25% EtOAc / hexanes). The reaction was quenched by addition of an ice / water mixture. The organic layer was washed thoroughly with water (2×1 L), followed by cold saturated aqueous NaHCO3 (1 L), water (1 L) and brine (1 L). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product, which was purified by silica gel flash chromatography (0-20% EtOAc in hexanes) to give the desired product (2S,3R,4S)-4,6-bis(acetyloxy)-2-methyloxan-3-yl acetate, compound (208), as a white solid (83 g, 86.2% yield).

[0236] (2S,3R,4S,6S)-4-(Acetyloxy)-6-bromo-2-methyloxan-3-yl acetate (209) [ka] To a solution of (2S,3R,4S)-4,6-bis(acetyloxy)-2-methyloxan-3-yl acetate (208) (82 g, 0.3 mol) in anhydrous dichloromethane (700 mL) was added 33% HBr in AcOH (80 mL) at 0° C. The reaction mixture was stirred for 15 min, and then ice-cold water (300 mL) was added to quench the reaction. The aqueous phase was extracted with dichloromethane (3×700 mL) and the combined organic layers were washed with brine (2×500 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give (2S,3R,4S,6S)-4-(acetyloxy)-6-bromo-2-methyloxan-3-yl acetate, compound (209), as a sticky oil. The crude material was carried on to the next step as soon as possible without further purification.

[0237] (2S,3R,4S,6S)-4-(Acetyloxy)-6-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)oxy]-2-methyloxan-3-yl acetate (210) [ka] To a solution of crude (2S,3R,4S,6S)-4-(acetyloxy)-6-bromo-2-methyloxan-3-yl acetate (209) and N-hydroxyphthalimide (54 g, 0.33 mol) in anhydrous dichloromethane (600 mL) was added triethylamine (55 mL, 0.33 mol) followed by BF3·OEt2 (92 mL, 0.75 mol) at 0 °C. The reaction mixture was allowed to reach room temperature and stirred for 1 h until the color became greenish grey. Cold saturated aqueous NaHCO3 (500 mL) was added and the organic layer was separated. The aqueous layer was extracted with dichloromethane (3 × 500 mL) and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude material. Silica gel flash chromatography (10% to 60% EtOAc in hexanes) afforded (2S,3R,4S,6S)-4-(acetyloxy)-6-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)oxy]-2-methyloxan-3-yl acetate (210) as a white foamy solid (75 g, 66% yield over two steps). LC / MS (Method B): RT=4.32 min; m / z=377.1, found=378.2 [M + H] + Total time = 12 minutes. 1 H NMR (500MHz, chloroform d) δ7.85 (ddd, J=5.5, 3.3, 0.6Hz, 2H), 7.76 (ddd, J=5.9, 2.9, 0.8Hz, 2H), 5.62-5.52 (m, IH), 5.43 (ddd, J=12.5, 5.3, 3.0Hz) , IH), 5.38-5.23(m, IH), 4.97(td, J=6.7, 6.7, 5.6Hz, IH), 2.35-2.18(m, 2H), 2.17(s, 3H), 2.03(d, J=0.6Hz, 3H), 1.14(dd, J=6.5, 0.6Hz, 3H).

[0238] (2S,3R,4S,6S)-4-(Acetyloxy)-6-(aminooxy)-2-methyloxan-3-yl acetate (211) [ka] A solution of (2S,3R,4S,6S)-4-(acetyloxy)-6-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)oxy]-2-methyloxan-3-yl acetate (210) (25 g, 0.066 mol) in methanol (500 mL) was cooled to 0° C. under ice / water bath. Hydrazine hydrate (5.5 mL, 0.066 mol) was added slowly and the resulting reaction mixture was stirred at 0° C. for another 30 min. The precipitate was filtered and the filtrate was diluted with dichloromethane (500 mL) and washed with cold aqueous NaHCO3 (2×350 mL), water (350 mL) and brine (350 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to give crude (2S,3R,4S,6S)-4-(acetyloxy)-6-(aminooxy)-2-methyloxan-3-yl acetate, compound (211) (12 g, 78% yield) as an off-white foamy solid. LC / MS (Method A): RT=1.22 min; m / z=247.2, found=248.3 [M+H]. + Total time = 6 minutes.

[0239] [ka] Scheme 3 illustrates the preparation of compound 101.

[0240] Preparation of (2S,3R,4S,6S)-3-(acetyloxy)-6-{[(2E)-3-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}-2-methyloxan-4-yl acetate (212) [ka] (E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (203) (710 mg, 1.27 mmol, 1.0 equiv.) was dissolved in DMF (7 mL). EDCI (277 mg, 1.77 mmol) and HOBt (240 mg, 1.77 mmol) were added and the resulting mixture was stirred at room temperature for 10 min, followed by the addition of a solution of (2S,3R,4S,6S)-4-(acetyloxy)-6-(aminooxy)-2-methyloxan-3-yl acetate (211) (437 mg, 1.4 mmol) in dichloromethane (1.0 mL) and DIPEA (0.29 mL). After 2 h, the reaction mixture was quenched by the addition of saturated ammonium chloride (5 mL) and extracted with EtOAc (2×50 mL). The combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product. Purification on a flash silica gel column (10-70% EtOAc in hexanes) gave (2S,3R,4S,6S)-3-(acetyloxy)-6-{[(2E)-3-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}-2-methyloxan-4-yl acetate (212) (540 mg, 54% yield). LC / MS (Method B): RT=6.19 min; m / z=785.3, found=786.7 [M + H], total time = 12 min.

[0241] Example 1: (2S,3R,4S,6S)-3-(acetyloxy)-2-methyl-6-{[(2E)-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}oxan-4-yl acetate, (101) [ka] To a solution of (2S,3R,4S,6S)-3-(acetyloxy)-6-{[(2E)-3-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}-2-methyloxan-4-yl acetate (212) (700 mg, 0.89 mmol, 1.0 equiv) in DMF (3.0 mL) was added triethylamine (3.0 mL). The reaction mixture was stirred at room temperature until complete consumption of starting material was indicated by LCMS. Cold saturated sodium bicarbonate solution (20 mL) was added and the reaction mixture was extracted with ethyl acetate (3×30 mL). The combined organic layers were concentrated under reduced pressure to give the crude product, which was purified by reverse phase HPLC using ammonium bicarbonate buffer to give (2S,3R,4S,6S)-3-(acetyloxy)-2-methyl-6-{[(2E)-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}oxan-4-yl acetate (101). LC / MS (Method C): RT=1.98 min; m / z=563.2, found=564.4 [M + H] + Total time = 6 minutes.

[0242] [ka] Scheme 4 illustrates the preparation of compound 102.

[0243] Example 2: (2E)-N-{[(2S,4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamide (102) [ka] To a solution of (2S,3R,4S,6S)-3-(acetyloxy)-2-methyl-6-{[(2E)-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamido]oxy}oxan-4-yl acetate (101) (230 mg, 0.4 mmol, 1.0 equiv.) in methanol (4.0 mL) was added 25% sodium methoxide in methanol (0.06 mL, 0.28 mmol) on an ice / water bath. The reaction mixture was gradually brought to room temperature and stirred until completion as indicated by LCMS. The reaction mixture was cooled in an ice / water bath and quenched by the addition of 1N aqueous HCl (0.1 mL) until pH 7 was achieved. The reaction mixture was concentrated under reduced pressure and the crude material was purified by reverse phase HPLC using ammonium bicarbonate buffer to give the desired product (2E)-N-{[(2S,4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamide (102) (70 mg, 36% yield). LC / MS (Method C): RT=1.44 min; m / z=479.2, found=480.1 [M+H] + Total time = 6 minutes. 1 H NMR (500MHz, DMSO-d6) δ11.10(d, J=62.0Hz, 1H), 10.64(s, 1H), 7.53-7.40(m, 3H), 7.34(dd, J=8.0, 2.7Hz, 3H), 7.19( d, J=7.9Hz, 1H), 6.97-6.91(m, 1H), 6.90-6.84(m, 1H), 6.44(d, J=15.9Hz, 1H), 5.01(d, J=3.8Hz, 1H), 4.64(d, J=6.0H) z, 1H), 4.37(d, J=4.7Hz, 1H), 4.04(d, J=7.0Hz, 1H), 3.73(s, 2H), 3.42(d, J=8.0Hz, 1H), 2.77(t, J=7.4, 7.4Hz, 2H), 2 .66(t, J=7.4, 7.4Hz, 2H), 2.29(s, 3H), 1.83(td, J=12.7, 12.5, 4.0Hz, 1H), 1.76-1.64(m, 1H), 1.11(d, J=6.5Hz, 3H).

[0244] [ka] Scheme 5 illustrates the preparation of compound 109.

[0245] tert-Butyl N-({4-[(1E)-2-({[(4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate (213) [ka] To a solution of (2E)-N-{[(2S,4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamide (101), (100 mg, 0.208 mmol) in DCM (2 mL) was added (Boc)O (64 mg, 0.292 mmol) and triethylamine (0.072 mL, 0.416 mmol). The reaction mixture was stirred at 40° C. for 3 h and the solvent was removed under reduced pressure to give crude tert-butyl N-({4-[(1E)-2-({[(4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate (213), which was used in the next step without further purification. LC / MS (Method A): RT=2.81 min; m / z=579.6, found=580.5 [M + H], total time = 6 min.

[0246] tert-Butyl N-({4-[(1E)-2-({[(3aR,4S,7aS)-4-methyl-2-oxo-hexahydro-[1,3]dioxolo[4,5-c]pyran-6-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate (214) [ka] To a solution of tert-butyl N-({4-[(1E)-2-({[(4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate, (213) (crude from previous step, 0.208 mmol) in THF (2 mL) was added CDI (50 mg, 0.308 mmol). The reaction mixture was stirred at 55° C. for 18 h and concentrated under reduced pressure to give crude tert-butyl N-({4-[(1E)-2-({[(3aR,4S,7aS)-4-methyl-2-oxo-hexahydro-[1,3]dioxolo[4,5-c]pyran-6-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate (214), which was used for the next step without purification. LC / MS (Method A): RT=3.2 min; m / z=605.7, found=606.7 [M+H] + Total time = 6 minutes.

[0247] Example 3: (2E)-N-{[(3aR,4S,7aS)-4-methyl-2-oxo-hexahydro-[1,3]dioxolo[4,5-c]pyran-6-yl]oxy}-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamide (109) [ka] The crude tert-butyl N-({4-[(1E)-2-({[(3aR,4S,7aS)-4-methyl-2-oxo-hexahydro-[1,3]dioxolo[4,5-c]pyran-6-yl]oxy}carbamoyl)eth-1-en-1-yl]phenyl}methyl)-N-[2-(2-methyl-1H-indol-3-yl)ethyl]carbamate (214) (0.208 mmol) obtained in the previous step was dissolved in 20% TFA in DCM (2 mL). The reaction mixture was heated at 50° C. for 1 h and concentrated under reduced pressure. The crude residue was purified by reverse phase HPLC to give (2E)-N-{[(3aR,4S,7aS)-4-methyl-2-oxo-hexahydro-[1,3]dioxolo[4,5-c]pyran-6-yl]oxy}-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]prop-2-enamide (109) as a white solid (12.5 mg, 12% yield for 3 steps). LC / MS (Method C): RT=2.1 min; m / z=505.2, found=506.2 [M+H]. + Total time = 6 minutes. 1 H NMR (500MHz, DMSO-d6) δ10.63(s, 1H), 7.48(t, J=14.8, 14.8Hz, 3H), 7.36-7.31(m, 3H), 7.19(d, J=7.9 Hz, 1H), 6.96-6.90(m, 1H), 6.87(t, J=7.0, 7.0Hz, 1H), 6.44(d, J=16.0Hz, 1H), 5.13(d, J=8.5Hz, 1H), 5 .04(t, J=7.0, 7.0Hz, 1H), 4.74(dd, J=8.7, 1.8Hz, 1H), 4.18(d, J=6.1Hz, 1H), 3.72(s, 2H), 2.77(t, J=7 .4, 7.4Hz, 2H), 2.66(t, J=7.4, 7.4Hz, 2H), 2.28(s, 3H), 1.91(d, J=17.4Hz, 1H), 1.16(d, J=6.6Hz, 3H).

[0248] [ka] Scheme 6 illustrates the preparation of compound 107.

[0249] 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-5-carboxylic acid methyl ester (217) [ka] Tert-butyl(piperidin-4-ylmethyl)carbamate (215) (5 g, 23 mmol, 1.0 equiv.) and methyl 2-chloropyrimidine-5-carboxylate (216) (4.8 g, 28 mmol) in dioxane (90 mL) were treated with cesium carbonate (18 g, 57 mmol) and Pd(dba)2acetone (1.56 g, 1.7 mmol). The solution was purged with nitrogen (3×) and Xantphos (1.99 g, 3.45 mmol) was added in one portion. The suspension went from dark red to yellow-green within minutes. The reaction mixture was then heated at 70° C. for 30 min, at which point LCMS analysis indicated the presence of the desired product. The mixture was cooled to room temperature and filtered through a pad of Celite, washing with dichloromethane (3×40 mL). The solvent was removed and the residue was subjected to normal phase purification eluting with hexane-ethyl acetate (40-100%). The product fractions were collected, combined and concentrated to give 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-5-carboxylic acid methyl ester as an off-white solid (217) (5 g, 61% yield). LC / MS (Method A): RT=3.23 min; m / z=350.4, found=351.6 [M+H] + Total time = 6 minutes.

[0250] 2-(4-Aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester hydrochloride (218) [ka] 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-5-carboxylic acid methyl ester (217) (2.8 g, 8 mmol) was dissolved in THF (30 mL). 4N HCl / dioxane (10 mL) was added and the solution was heated at 70° C. for 2 h, during which time a solid precipitated. The precipitate was filtered, washed with ether / hexanes (3×) and dried under high vacuum to give 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester hydrochloride (218) as a white solid (2.3 g, quantitative). LC / MS (Method A): RT=1.89 min; m / z=250.3, found=251.4 [M + H], total time = 6 min.

[0251] Methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (220) [ka] To 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester (218) (2.3 g, 8 mmol) and triethylamine (4 mL, 28 mmol, 3.5 equiv.) in THF:DCE (1:1) 5% methanol (30 mL) was added 1-methyl-1H-indole-3-carbaldehyde (219) (1.2 g, 7.6 mmol) in one portion. Sodium triacetoxyborohydride (9.8 g, 48 mmol) was added followed by acetic acid (0.5 mL). NMP (1.1 mL) was then added and the mixture was stirred at room temperature for 2 days until LCMS analysis showed the formation of the desired product. Water was added, the pH was adjusted to 7 with sodium bicarbonate and the white solid was filtered and washed with water (10 mL) and ethyl acetate (20 mL). The product was dried under high vacuum to give methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (220) as a white solid (3 g, 95% yield). This material was used in the next step without further purification. LC / MS (Method A): RT=2.3 min; m / z=393.4, found=394.5 [M + H], total time = 6 min.

[0252] 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (221) [ka] Crude methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (220) (3.3 g, 8.4 mmol) and sodium hydroxide (2.76 mg, 69 mmol) were suspended in dioxane / water (3:1) (30.0 mL). The solution was heated at 70° C. for 2 h until LCMS analysis indicated complete reaction. Dioxane was removed and the mixture was acidified to pH ∼5. The precipitate was washed with water followed by hexanes. The grey solid was dried under high vacuum to give pure 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (221) (2.1 g, 66% yield). LC / MS (Method A): RT = 2.41 min; m / z = 379.4, found = 380.6 [M + H], total time = 6 min.

[0253] 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (222) [ka] 2-(4-((((1-Methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (221) (1.3 g, 3.43 mmol) and sodium bicarbonate (720 mg, 8.5 mmol) were suspended in THF:water (3:1) (20 mL). Fmoc-OSu (1.21 g, 3.63 mmol) was added in portions over the course of 1 h, followed by N-methyl-2-pyrrolidone (1.2 mL). The reaction was stirred until LCMS analysis showed the reaction was complete, concentrated in vacuo, and diluted with water (10 mL). Solid sodium bicarbonate was added to adjust the pH to approx. 8, and the aqueous solution was extracted with ethyl acetate (2×25 mL). The combined organic layers were discarded. The aqueous layer was acidified to pH approx. 2 with 1 N HCl and extracted with ethyl acetate (3×30 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the desired product 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)-methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (222) as a white foam (1.55 g, 76% yield). LC / MS (Method A): RT=3.96 min; m / z=601.7, found=602.3 [M + H], total time = 6 min.

[0254] (2S,3R,4S,6S)-3-(Acetyloxy)-6-[({2-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]-2-methyloxan-4-yl acetate (223) [ka] To a solution of 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)-methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (222) (250 mg, 0.41 mmol) in DMF (1.3 mL) was added EDCI (109 mg, 0.57 mmol) and HOBt (87 mg, 0.41 mmol). The resulting mixture was stirred at room temperature for 15 min. A solution of (2S,3R,4S,6S)-4-(acetyloxy)-6-(aminooxy)-2-methyloxan-3-yl acetate, compound (211) (100 mg, 0.404 mmol) in DCM / DMF (1:1; 0.4 mL) and DIPEA (0.1 mL) was added and the reaction mixture was stirred for 2 h. The reaction was quenched by the addition of a saturated aqueous solution of ammonium chloride (5 mL) and extracted with ethyl acetate (2×15 mL). The combined organic layers were washed with a 10% aqueous solution of sodium bicarbonate (10 mL) and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to give the crude product, which was purified by silica gel flash chromatography (30-70% ethyl acetate in hexanes) to give (2S,3R,4S,6S)-3-(acetyloxy)-6-[({2-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]-2-methyloxan-4-yl acetate (223) as a yellow foam (200 mg, 58% yield). LC / MS (Method B): RT = 7.06 min; m / z = 830.4, found = 831.1 [M+H] + Total time = 12 minutes.

[0255] Example 4: (2S,3R,4S,6S)-3-(acetyloxy)-2-methyl-6-[({2-[4-({[(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]oxan-4-yl acetate (107) [ka] (2S,3R,4S,6S)-3-(acetyloxy)-6-[({2-[4-({[(9H-fluoren-9-ylmethoxy)carbonyl][(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]-2-methyloxan-4-yl acetate (223) (200 mg, 0.24 mmol) was dissolved in DMF (2.0 mL). Triethylamine (2.0 mL) was added in one portion and the resulting reaction mixture was stirred at room temperature overnight. Saturated aqueous sodium bicarbonate (10 mL) was then added and the reaction mixture was extracted with ethyl acetate (3×20 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product (120 mg). A 40 mg portion of this crude material was purified by reverse phase HPLC using ammonium bicarbonate buffer to give (2S,3R,4S,6S)-3-(acetyloxy)-2-methyl-6-[({2-[4-({[(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]oxan-4-yl acetate (107) (19 mg, 39% yield). LC / MS (Method C): RT=2.31 min; m / z=608.3, found=609.4 [M + H], total time = 6 min. 1H NMR (500MHz, DMSO-d6) δ8.62(s, 2H), 7.59(dt, J=7.9, 1.0, 1.0Hz, 1H), 7.35(dd, J=8.2, 0.9Hz, 1H), 7.17(s, 1H), 7.11(ddd, J=8. 3, 7.1, 1.3Hz, 1H), 6.98(ddd, J=7.9, 7.0, 1.1Hz, 1H), 5.21(t, J=2.7, 2.7Hz, 1H), 5.14(ddd, J=11.0, 6.8, 3.0Hz, 1H), 5.11-5.07 (m, 1H), 4.68(d, J=13.2Hz, 2H), 4.46(q, J=6.2, 6.2, 6.2Hz, 1H), 3.81(s, 2H), 3.72(s, 3H), 2.91(td, J=13.1, 12.8, 2.7Hz, 2H), 2 .44(d, J=6.5Hz, 2H), 2.10(s, 3H), 1.99-1.94(m, 2H), 1.93(s, 3H), 1.81-1.70(m, 3H), 1.09-1.02(m, 2H), 1.01(d, J=6.5Hz, 3H).

[0256] [ka] Scheme 7 illustrates the preparation of compound 108.

[0257] Example 5: N-{[(2S,4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}-2-[4-({[(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidine-5-carboxamide (108) [ka] (2S,3R,4S,6S)-3-(Acetyloxy)-2-methyl-6-[({2-[4-({[(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidin-5-yl}formamido)oxy]oxan-4-yl acetate (107) (80 mg, 0.131 mmol) was suspended in methanol:water (2:1; 2.0 mL). Triethylamine (0.4 mL) was added and the resulting mixture was heated at 55° C. for 6 h. The reaction mixture was concentrated in vacuo and subsequently purified by reverse phase HPLC using ammonium bicarbonate buffer to give N-{[(2S,4S,5S,6S)-4,5-dihydroxy-6-methyloxan-2-yl]oxy}-2-[4-({[(1-methyl-1H-indol-3-yl)methyl]amino}methyl)piperidin-1-yl]pyrimidine-5-carboxamide (108) (60.5 mg, 88% yield). LC / MS (Method C): RT=1.82 min; m / z=524.3, found=525.7 [M + H], total time = 6 min. 1 H NMR (500MHz, DMSO4) δ8.62(s, 2H), 7.59(dt, J=7.9, 1.1, 1.1Hz, 1H), 7.35(dd, J=8.3, 1.0Hz, 1H), 7.17(s, 1H), 7.11(ddd, 8.2, 7. 0, 1.3Hz, 1H), 6.98(ddd, J=8.1, 7.0, 1.1Hz, 1H), 5.05(d, J=3.5Hz, 1H), 4.68(dt, J=12.3, 3.0, 3.0Hz, 2H), 4.64(d, J=6.3Hz, 1H) , 4.35(d, J=4.6Hz, 1H), 4.11(d, J=6.6Hz, 1H), 3.81(s, 2H), 3.79-3.74(m, 1H), 3.72(s, 3H), 3.42(t, J=3.9, 3.9Hz, 1H), 2.92(td , J=13.1, 12.9, 2.7Hz, 2H), 2.44(d, J=6.5Hz, 2H), 1.88-1.66(m, 5H), 1.08(d, J=6.6Hz, 3H), 1.02(ddd, J=15.6, 8.4, 3.6Hz, 2H).

[0258] [ka] Scheme 8 illustrates the preparation of compound 121.

[0259] (1S,2R,6R,8S,9R)-8-(Fluoromethyl)-4,4,11,11-tetramethyl-3,5,7,10,12-pentaoxatricyclo[7.3.0.02,6]dodecane (225) [ka] To a solution of 1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose (224) (1.7 mL, 7.68 mmol, 1.00 equiv.) in dichloromethane (20 mL) was added 2,4,6-trimethylpyridine (2.4 mL, 18.4 mmol, 2.40 equiv.). The mixture was cooled to 0° C. and treated with (diethylamino)sulfur trifluoride (1.2 mL, 9.22 mmol, 1.20 equiv.). The reaction mixture was stirred at room temperature under nitrogen and monitored by TLC (ethyl acetate:cyclohexane 1:1). After 18 h, the reaction mixture was diluted with dichloromethane, washed with saturated NaHCO3, brine (25 mL), dried (Na2SO4), filtered, and the volatiles were evaporated. The residue was purified by silica flash chromatography eluting with ethyl acetate:cyclohexane (0-20%) to give the title compound (225) (913 mg, 45%) as a colorless syrup. 1 H NMR (300MHz, CDCl3): d, 5.55 (d, J=4.9Hz, 1H), 4.69-4.58 (m, 2H), 4.48 (dq, J=6.1, 8.9Hz, 1H), 4.35 (ddd, J= 2.5, 2.5, 2.5Hz, 1H), 4.27(dd, J=2.0, 8.0Hz, 1H), 4.13-4.03(m, 1H), 1.55(s, 3H), 1.45(s, 3H), 1.34(s, 6H).

[0260] (3R,4S,5R,6S)-6-(fluoromethyl)tetrahydropyran-2,3,4,5-tetrol (226) [ka] (1S,2R,6R,8S,9R)-8-(fluoromethyl)-4,4,11,11-tetramethyl-3,5,7,10,12-pentaoxatricyclo[7.3.0.02,6]dodecane (225) (913 mg, 3.48 mmol, 1.00 equiv) was treated with a mixture of trifluoroacetic acid (8.0 mL, 0.104 mol, 30.0 equiv) and water (0.92 mL). The reaction mixture was stirred at room temperature and monitored by TLC (ethyl acetate:cyclohexane 1:1). After 0.5 h, the reaction was diluted with toluene and concentrated under reduced pressure to give the crude title compound (226) (1.0 g) as a pale beige syrup, which was carried on directly to the next synthetic step.

[0261] [(2S,3R,4S,5R)-4,5,6-triacetoxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (227) [ka] (3R,4S,5R,6S)-6-(fluoromethyl)tetrahydropyran-2,3,4,5-tetrol (634 mg, 3.48 mmol, 1.00 equiv) (226) was dissolved in dry pyridine (10 mL), cooled to 0° C., and treated with acetic anhydride (3.3 mL, 34.8 mmol, 10.0 equiv). The reaction mixture was stirred at room temperature under nitrogen and monitored by TLC (1:4 ethyl acetate:dichloromethane). After 5 h, the reaction was diluted with toluene (3×) and concentrated under reduced pressure to remove excess reagent. The oily residue was purified by flash chromatography (12 g cartridge eluting with ethyl acetate:dichloromethane (1:9)) to give the title compound (227) (960 mg, 79%) (mixture of anomers) as a colorless oil.

[0262] [(2S,3R,4S,5R,6R)-4,5-diacetoxy-6-bromo-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (228) [ka] In a reaction vessel protected from light, a solution of [(2S,3R,4S,5R)-4,5,6-triacetoxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (227) (300 mg, 0.856 mmol, 1.00 equiv) in dry dichloromethane (7 mL) was slowly treated with hydrogen bromide in acetic acid (33%) (1.4 mL) at 0° C. under nitrogen atmosphere. The reaction was allowed to warm to room temperature and monitored by TLC. After 1 h, the reaction mixture was slowly poured into a solution of NaHCO3 (2.10 g) in ice water (15 mL) and stirred for 15 min. The organic layer was passed through a phase separation cartridge and concentrated to give the title compound (229) (300 mg, 94%) as a white solid. 1 H NMR (300MHz, CDCl3): d6.72 (d, J=4.2Hz, 1H), 5.58 (d, J=3.5Hz, 1H), 5.42 (dd, J=3.2, 10.9Hz, 1H), 5.08 (dd, J=4.6, 10.6Hz, 1H), 4.58-4.49(m, 2H), 4.39-4.35(m, 1H), 2.15(s, 3H), 2.12(s, 3H), 2.02(s, 3H).

[0263] [(2S,3R,4S,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (229) [ka] A suspension of red N-hydroxyphthalimide (114 mg, 0.700 mmol, 1.00 equiv.), tetrabutylammonium bromide (113 mg, 0.350 mmol, 0.500 equiv.) in dichloromethane (0.8 mL) was added to a solution of potassium carbonate (106 mg, 0.770 mmol, 1.10 equiv.) in water (0.8 mL) and a solution of [(2S,3R,4S,5R,6R)-4,5-diacetoxy-6-bromo-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (228) (260 mg, 0.700 mmol, 1.00 equiv.) in dichloromethane (0.8 mL). The reaction mixture was stirred at room temperature for 24 h and monitored by LCMS. The reaction mixture was then partitioned between dichloromethane and saturated aqueous NaHCO3 solution. The organic layer was separated, dried (Na2SO4), filtered and the volatiles were evaporated to give the crude product which was purified by flash chromatography eluting with cyclohexane:ethyl acetate (2-60%) to give an impure solid (85 mg) which was further purified by dissolving in dichloromethane and separating the impurities by filtration. The filtrate was concentrated to give the title compound (229) (65 mg, 18%) as a dusty solid. 1 H NMR (300MHz, CDCl3) d7.88-7.83(m, 2H), 7.81-7.75(m, 2H), 5.53-5.46(m, 2H), 5.14(dd, J=3.7, 9.9Hz, 1 H), 5.03(d, J=8.8Hz, 1H), 4.66-4.36(m, 2H), 4.04-3.95(m, 1H), 2.23(s, 3H), 2.20(s, 3H), 2.03(s, 3H). LC / MS: Rt=1.53 min; m / z=476[M+Na]+.

[0264] [(2S,3R,4S,5R,6S)-4,5-diacetoxy-6-aminooxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (230) [ka] To a suspension of [(2S,3R,4S,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (229) (280 mg, 0.618 mmol, 1.00 equiv) in methyl alcohol (4 mL) was added hydrazine monohydrate (98%, 0.031 mL, 0.618 mmol, 1.00 equiv) slowly at 0° C. The reaction mixture was stirred at 0° C. for 30 min and monitored by TLC (1:1 ethyl acetate:cyclohexane). The solid was removed by filtration and discarded. The filtrate was diluted with dichloromethane, washed with cold aqueous NaHCO3, water and brine, dried (Na2SO4), filtered and the volatiles were evaporated to give a solid (169 mg) which was purified by flash chromatography (12 g cartridge eluting with cyclohexane:ethyl acetate (5-80%)) to give the title compound (230) (158 mg, 79%) as a white solid. 1 H NMR: (300MHz, CDCl3): 5.84(s, 2H), 5.46(d, J=3.2Hz, 1H), 5.27(dd, J=8.3, 10.4Hz, 1H), 5.06(dd, J=3.4, 10. 4Hz, 1H), 4.72(d, J=8.5Hz, 1H), 4.64-4.34(m, 2H), 4.06-3.96(m, 1H), 2.17(s, 3H), 2.09(s, 3H), 2.00(s, 3H).

[0265] [(2S,3R,4S,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoyl]amino]oxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (231) [ka] To a solution of (E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoic acid (189 mg, 0.340 mmol, 1.00 equiv) (203) in N,N-dimethylformamide (6.0 mL) was added in one portion 1-hydroxybenzotriazole hydrate (69 mg, 0.453 mmol, 1.33 equiv) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (87 mg, 0.453 mmol, 1.33 equiv). After 15 min, the mixture was cooled to 0° C. and a solution of [(2S,3R,4S,5R,6S)-4,5-diacetoxy-6-aminooxy-2-(fluoromethyl)tetrahydropyran-3-yl]acetate (110 mg, 0.340 mmol, 1.00 equiv.) (230) in N,N-dimethylformamide (1 mL) was added slowly containing N,N-diisopropylethylamine (0.079 mL, 0.453 mmol, 1.33 equiv.). The resulting mixture was allowed to reach room temperature and stirred for an additional 18 h. The reaction mixture was cooled to 0° C. and quenched by the slow addition of cold saturated aqueous NH4Cl solution to give a pale yellow precipitate, which was collected by filtration and rinsed with water. The collected solid was dissolved in ethyl acetate and washed with water, saturated aqueous NaHCO3 solution, and brine. The organic layer was dried (Na2SO4), filtered and the volatiles were evaporated to give the crude product (254 mg) which was purified by flash chromatography (12 g silica cartridge eluted with cyclohexane:ethyl acetate (0-70%) to give the title compound (231) (182 mg, 62%) as a yellow solid. LC / MS: Rt=1.92 min; m / z=862 [M+H] + .

[0266] Example 6: [(2S,3R,4S,5R,6S)-4,5-diacetoxy-2-(fluoromethyl)-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-3-yl]acetate (121) [ka] To a solution of [(2S,3R,4S,5R,6S)-4,5-diacetoxy-2-(fluoromethyl)-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-3-yl]acetate (231) (85 mg, 0.120 mmol, 57%) in N,N-dimethylformamide (1.6 mL) was added triethylamine (0.81 mL, 5.82 mmol, 27.5 equiv.) at 0° C. The reaction mixture was stirred at 0° C. for 10 min and then at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure and the oily residue was dissolved in ethyl acetate and washed with saturated aqueous NH4Cl solution. The organic layer passed through the phase separator cartridge and the filtrate were concentrated to give a beige solid (140 mg) which was purified by silica chromatography 12 g (silica cartridge (50 micron) eluting with dichloromethane:MeOH (0-10%)) to give a beige solid (110 mg). Further purification by silica chromatography (12 g silica cartridge, 15 micron) eluting with c-hexane:(ethyl acetate:IPA 3:1) gave the title compound (121) (85 mg, 57%) as a white solid. 1 H NMR (400MHz, DMSO) d10.68-10.65(m, 1H), 7.54-7.47(m, 3H), 7.39-7.34(m, 3H), 7.22 -7.19(m, 1H), 6.97-6.86(m, 2H), 6.50-6.40(m, 1H), 5.34-5.25(m, 2H), 5.07-5.02(m , 2H), 4.62-4.35(m, 3H), 3.78-3.75(m, 2H), 3.33(m, 2H, under water signal), 2.79(t, J=7.2Hz, 2 H), 2.69(t, J=7.0Hz, 2H), 2.30(s, 3H), 2.13(s, 3H), 2.10(s, 3H), 1.95-1.94(m, 3H). LC / MS: Rt=3.37 min, m / z=640.2[M+H] + .

[0267] [ka] Scheme 8 illustrates the preparation of compound 122.

[0268] Example 7: (E)-N-[(2S,3R,4S,5R,6S)-6-(fluoromethyl)-3,4,5-trihydroxy-tetrahydropyran-2-yl]oxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide (122) [ka] To a solution of [(2S,3R,4S,5R,6S)-4,5-diacetoxy-2-(fluoromethyl)-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-3-yl]acetate (121) (90%, 75 mg, 0.106 mmol, 1.00 equiv) in methyl alcohol (2.50 mL) was added water (0.35 mL) and triethylamine (0.37 mL, 2.65 mmol, 25.1 equiv). The reaction mixture was stirred at room temperature for 24 h. The crude product was concentrated to dryness, dissolved in 1:1 CH3CN:water, and lyophilized overnight to give the title compound (122) (57 mg, 99%) as an off-white solid. 1H NMR (400MHz, MeOD): d7.60-7.49(m, 3H), 7.38(d, J=7.8Hz, 1H), 7.29(d, J=8.0Hz, 2H), 7.24-7.21(m , 1H), 7.00(ddd, J=1.0, 7.1, 8.1Hz, 1H), 6.92(ddd, J=1.0, 7.0, 7.8Hz, 1H), 6.49(d, J=15.3Hz, 1H), 4 .68-4.65(m, 1H), 4.60(d, J=8.3Hz, 1H), 4.57-4.53(m, 1H), 3.92-3.85(m, 1H), 3.84(s, 3H), 3.69(dd , J=7.9, 9.7Hz, 1H), 3.58(dd, J=3.4, 9.7Hz, 1H), 2.98-2.92(m, 2H), 2.89-2.85(m, 2H), 2.34(s, 3H). 19F NMR (400MHz, MeOD) 231.52ppm. LC / MS:Rt=2.61min;m / z=514[M+H] + .

[0269] [ka] Scheme 9 illustrates the preparation of compound 123.

[0270] (2R,3R,4S,6S)-6-(acetoxymethyl)tetrahydro-2H-pyran-2,3,4-triyl triacetate (233) [ka] To a solution of (3R,4S,6S)-6-(hydroxymethyl)tetrahydropyran-2,3,4-triol (232) (500 mg, 3.05 mmol, 1.00 equiv.) and 4-(dimethylamino)pyridine (37 mg, 0.305 mmol, 0.100 equiv.) in pyridine (10 mL) at 0° C., acetic anhydride (4.3 mL, 45.7 mmol, 15.0 equiv.) was added over a period of 10 min and the reaction mixture was stirred at 0° C. for 2.5 h. The reaction mixture was concentrated to a minimum volume and the remaining pyridine was coevaporated with toluene. The oily residue was redissolved in toluene and washed with 1M HCl, water and brine. The organic phase was dried (Na2SO4), filtered and concentrated to give the title compound (233) (993 mg, 98%). 1 H NMR (300MHz, CDCl3): d, 5.69-5.65(m, 1H), 5.09-4.99(m, 2H), 4.19-4.15(m, 2H), 3.96-3 .87(m, 1H), 2.23-2.16(m, 1H), 2.12(s, 3H), 2.10(s, 3H), 2.06(s, 6H), 1.73-1.59(m, 1H).

[0271] (2R,3R,4S,6S)-6-(acetoxymethyl)-2-bromotetrahydro-2H-pyran-3,4-diyl diacetate (234) [ka] To a reaction vessel protected from light was added [(2S,4S,5R)-4,5,6-triacetoxytetrahydropyran-2-yl]methyl acetate (233) (200 mg, 0.602 mmol, 1.00 equiv) and dichloromethane (5 mL). The flask was kept at 0° C. and hydrogen bromide in acetic acid (33%) (0.6 mL) was added slowly under a nitrogen atmosphere. The reaction mixture was stirred at room temperature and monitored by TLC. After 3 h, TLC (1:1 cyclohexane:ethyl acetate) showed R fThe expected product was shown at 0.60. The crude reaction mixture was added portionwise to a beaker containing a mixture of sodium bicarbonate (1.1 g) and ice water (8 mL) and mixed vigorously (evolving gas) for 5 min. The organic phase was separated and the aqueous phase was further extracted with dichloromethane (30 mL). The combined organic phase was dried (Na2SO4), filtered and the volatiles were evaporated to give the title compound (234) (200 mg, 94%) as a colorless oil. 1 H NMR (300MHz, CDCl3): d, 6.66 (d, J=3.9Hz, 1H), 4.79 (dd, J=3.9, 9.9Hz, 1H), 4.43-4.34 (m, 1H), 4.18 (d, J=4 .6Hz, 2H), 2.34-2.25(m, 1H), 2.13(s, 3H), 2.12(s, 3H), 2.07(s, 3H), 1.71(ddd, J=12.0, 12.0, 12.0Hz, 1H).

[0272] [(2S,4S,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-tetrahydropyran-2-yl]methyl acetate (235) [ka] To a solution of N-hydroxyphthalimide (93 mg, 0.570 mmol, 0.950 equiv) and tetrabutylammonium bromide (97 mg, 0.300 mmol, 0.500 equiv) in dichloromethane (0.6 mL) was added a solution of potassium carbonate (91 mg, 0.660 mmol, 1.10 equiv) in water (1.2 mL), followed by a solution of [(2S,4S,5R,6R)-4,5-diacetoxy-6-bromo-tetrahydropyran-2-yl]methyl acetate (234) (212 mg, 0.600 mmol, 1.00 equiv) in dichloromethane (1.0 mL). The reaction mixture was stirred in the dark for 24 h and monitored by LCMS. The reaction mixture was then partitioned between dichloromethane and saturated aqueous NaHCO3. The organic layer was separated and the aqueous layer was extracted with additional dichloromethane. The combined organic extracts were separated using a phase separation cartridge and the volatiles were evaporated to give a light brown oil (322 mg) which was purified by silica chromatography (12 g cartridge eluting with cyclohexane:ethyl acetate (2-50%)) to give the title compound (235) (172 mg, 66%) as a white foam. 1 H NMR (300MHz, CDCl3) d7.88-7.83(m, 2H), 7.81-7.74(m, 2H), 5.21(dd, J=7.7, 9.1Hz, 1H), 5.13-5.03(m, 1H), 5.01(d, J=7.9Hz, 1H), 4.28(dd, J=5.9, 11 .6Hz, 1H), 4.13(d, J=5.1Hz, 1H), 3.85-3.76(m, 1H), 2.25-2.17(m, 1H), 2. 21(s, 3H), 2.07(s, 3H), 2.04(s, 3H), 1.76(ddd, J=12.2, 12.2, 12.2Hz, 1H). LC / MS:Rt=1.49 min; m / z=458[M+Na] + .

[0273] [(2S,4S,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-tetrahydropyran-2-yl]methyl acetate (236) [ka] To a suspension of [(2S,4S,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-tetrahydropyran-2-yl]methyl acetate (235) (270 mg, 0.620 mmol, 1.00 equiv.) in methyl alcohol (2 mL) was added hydrazine monohydrate (98%, 0.031 mL, 0.620 mmol, 1.00 equiv.). The reaction mixture was stirred at 0° C. and monitored by TLC. After 35 min, the resulting solid was filtered, washed with cold methanol and discarded. The filtrate was diluted with dichloromethane and washed with saturated aqueous NaHCO3, water and brine. The organic layer was dried (Na2SO4), filtered and the volatiles were evaporated to give the crude product. Purification by flash chromatography, (12 g silica cartridge eluting with cyclohexane:ethyl acetate (10-70%)) afforded the title compound (236) (180 mg, 95%) as a white solid. 1 H NMR (300MHz, CDCl3): d, 5.76-5.74(m, 2H), 5.04-4.95(m, 2H), 4.62(d, J=8.8Hz, 1H), 4.20-4.16(m, 2 H), 3.85-3.76(m, 1H), 2.18-2.11(m, 1H), 2.10(s, 3H), 2.08(s, 3H), 2.03(s, 3H), 1.66-1.57(m, 1H).

[0274] [(2S,4S,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-2-yl]methyl acetate (237) [ka] To a solution of (E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoic acid (237 mg, 0.426 mmol, 1.00 equiv) (203) in N,N-dimethylformamide (4.5 mL) was added 1-hydroxybenzotriazole hydrate (87 mg, 0.566 mmol, 1.33 equiv) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (109 mg, 0.566 mmol, 1.33 equiv). After stirring at room temperature for 10 min, the solution was cooled to 0° C. and a solution of [(2S,4S,5R,6S)-4,5-diacetoxy-6-aminooxy-tetrahydropyran-2-yl]methyl acetate (236) (130 mg, 0.426 mmol, 1.00 equiv.) in N,N-dimethylformamide (1.5 mL) containing N,N-diisopropylethylamine (0.099 mL, 0.566 mmol, 1.33 equiv.) was slowly added. The resulting solution was stirred at room temperature overnight. The reaction mixture was then concentrated to a minimum volume, cooled to 0° C., and quenched with saturated aqueous NH4Cl. The yellow precipitate was collected by filtration, washed with water, redissolved in ethyl acetate, washed with water, aqueous NaHCO3 (sat.), and brine. The organic layer was separated, dried (Na2SO4), filtered, and the volatiles were evaporated to give the crude product. Purification by flash chromatography (12 g silica cartridge eluted with cyclohexane:ethyl acetate (5-70%)) afforded the title compound (237) (270 mg, 75%) as a yellow solid. LC / MS: Rt=1.93 min; m / z=844 [M+H] + .

[0275] Example 8: [(2S,4S,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-2-yl]methyl acetate (123) [ka] To a solution of [(2S,4S,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-2-yl]methyl acetate (237) (250 mg, 0.296 mmol, 1.00 equiv.) in N,N-dimethylformamide (2.25 mL) was slowly added triethylamine (1.1 mL, 8.15 mmol, 27.5 equiv.) at 0° C. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the residue was partitioned between ethyl acetate and saturated aqueous NH4Cl. The organic layer was separated, dried (Na2SO4), filtered and concentrated to give the crude product, which was purified by flash chromatography (12 g, 50 um silica cartridge, eluting with [cyclohexane:(EA:IMS 3:1)] (1-60%)) to give the title compound (123) (142 mg, 77%) as a white solid. 1 H NMR (400MHz, MeOD): d7.58-7.47(m, 3H), 7.38(d, J=7.8Hz, 1H), 7.29(d, J=8.8Hz, 2H) , 7.22(d, J=7.7Hz, 1H), 7.02-6.89(m, 2H), 6.49-6.42(m, 1H), 5.16-5.09(m, 1H), 4.9 7-4.88(m, 2H), 4.20-4.17(m, 2H), 3.97-3.92(m, 1H), 3.84-3.82(m, 2H), 2.97-2.84( m, 4H), 2.35-2.34 (m, 3H), 2.17-2.00 (m, 10H), 1.63 (ddd, J=12.1, 12.1, 12.1Hz, 1H). LC / MS:Rt=3.34min;m / z=622.2[M+H] + .

[0276] [ka] Scheme 10 illustrates the preparation of compound 124.

[0277] Example 9: (E)-N-[(2S,3R,4S,6S)-3,4-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide [ka] To a solution of [(2S,4S,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-2-yl]methyl acetate (110 mg, 0.177 mmol, 1.00 equiv) in methyl alcohol (4.40 mL) (123) was added water (0.60 mL) and triethylamine (0.62 mL, 4.42 mmol, 25.0 equiv). The reaction mixture was stirred at room temperature for 24 hours. LCMS showed starting material and intermediates. The reaction mixture was stirred at room temperature for 48 hours. Additional triethylamine (148 uL) and water (148 uL) were added and the mixture was stirred for an additional 6 hours. The reaction mixture was concentrated under reduced pressure and the crude product was purified by flash chromatography (12 g, 15 micron cartridge eluting with dichloromethane:MeOH (1-20%)) to give the title (124) compound (49 mg, 53%) as a white solid. 1H NMR (400MHz, DMSO): d10.68 (s, 1H), 7.56-7.50 (m, 3H), 7.36 (d, J=7.9Hz, 3H), 7.20 (d, J=7.9Hz, 1H), 6.97-6.86(m, 2H), 6.48(d, J=16.0Hz, 1H), 5.05-4.99(m, 1H), 4.72(s, 1H), 4.50(d, J=7.9Hz, 1H), 3. 75-3.73(m, 2H), 3.57-3.43(m, 3H), 3.43-3.39(m, 1H), 3.03-2.98(m, 1H), 2.78(t, J=7.3Hz, 2H), 2.6 7(t, J=7.4Hz, 2H), 2.30(s, 3H), 1.81(dd, J=4.3, 12.1Hz, 1H), 1.18(ddd, J=12.0, 12.0, 12.0Hz, 1H). LC / MS:RT=2.47min;m / z=496.2[M+H] + .

[0278] [ka] Scheme 11 illustrates the preparation of compound 125.

[0279] (1R,2R,6S,7R,8R)-4,4-Dibutyl-3,5,10,11-tetraoxa-4-stannatricyclo[6.2.1.02,6]undecane-7-ol (239) [ka] A mixture of 1,6-anhydro-β-D-glucose (238) (5.00 g, 30.8 mmol, 1.00 equiv.) and dibutyltin(IV) oxide (7.68 g, 30.8 mmol, 1.00 equiv.) in toluene (150 mL) was refluxed for 12 h in an apparatus equipped for azeotropic removal of water (see Grindley et al., C-arbohydrate Res. 1988, 172, 311). The cooled mixture was evaporated under reduced pressure to give the crude stannylene derivative (239) as a white semi-solid, which was used without purification.

[0280] 1,6-Anhydro-4-Op-tolylsulfonyl-β-D-glucopyranose (240) [ka] To a solution of (1R,2R,6S,7R,8R)-4,4-dibutyl-3,5,10,11-tetraoxa-4-stannatricyclo[6.2.1.02,6]undecan-7-ol (239) (12.54 g, 31.9 mmol, 1.00 equiv.) in tetrahydrofuran (300 mL) was added triethylamine (4.9 mL, 35.1 mmol, 1.10 equiv.) and powdered 4A molecular sieves (3 g). p-Toluenesulfonyl chloride (6.69 g, 35.1 mmol, 1.10 equiv.) was added and the mixture was stirred vigorously for 2 days and then filtered through Celite. The filtrate was evaporated and the residue was diluted with dichloromethane (150 mL). The organic solution was washed with water (2×50 mL), dried (sodium sulfate) and evaporated. The crude material was purified by column chromatography on silica gel using 7:3 dichloromethane:2-methyltetrahydrofuran as the eluent. The first component to elute was 1,6-anhydro-2,4-di-Op-tolylsulfonyl-β-D-glucopyranose, which was easily separated. The second component was the desired product (240) (approximately 8 g of a colorless oil), which was contaminated with the other regioisomer 1,6-anhydro-2-Op-tolylsulfonyl-β-D-glucopyranose, which was difficult to separate. The mixture was recrystallized from a mixture of acetone, ether, and petroleum ether (boiling point 30-60 °C) to give the desired product (240) as white needles. A second recrystallization gave the pure product (2.2 g, 22%) as a single regioisomer. 1H NMR (400MHz, CDCl3): d7.83(d, J=8.3Hz, 2H), 7.38(d, J=8.1Hz, 2H), 5.48(s, 1H), 4.65(d, J=5.4Hz, 1H), 4.42(s, 1H), 4.13(d , J=8.1Hz, 1H), 3.79-3.71(m, 2H), 3.49(dd, J=0.9, 11.3Hz, 1H), 2.50(d, J=7.5Hz, 1H), 2.47(s, 3H), 2.32(d, J=11.4Hz, 1H).

[0281] 1,6-Anhydro-2,3-bis(O-methoxymethyl)-4-O-(4-toluenesulfonyl)-β-D-glucopyranose (241) [ka] To a stirred solution of [(1R,2S,3R,4R,5R)-3,4-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-2-yl]4-methylbenzenesulfonate (240) (2.20 g, 6.95 mmol, 1.00 equiv.) in dichloromethane (50 mL) was added N,N-diisopropylethylamine (13 mL, 76.5 mmol, 11.0 equiv.) and chloromethyl methyl ether (5.3 mL, 69.5 mmol, 10.0 equiv.). The mixture was stirred at 40° C. for 4 h to give a brown solution. The solution was cooled and then quenched with water (50 mL). The mixture was extracted with dichloromethane (2×50 mL) and the combined organic phase was washed with brine (100 mL). The organic solution was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel 40 g, ethyl acetate / hexane, (5-50%)) to give [(1R,2R,3R,4R,5R)-3,4-bis(methoxymethoxy)-6,8-dioxabicyclo[3.2.1]octan-2-yl]4-methylbenzenesulfonate (241) (2.10 g, 5.19 mmol, 75%) as a colorless oil, Rf=0.5 (silica, ethyl acetate / cyclohexane 1:1). 1H NMR (400MHz, CDCl3): d7.84(d, J=8.3Hz, 2H), 7.36(d, J=8.1Hz, 2H), 5.46(s, 1H), 4.68-4.63(m, 2H), 4.59(s, 2H), 4.58-4.53(m, 1H), 4.44 (s, 1H), 4.04(d, J=7.7Hz, 1H), 3.86-3.84(m, 1H), 3.71(dd, J=6.0, 7.5Hz, 1H), 3.52-3.50(m, 1H), 3.37(s, 3H), 3.32(s, 3H), 2.45(s, 3H).

[0282] 1,6-anhydro-4-deoxy-4-fluoro-2,3-bis(O-methoxymethyl)-β-D-galactopyranose (242) [ka] [(1R,2R,3R,4R,5R)-3,4-bis(methoxymethoxy)-6,8-dioxabicyclo[3.2.1]octan-2-yl]4-methylbenzenesulfonate (241) (2.10 g, 5.19 mmol, 1.00 equiv.) was stirred at reflux in tetrabutylammonium fluoride (1 M in THF, 55 mL, 10 equiv.) for 5 days. The black mixture was cooled and evaporated. The residue was diluted with water (100 mL) and the mixture was extracted with ethyl acetate (3×50 mL). The combined organic phases were washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by flash column chromatography (silica gel, ethyl acetate / cyclohexane, 0-30%) to give (1R,2S,3R,4R,5R)-2-fluoro-3,4-bis(methoxymethoxy)-6,8-dioxabicyclo[3.2.1]octane (242) as a pale yellow oil (470 mg, 25% yield, 70% purity). This inseparable mixture containing the desired fluoro product and other unknown products was used without further purification for the next step. Rf=0.51 (silica, ethyl acetate / cyclohexane 2:3). NMR (400 MHz, CDCl3) was consistent with product (242) as the major component (approximately 70% purity).

[0283] 1,2,3,6-Tetra-O-acetyl-4-deoxy-4-fluoro-α / β-D-galactopyranose (243) [ka] To a stirred solution of a mixture containing compound (1R,2S,3R,4R,5R)-2-fluoro-3,4-bis(methoxymethoxy)-6,8-dioxabicyclo[3.2.1]octane (242) (470 mg, 1.86 mmol, 1.00 equiv.) (70% purity) in acetic anhydride (5.3 mL, 55.9 mmol, 30.0 equiv.) was added sulfuric acid (0.99 mL, 18.6 mmol, 10.0 equiv.) dropwise at 0° C. The mixture was stirred at room temperature for 72 h. The mixture was then cooled to 0° C. and sodium acetate (3.06 g, 37.3 mmol, 20.0 equiv.) was added and stirred for an additional 20 min, then quenched with water (20 mL). The mixture was extracted with dichloromethane (3×15 mL). The combined organic phase was washed successively with water (3×30 mL) and brine (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, 12 g, 15 μm, ethyl acetate in cyclohexane, 1-40%) to give the product [(2R,3S,4R,5R)-4,5,6-triacetoxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (243) (360 mg, 0.925 mmol, 50%) a mixture of anomers (α / β=4:1) as a colorless oil (360 mg, 90% purity, ca. 50% yield). Rf=0.4 (silica, AcOEt / hexane, 1:1). 1 H NMR (400MHz, CDCl3): δ6.39(d, J=3.5Hz, 1H), 5.43-5.39(m, 1H), 5.32-5.21(m, 1H), 4.97(dd , J=2.7, 50.2Hz, 1H), 4.32-4.16(m, 3H), 2.16(s, 3H), 2.14(s, 3H), 2.09(s, 3H), 2.03(s, 3H).

[0284] [(2R,3S,4R,5R)-4,5-Diacetoxy-6-bromo-3-fluoro-tetrahydropyran-2-yl]methyl acetate (244) [ka] To a stirred solution of [(2R,3S,4R,5R)-4,5,6-triacetoxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (243) (360 mg, 1.03 mmol, 1.00 equiv.) in dichloromethane (6.00 mL) was added 6 M hydrogen bromide (4.0 mL, 24.0 mmol, 23.4 equiv.) as a 33 wt.% solution in AcOH at 0° C. The mixture was stirred at room temperature for 1 h and then quenched with saturated aqueous NaHCO3 (20 mL) at 0° C. The dichloromethane layer was filtered through a hydrophobic frit and not evaporated. Most of the SM had reacted, although TLC (50:50 ethyl acetate:cyclohexane) showed a less polar spot. The crude bromide (244) was used as a solution in dichloromethane for the next step without further purification.

[0285] [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (245) [ka] To a solution of N-hydroxyphthalimide (224 mg, 1.37 mmol, 0.950 equiv), tetrabutylammonium bromide (233 mg, 0.721 mmol, 0.500 equiv), dichloromethane (2.0 mL) and potassium carbonate (219 mg, 1.59 mmol, 1.10 equiv) in water (3.9 mL) was added a solution of [(2R,3S,4R,5R)-4,5-diacetoxy-6-bromo-3-fluoro-tetrahydropyran-2-yl]methyl acetate (244) (535 mg, 1.44 mmol, 1.00 equiv) in dichloromethane (2.0 mL). The solution was stirred at room temperature and monitored by LCMS. After 24 h, the reaction mixture was partitioned between dichloromethane and saturated aqueous NaHCO3. The organic layer was separated and the aqueous layer was extracted with additional dichloromethane. The combined organic extracts were dried (MgSO4), filtered and the volatiles were evaporated to give the crude product which was purified by flash chromatography, (20 g silica cartridge eluting with cyclohexane:ethyl acetate (2-40%)) to give the title compound (245) (443 mg, 68%). 1 H NMR (300MHz, CDCl3): d7.91-7.77(m, 4H), 5.55(dd, J=8.6, 9.8Hz, 1H), 5.14-4.80(m, 3H), 4.45(dd, J=6. 0, 11.3Hz, 1H), 4.28(dd, J=7.3, 11.2Hz, 1H), 3.92-3.78(m, 1H), 2.24(s, 3H), 2.17(s, 3H), 2.06(s, 3H). LC / MS;Rt=1.58 min;m / z=476[M+Na] + .

[0286] [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-aminooxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (246) [ka] To a suspension of [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-(1,3-dioxoisoindolin-2-yl)oxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (246) (440 mg, 0.971 mmol, 1.00 equiv) in methyl alcohol (6.00 mL) at 0° C., hydrazine monohydrate (0.047 mL, 0.971 mmol, 1.00 equiv) was added slowly. The reaction mixture was stirred at 0° C. for 45 min and monitored by TLC (1:1 ethyl acetate:cyclohexane) and LCMS. The solid was separated by filtration, dried, and kept aside. The filtrate was diluted with dichloromethane and washed with cold saturated aqueous NaHCO3 and water. The organic layer was passed through a phase separation cartridge and concentrated to give a crude oil. Both the impure solid and the crude oil were purified by flash chromatography (12 g silica cartridge eluting with cyclohexane:ethyl acetate (10-80%)) to afford the title compound (247) (230 mg, 73%) as a white solid. 1 H NMR (300MHz, CDCl3): d, 5.82(s, 2H), 5.39(t, J=8.7Hz, 1H), 5.07-4.78(m, 2H), 4.71(d, J=8.2Hz, 1 H), 4.43-4.25(m, 2H), 3.87(ddd, J=6.7, 6.7, 26.3Hz, 1H), 2.14(s, 3H), 2.12(s, 3H), 2.10(s, 3H).

[0287] [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoyl]amino]oxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (247) [ka] To a solution of (E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoic acid (203) (250 mg, 0.449 mmol, 1.00 equiv) in N,N-dimethylformamide (4.0 mL) at room temperature was added 1-hydroxybenzotriazole hydrate (91 mg, 0.597 mmol, 1.33 equiv) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (114 mg, 0.597 mmol, 1.33 equiv) in one portion. After stirring for 10 min, the solution was cooled to 0° C. and a solution of [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-aminooxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (246) (145 mg, 0.449 mmol, 1.00 equiv.) in N,N-dimethylformamide (2.0 mL) containing N,N-diisopropylethylamine (0.10 mL, 0.597 mmol, 1.33 equiv.) was slowly added. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated to a minimum volume, cooled to 0° C., and quenched by the slow addition of saturated aqueous NH4Cl (15 mL). The resulting yellow precipitate was collected by filtration and washed with water. The solid was redissolved in ethyl acetate and washed with water, aqueous NaHCO3 (saturated), and brine. The organic layer was separated, dried (Na2SO4), filtered and the volatiles were evaporated to give the crude product (443 mg) which was purified by flash chromatography (12 g silica cartridge eluted with cyclohexane:ethyl acetate (5-50%)) to give the title compound (247) (236 mg, 61%) as a white solid. LC / MS: Rt=1.97 min; m / z=862 [M+H] + .

[0288] Example 10: [(2S,3R,4S,5R,6S)-4,5-diacetoxy-2-(fluoromethyl)-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-3-yl]acetate (125) [ka] To a solution of [(2R,3S,4R,5R,6S)-4,5-diacetoxy-6-[[(E)-3-[4-[[9H-fluoren-9-ylmethoxycarbonyl-[2-(2-methyl-1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enoyl]amino]oxy-3-fluoro-tetrahydropyran-2-yl]methyl acetate (247) (236 mg, 0.274 mmol, 1.00 equiv.) in N,N-dimethylformamide (2.5 mL) was added triethylamine (1.1 mL, 7.54 mmol, 27.5 equiv.) at 0° C. The reaction mixture was stirred at room temperature and after 18 h the solution was concentrated to a small volume. The oily residue was dissolved in ethyl acetate and washed with saturated aqueous NH4Cl. The organic layer was passed through a phase separator cartridge and the filtrate was concentrated to give the crude product, which was purified by flash chromatography (12 g silica cartridge, 15 micron, eluting with cyclohexane:ethyl acetate (15-60%)) to give the title compound (125) (48 mg, 26%) as a white solid. 1 H NMR (400MHz, DMSO): d10.65(s, 1H), 7.52-7.46(m, 3H), 7.37-7.33(m, 3H), 7. 21-7.18(m, 1H), 6.97-6.86(m, 2H), 6.45-6.40(m, 1H), 5.41-5.29(m, 1H), 5. 09-4.92(m, 3H), 4.24-4.19(m, 3H), 3.75-3.73(m, 2H), 3.33(m, 2H, under water signal), 2.78(t, J=7.3Hz, 2H), 2.67(t, J=7.2Hz, 2H), 2.30(s, 3H), 2.09-2.03(m, 9H). LC / MS: Rt=3.37 min; m / z=640 [M+H] + .

[0289] [ka] Scheme 12 illustrates the preparation of compound 126.

[0290] Example 11: (E)-N-[(2S,3R,4S,5R,6S)-6-(fluoromethyl)-3,4,5-trihydroxy-tetrahydropyran-2-yl]oxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide (126) [ka] To a solution of [(2R,3S,4R,5R,6S)-4,5-diacetoxy-3-fluoro-6-[[(E)-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enoyl]amino]oxy-tetrahydropyran-2-yl]methyl acetate (125) (58 mg, 0.0907 mmol, 1.00 equiv) in methyl alcohol (2.50 mL) was added water (0.35 mL) and triethylamine (0.32 mL, 2.27 mmol, 25.1 equiv). The reaction mixture was stirred at room temperature and monitored by LCMS. After 48 h, the crude reaction mixture was concentrated to dryness and purified by flash chromatography (12 g silica cartridge, 15 micron eluting with dichloromethane:MeOH (1-20%)) to give the title compound (126) (18 mg, 38%) as a white solid. 1 H NMR (400MHz, DMSO): d10.65(s, 1H), 7.57-7.48(m, 3H), 7.38-7.34(m, 3H), 7.20(d, J=7 .9Hz, 1H), 6.98-6.86(m, 2H), 6.48(d, J=16.2Hz, 1H), 5.38-5.38(m, 1H), 4.95-4.88(m , 1H), 4.72-4.51(m, 2H), 3.76-3.53(m, 6H), 3.43(t, J=8.9Hz, 1H), 3.33(m, 2H, under water signal ), 2.79(t, J=7.3Hz, 2H), 2.67(t, J=7.3Hz, 2H), 2.31-2.29(m, 3H), 1.25-1.22(m, 1H). LC / MS:Rt=2.52min;m / z=514[M+H] + .

[0291] [ka] Scheme 13 illustrates the preparation of compound 127.

[0292] [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-(4-formylphenoxy)-2-methyl-tetrahydropyran-3-yl]acetate (248) [ka] A mixture of 1,2,3,4-tetra-o-acetyl-alpha-1-fucopyranose (205) (3.00 g, 9.03 mmol, 1.00 equiv.) and 4-hydroxybenzaldehyde (2.20 g, 18.1 mmol, 2.00 equiv.) was suspended in 1,2-dichloroethane (40 ml) under argon, 4-(dimethylamino)pyridine (4.41 g, 36.1 mmol, 4.00 equiv.) was added, and the mixture was stirred for 15 minutes to ensure dissolution. The solution was cooled in ice-water under argon. Boron trifluoride diethyl etherate (14 mL, 0.112 mol, 12.4 equiv.) was added dropwise to give a light brown solution. The resulting solution was heated at 63° C. (external) for 3 hours until TLC (20% ethyl acetate in toluene) showed product. The brown solution was cooled and neutralized by slow addition to saturated aqueous NaHCO3 until bubbling ceased. The product was extracted with dichloromethane. The dichloromethane extract was washed with 1N NaOH to remove unreacted phenol along with brine, dried (PTFE), and concentrated. The crude product was purified by chromatography on silica ((40 g, 50 μm), eluting with 0-20% ethyl acetate in toluene) to elute the first desired product (248) (1.30 g, 2.64 mmol, 29%) as a yellow oil that semi-crystallized on standing. 1H NMR (400MHz, CDCl3) d9.93(s, 1H), 7.86(d, J=9.0Hz, 2H), 7.21-7.17(m, 2H), 5.86(d, J=3.7Hz, 1H), 5.58(dd, J=3.3, 11.0Hz, 1H), 5.37(d d.

[0293] [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-(hydroxymethyl)phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (249) [ka] A solution of [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-(4-formylphenoxy)-2-methyl-tetrahydropyran-3-yl]acetate (248) (80%, 1.30 g, 2.64 mmol, 1.00 equiv.) in dichloromethane (2.00 mL) and methyl alcohol (18.00 mL) was cooled in ice water. Sodium borohydride (100 mg, 2.64 mmol, 1.00 equiv.) was added and the solution was stirred for 30 min; the yellow color disappeared. TLC (ethyl acetate:cyclohexane 1:1) showed disappearance of starting material with the appearance of a more polar spot. The mixture was quenched by adding 1 M hydrogen chloride (2.6 mL, 2.64 mmol, 1.00 equiv.). The solvent was evaporated and the crude material was dispersed between water and dichloromethane. The organic extract was washed with brine, dried (PTFE) and evaporated to give the product as a white foam and dried in vacuo. The crude material was purified on silica using 0-50% ethyl acetate in cyclohexane to give the product (249) (880 mg, 2.22 mmol, 84%) as a white foam. 1H NMR (400MHz, CDCl3) d7.32(d, J=8.4Hz, 2H), 7.05(d, J=8.7Hz, 2H), 5.74(d, J=3.7Hz, 1H), 5.58(dd, J=3.4, 10.9Hz, 1H), 5.36(d, J=3.1Hz, 1H), 5.28(dd , J=3.6, 10.9Hz, 1H), 4.64(d, J=5.8Hz, 2H), 4.27(q, J=6.6Hz, 1H), 2.20(s, 3H), 2.06(s, 3H), 2.03(s, 3H), 1.60(t, J=5.9Hz, 1H), 1.12(d, J=6.5Hz, 3H).

[0294] [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-(bromomethyl)phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (250) [ka] A solution of [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-(hydroxymethyl)phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (249) (200 mg, 0.505 mmol, 1.00 equiv) in dry diethyl ether (14 mL) was cooled to 0° C. and phosphorus tribromide (0.024 mL, 0.252 mmol, 0.500 equiv) was added. The solution was stirred at 0° C. and monitored by TLC (ethyl acetate:cyclohexane 1:1). After 45 min, the reaction was quenched with saturated aqueous NaHCO3. The product was extracted into diethyl ether, dried (Na2SO4), filtered, and the solvent was evaporated to give the title compound (250) (185 mg, 80%) as a white solid. 1H NMR (300MHz, CDCl3) d7.35(td, J=2.5, 9.5Hz, 2H), 7.04(td, J=2.5, 9.6Hz, 2H), 5.76(d, J=3.6Hz, 1H), 5.59(dd, J=3.4, 10.9Hz, 1H), 5.38(dd, J= 1.2, 3.5Hz, 1H), 5.29(dd, J=3.7, 10.8Hz, 1H), 4.50(s, 2H), 4.26(q, J=6 .4Hz, 1H), 2.21(s, 3H), 2.07(s, 3H), 2.04(s, 3H), 1.14(d, J=6.5Hz, 3H).

[0295] Example 12: [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-[[[4-[[2-(4-chlorophenyl)-5,5-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]benzoyl]-[4-[[3-morpholino-1-(phenylsulfanylmethyl)propyl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonyl-amino]methyl]phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (127) [ka] Potassium hydroxide (36 mg, 0.637 mmol, 1.80 equiv.) was dissolved in 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[3-morpholino-1-(phenylsulfanylmethyl)propyl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonyl-benzamide in dry toluene (14 mL). To a stirred mixture of (251) (345 mg, 0.354 mmol, 1.00 equiv.), [(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-(bromomethyl)phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (250) (183 mg, 0.398 mmol) and tetrabutylammonium bromide (23 mg, 0.0708 mmol, 0.200 equiv.) was added. The mixture was stirred overnight at 80° C. under nitrogen and monitored by LCMS and TLC (dichloromethane:methanol 9.5:0.5). After 48 h, the reaction mixture was cooled to room temperature and the toluene was removed by evaporation. The residue was partitioned between water and ethyl acetate. The organic layer was passed through a phase separation cartridge and the volatiles were evaporated to give the crude product (450 mg). Purification by flash chromatography (40 g, 15 micron silica cartridge eluting with dichloromethane:MeOH 0-3%) gave an impure product which was repurified by flash chromatography (25 g, 15 micron silica cartridge eluting with cyclohexane:[EA:IMS (3:1)] 0-50%) to give an impure product which was purified by SFC using a YMC Cellulose-SC, 10×250 mm column, 5 um, 55 / 45 MeOH (0.1% NH4OH) / CO2, 15 ml / min, 120 bar, 40° C., DAD 330 nm to give the title compound (127) in two batches: (19 mg, 99% purity) as a white solid and (50 mg, 80% purity) as a beige solid. 1H NMR (400MHz, CDCl3)d7.89-7.84(m, 2H), 7.54(d, J=9.0Hz, 2H), 7.40-7.36( m, 5H), 7.35-7.27(m, 5H), 7.10(d, J=9.2Hz, 2H), 7.04(d, J=9.2Hz, 1H), 6.99 (d, J=8.4Hz, 2H), 6.93(d, J=8.4Hz, 2H), 6.78-6.74(m, 2H), 6.57(d, J=9.3Hz , 1H), 5.64(d, J=3.7Hz, 1H), 5.54(dd, J=3.4, 10.9Hz, 1H), 5.34(dd, J=0.9, 3 .4Hz, 1H), 5.26(dd, J=3.6, 10.9Hz, 1H), 4.85(s, 2H), 4.22(q, J=6.5Hz, 1H), 3.94-3.85(m, 1H), 3.68-3.63(m, 4H), 3.26(t, J=4.9Hz, 4H), 3.13-3.00(m, 2 H), 2.79(s, 2H), 2.43-2.22(m, 12H), 2.16-2.07(m, 1H), 2.02(d, J=8.2Hz, 8H ), 1.72-1.63(m, 1H), 1.46(t, J=6.5Hz, 2H), 1.10-1.08(m, 3H), 0.99(s, 6H). LC / MS:Rt=6.87min;m / z=677.6[M+H] + .

[0296] [ka] Scheme 14 illustrates the preparation of compound 128.

[0297] Example 13: 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[3-morpholino-1-(phenylsulfanylmethyl)propyl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonyl-N-[[4-[rac-(2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyl-tetrahydropyran-2-yl[oxyphenyl]methyl]benzamide [ka] To a suspension of [rac-(2S,3R,4R,5S,6S)-4,5-diacetoxy-6-[4-[[[4-[[2-(4-chlorophenyl)-5,5-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]benzoyl]-[4-[[3-morpholino-1-(phenylsulfanylmethyl)propyl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonyl-amino]methyl]phenoxy]-2-methyl-tetrahydropyran-3-yl]acetate (127) (40 mg, 0.0296 mmol, 1.00 equiv) in methyl alcohol (1.6 mL) was added water (228 uL) and triethylamine (25 uL, 0.177 mmol, 6.00 equiv) at 0 °C. The reaction was monitored by TLC 1:1 cyclohexane:[3:1 EA:IMS] and stirred at room temperature overnight to give a solid suspended in solution. The reaction was concentrated to remove most of the volatiles and the residue was dry loaded into HMN for purification by flash chromatography (12 g, 15 micron silica cartridge, eluting with cyclohexane:[3:1 EA:IMS] (5-100%) to give unreacted starting material (10 mg) as a white solid and the title compound (11.6 mg) (128) as a white solid. 1H NMR (400MHz, DMSO) d7.97(d, J=2.3Hz, 1H), 7.79(dd, J=2.1, 9.4Hz, 1H), 7.43(d, J=8.9Hz, 2H), 7.39-7.26(m, 6H), 7.23-7.18(m, 1H), 7.12-7.05(m, 3) H), 7.01(d, J=9.9Hz, 1H), 6.97(d, J=8.7Hz, 2H), 6.90-6.83(m, 4H), 5.29( d, J=2.8Hz, 1H), 4.84(s, 2H), 4.77(d, J=5.9Hz, 1H), 4.64(d, J=5.4Hz, 1H) , 4.55(d, J=4.5Hz, 1H), 4.17-4.08(m, 1H), 3.83(q, J=6.5Hz, 1H), 3.76-3. 67(m, 2H), 3.56-3.43(m, 5H), 3.41-3.32(m, 2H), 3.27-3.20(m, 4H), 2.75- 2.71(m, 2H), 2.34-2.19(m, 10H), 2.17-2.12(m, 2H), 2.01-1.87(m, 3H), 1. 78-1.70(m, 1H), 1.43(t, J=6.4Hz, 2H), 1.02(d, J=6.6Hz, 3H), 0.97(s, 6H). LC / MS:Rt=1.41min;m / z=614[M+H] + / 2.

[0298] LCMS method: Method A: Chromlith, C-18, 50 x 4.6 mm; flow rate of 1.5 mL / min, ELSD and UV detection at 254 nm; Mobile phase A: 0.1% TFA in water; Mobile phase B: 0.1% TFA in acetonitrile; 5 to 100% mobile phase B over 6 min; ambient temperature.

[0299] Method B: Chromlith, C-18, 50 x 4.6 mm; flow rate of 1.5 mL / min, ELSD and UV detection at 254 nm; Mobile phase A: 0.1% TFA in water; Mobile phase B: 0.1% TFA in acetonitrile; 5 to 100% mobile phase B over 12 min; ambient temperature.

[0300] Method C: Water Cortex, C18, 3.0mm x 50mm, 2.7um column, 3uL injection, 1.2mL / min flow rate, UV detection at 220 and 254nm, 5% ACN (0.1% TFA) over 4 minutes to 100% water (0.1% TFA), stay at 100% (ACN, 0.1% TFA) for 0.5 minutes, then equilibrate to 5% (ACN, 0.1% TFA) over 1.5 minutes.

[0301] Cell hydrolysis assay T47D breast cancer cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum. Cell lines were infected with lentiviral constructs containing S.pyogenes Cas9 and sgRNA targeting the gene of interest. Infected cells were selected by antibiotic treatment. To assess cellular hydrolysis of compounds, cells were seeded in 96-well plates. The following day, 30uM of compound was added to the cells. Relative fluorescence (excitation 330nm / emission 450nm) was recorded at baseline and monitored every 24 hours for 2-4 days using a Molecular Devices SpectraMax M5 plate reader. The average relative fluorescence of each compound in medium without cells at each time point was subtracted from the relative fluorescence produced by wells containing cells. Product ladder

[0302] Experimental procedure for CRISPR engineered cancer cell line viability assay T47D and HCC1954 breast cancer cells were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum. Cell lines were infected with lentiviral constructs containing S. pyogenes Cas9 and sgRNA targeting the gene of interest. Infected cells were selected by antibiotic treatment. To assess cell viability following compound treatment, cells were seeded in 96-well plates. The next day, serial dilutions covering 10 concentrations of compound were added to the cells. Cells were treated for 3 days. Cell viability was determined by mitochondrial dehydrogenase activity (XTT assay, Cayman Chemical). To generate dose-response curves, the data were fitted to a 4-parameter Hill function and absolute IC50 was determined at Y=0.5 viability.

[0303] Cytotoxicity assay Proliferating and senescent cells were passaged when they reached approximately 90% confluency and maintained in T175 flasks under the conditions specified below. Prior to use in the cytotoxicity assay, cells were visually inspected under a phase contrast microscope for contamination and health; cells were not used if contamination or significant cell debris was noted. Healthy cells were plated in the central 60 wells of a 96-well plate at a concentration of 5,000 or 10,000 cells / well (for proliferation and senescence conditions, respectively). The outer 36 wells in each plate contained DPBS to prevent both drying of the inner wells and edge effects from spectrophotometric readings. After seeding in the 96-well plate, cells were allowed 24 hours to attach before the addition of drugs. Drugs were added in triplicate across 10 different concentrations spanning roughly 4 Log10 units. Plates were incubated with drugs for 72 hours, at which point the drug-containing medium was aspirated and replaced with XTT medium. The XTT reagent undergoes an absorbance shift upon reduction by an NAD(P)H-dependent metabolic reaction, and the resulting shift can be used to quantify remaining cell viability following drug treatment. Once an appropriate dynamic range was achieved, absorbance readings for each test article were taken (typically 0.7-1.4 absorbance units). The data thus obtained were then background corrected and logarithmic to generate a concentration-response curve.

[0304] A549 senescence induction protocol Wild-type A549 cells were thawed in DMEM (high glucose, 4 mM L-glutamine, no sodium pyruvate) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin antibiotic cocktail. Cells were cultured at 37C, 5% CO2, and air oxygen. For senescence induction, only A549 at passage 20 or less were used. Cells were grown to 60-70% confluency, after which the medium was aspirated and replaced with fresh medium containing 25 μM gemcitabine. Cells were cultured for 72 hours without refreshing the medium. After treatment, drug-containing medium was aspirated, cells were gently washed with 1×DPBS, and fresh drug-free medium was added to the flask, at which point the cells were left undisturbed for an additional 72 hours. Following this period of disturbance, senescence induction was assessed by morphology, SA-β-gal activity, and EdU incorporation. NOTE: Senescence induction with gemcitabine may occasionally cause spontaneous re-entry into the cell cycle in A549. For this reason, cells should be used within 14 days of induction in various assays to avoid the growth of non-senescent populations. In cytotoxicity assays, proliferated cells at passage 20 or less were included as a comparative control. A representative A549 image EdU incorporation assay (EdU fluorophore visualized in the FITC channel, counterstained with DAPI) is illustrated in Figure 2.

[0305] IMR90 senescence induction protocol IMR90 primary lung fibroblasts were thawed in DMEM (high glucose, 4 mM L-glutamine, no sodium pyruvate) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin antibiotic cocktail. Cells were cultured under hypoxic (5% O2) conditions at 37C and 5% CO2. For senescence induction, only IMR90 cells at or below approximately 50 population doublings were used to avoid confounding effects as a result of replicative senescence. Cells were grown to 60-70% confluency, after which the medium was aspirated and replaced with fresh medium containing 300 nM doxorubicin. Cells were cultured in drug-containing medium for 48 hours, following which one-third of the medium was aspirated and replaced with drug-free medium. The cells were then cultured for an additional 24 hours, after which all medium was aspirated, gently washed with 1xDPBS, refreshed with drug-free medium, and the cells were returned to the incubator for 72 hours. Following this resting period, senescence induction was assessed by morphology, SA-β-gal activity, and EdU incorporation. Cells were used for downstream assays within 21 days of induction. Representative IMR90 images (from left to right: SA-β-Gal, SA-α-Fuc, EdU incorporation assay [EdU fluorophore visualized in FITC channel, counterstained with DAPI]) are illustrated in Figure 1.

[0306] Table 2 below reports the biological activity of selected compounds as measured by T47 / DsgNTC, T47D / sgFUCA1, T47D / sgGLBsgGALC, IMR90SEN0, A549SEN.

[0307] [Table 1]

Claims

1. Formula (I) or a compound of formula (I): 【Chemical 1】 [In the formula, R １ is R １８ C(O)NH—, and R １８ is a residue of a hydroxamic acid histone deacetylase inhibitor, a residue of an Hsp90 inhibitor, a residue of a topoisomerase inhibitor, a residue of an Akt1 inhibitor, a residue of a DNA alkylating agent, a residue of a proteasome inhibitor, or a residue of a Bcl2 inhibitor; L is a linker; n is 0 or 1; R ２ is -H, -F, -OH, -OC(O)R ９ or -OC(O)OR １０ and R ３ is -H, -F, -OH, -OC(O)R １１ or -OC(O)OR １２ and R ４ is -H, -F, -OH, -OC(O)R １３ or -OC(O)OR １４ Alternatively, R ３ and R ４ together with the atom to which they are attached form R at the acetal carbon atom. １７ forming a 5-membered cyclic acetal substituted with Instead, R ３ and R ４ together with the atoms to which they are attached form a five-membered cyclic carbonate; R ５ is -CH ３ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ６ is —H or —F; R ７ is —H or —F; R ８ is —H or —F; R ９ ~R １７ is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, or substituted heteroaryl; However, R ５ Ga-CH ２ F, -CHF ２ or -CF ３ When R ２ , R ３ or R ４ is —H or —F; However, R ５ Ga-CH ３ , -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ When R ２ , R ３ or R ４ one or two of are —H or —F; However, R ４ R only if -F or -H ６ is -F; R ３ R only if -F or -H ７ is -F; R ２ R only if -F or -H ８ is -F; R ６ R only if -F or -H ４ is -F; R ７ R only if -F or -H ３ is -F; R ８ R only if -F or -H ２ is -F] or pharmaceutically acceptable salts, hydrates and solvates thereof.

2. R ５ But -CH ３ and R ２ The compound of claim 1, wherein is —H or —F.

3. R ５ But -CH ３ and R ３ The compound of claim 1, wherein is —H or —F.

4. R ５ But -CH ３ and R ４ The compound of claim 1, wherein is —H or —F.

5. R ５ But -CH ３ and R ２ is -F and R ８ The compound of claim 1 , wherein is —F.

6. R ５ But -CH ３ and R ３ is -F and R ７ The compound of claim 1 , wherein is —F.

7. R ５ But -CH ３ and R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

8. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ２ The compound of claim 1, wherein is —H or —F.

9. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ３ The compound of claim 1, wherein is —H or —F.

10. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ４ The compound of claim 1, wherein is —H or —F.

11. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ２ is -F and R ８ The compound of claim 1 , wherein is —F.

12. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ３ is -F and R ７ The compound of claim 1 , wherein is —F.

13. R ５ But -CH ２ OH, -CH ２ O.C.(O.)R. １５ or -CH ２ OC(O)OR １６ and R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

14. R ５ But -CH ２ F, -CHF ２ or -CF ３ and R ２ The compound of claim 1, wherein is —H or —F.

15. R ５ But -CH ２ F, -CHF ２ or -CF ３ and R ３ The compound of claim 1, wherein is —H or —F.

16. R ５ But -CH ２ F, -CHF ２ or -CF ３ and R ４ The compound of claim 1, wherein is —H or —F.

17. R ５ But -CH ２ F, -CHF ２ or -CF ３ and R ２ is -F and R ８ The compound of claim 1 , wherein is —F.

18. R ５ But -CH ２ F, -CHF ２ or CF ３ and R ３ is -F and R ７ The compound of claim 1 , wherein is —F.

19. R ５ But -CH ２ F, -CHF ２ or CF ３ and R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

20. R ２ is —H or —F, and R ３ The compound of claim 1, wherein is —H or —F.

21. R ２ is —H or —F, and R ４ The compound of claim 1, wherein is —H or —F.

22. R ３ is —H or —F, and R ４ The compound of claim 1, wherein is —H or —F.

23. R ２ is —H or —F, and R ３ is -F and R ７ The compound of claim 1 , wherein is —F.

24. R ２ is —H or —F, and R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

25. R ３ is —H or —F, and R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

26. R ２ is -F and R ８ is -F and R ３ The compound of claim 1, wherein is —H or —F.

27. R ２ is -F and R ８ is -F and R ４ The compound of claim 1, wherein is —H or —F.

28. R ３ is -F and R ７ is -F and R ４ The compound of claim 1, wherein is —H or —F.

29. R ２ is -F and R ８ The compound of claim 1 , wherein is —F.

30. R ３ is -F and R ７ The compound of claim 1 , wherein is —F.

31. R ４ is -F and R ６ The compound of claim 1 , wherein is —F.

32. R ２ The compound of claim 1, wherein is —H or —F.

33. R ３ The compound of claim 1, wherein is —H or —F.

34. R ４ The compound of claim 1, wherein is —H or —F.

35. R ９ ~R １７ The compound of any one of claims 1 to 34, wherein is independently alkyl, alkenyl, alkynyl, aryl, substituted aryl, cycloalkyl, cycloheteroalkyl, or heteroaryl.

36. R ９ ~R １７ The compound of any one of claims 1 to 34, wherein is independently alkyl, alkenyl, aryl, substituted aryl, or cycloheteroalkyl.

37. R ９ ~R １７ But independently, (C １ ~C ４ ) alkyl, (C １ ~C ４ ) alkenyl, phenyl, substituted phenyl or (C ５ ~C ７ 35. The compound of any one of claims 1 to 34, wherein:

38. 35. The compound of any one of claims 1 to 34, wherein the anomeric carbon is the S stereoisomer.

39. R １ But, R １８ C(O)NH—, and R １７ 35. The compound of any one of claims 1 to 34, wherein is a residue of a hydroxamic acid histone deacetylase inhibitor.

40. R １ The compound of any one of claims 1 to 34, wherein is a residue of an Hsp90 inhibitor.

41. R １ The compound of any one of claims 1 to 34, wherein is a residue of a topoisomerase inhibitor.

42. R １ The compound of any one of claims 1 to 34, wherein is a residue of an Akt1 inhibitor.

43. R １ 35. The compound of any one of claims 1 to 34, wherein is a residue of a DNA alkylating agent.

44. R １ The compound of any one of claims 1 to 34, wherein is a residue of a proteasome inhibitor.

45. R １ The compound of any one of claims 1 to 34, wherein is a residue of a Bcl2 inhibitor.

46. A pharmaceutical composition comprising a compound according to any one of claims 1 to 34 and a pharmaceutically acceptable excipient.

47. 36. A method for treating a disease or disorder associated with aging, comprising administering to a non-human mammalian subject in need thereof a therapeutically effective amount of a compound of any one of claims 1 to 34.

48. 47. A method for treating a disease or disorder associated with aging, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 46 to a non-human mammalian subject in need thereof.

Images