Senolytic compositions and uses thereof

Abstract

To provide senolytic agents for selectively killing senescent cells that are associated with numerous pathologies and diseases, including age-related pathologies and diseases.SOLUTION: The invention provides a compound represented by formula (IV) or (V) in the figure or pharmaceutically acceptable salts, hydrates or solvates thereof. A method for treating a senescence-associated disease or disorder comprises administering to a subject in need thereof a therapeutically effective amount of the compound or a pharmaceutical composition thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 696,486, filed July 11, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Provided herein are senolytic agents for selectively killing senescent cells associated with many pathologies and diseases, including age-related pathologies and diseases. As disclosed herein, senescent cell-associated diseases and disorders can be treated or prevented by administering at least one senolytic agent or a 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, cardiovascular diseases and disorders associated with arteriosclerosis such as atherosclerosis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory diseases or disorders, autoimmune diseases or disorders, pulmonary diseases or disorders, neurological 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, disorders, and ill health. Senescent cells, which are cells that have stopped replicating, accumulate in individuals with age and can partially or significantly contribute to the cell and tissue deterioration that underlies 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 damage or as a result of disease (see, e.g., Demaria et al., Cancer Discovery 7 (2017) 165-176; and Schafer et al., Nat. Commun. 8 (2017) doi:10.1038 / ncomms14532).

[0004] Senocyte-eliminating agents with a variety of pharmacological mechanisms are known in the art. Senocyte-eliminating agents can be specific inhibitors of one or more Bcl-2 anti-apoptotic protein family members, which inhibit 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, see, e.g., Zhu et al., Aging Cell 9 (2017) 955-965); 14(2015)654-658); CDK4 / 6 inhibitors (e.g., palbocyclib, (see, e.g., Whittaker et al., Pharmacol. Ther. 173(2017)83-105)); mTOR inhibitors (e.g., rapamycin, (see, e.g., Laberge et al., Nat. Cell Biol. 17(2015)1049-1061)); MDM2 inhibitors (e.g., Nutlin-3; and RG-7112, see, e.g., U.S. Patent Application Publication No. 2016 / 0339019)); Hsp90 inhibitors (e.g., 17-DMAG; and ganetespib, see, e.g., Fuhrmann-Stroissnigg et al., al., Nat. Commun. 8 (2017) doi:10.1038 / s41467-017-00314-z); flavones (e.g., quercetin; and fisetin, (see, e.g., 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, e.g., Samaraweera et al., Sci. Rep. 7 (2017) 1900. doi:10.1038 / s41598-017-01964-1)).

[0005] A key challenge has been identifying senolytic agents that selectively kill senescent cells while sparing non-senescent cells. Furthermore, many known senolytic agents were initially developed as cytotoxic anticancer agents and then repurposed for the "selective" elimination of senescent cell populations. Because proliferating cells are often more sensitive to the cytotoxic or cytostatic effects of anticancer drugs, dose-limiting toxicity in hematopoietic cells is a common side effect that limits the clinical usefulness of anti-senescence therapies (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). Pulse administration of such senolytic agents has been proposed as a mechanism to minimize the exposure of non-senescent cells to these molecules and potentially limit off-target effects. Therefore, there is a need for senolytic agents that have improved selectivity for killing senescent cells with minimal toxicity to non-senescent cells. Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein are non-toxic prodrugs of senolytic agents that are activated by hydrolase enzymes that preferentially accumulate in senescent cells, satisfying these and other needs. In one aspect, the hydrolase enzyme is a glycosidase, and senescence-associated increased intracellular glycosidase activity is utilized to convert a non-toxic prodrug derivative of a pro-apoptotic agent (I) into the toxic pro-apoptotic parent compound (II), resulting in the specific killing of senescent cells. [ka]

[0007] In certain embodiments, Compound (II) is capable of promoting apoptosis in non-proliferating cells.

[0008] In another aspect, non-toxic prodrugs of toxic senolytic agents are provided that, upon cleavage into the active senolytic agent in senescent cells, specifically result in senescent cell death. In certain 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 Aktl inhibitors are provided. In still other embodiments, prodrugs of proteasome inhibitors are provided. Derivatives, including salts, solvates, hydrates, and metabolites, of the prodrugs described herein are also provided. Compositions comprising the prodrugs provided herein and a vehicle are further provided.

[0009] In another aspect, also provided herein are methods for treating, preventing, or ameliorating the symptoms of a medical disorder in a subject, such as, for example, a cardiovascular disease or disorder, atherosclerosis-related cardiovascular disease and disorder such as atherosclerosis, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, an inflammatory disease or disorder, an autoimmune disease or disorder, a pulmonary disease or disorder, a neurological disease or disorder, a skin disease or disorder, a chemotherapy side effect, a radiation therapy side effect, metastasis, and a metabolic disease. In practicing this method, a therapeutically effective amount of a senolytic agent or a pharmaceutical composition thereof is administered to the subject.

[0010] In yet another aspect, a method for treating an age-related disease or condition is provided. The method comprises administering a therapeutically effective amount of a senolytic agent or a pharmaceutical composition thereof to a subject. In yet another aspect, a method for delaying at least one characteristic of aging in a subject is provided. The method comprises administering a therapeutically effective amount of a senolytic agent or a pharmaceutical composition thereof to a subject.

[0011] In yet another aspect, a method for killing therapy-induced senescent cells is provided, comprising administering a therapeutically effective amount of a senolytic agent or a pharmaceutical composition thereof to a subject that has undergone a DNA-damaging therapy. [Brief explanation of the drawings]

[0012] [Figure 1A] Figure 1 shows the viability of proliferating mouse embryonic fibroblasts (MEFs) treated with various drug concentrations of 5-fluorouridine (FUR) (102) or 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101). [Figure 1B] Figure 1 shows the viability of senescent mouse embryonic fibroblasts (MEFs) treated with various drug concentrations of 5-fluorouridine (FUR) (102) or 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101). [Figure 2A] Quantification of blood cell counts from wild-type (wt) C57BL / 6 mice (N=3 mice / group) administered a single intraperitoneal injection of FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) after 6 days of treatment. [Figure 2B] Quantification of bone marrow cell numbers from femurs from wild-type (wt) C57BL / 6 mice (N=3 mice / group) administered a single intraperitoneal injection of FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) after 6 days of treatment. [Figure 2C] Quantification of total spleen weight from wild-type (wt) C57BL / 6 mice (N=3 mice / group) administered a single intraperitoneal injection of FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) after 6 days of treatment is shown. [Figure 3A] Representative images of liver sections from C57BL / 6 mice injected with doxorubicin (25 mg / kg) are shown. [Figure 3B] Representative images of liver sections from C57BL / 6 mice injected with doxorubicin (25 mg / kg) and FURGal (140 mg / kg) are shown. [Figure 3C] Quantification of liver sections is shown in Figures 3A and 3B along with controls. [Figure 3D] The average body weight of C57BL / 6 mice on the day of analysis is shown. [Figure 4A] 1 shows a protocol for the induction of senescence in C57BL / 6 mouse hepatocytes and subsequent treatment with compound (113). [Figure 4B] Representative images of liver sections from C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113) are compared. [Figure 4C] Quantification of SA-β-Gal in liver sections from Figure 4B is shown along with controls. [Figure 4D] Quantification of Cdkn2a expression in the liver of C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113). [Figure 4E] Quantification of IL-6 expression in the liver of C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113). [Figure 5A] This shows the protocol for observing the senescent cell-eliminating effect of compound (119) in the lung tissue of C57BL / 6 mice. [Figure 5B] Representative images of lung sections from C57BL / 6 mice injected with doxorubicin (15 mg / kg) followed by 20 mg / kg of vehicle or compound (119) are compared. [Figure 5C] Quantification of SA-β-Gal in lung sections following intravenous (iv) administration of Compound (119) at 10 mg / kg, 20 mg / kg, or 40 mg / kg, along with controls, is shown. [Figure 5D] Quantification of Cdkn2a expression in the lungs of C57BL / 6 mice injected with doxorubicin (15 mg / kg) followed by vehicle or compound (119) at 10 mg / kg, 20 mg / kg, or 40 mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0013] definition "Hallmarks of aging" as used herein include, but are not limited to, generalized weakening of the immune system, muscle atrophy and weakness, decreased skin elasticity, delayed wound healing, retinal atrophy, decreased lens transparency, hearing loss, osteoporosis, sarcopenia, graying of hair, facial wrinkling, decreased vision, frailty, and cognitive impairment.

[0014] "Acyl" means an H-CO-, alkyl-CO-, alkenyl-CO- or cycloalkyl-CO- group, where the alkyl, alkenyl, or cycloalkyl group is as defined herein.

[0015] "Acylamino" refers to the group acyl-NH-, where acyl is as defined herein.

[0016] As used herein, "age-related diseases or conditions" includes, but is 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 impairment, atherosclerosis, acute coronary syndrome, 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, disorders associated with cancer treatment, such as atrophy and fibrosis in various tissues, brain and and cardiac injury, and therapy-related myelodysplastic syndromes, as well as diseases associated with accelerated aging and / or defects in DNA damage repair and telomere maintenance, such as progeria (i.e., Hutchinson-Gilford progeria syndrome, Werner syndrome, Bloom syndrome, Rothmund-Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum, sulfur deficiency hair dysgenesis, xeroderma pigmentosum-Cockayne syndrome combined, restrictive skin disorder), ataxia-telangiectasia, Fanconi anemia, Friedreich's ataxia, dyskeratosis congenita, aplastic anemia, and others.

[0017] "Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond and having 2 to 20 carbon atoms in the chain, which may be straight or branched. In some embodiments, an alkenyl group has 2 to 12 carbon atoms in the chain. In other embodiments, an alkenyl group has about 2 to 6 carbon atoms in the chain. In yet other embodiments, an alkenyl group has 2 to 4 carbon atoms in the chain. As used throughout this specification, "branched" means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are attached to a straight chain; here, a straight alkenyl chain. "Lower alkenyl" refers to about 2 to about 4 carbon atoms in the chain, which may be straight or branched. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl, and decenyl.

[0018] "Alkenylene" means an aliphatic divalent radical derived from a straight-chain or branched alkenyl group, where the alkenyl group is as described herein. Exemplary alkenylene radicals include, but are not limited to, vinylene and propylene.

[0019] "Alkoxy" means an alkyl-O- group, where the alkyl group is as described herein. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy.

[0020] "Alkoxycarbonyl" means an alkyl-O-CO- group, where the alkyl group is as described herein. Exemplary alkoxycarbonyl groups include, but are not limited to, methoxy and ethoxycarbonyl.

[0021] "Alkyl," unless otherwise specified, refers to an aliphatic hydrocarbon group which may be straight or branched and having 1 to 20 carbon atoms in the chain. In certain embodiments, an alkyl group has 1 to 6 carbon atoms. "Lower alkyl," as a group or part of a lower alkoxy, lower alkylthio, lower alkylsulfinyl, or lower alkylsulfonyl group, refers to an aliphatic hydrocarbon group which may be straight or branched and having 1 to 4 carbon atoms in the chain, unless otherwise specified. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, 3-pentyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0022] "Alkylene" means an aliphatic divalent radical derived from a straight-chain or branched-chain alkyl group, where the alkyl group is as defined herein. Exemplary alkylene radicals include, but are not limited to, methylene, ethylene, and trimethylene.

[0023] "Alkylenedioxy" means an -O-alkylene-O- group, where alkylene is defined as above. Exemplary alkylenedioxy groups include, but are not limited to, methylenedioxy and ethylenedioxy.

[0024] "Alkylsulfinyl" means an alkyl-SO- group, in which the alkyl group is as defined above. Exemplary alkylsulfinyl groups include, but are not limited to, those in which the alkyl group is C 1~4 Examples of the alkyl group include alkyl.

[0025] "Alkylsulfonyl" means an alkyl-SO2- group, where the alkyl group is as described above. In certain embodiments, an alkylsulfonyl group is an alkyl group where the alkyl group is C 1~4 It is an alkyl.

[0026] "Alkylthio" means an alkyl-S- group in which the alkyl group is as previously described. Exemplary alkylthio groups include, but are not limited to, methylthio, ethylthio, isopropylthio, and heptylthio.

[0027] "Alkynyl" means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and having about 2 to about 20 carbon atoms in the chain, which may be straight or branched. In some embodiments, an alkynyl group has 2 to 12 carbon atoms in the chain. In other embodiments, an alkynyl group has 2 to 6 carbon atoms in the chain. In yet other embodiments, an alkynyl group has 2 to 4 carbon atoms in the chain. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, n-butynyl, i-butynyl, 3-methylbut-2-ynyl, and n-pentynyl.

[0028] "Alkynylene" means an aliphatic divalent radical derived from a straight-chain or branched alkynyl group, where alkynyl group is as described herein. Exemplary alkynylene radicals include, but are not limited to, ethynylene and propynylene.

[0029] "Amino acid side chain" means a substituent found on the carbon between the amino and carboxy groups in α-amino acids. For examples of "corresponding protected derivatives" of amino acid side chains, see T.W. Greene and P.G.M. Buts in "Protective Groups in Organic Chemistry," John Wiley and Sons, 1991.

[0030] "Aroyl" means an aryl-CO- group in which the aryl group is as described herein. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

[0031] "Aryl" as a group or part of a group refers to (i) an optionally substituted monocyclic or polycyclic aromatic carbocyclic moiety of about 6 to about 14 carbon atoms, e.g., phenyl or naphthyl; or (ii) an optionally substituted partially saturated polycyclic aromatic carbocyclic moiety in which aryl and cycloalkyl or cycloalkenyl groups are fused together to form a ring structure, e.g., a tetrahydronaphthyl, indenyl, or indanyl ring.

[0032] "Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties are as previously described. Exemplary arylalkyl groups include, but are not limited to, benzyl, 2-phenethyl, and naphthalenemethyl.

[0033] "Aryldiyl" means an optionally substituted divalent radical derived from an aryl group. Exemplary aryldiyl groups include, but are not limited to, optionally substituted phenylene, naphthylene, and indanylene. Suitable substituents include one or more "aryl group substituents" as defined above, particularly halogen, methyl, or methoxy.

[0034] "Aryloxy" means an aryl-O- group, where the aryl group is as previously described. Exemplary aryloxy groups include, but are not limited to, optionally substituted phenoxy and naphthoxy.

[0035] "Aryloxycarbonyl" means an aryl-O-C(=O)- group, where the aryl group is as previously described. Exemplary aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthoxycarbonyl.

[0036] "Arylsulfinyl" means an aryl-SO- group in which the aryl group is as previously described.

[0037] "Arylsulfonyl" means an aryl-SO2- group in which the aryl group is as previously described.

[0038] "Arylthio" means an aryl-S- group in which the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

[0039] "Azaheteroaryl" means an aromatic carbocyclic moiety of about 5 to about 10 ring members, where one ring member is nitrogen and the other ring members are selected from carbon, oxygen, sulfur, or nitrogen. Examples of azaheteroaryl groups include pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl, quinazolinyl, imidazolyl, and benzimidazolyl.

[0040] "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 either their chemical structure and / or chemical name. In the event of a conflict between the chemical structure and the chemical name, the chemical structure is determinative of the identity of the compound. The compounds described herein may contain one or more chiral centers and / or double bonds and may therefore exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Thus, chemical structures depicted herein encompass stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) depicted in the structure. Chemical structures depicted herein also encompass enantiomers and stereoisomeric derivatives of the depicted compounds, unless expressly specified otherwise. Enantiomeric and stereoisomeric mixtures may be resolved into their component enantiomers or stereoisomers using separation or chiral synthesis techniques well known to those skilled in the art. Compounds may also exist in several tautomeric forms, including enol forms, keto forms, and mixtures thereof. Thus, the chemical structures depicted herein encompass all possible tautomeric forms of the depicted compounds. The compounds described also include isotopically labeled compounds, where one or more atoms have an atomic mass different from the atomic mass usually 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. 17 O and the like. Compounds can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, compounds can be hydrated or solvated. Certain compounds may exist in polycrystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present disclosure. Furthermore, when a partial structure of a compound is shown, it should be understood that parentheses indicate the point of attachment of the partial structure to the remainder of the molecule.

[0041] "Cyclic amine" means a 3- to 8-membered monocyclic cycloalkyl ring system in which one of the ring carbon atoms is replaced with nitrogen and which may also contain a heteroatom selected from O, S, SO, or NY (where Y is hydrogen, alkyl, aryl, arylalkyl, acyl, acyloxyalkyl, cycloalkyl, heteroaryl, heterocycloalkyl, or sulfonyl). Exemplary cyclic amines include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, indolinyl, pyridolinyl, and tetrahydroquinolinyl.

[0042] "Cycloalkenyl" means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having 3 to 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include, but are not limited to, cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0043] "Cycloalkyl" means a saturated monocyclic or bicyclic ring system of 3 to 10 carbon atoms optionally substituted with oxo. Exemplary monocyclic cycloalkyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0044] "Cycloalkylene" means a divalent radical derived from a cycloalkyl group. Exemplary cycloalkenylene radicals include, but are not limited to, cyclopentylene and cyclohexylene.

[0045] "DNA damaging therapy" as used herein includes, but is not limited to, gamma irradiation, alkylating agents such as nitrogen mustards (e.g., chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (streptozocin, carmustine, lomustine), alkylsulfonates (e.g., busulfan), triazines (dacarbazine, temozolomide) and ethylenimines (e.g., thiotepa, altretamine), platinum agents such as cisplatin, carboplatin, oxaliplatin, antimetabolites such as 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxybenzoates, urea, 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.

[0046] "Halo" or "halogen" means fluoro, chloro, bromo, or iodo.

[0047] "Heteroaroyl" means a heteroaryl-C(=O)- group, where the heteroaryl group is as described herein. An exemplary group is pyridylcarbonyl.

[0048] "Heteroaryl" as a group or part of a group refers to (i) an aromatic monocyclic or polycyclic organic moiety of about 5 to about 10 ring members, in which one or more of the ring members is an element other than carbon, e.g., nitrogen, oxygen, or sulfur (examples of such groups include benzimidazolyl, benzthiazolyl, furyl, imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl, and triazolyl groups, optionally substituted with one or more aryl group substituents as defined above); or (ii) an optionally substituted partially saturated polycyclic heterocarbocyclic moiety in which a heteroaryl and a cycloalkyl or cycloalkenyl group are fused together to form a ring structure (an example of such a group is the pyrindanyl group).

[0049] "Heteroaryldiyl" means a divalent radical derived from a heteroaryl group.

[0050] "Heteroaryloxy" means a heteroaryl-O- group in which the heteroaryl group is as previously described. An exemplary heteroaryloxy group is optionally substituted pyridyloxy.

[0051] "Heterocycle" refers to a 5- or 6-membered, optionally substituted, saturated, partially saturated, or fully unsaturated, monocyclic organic moiety in which one or more of the ring members is an element other than carbon, such as nitrogen, oxygen, or sulfur. Exemplary 5- or 6-membered heterocycles include furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, oxazinyl, piperidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, pyrrolidinyl, pyrrolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl, and triazolyl groups.

[0052] "Heterocycloalkyl" means (i) a cycloalkyl group of about 3 to 7 ring members containing one or more heteroatoms selected from O, S, or N, and optionally substituted with oxo; (ii) a partially saturated polycyclic heterocarbocyclic moiety in which an aryl (or heteroaryl ring) and a heterocycloalkyl group are fused together to form a ring structure (examples of such groups include chromanyl, dihydrobenzofuranyl, indolinyl, and pyrindolinyl groups).

[0053] As used herein, a "histone deacetylase inhibitor" or "HDAC inhibitor" is a compound capable of inhibiting histone deacetylation in vivo, in vitro, or both (see, for example, Mottamal et al., Molecules 20 (2015) 3898-3941; Roche and Bertrand, Eur. J. Med. Chem. 121 (2016) 451-483). Thus, an HDAC inhibitor inhibits the activity of at least one histone deacetylase. Inhibiting the deacetylation of at least one histone results in an increase in acetylated histones, and the accumulation of acetylated histones is a suitable biological marker for evaluating the activity of an HDAC inhibitor. Therefore, procedures that can assay the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of a compound of interest. It is understood that compounds that can inhibit histone deacetylase activity can also bind to other substrates and thus inhibit other biologically active molecules, such as enzymes.

[0054] "Hydrate" refers to the incorporation of water into the crystal lattice of a compound described herein in a stoichiometric proportion to form an adduct. Methods for producing hydrates include, but are not limited to, storage in an atmosphere containing water vapor, a dosage form containing water, or routine formulation processes such as crystallization (i.e., from water or a mixed aqueous solvent), lyophilization, wet granulation, aqueous film coating, or spray drying. Hydrates can also form from crystalline solvates under certain circumstances upon exposure to water vapor or upon suspension of anhydrous materials in water. Hydrates can also crystallize in more than one form, 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 capabilities of those skilled in the art and are completely conventional, requiring no experimentation beyond that typical in the art. Hydrates can be characterized and / or analyzed by methods well known to those skilled in the art, such as, for example, single crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). Furthermore, many commercial companies routinely offer services that include the preparation and / or characterization of hydrates, such as, for example, HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100 Val de Reuil, France (http: / / www.holodiag.com).

[0055] "Hydroxamic acid derivative histone deacetylase inhibitors" as used herein refers to the class of histone deacetylase inhibitors that are hydroxamic acid derivatives.

[0056] As used herein, a "residue of a hydroxamic acid derivative histone deacetylase inhibitor" refers to the entire portion of a hydroxamic acid derivative histone deacetylase inhibitor, excluding the hydroxamic acid moiety.

[0057] As used herein, a "pharmaceutical composition" refers to at least one compound and a pharmaceutically acceptable vehicle with which the compound is administered to a patient.

[0058] As used herein, "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, or with 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-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbis(2-methylpropional) ... (2) acid addition salts formed with organic acids such as 2[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (3) salts formed when an acidic proton present in the parent compound is replaced with a metal ion, e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or coordinates with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.

[0059] As used herein, "pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound is administered.

[0060] A "patient" includes a human. The terms "human" and "patient" are used interchangeably.

[0061] As used herein, "preventing" or "prevention" refers to reducing the risk of acquiring a disease or disorder (i.e., preventing at least one clinical symptom of the disease from occurring in a patient who may be exposed to or predisposed to the disease but who has not yet experienced or exhibited symptoms of the disease).

[0062] As used herein, "prodrug" refers to a derivative of a drug molecule that requires a transformation within the body to release the active drug. Prodrugs are often, though not necessarily, pharmacologically inactive until converted to the parent drug.

[0063] As used herein, a "promoiety" 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 non-enzymatic means.

[0064] As used herein, a "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, reduces or prevents the reactivity of the functional group. Examples of protecting groups are described in Green et al., "Protective Groups in Organic Chemistry," (Wiley, 2009). nded. 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"), nitro-veratryloxycarbonyl ("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.

[0065] As used herein, "senescence" or "senescent cells" refers to a state in which cells acquire one or more markers of senescence in response to certain cellular stresses. Such markers typically include permanent withdrawal from the cell cycle, expression of a bioactive secretome of inflammatory factors, altered methylation, senescence-associated heterochromatin formation (SAHF), expression of markers of oxidative stress, expression of markers of DNA damage, protein and lipid modifications, morphological hallmarks of senescence, altered lysosomes / vacuoles, and expression of senescence-associated β-galactosidase (see Lorenzo Galluzzi et al. (eds.), Cell Senescene: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007 / 978-1-62703-239-1_4, © Springer Science+Business Media, LLC 2013).

[0066] As used herein, a "senolytic agent" refers to an agent that "selectively" (preferentially or to a greater extent) destroys, kills, removes, or promotes 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 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 insufficiently to kill non-senescent cells in a clinically or biologically significant manner. In certain embodiments, the senolytic agents described herein induce (i.e., initiate, stimulate, cause, activate, promote) the death of senescent cells and alter at least one signaling pathway to result in the death of senescent cells.

[0067] "Hydrate" refers to the incorporation of water into the crystal lattice of a compound described herein in a stoichiometric proportion to form an adduct. Methods for producing hydrates include, but are not limited to, storage in an atmosphere containing water vapor, a dosage form containing water, or routine formulation processes such as crystallization (i.e., from water or a mixed aqueous solvent), lyophilization, wet granulation, aqueous film coating, or spray drying. Hydrates can also form from crystalline solvates under certain circumstances upon exposure to water vapor or upon suspension of anhydrous materials in water. Hydrates can also crystallize in more than one form, 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 capabilities of those skilled in the art and are completely conventional, requiring no experimentation beyond that typical in the art. Hydrates can be characterized and / or analyzed by methods well known to those skilled in the art, such as, for example, single crystal X-ray diffraction, X-ray powder diffraction, polarized optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999). Furthermore, many commercial companies routinely offer services that include the preparation and / or characterization of hydrates, such as, for example, HOLODIAG, Pharmaparc II, Voie de l'Innovation, 27 100 Val de Reuil, France (http: / / wwvv.holodiag.com).

[0068] As used herein, "substituted," when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each replaced, independently of one another, with the same or different substituent(s).

[0069] Substituents useful for replacing saturated carbon atoms in particular groups or radicals include, but are not limited to, -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)R b, -OC(S)R b , -OC(O)O-, -OC(O)OR b , -OC(O)NR c R c , -OC(NCN)NR c R c -OC(S)OR b , -NR b C(O)R b , -NR b C(S)R b , -NR b C(O)O-, -NR b C(O)OR b , -NR b C(NCN)OR b , -NR b S(O)NR 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 R a are independently alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; each R b are independently hydrogen, R a , substituted alkyl, substituted heteroalkyl, substituted aryl, substituted arylalkyl, substituted heteroaryl, and substituted heteroarylalkyl; each R cBut independently, R b Or, two R c taken together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered cycloheteroalkyl, a substituted cycloheteroalkyl, or a 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. Similarly, useful substituents for replacing unsaturated carbon atoms in particular groups or radicals include, but are not limited to, -R a , halo, -O-, -OR b , -SR b , -S-, -NR c R c , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -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)O-, -C(O)OR b , -C(S)OR b , -C(O)NR c R c , -C(NR b )NR c R c , -OC(O)R b , -OC(S)R b , -OC(O)O-, -OC(O)OR b , -OC(S)OR b , -OC(O)NR c R c, -OSO(O)NR 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 are as described above. 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 c are as described above. Substituents from the above list that are useful for substituting other specific groups or atoms will be apparent to those skilled in the art. In certain embodiments, the substituents used to substitute a specific group can typically be further substituted with one or more of the same or different groups selected from the various groups defined above.

[0070] As used herein, "treating" or "treatment" of any disease or disorder refers, in certain embodiments, to ameliorating the disease or disorder (i.e., halting or alleviating the development of the disease or at least one of its clinical symptoms). In other embodiments, "treating" or "treatment" refers to improving at least one physical parameter, which 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.

[0071] As used herein, a "therapeutically effective amount" refers to the amount of a compound that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the patient being treated.

[0072] Reference will now be made in detail to specific embodiments of the compounds and methods. The disclosed embodiments are not intended to limit the scope of the claims. On the contrary, the claims are intended to cover all alternatives, modifications, and equivalents.

[0073] Senolytic agent Hydroxamic acid derivative HDAC inhibitors approved for the clinical treatment of hematologic cancers (such as T-cell lymphoma, leukemia, and multiple myeloma) include vorinostat (suberoylanilide hydroxamic acid or SAHA (1)), belinostat (2), and panobinostat (3). Several other hydroxamic acid derivative HDAC inhibitors (e.g., compounds (4)–(13)) are under clinical investigation for the treatment of both hematologic and solid tumors, either as single agents or in combination with other oncolytic compounds. In addition to variably inhibiting enzymes within HDAC classes I, II, and IV, hydroxamic acid derivatives have been designed to simultaneously inhibit other therapeutic targets, such as CUDC-101 (12) (potently inhibiting EGFR and HER-2 kinases) and CUDC-907 (13) (which additionally inhibit various PI3K isoforms). Many other hydroxamic acid derivative HDAC inhibitors have been disclosed, including the natural product trichostatin A (14), isolated from Streptomyces, and many synthetically derived compounds, such as compounds (15)-(21) and others disclosed by 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] [ka] [ka] [ka]

[0074] Senescent cell-cleaving activity has previously been reported for the pan-HDAC inhibitor panobinostat (3) (Samaraweera et al., supra), and senescence has been shown to be associated with decreased global histone acetylation (Li et al., Proteomics 13 (2013) 2585-2596). Several reports have demonstrated HDAC inhibitor-mediated reductions in Bcl-xL expression (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; and Frys et al., Br. J. Haematol. 169 (2015) 506-519). Without wishing to be bound by theory, it is possible that one pharmacological basis for the senolytic activity of HDAC inhibitors is mediated by a reduction in anti-apoptotic Bcl-xL protein levels.

[0075] In certain embodiments, a compound effective as a senolytic agent has formula (IV) or (V): [ka] wherein R is a residue of a hydroxamic acid derivative histone deacetylase inhibitor; and each R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are independently hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): [ka] and each R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are independently hydrogen or —C(O)—R 2and each R 1 But independently, C 1~4 alkyl or phenyl, provided that R 13 , R 14 , R 15 or R 16 is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 and each R 2 are independently hydrogen, C 1~4 alkyl or phenyl, provided that R 23 , R 24 or R 25 is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or -C(O)-R 1 where R 13 , R 14 , R 15 and R 16 is hydrogen, then R is not 7-heptanoylphenylamido.

[0076] In certain embodiments of the compound of Formula (IV) or (V), the pyranose ring (labeled * The aromatic carbon of ) is of the S configuration, and the compounds are β-D-galactoside and α-L-fucoside conjugates of hydroxamic acid derivative histone deacetylase inhibitors, respectively.

[0077] In certain embodiments of a compound of Formula (IV) or (V), R is a residue of a hydroxamic acid derivative histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected from the group consisting of panobinostat, xinostat, vorinostat, dacinostat, gibinostat, CUDC-907, CUDC-101, abexinostat, belinostat, pracinostat, resminostat, ricolinostat, piroxamide, APHA, trichostatin A, oxamflatin, and AR-42.

[0078] In certain embodiments of the compound of Formula (IV), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (V), R 23 , R 24 and R 25 Each of these is hydrogen.

[0079] In other embodiments of the compound of formula (IV), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In another embodiment of the compound of formula (V), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 It is alkyl or phenyl.

[0080] In still other embodiments of compounds of formula (IV), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 is methyl. In still other embodiments of compounds of formula (V), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl.

[0081] In still other embodiments of compounds of formula (IV), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R1 where R 1 is ethyl. In still other embodiments of compounds of formula (V), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl.

[0082] In a further embodiment, the compound of formula (IV) has the structure: [ka] [ka] It is a compound having any one of the following:

[0083] In a further embodiment, the compound of formula (V) has the structure: [ka] [ka] It is a compound having any one of the following:

[0084] In still other embodiments, the compound of formula (IV) has the structure: [ka] [ka] It is a compound having any one of the following:

[0085] In still other embodiments, the compound of formula (V) has the structure: [ka] [ka] It is a compound having any one of the following:

[0086] In still other embodiments, the compound of formula (IV) has the structure: [ka] [ka] [ka] It is a compound having any one of the following:

[0087] In still other embodiments, the compound of formula (V) has the structure: [ka] [ka] [ka] It is a compound having any one of the following:

[0088] Compounds of formula (IV) or (V) can be synthesized by coupling the carboxylic acid precursor of a hydroxamic acid HDAC inhibitor, RCOH (VIII), with sugar oxamic compounds (IX) and (X), respectively, in the presence of an acyl coupling reagent such as a carbodiimide (e.g., EDC) or, after activation, an acyl chloride or mixed anhydride acylating agent, as shown below. [ka]

[0089] Specifically, the O-β-galactoside derivative of SAHA, compound (22), is a bromogalactoside analogue of the known bromogalactoside (23): [ka] [ka] It can be prepared from according to the method of Thomas et al. Bioorg. Med. Chem. Lett. 17 (2007) 983-986.

[0090] Incubation of compound (22) with β-galactosidase showed quantitative conversion to (1). It is well known that the HDAC inhibitory activity of hydroxamic acid derivative compounds is highly dependent on the zinc-chelating activity of the free hydroxamic acid moiety (e.g., Roche and Bertrand, supra). Therefore, masking the hydroxamic acid functionality as a glycoside derivative in compounds of formula (IV) or (V) ensures that these prodrugs are inactive as HDAC inhibitors but are activated by hydrolysis in the lysosomes of senescent cells.

[0091] Similarly, the O-β-galactoside derivative of panobinostat, compound 26, can be synthesized by reductive amination of 4-formylcinnamic acid 27 with 2-(2-methyl-1H-indol-3-yl)ethylamine 28 (described in International Application No. WO 02 / 22577) and the basic nitrogen of the resulting protected amino acid, for example, as the fluorenylmethyloxycarbamate (Fmoc) derivative 29. Coupling with compound 24, as described above, followed by subsequent deprotection, affords the panobinostat prodrug 26. [ka] [ka]

[0092] In an alternative route to compound 26, compound 24 is first coupled with 27 (e.g., using EDC, HOBt), and the resulting aldehyde is treated with tryptamine derivative 28 in a reductive amination reaction, followed by sodium methoxide-mediated removal of the acetyl protecting group to afford prodrug 26.

[0093] Certain α-L-fucoside conjugates of hydroxamic acid derivative histone deacetylase inhibitors can be prepared in a similar manner, starting from appropriately protected and activated fucose derivatives prepared as described in Hou et al., Mater. Chem. Front. 1 (2017) 660-667 or U.S. Patent Application Publication No. 2015 / 0168374. For example, 1-fluoro-2,3,4-tri-O-acetyl-fucose (30) is treated with N-hydroxyphthalimide to give a mixture of α- and β-L-fucosyloxime derivatives, where the desired α-anomer (31) is the less polar product. Subsequent deprotection with hydrazine affords α-L-fucosyloxime (32), which can be further converted into α-L-fucoside prodrugs of SAHA and panobinostat, compounds (33) and (35), respectively: [ka] [ka] can be further synthesized into

[0094] Hsp90 inhibitors are exemplified by the resorcinol compounds AT13387 (onarespib, (36)), NYP-AUY922 (luminespib, (37)), ganetespib (38), VER-50589 (39), VER-49009 (40), CCT018159 (41), and KW-2478 (42), and 2-(4-aminocyclohexanol)-benzamide derivatives are exemplified by SNX-2112 (43) and (SNX-7081) (44). In certain embodiments, O-galactoside or O-fucoside conjugates of Hsp90 inhibitors are senolytic compounds. In other embodiments, O-β-D-galactoside or O-α-L-fucoside conjugates of Hsp90 inhibitors are senolytic compounds. [ka] [ka] [ka]

[0095] In certain embodiments, the compound of Formula (XI), (XII), (XIII), or (XIV) is a senolytic agent: [ka] wherein Y is carbonyl or absent; A is a substituted or benzo-fused 5-membered heteroaryl or heterocyclic group containing at least one nitrogen atom; B is selected from the group consisting of ethyl, isopropyl, or chloro; and each R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are independently hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII); [ka] and each R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are independently hydrogen or —C(O)—R 2 and each R 1 But independently, C 1~4 alkyl or phenyl, provided that R 13 , R 14 , R 15 or R 16 is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 and each R 2 But independently, C 1~4 alkyl or phenyl, provided that R 23 , R24 or R 25 is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or -C(O)-R 1 is.

[0096] In certain embodiments of compounds of Formula (XI), (XII), (XIII), or (XIV), the anomeric carbon of the pyranose ring (labeled * ) are in the S configuration, which are β-D-galactoside and α-L-fucoside conjugates of the resorcinol Hsp90 inhibitor, respectively.

[0097] In some embodiments of a compound of Formula (XI), (XII), (XIII), or (XIV), the moiety AY-C6H2(OH)2-B is an Hsp90 inhibitor selected from the group consisting of luminespib (NVP-AUY922), ganetespib, VER-50589, AT13387, and KW-2478.

[0098] In some embodiments of the compound of Formula (XI), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XII), R 23 , R 24 and R 25 is hydrogen. In certain embodiments of a compound of Formula (XIII), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XIV), R 23 , R 24 and R 25 Each of these is hydrogen.

[0099] In some embodiments of the compound of Formula (XI), R 13 , R 14 , R 15 and R 16each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XII), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XIII), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of a compound of Formula (XIV), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 It is alkyl or phenyl.

[0100] In some embodiments of the compound of Formula (XI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1is methyl. In certain embodiments of a compound of Formula (XIV), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl.

[0101] In some embodiments of the compound of Formula (XI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XIV), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl.

[0102] In a further embodiment, the compound of formula (XI) has the structure: [ka] [ka] It is a compound having any one of the following:

[0103] In a further embodiment, the compound of formula (XII) has the structure: [ka] [ka] It is a compound having any one of the following:

[0104] In a further embodiment, the compound of formula (XIII) has the structure: [ka] [ka] It is a compound having any one of the following:

[0105] In a further embodiment, the compound of formula (XIV) has the structure: [ka] [ka] It is a compound having any one of the following:

[0106] Compounds of formula (XI) and (XIII) can be prepared by reaction of compound of formula (XV) with a protected D-galactosyl donor moiety under classical BF3-mediated glycosylation or König-Knorr coupling conditions, where the resulting regioisomers are separated by chromatographic means. Alternatively, the phenolic hydroxyl group of resorcinol compound (XV) can first be selectively protected to allow regioselective glycosylation. [ka]

[0107] Specifically, the O-β-galactoside conjugates of AT13387 (36) (prepared as described in U.S. Pat. No. 8,779,132), i.e., compounds (46) and (47), can be prepared by the reaction of (36) with (23) according to the method of Shie et al., Carbohydrate Res. 341 (2006) 443-456. [ka]

[0108] Similarly, the O-β-galactoside conjugates of NVP-AUY922 (37) (prepared as described in Broough et al., J. Med. Chem. 51 (2008) 196-218), i.e., compounds (48) and (49), can be prepared by the reaction of (37) with (23). [ka]

[0109] Specific α-L-fucoside conjugates of Hsp90 inhibitors 36 and 37, compounds 50–53, can be prepared in an analogous manner starting from the protected fucose derivative 45. [ka] [ka]

[0110] The O-β-galactoside conjugates of KW-2478 (42) (i.e., compounds (54) and (55)), SNX-2112 (43) (i.e., compound (56)), and SNX-7081 (44) (i.e., compound (57)), as well as the O-α-fucoside conjugates of KW-2478 (42) (i.e., compounds (58) and (59)), SNX-2112 (43) (i.e., compound (60)), and SNX-7081 (44) (i.e., compound (61)), can be prepared in a similar manner. [ka] [ka] [ka]

[0111] Yet another embodiment relates to OD-galactosyl and OL-fucosyl conjugates of topoisomerase I (TOP1) inhibitory compounds as senolytic compounds. Camptothecin (62), a cytotoxic pentacyclic quinoline alkaloid natural product, is the prototypical TOPOI inhibitor, and many synthetic analogs, including SN-38 (63) and topotecan (64), have been investigated clinically or preclinically as anticancer agents (see, e.g., Jain et al., Current Genomics 18 (2017) 75-92; and Liu et al., Med. Res. Rev. 35 (2015) 753-789). Other important structural classes of TOP1 inhibitors include indenoisoquinolines (exemplified by compounds 65-70 (see, e.g., Cinelli et al., J. Med. Chem. 55 (2012) 10844-10862; and Lv et al., J. Med. Chem. 59 (2016) 4890-4899)) and dibenzonaphthyridones (exemplified by compounds 71-73 (see, e.g., Sooryakumar et al., Mol. Cancer Ther. 10 (2011) 1490-1499)). [ka]

[0112] The O-β-D-galactoside conjugate of SN-38 (74) (Chinese Patent No. CN1534046, 2004) and the O-β-L-fucoside conjugate of SN-38 (75) (Japanese Patent No. JP6328098, 1988) have previously been disclosed as anticancer agents. [ka]

[0113] In certain embodiments, the compound of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), or (XXV) is a senolytic agent: [ka] [ka] [ka] [ka] [ka] where R 8 is a heteroaryl or heterocyclic group containing at least one nitrogen atom; each R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are independently hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): [ka] and each R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R45 are independently hydrogen or —C(O)—R 2 and each R 1 But independently, C 1~4 alkyl or phenyl, provided that R 13 , R 14 , R 15 or R 16 is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 and each R 2 But independently, C 1~4 alkyl or phenyl, provided that R 23 , R 24 or R 25 is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or -C(O)-R 1 is.

[0114] In certain embodiments of a compound of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), or (XXV), the anomeric carbon of the pyranose ring (labeled * ) is in the S configuration, and the compounds are β-D-galactoside and α-L-fucoside conjugates of TOP1 inhibitors, respectively. In other embodiments of the compounds of formula (XVIII), (XIX), (XX) or (XXI), R 8 is 4-morpholinyl or 1-imidazolyl.

[0115] In certain embodiments of a compound of Formula (XVI), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XVII), R 23 , R 24 and R 25is hydrogen. In certain embodiments of a compound of Formula (XVIII), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XIX), R 23 , R 24 and R 25 is hydrogen. In certain embodiments of a compound of Formula (XX), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XXI), R 23 , R 24 and R 25 is hydrogen. In certain embodiments of a compound of Formula (XXII), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XXIII), R 23 , R 24 and R 25 is hydrogen. In certain embodiments of a compound of Formula (XXIV), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XXV), R 23 , R 24 and R 25 Each of these is hydrogen.

[0116] In certain embodiments of a compound of Formula (XVI), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XVII), R 23 , R 24 and R 25 each independently represents -C(O)-R1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XVIII), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XIX), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of a compound of Formula (XX), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XXI), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XXII), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XXIII), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C1~4 In certain embodiments of a compound of Formula (XXIV), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of a compound of Formula (XXV), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 It is alkyl or phenyl.

[0117] In certain embodiments of a compound of Formula (XVI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XVII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XVIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XIX), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XX), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXI), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XIII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXIV), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXV), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl.

[0118] In certain embodiments of a compound of Formula (XVI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XVII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1is ethyl. In certain embodiments of a compound of Formula (XVIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XIX), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XX), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXI), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXIII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXIV), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXV), R 23 , R 24 and R 25Each of these is -C(O)-R 1 where R 1 is ethyl.

[0119] In yet another embodiment, OD-galactosyl and OL-fucosyl conjugates of DNA alkylating agents based on the cytotoxic duocarmycin family of antibiotics are senolytic agents. Duocarmycin SA (76), isolated from Streptomyces DO-113, contains a highly reactive spirocyclopropylcyclohexadienone moiety and has been used as inspiration for the design of mono- and disaccharide derivatives such as galactosyl compound (77) (see, e.g., Tietze et al., Angew. Chem. Int. Ed. 45 (2006) 6574-6577; Tietze et al., J. Med. Chem. 52 (2009) 537-543). Compound (77) is over 4000-fold less cytotoxic than its hydrolyzed seco product (78), which undergoes in situ so-called Winstein cyclization to give the DNA-reactive spirocyclopropylcyclohexadienone (79). [ka]

[0120] In certain embodiments, the compound of Formula (XXVI) or (XXVII) is a senolytic agent: [ka] where R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 each independently represents hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): [ka] and each R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are independently hydrogen or —C(O)—R 2 and each R 1 But independently, C 1~4 alkyl or phenyl, provided that R 13 , R 14 , R 15 or R 16 is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 and each R 2 But independently, C 1~4 alkyl or phenyl, provided that R 23 , R 24 or R 25 is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or -C(O)-R 1 provided that in the compound of formula (XXVI), R 13 , R 14 , R 15 , R 16 cannot be hydrogen or acetyl at the same time.

[0121] In certain embodiments of compounds of Formula (XXVI) or (XXVII), the anomeric carbon of the pyranose ring (labeled * ) are in the S configuration, and these compounds are the β-D-galactoside and α-L-fucoside conjugates of duocarmycin analogues, respectively.

[0122] In certain embodiments of the compound of Formula (XXVI), R 13 , R 14 , R 15 and R 16is hydrogen. In certain embodiments of a compound of Formula (XXVII), R 23 , R 24 and R 25 Each of these is hydrogen.

[0123] In certain embodiments of the compound of Formula (XXVI), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of the compound of Formula (XXVII), R 23 , R 24 and R 25 each independently represents -C(O)-R 1 where R 1 But C 1~4 It is alkyl or phenyl.

[0124] In certain embodiments of the compound of Formula (XXVI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXVII), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl.

[0125] In certain embodiments of the compound of Formula (XXVI), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXVII), R 23 , R 24 and R 25Each of these is -C(O)-R 1 where R 1 is ethyl.

[0126] In certain embodiments of the compound of Formula (XXVII), compound (80) can be prepared according to the method of Tietze et al., supra, as compound (81): [ka] It is synthesized from

[0127] Yet another embodiment relates to OD-galactosyl and OL-fucosyl 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 (85). These compounds 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, e.g., Antonow and Thurston, Chem. Rev. 111 (2011) 2815-2864; and Mantaj et al., Angew. Chem. Int. Ed. 56 (2017) 462-488). PBD monomers exhibit significant cytotoxicity, and linking two PBD monomers via a linker generates PBD dimers capable of interstrand DNA cross-linking. SJG-136 (86) is one such dimer with high cytotoxic potency that has been used to construct antibody-drug conjugates with clinical utility. [ka]

[0128] Kamal and coworkers have described both monomeric and dimeric β-galactoside analogs of PBD as anticancer agents (see, e.g., compounds 87 and 88 (Kamal et al., ChemMedChem 3 (2008) 794-802)). [ka] [ka]

[0129] In certain embodiments, the compound of Formula (XXVIII) or (XXIX) is a senolytic agent: [ka] where R 9 But hydrogen, C 1~4 alkyl, CF3, CN or NO2; R 10 But hydrogen, C 1~4 alkyl or arylalkyl; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 each independently represents hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): [ka] and each R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are independently hydrogen or C(O)-R 2 and each R 1 But independently, C 1~4 alkyl or phenyl, provided that R 13 , R 14 , R 15 , or R 16 is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 and each R 2 But independently, C 1~4alkyl or phenyl, provided that R 23 , R 24 or R 25 is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or C(O)-R 1 provided that in the compound of formula (XXVIII), R 9 is NO2 and R 10 is benzyl, R 13 , R 14 , R 15 , R 16 cannot be hydrogen or acetyl at the same time.

[0130] In certain embodiments of compounds of Formula (XXVIII) or (XXIX), the anomeric carbon of the pyranose ring (labeled * ) are in the S configuration, and these compounds are β-D-galactoside and α-L-fucoside conjugates of pyrrolo[2,1-c][1,4]benzodiazepine analogs, respectively.

[0131] In certain embodiments of a compound of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 is hydrogen. In certain embodiments of a compound of Formula (XXIX), R 23 , R 24 and R 25 Each of these is hydrogen.

[0132] In certain embodiments of a compound of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 each independently represents -C(O)-R 1 where R 1 But C 1~4 In certain embodiments of a compound of Formula (XXIX), R 23 , R 24 and R 25each independently represents -C(O)-R 1 where R 1 But C 1~4 It is alkyl or phenyl.

[0133] In certain embodiments of a compound of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is methyl. In certain embodiments of a compound of Formula (XXIX), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is methyl.

[0134] In certain embodiments of a compound of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 Each of these is -C(O)-R 1 where R 1 is ethyl. In certain embodiments of a compound of Formula (XXIX), R 23 , R 24 and R 25 Each of these is -C(O)-R 1 where R 1 is ethyl.

[0135] In some embodiments of the compound of Formula (XXVIII), compound (89) can be prepared according to the method of compound (90) (Kamal et al., ibid.): [ka] [ka] It is synthesized from

[0136] Specifically, as an embodiment of the compound of formula (XXIX), compound (91) is prepared from compound (90) using an equivalent synthetic method. [ka]

[0137] In yet other embodiments, an O-galactoside or O-fucoside conjugate of an Akt inhibitor is the senolytic agent. Akt inhibitors useful for preparing such conjugates are exemplified by compounds such as ipatasertib (or GDC-0068) (92), AZD5363 (93), and triciribine (94). In certain embodiments, an O-β-D-galactoside or O-α-L-fucoside conjugate of an Akt inhibitor is the senolytic agent. [ka] [ka]

[0138] Certain compounds are prepared according to the methods previously disclosed herein, including compounds (95) to (100), where each R 46 is either hydrogen, acetyl or propionyl): [ka] [ka] [ka] This is exemplified by:

[0139] In certain embodiments, compounds of formula (I), exemplified by 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101), are converted to pro-apoptotic compounds of formula (II), particularly the cytotoxin 5-fluorouridine (FUR) (102), by the action of intracellular β-galactosidase (i.e., SA-β-Gal), which is abundant in senescent cells. [ka]

[0140] In another embodiment, compounds of formula (I), exemplified by 5-fluorouridine-5'-O-α-L-fucopyranoside (FURFuc) (106), are converted to pro-apoptotic compounds of formula (II), particularly the cytotoxin 5-fluorouridine (FUR) (102), by the action of intracellular α-fucosidases, which are abundant in senescent cells. [ka]

[0141] In yet other embodiments, O-galactoside or O-fucoside conjugates of proteasome inhibitors are senolytic agents. Proteasome inhibitors useful for preparing such conjugates are exemplified by compounds such as delanzomib (103). In certain embodiments, O-β-D-galactoside or O-α-L-fucoside conjugates (104) and (105) are senolytic agents. [ka] [ka]

[0142] Methods for characterizing and identifying senolytic agents Characterization of senolytic agents can be determined using one or more cell-based assays and one or more animal models described herein or in the art and known to those skilled in the art. The senolytic agent may selectively kill one or more types of senescent cells (e.g., senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes). In certain embodiments, the senolytic agent may selectively kill at least senescent fibroblasts.

[0143] Characterizing a compound as a senolytic agent can be performed 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 understand that characterizing a compound as a senolytic agent and determining the level of killing caused by the compound can be performed by comparing the activity of the test agent to an appropriate negative control (e.g., vehicle or diluent alone and / or a composition or compound known in the art not to kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing senolytic agents also include controls to determine the effect of the agent on non-senescent cells (e.g., quiescent or proliferating cells). A senolytic agent reduces (i.e., decreases) the percent viability of a plurality of senescent cells (i.e., reduces 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 factors that maintain the integrity of the test agents and reagents used in the assay and are well known to those of skill in the art and / or can be readily determined by routine experimentation.

[0144] The source of senescent cells for use in the assays can be primary cell cultures or cell lines adapted for culture, including, but not limited to, genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid cell lines, differentiated or differentiable cell lines, transformed cell lines, etc. In certain embodiments, senescent cells are isolated from a biological sample obtained from a host or subject that has senescent cells associated with a disease or disorder. In other embodiments, non-senescent cells are used (e.g., primary cells obtained from a subject or cell lines adapted to grow in culture) to induce senescence by methods described herein and in the art, such as by exposure to radiation or a chemotherapeutic agent (e.g., doxorubicin). The biological sample can be, for example, a blood sample, a biopsy, a bodily fluid (e.g., lung lavage, ascites, mucosal washings, 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 can be a tissue or cell preparation whose morphological integrity or physical state has been disrupted by, for example, dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication, or any other means for processing a sample derived from a subject or biological source. The subject can be a human or a non-human animal. By way of example, the senolytic effect of certain compounds of the present invention on human fibroblasts in culture medium is characterized in Example 23 herein, and their effect on mouse embryonic fibroblasts is characterized in Example 24 herein. Examples 26 and 27 demonstrate that in vivo administration of senolytic compounds of the present invention results in a reduction of senescent liver cells in mice. Example 28 demonstrates that in vivo administration of senolytic compounds of the present invention results in a reduction of senescent lung cells in mice.

[0145] The transgenic animal models described herein or in the art can be used to determine the death or removal of senescent cells (see, e.g., Baker et al., supra; Nature, 479 (2011) 232-236; International Application No. WO 2012 / 177927; International Application No. WO 2013 / 090645). An exemplary transgenic animal model contains a transgene comprising a nucleic acid that allows for controlled clearance of senescent cells (e.g., pl6INK4a-positive senescent cells) as a positive control. The presence and level of senescent cells in a transgenic animal can be determined by measuring the level of a detectable label expressed in the animal's senescent cells. The nucleotide sequence of the transgene includes one or more detectable labels, such as red fluorescent protein; green fluorescent protein; and one or more luciferases, to detect the clearance of senescent cells.

[0146] The animal models described herein or in the art include art-recognized models for determining the efficacy of senolytic agents for treating or preventing (i.e., reducing the likelihood of developing) specific aging-related diseases or disorders, such as atherosclerosis models, osteoarthritis models, COPD models, and IPF models. As described herein, mouse models of lung disease, such as the bleomycin pulmonary fibrosis model, and chronic smoking models, are applicable to diseases such as COPD and can be routinely performed by those skilled in the art. Animal models for determining the efficacy of senolytic agents for treating and / or preventing (i.e., reducing the likelihood of developing) side effects of chemotherapy and radiotherapy, or for treating or preventing (i.e., reducing the likelihood of developing) metastasis, are described in International Application 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; and Chavala et al., J. Clin. Invest. 123 (2013) 4170-4181).

[0147] As a non-limiting example and as described herein, osteoarthritis animal models have been developed. Osteoarthritis can be induced in animals by causing damage to the joint, e.g., in the knee, for example, by incomplete or complete surgical transection of the anterior cruciate ligament. Osteoarthritis animal models can be used to evaluate the effectiveness of senolytic agents for treating or preventing osteoarthritis (i.e., reducing the likelihood of its development), reducing proteoglycan erosion, inducing (i.e., stimulating or enhancing) collagen (such as type II collagen) production, and alleviating pain in animals undergoing ACL surgery. Immunohistology can be performed to examine the integrity and composition of tissues and cells in the joint. Immunochemical and / or molecular biology techniques, such as assays for determining levels of inflammatory molecules (e.g., IL-6) and assays for determining levels of senescence markers as described above, can also be performed using the methods and techniques described herein that can be routinely performed by those skilled in the art.

[0148] As another non-limiting example and as described herein, animal models of atherosclerosis have been developed. Atherosclerosis can be induced in animals, for example, by feeding animals a high-fat diet or by using transgenic animals highly susceptible to atherosclerosis. Animal models can be used to determine the effectiveness of senolytic agents for reducing plaque volume or inhibiting plaque formation in atherosclerotic arteries, reducing the lipid content of atherosclerotic plaques (i.e., reducing the amount of lipids in the plaque), and increasing or enhancing the fibrous cap thickness of the plaque. Sudan staining can be used to detect lipid levels in atherosclerotic blood vessels. Immunohistochemistry 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 such as those described above) can all be performed according to methods routinely practiced in the art and described herein.

[0149] As yet another non-limiting example, and as described herein, a mouse model in which animals are treated with bleomycin has been described to determine the efficacy of drugs for treating 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.), respiratory measurements can be taken 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 according to methods routinely practiced in the art and described herein.

[0150] Determining the effectiveness of a senolytic agent for selectively killing senescent cells as described herein in an animal model can be performed using one or more statistical analyses that would be familiar to one of skill in the art. For example, a statistical analysis such as a two-way analysis of variance (ANOVA) can be used to determine the statistical significance of differences between a group of animals treated with a drug and a group of animals not treated with the drug (i.e., a negative control group that may contain vehicle only and / or a non-senolytic agent). Statistical packages such as SPSS, MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are readily available and are routinely used by those skilled in the art of animal models.

[0151] Those skilled in the art will readily understand that characterizing a senolytic agent and determining the level of killing by the senolytic agent can be performed by comparing the activity of the test agent to 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 viability of senescent cells compared to one or more negative controls (i.e., in some methods, reduce the amount of viable senescent cells in an animal or in a cell-based assay). Thus, a senolytic agent selectively kills senescent cells compared to the killing of non-senescent cells (this may be referred to herein as selectively killing senescent cells over non-senescent cells).

[0152] In certain embodiments (either 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 (either 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 10% or less of non-senescent cells. In still other embodiments (either 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 no more than about 5%, 10%, or 15% of non-senescent cells. In still other embodiments (either 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 no more than about 5%, 10%, 15%, 20%, or 25% of non-senescent cells. In still other embodiments (either in an in vitro assay 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 no more than about 5%, 10%, 15%, 20%, 25%, or 30% of non-senescent cells. In other words, 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.

[0153] With respect to certain embodiments of the methods described herein for treating an aging-related disease or disorder, the percentage of senescent cells killed may refer to the percentage of senescent cells killed in a tissue or organ containing senescent cells that contribute to the onset, progression, and / or worsening of the disease or disorder. By way of non-limiting example, brain tissue, eye tissues and areas, lung tissue, heart tissue, arteries, joints, skin, and muscle may contain senescent cells that can be reduced by the percentages described above by the senolytic agents described herein, thereby providing a therapeutic effect. Furthermore, selective removal of at least 20% or at least 25% of senescent cells from an affected tissue or organ may have a clinically significant therapeutic effect.

[0154] For certain embodiments of the methods described herein for treating a cardiovascular disease or disorder associated with arteriosclerosis, such as atherosclerosis, by administering a senolytic agent (i.e., for the in vivo methods described above), the percentage of senescent cells killed may refer to the percentage of senescent cells killed in the diseased artery containing the plaque relative to the non-senescent cells killed in the arterial plaque. In certain embodiments, in methods for treating a cardiovascular disease, such as 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.

[0155] In certain 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 no more than 5% 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.

[0156] In certain 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 senescent cells killed may refer to the percentage of senescent cells killed in the diseased lung tissue relative to the non-senescent cells killed in the diseased 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 no more than 5% of non-senescent cells in the diseased lung tissue. In other embodiments, the senolytic agent selectively kills at least 25% of senescent cells in the diseased lung tissue.

[0157] 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 developing) senescence-associated diseases or disorders. In certain embodiments, methods for identifying senolytic agents for treating such diseases and disorders include inducing senescence in cells to provide established senescent cells. Methods for inducing senescence in cells are described herein and in the art and include, for example, exposure to radiation (e.g., 10 Gy is typically sufficient) or chemotherapeutic agents (e.g., doxorubicin or other anthracyclines). After 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 establish senescence. As described herein, cellular senescence can be determined by determining any number of characteristics, such as changes in morphology (e.g., as seen by microscopy); for example, production of senescence-associated β-galactosidase (SA-β-gal), p16INK4a, p21, or any one or more SASP factors (e.g., IL-6, MMP3). A sample of senescent cells is then contacted (i.e., mixed, combined, or otherwise allowed to interact with the cells and agent) with a candidate agent. Those skilled in the art will recognize that assays, whether performed retrospectively or presently, include appropriate negative and positive controls. For example, a sample of control non-senescent cells cultured similarly to the senescent cells but not 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 non-senescent cells. A senolytic agent is identified when the level of survival of senescent cells is less than the level of survival of non-senescent cells.

[0158] In certain embodiments, the above-described method for identifying a senolytic agent may further include the steps of identifying whether the senolytic agent is useful for treating osteoarthritis. The method may further include the steps of contacting the identified senolytic agent with cells capable of producing collagen; and determining the level of collagen produced by the cells. In one embodiment, the cells are chondrocytes and the collagen is type II 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 animal's physical function; (c) the level of one or more markers of inflammation; (d) the histology of the joint; and (e) the level of type II collagen produced, thereby determining the therapeutic effect 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) 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 joints of the treated animal; and (v) an increase in the level of type II 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 limb to induced or natural osteoarthritis, for example, by the animal's resistance to bearing weight on the affected limb or the animal's ability to escape 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 will be familiar to those skilled in the art. Statistical analyses described herein and routinely performed in the art can be applied to analyze the data.

[0159] In other embodiments, the above-described methods for identifying senolytic agents may further include a step of identifying whether the senolytic agent is useful for treating cardiovascular diseases caused by or associated with arteriosclerosis. Thus, the method may further include a step of administering a candidate senolytic agent to a non-human animal or animal model to determine the effectiveness of the agent for reducing plaque volume, inhibiting plaque formation in atherosclerotic arteries, reducing the lipid content of atherosclerotic plaques (i.e., reducing or reducing the amount of lipids in plaques), and / or increasing or enhancing the fibrous cap thickness of plaques. Sudan staining may be used to detect lipid levels in atherosclerotic blood vessels. Immunohistology, assays for determining levels of inflammatory molecules (e.g., IL-6), and / or immunohistological tests, such as those described above, may all be performed according to methods described herein and routinely practiced in the art.

[0160] 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 atherosclerotic plaques and determining one or more of: (a) the level of senescent cells in the arteries; (b) the animal's physical function; (c) the level of one or more markers of inflammation; or (d) the histology of the affected blood vessel (e.g., artery); thereby determining the therapeutic effect 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 arteries 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; or (iv) an increase in histological normality in the arteries of the treated animal. As described herein and in the art, the physical function of the animal can be determined by measuring physical activity. Statistical analyses described herein and routinely performed in the art can be applied to analyze the data.

[0161] In certain embodiments, the methods described herein for identifying senolytic agents may further include administering a candidate senolytic agent to a non-human animal lung disease model, such as a bleomycin model or an animal model of smoke exposure, and determining one or more of: (a) the level of senescent cells in the lungs; (b) the animal's lung function; (c) the level of one or more markers of inflammation; and (d) the histology of the lung tissue, thereby determining the therapeutic effect 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 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) increased histological normality in the lung tissue of the treated animal. Respiration measurements may be taken to determine elastance, compliance, static compliance, and peripheral capillary oxygen saturation (SpO2). Pulmonary function can be assessed by determining any 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 / FEV1 ratio, forced expiratory flow rate 25%-75%, and maximal ventilation (MVV), peak expiratory flow rate (PEF), slow 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 diffusing capacity for carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO2) can also be measured. Statistical analyses described herein and routinely performed in the art can be applied to analyze the data.

[0162] Methods for treating and preventing age-related diseases and disorders Provided herein are methods for treating conditions, diseases, or disorders related to, associated with, or caused by cellular senescence, including age-related diseases and disorders, in a subject in need thereof. Age-related diseases or disorders may also be referred to herein as diseases or disorders associated with senescent cells. Age-related diseases and disorders include, for example, age-related diseases and disorders caused by senescence; pulmonary diseases and disorders; neurological diseases and disorders (e.g., neurodegenerative diseases and disorders); ocular diseases and disorders; metabolic diseases and disorders; cardiovascular diseases and disorders; inflammatory diseases and disorders; autoimmune diseases and disorders; skin diseases and disorders; skin diseases; age-related diseases; and transplant-related diseases and disorders. A hallmark of aging is the gradual functional decline or degeneration that occurs at the molecular, cellular, tissue, and organismal levels. Age-related degeneration leads 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 other pathologies. Although different mammalian species differ in their susceptibility to specific age-related pathologies, collectively, age-related pathologies generally increase with approximately exponential kinetics starting approximately at the midpoint of the species-specific lifespan (e.g., 50-60 years in humans) (see, e.g., Campisi, Annu. Rev. Physiol. 75 (2013) 685-705; Naylor et al., Clin. Pharmacol. Ther. 93 (2013) 105-116).

[0163] Examples of aging-associated conditions, diseases, or disorders that can be treated by administering the senolytic agents described herein in accordance with the methods described herein include aging-associated diseases and disorders (e.g., kyphosis, renal dysfunction, frailty, hair loss, hearing loss, muscle wasting, skin disorders, sarcopenia, and herniated discs) and other age-related diseases caused by aging (e.g., diseases / disorders resulting from radiation, chemotherapy, smoking, consumption of a high-fat / high-carbohydrate diet, and environmental factors); pulmonary diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiolitis obliterans, asthma); proliferative diseases, including cancer and metastasis; side effects associated with chemotherapy or radiation therapy; fibrosis and fibrotic disorders (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucous fibrosis, myocardial fibrosis, and pancreatic fibrosis); cognitive diseases (e.g., mild cognitive impairment, motor function diseases and disorders (e.g., Parkinson's disease, motor neuron dysfunction (MND); Huntington's disease); cerebrovascular disease; emphysema; osteoarthritis; benign prostatic hyperplasia; eye diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, decreased vision, presbyopia); metabolic diseases and disorders (e.g., obesity, diabetes, metabolic syndrome); cardiovascular diseases (e.g., atherosclerosis, cardiac diastolic dysfunction, aortic aneurysm, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress tolerance, myocardial fibrosis); inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); skin diseases, such as diabetic ulcers, wound healing, and skin nevi. In certain embodiments, any one or more of the diseases or disorders listed above or described herein may be excluded.

[0164] In certain embodiments, methods are provided for treating a senescence-associated 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, wherein the disease or disorder is a disease associated with aging (e.g., frailty, muscle weakness, cognitive impairment); idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease (COPD); renal or hepatic fibrosis; metastasis or other proliferative disease; osteoarthritis; or atherosclerosis.

[0165] Age-related diseases and disorders The senolytic agents described herein selectively kill senescent cells. Thus, targeting senescent cells during the aging process can be a preventative approach. Thus, administration of the senolytic agents described herein to a subject can prevent comorbidities and delay death in elderly subjects. Furthermore, selective killing of senescent cells can enhance immune system function, extend healthy lifespan, and improve a subject's quality of life.

[0166] Senolytic agents may also be useful for treating or preventing (i.e., reducing the likelihood of developing) 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., radiation, chemotherapy, smoking, a high-fat / high-carbohydrate diet, other environmental factors). The age-related disorder or disease or age-sensitive trait 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 of an age-related disorder or age-sensitive trait associated with a senescence-inducing stimulus, by reducing the severity of one or more symptoms, or by slowing the progression of an age-related disorder or age-sensitive trait associated with a senescence-inducing stimulus. In another embodiment, preventing an age-related disorder or age-sensitive characteristic associated with an age-inducing stimulus refers to preventing (i.e., reducing the likelihood of) or delaying the onset of an age-related disorder or age-sensitive characteristic associated with an age-inducing stimulus, or the recurrence of one or more age-related disorders or age-sensitive characteristics associated with an age-inducing stimulus. Age-related diseases or conditions include, for example, renal dysfunction, kyphosis, herniated disc, frailty, cognitive impairment, hair loss, hearing loss, vision loss (blindness or visual impairment), muscle fatigue, skin disorders, skin nevi, diabetes, metabolic syndrome, and sarcopenia. Vision loss refers to the lack of vision that a subject previously had. Various scales have been developed to express the degree of vision and vision loss based on visual acuity. Age-related diseases and conditions also include, but are not limited to, skin diseases, such as the treatment of one or more of the following conditions: wrinkles, including superficial fine lines; hyperpigmentation; scars; keloids; dermatitis; psoriasis; eczema (including seborrheic eczema); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis. Frailty is defined as a clinically recognizable state of increased vulnerability due to aging-related declines in reserve and function across multiple physiological systems, which impairs a subject's ability to cope with daily or acute stressors. Frailty can be characterized by impaired energy characteristics, such as low grip strength, low energy, slow walking speed, low physical activity, and / or unintentional weight loss.Research suggests that a patient may be diagnosed with frailty when three out of five of the above characteristics are observed (see, e.g., Fried et al., J. Gerontol. A Biol. Sci. Med, Sci. 56(3)(2001)M146-M156; Xue, Clin. Geriatr. Med. 27(1)(2001)1-15). In certain embodiments, aging and aging-related diseases and disorders may be treated or prevented (i.e., the likelihood of occurrence is reduced) by administering a senolytic agent. The senolytic agent may inhibit the senescence of adult stem cells or inhibit the accumulation of, kill, or promote the removal of senescent adult stem cells. The importance of preventing senescence in stem cells to maintain tissue regenerative capacity is described, for example, in Park et al., J. Clin. Invest. 113 (2004) 175-179; and Sousa-Victor, Nature 506 (2014) 316-321.

[0167] Methods for measuring aging are known in the art. For example, aging can be measured in bone by measuring the incidence of non-vertebral fractures, hip fractures, total fractures, vertebral fractures, recurrent fractures, functional recovery after fractures, bone mineral density loss in the lumbar spine and hip, knee flexion velocity, NSAID use, number of painful joints, and osteoarthritis. Aging can also be measured in muscle by measuring functional decline, fall rate, reaction time and grip strength, muscle mass loss in the upper and lower limbs, and dual-task 10-meter walking speed. Furthermore, aging can be measured in the cardiovascular system by measuring changes in systolic and diastolic blood pressure, the incidence of hypertension, major cardiovascular events such as myocardial infarction, stroke, congestive heart disease, and cardiovascular mortality. Furthermore, aging can be measured in the brain by measuring cognitive decline, the incidence of depression, and the incidence of dementia. Aging can also be measured in the immune system by infection rates, upper respiratory tract infection rates, flu-like illness rates, incidence of severe infections leading to hospitalization, cancer incidence, transplant infection rates, and gastrointestinal infection rates. Other indications of aging include, but are not limited to, poor oral hygiene, tooth loss, incidence of gastrointestinal (GI) symptoms, fasting blood glucose and / or insulin levels, changes in body composition, decline in renal function, quality of life, incidence of disability related to activities of daily living, and incidence of nursing home admission. Methods for measuring skin aging are known in the art and may include transepidermal water loss (TEWL), skin hydration, skin elasticity, crow's feet wrinkle area ratio analysis, sensitivity, radiance, roughness, blemishes, sagging, evenness of skin tone, softness, and contouring (depth variation).

[0168] Administration of the senolytic agents described herein can prolong survival compared to the expected survival if the subject had not received treatment. Subjects in need of treatment include those who already have a disease or disorder, as well as those prone to or at risk of developing a disease or disorder, and subjects for whom a disease, condition, or disorder is to be treated prophylactically. A subject may have a genetic predisposition to developing a disease or disorder that would benefit from clearance of senescent cells, or may be of a particular age where ingestion of a senolytic agent may provide a clinical effect of delaying the onset or reducing the severity of a disease, including an age-related disease or disorder.

[0169] In other embodiments, methods for treating a senescence-related disease or disorder are provided, further comprising identifying a subject who would benefit from treatment (i.e., phenotyping; individualized treatment) with a senolytic agent described herein. The method comprises first detecting the level of senescent cells in the subject, such as in a specific organ or tissue of the subject. A biological sample, e.g., a blood sample, serum or plasma sample, biopsy material, body fluid (e.g., lung lavage, peritoneal fluid, mucosal lavage, synovial fluid, vitreous humor, cerebrospinal fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation from the subject, is obtained from the subject. The level of senescent cells can be determined according to any of the in vitro assays or techniques described herein. For example, senescent cells can be detected by morphology (e.g., as seen by microscopy); or by production of senescence-associated markers, such as senescence-associated β-galactosidase (SA-β-gal), p16INK4a, p21, PAI-1, or any one or more SASP factors (e.g., IL-6, MMP3). Senescent and non-senescent cells from a biological sample can also be used in in vitro cellular assays, in which cells are exposed to any one of the senolytic agents described herein to determine the ability of the agent to kill the subject's senescent cells without undesirable toxicity to non-senescent cells. Furthermore, these methods can 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 can be detected (e.g., by determining the level of mRNA senescent cell marker expression), and treatment and / or non-treatment intervals can be adjusted accordingly.

[0170] Pulmonary Diseases and Disorders In certain embodiments, methods are provided for treating or preventing (i.e., reducing the likelihood of developing) 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.

[0171] COPD is a pulmonary disease defined by persistently inadequate airflow due to the destruction of lung tissue (emphysema) and dysfunction of small airways (obstructive bronchiolitis). Primary symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excessive sputum production. Elastase from neutrophils and macrophages activated by cigarette smoke degrades the extracellular matrix of alveolar structures, leading to enlargement of 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 smoke, secondhand smoke, and pipe smoke), 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.

[0172] Processes involved in causing lung damage include, for example, oxidative stress caused by high concentrations of free radicals in cigarette smoke; cytokine release due to inflammatory responses to irritants in the airways; and impairment of antiprotease enzymes by cigarette smoke and free radicals (thereby allowing proteases to damage the lungs). Genetic susceptibility may also contribute to the disease. In approximately 1% of people with COPD, the disease is caused by a genetic disorder that causes low levels of alpha-1-antitrypsin production in the liver. The enzyme is normally secreted into the bloodstream to help protect the lungs.

[0173] 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. Fibroblast activation leads to increased production of extracellular matrix proteins, and transdifferentiation into contractile myofibroblasts contributes to wound contraction. A provisional matrix seals the damaged epithelium and provides a scaffold for epithelial cell migration, including epithelial-mesenchymal transition (EMT). Blood loss associated with epithelial injury induces platelet activation, growth factor production, and an acute inflammatory response. Normally, the epithelial barrier heals and the inflammatory response resolves. However, in fibrosis, the fibroblast response continues, resulting in unresolved wound healing. The formation of fibroblastic nests is a hallmark of this disease, reflecting the location of ongoing fibrosis. As the name implies, the etiology of IPF is unknown. The involvement of cellular senescence in IPF has been suggested by the observation that the incidence of IPF increases with age and that lung tissue from IPF patients is enriched in SA-β-Gal-positive cells and contains increased levels of the senescence marker p21 (see, e.g., Minagawa et al., Am. J. Physiol. Lung Cell. Mol. Physiol. 300 (2011) L391-L401; see also, e.g., Naylor et al., supra). Short telomeres 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, a contribution of cellular senescence to IPF has been suggested by reports that SASP components of senescent cells, such as IL-6, IL-8, and IL-1β, promote fibroblast-to-myofibroblast differentiation and epithelial-to-mesenchymal transition, leading to extensive remodeling of the extracellular matrix of the alveolar and interstitial spaces (e.g., Minagawa et al., see above).

[0174] Subjects at risk of developing pulmonary fibrosis include subjects exposed to environmental or occupational pollutants, such as those suffering from asbestosis and silicosis; smokers; subjects suffering from several typical connective tissue diseases, such as rheumatoid arthritis, SLE, and scleroderma; subjects suffering from other diseases associated with connective tissue, such as sarcoidosis and Wegener's granulomatosis; subjects suffering from infectious diseases; subjects taking certain medications (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin); subjects undergoing chest radiation therapy; and subjects with a family history of pulmonary fibrosis.

[0175] Symptoms of COPD may include any one of the following: shortness of breath, especially during physical activity; wheezing; chest tightness; clearing the throat first thing in the morning due to excess mucus in the lungs; chronic cough that produces clear, white, yellowish or greenish saliva; blue coloring of lips or nail bases (cyanosis); frequent respiratory infections; lack of energy; unintentional weight loss (observed in the later stages of the disease).Subjects suffering from COPD may also experience exacerbations, during which symptoms worsen and last for days.Symptoms of pulmonary fibrosis are known in the art and include shortness of breath, especially during exercise; dry, hacking cough; rapid, shallow breathing; gradual unintentional weight loss; fatigue; joint and muscle pain; and clubbing (the spreading and curling of the tips of fingers or toes).

[0176] Subjects suffering from COPD or pulmonary fibrosis can be identified using standard diagnostic methods routinely practiced in the art. Monitoring the effectiveness of one or more senolytic agents administered to subjects suffering from or at risk of developing pulmonary disease can be performed using methods typically used in diagnosis. Generally, one or more of the following tests or examinations can be performed: physical examination, patient medical history, patient family medical history, chest X-ray, pulmonary function tests (such as spirometry), blood tests (e.g., arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CT scan, and exercise testing.

[0177] Other lung diseases or disorders that can be treated 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 can also be exacerbated by tobacco smoke (including cigarette smoke, cigar smoke, secondhand smoke, and pipe smoke), occupational exposures (e.g., exposure to dust, smoke, or fumes), infections, and / or pollutants, which induce cellular senescence and thereby contribute to inflammation. Emphysema is sometimes considered a subgroup of COPD.

[0178] Bronchiectasis is caused by damage to the airways, causing them to widen, sag, and become scarred. Bronchiectasis is usually caused by a 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). If only part of the lung is affected, the disorder may be caused by obstruction rather than a condition.

[0179] The methods described herein for treating or preventing (i.e., reducing the likelihood of developing) aging-related lung diseases or disorders can also be used to treat subjects who are aging and have decreased (or degenerated) lung function (i.e., decreased 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 aging. Structural changes include deformation of the chest wall and thoracic vertebrae, which can impair overall respiratory system compliance, increasing the effort of breathing. The respiratory system undergoes structural, physiological, and immunological changes with aging. Increased rates of neutrophils and lower percentages of macrophages are found in bronchoalveolar lavage (BAL) of elderly subjects compared to younger adults. Persistent low-grade inflammation in the lower respiratory tract can cause proteolytic and oxidant-mediated damage to the lung matrix, leading to the loss of alveolar units and impaired gas exchange across the alveolar membrane seen with aging. Persistent inflammation in the lower airways may predispose older adults to increased susceptibility to toxic environmental exposures and accelerated decline in lung function (see, e.g., Sharma et al., Clinical Interventions in Aging 1 (2006) 253-260). Oxidative stress exacerbates inflammation during 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 during 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). Constitutive activation and recruitment of macrophages, T cells, and mast cells promotes the release of proteases, which leads to extracellular matrix degradation, cell death, remodeling, and other events that can cause tissue and organ damage during chronic inflammation (see, e.g., Demedts et al., Respir. Res. 7 (2006) 53-63).By administering senolytic agents to aging subjects (including asymptomatic middle-aged adults), the decline in lung function can be slowed or prevented by killing and removing senescent cells from the airways.

[0180] The effectiveness of the senolytic agent can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of analytical tests and methods described herein, can be used to monitor a subject's health status. The therapeutic effect of a senolytic agent or a pharmaceutical composition containing the agent can be analyzed using techniques known in the art, such as comparing the symptoms of treated patients suffering from or at risk of a lung disease with the symptoms of patients who have not received such treatment or who have received a placebo. Additionally, methods and techniques for assessing lung mechanical function, such as measuring lung volume, elasticity, and airway hyperresponsiveness, can be performed. Throughout the procedure, any of a number of measurements may be obtained to determine and monitor pulmonary function, including expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in 1 second, FEV1), FEV1 / FEV1 ratio, forced expiratory flow rate 25%-75%, and maximum ventilation (MVV), peak expiratory flow rate (PEF), and slow 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 diffusing capacity for carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO2) may also be measured; normal oxygen levels are typically 95%-100%. An SpO2 level below 90% suggests the subject is suffering from hypoxemia. Values ​​below 80% are considered severe and require intervention to maintain brain and heart function and prevent cardiac or respiratory arrest.

[0181] metastasis In certain embodiments, methods are provided for treating or preventing (i.e., reducing the likelihood of onset or occurrence of) a senescent cell-associated disease (or disorder or condition) that 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 onset of) metastasis (i.e., the spread and dissemination of cancer or tumor cells) from one organ or tissue to another in the body.

[0182] Senescent cell-associated diseases or disorders include metastasis, and subjects suffering from cancer may benefit from administration of the senolytic agents described herein to inhibit metastasis. When administered to a subject suffering from cancer according to the methods described herein, such senolytic agents may inhibit tumor growth. Cancer metastasis occurs when cancer cells (i.e., tumor cells) spread beyond the anatomical site of origin and initial colonization to other sites throughout the subject's body. Tumor growth may be determined by tumor size, which may be measured by various methods known to those skilled in the art, such as by PET scan, MRI, CAT scan, biopsy, etc. The effect of a therapeutic agent on tumor growth may also be assessed by examining tumor cell differentiation.

[0183] As used herein and in the art, the term cancer or tumor is a clinically descriptive term that encompasses diseases that are typically characterized by cells exhibiting abnormal cell proliferation. The term cancer is generally used to refer to a malignant tumor or a disease state resulting from a malignant tumor. Alternatively, the abnormal growth may be referred to in the art as a neoplasm. With respect to tissue, the term tumor generally refers to an abnormal tissue growth characterized at least in part by excessive and abnormal cell proliferation. A tumor may be metastatic, capable of spreading beyond its anatomical site of origin and initial colonization to other sites throughout a subject's body. Cancer may include solid tumors or "liquid" tumors (e.g., leukemia and other blood cancers).

[0184] Cells are induced to senesce by cancer treatments such as radiation and certain chemotherapeutic agents. The presence of senescent cells increases the secretion of inflammatory molecules and promotes tumor progression, which may include promoting tumor growth, increasing tumor size, promoting metastasis, and altering differentiation. When senescent cells are destroyed, tumor progression is significantly inhibited, tumor size decreases, and little or no metastatic growth is observed (see, e.g., WO 2013 / 090645).

[0185] In certain embodiments, methods are provided for preventing (i.e., reducing the likelihood of its development), inhibiting, or delaying metastasis in a subject suffering from cancer by administering a senolytic agent described herein. In other embodiments, the senolytic agent is administered for one or more days within a treatment period (i.e., a treatment course) of 7 or 14 days or less. In yet other embodiments, the treatment course is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or less, or 21 days or less. In yet other embodiments, the treatment course is one day. In yet other embodiments, the senolytic agent is administered for two or more days within a treatment period of 7 or 14 days or less.

[0186] Because cells can be induced to senesce by cancer treatments such as radiation and certain chemotherapeutic agents (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 described herein and understood in the art, the establishment of senescence, indicated by, for example, the presence of senescence-associated secretory phenomena (SASP), occurs over several days; therefore, administration of a senolytic agent to kill senescent cells and thereby reduce the likelihood of or extent of metastasis is initiated once senescence is established. As described herein, the following therapeutic courses for administration of a senolytic agent can be used in the methods described herein to treat or prevent (i.e., reduce the likelihood of occurrence or reduce the severity of) the side effects of chemotherapy or radiation therapy:

[0187] In certain embodiments, when chemotherapy or radiotherapy is administered in a treatment cycle of at least one day on-therapy (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) off-therapy (i.e., no chemotherapy or radiotherapy), the senolytic agent is administered on one or more days during the off-therapy time interval (time period) beginning on or after day 2 of the off-therapy time interval and ending on or before the last day of the off-therapy time interval. Illustratively, where n is the number of days off-therapy, the senolytic agent is administered on at least 1 day and no more than n-1 days of the off-therapy time interval. In certain embodiments, when chemotherapy or radiation therapy is administered in a treatment cycle of at least one day on-treatment (i.e., chemotherapy or radiation therapy), followed by at least one week off-treatment, the senolytic agent is administered on one or more days during the off-treatment time interval beginning on or after day 2 of the off-treatment time interval and ending on or before the last day of the off-treatment time interval.

[0188] Chemotherapy can be called chemotherapy, chemotherapy drugs, or chemotherapy agents. Many chemotherapy drugs are compounds called small organic molecules. Chemotherapy is also a term used to describe a combination of chemotherapy drugs administered to treat a specific cancer. As understood by those skilled in the art, chemotherapy can also refer to a combination of two or more chemotherapy molecules that are administered in a coordinated manner, which can be called combination chemotherapy. Many chemotherapy drugs are used in the field of oncology, including, but not limited to, alkylating agents; antimetabolites; anthracyclines, plant alkaloids; and topoisomerase inhibitors.

[0189] Cancers that can metastasize can be solid tumors or liquid tumors (e.g., blood cancers, such as leukemia). Liquid tumor cancers are classified in the art as those that arise 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 lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia. Solid tumors that occur with significant frequency 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, laryngeal cancer (squamous cell carcinoma that occurs in 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 likelihood of occurrence or development 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, laryngeal 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.

[0190] The methods described herein are also useful for inhibiting, retarding, or slowing the progression of metastatic cancer of any one of the tumor types described in the medical arts.Cancer (tumor) types include: adrenocortical carcinoma, pediatric adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, basal cell carcinoma, pediatric basal cell carcinoma, bladder cancer, pediatric bladder cancer, osteosarcoma, brain tumor, pediatric astrocytoma, pediatric brain stem glioma, pediatric central nervous system atypical teratoid / rhabdomyosarcoma 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, cancer of unknown primary site, childhood cancer of unknown primary site, childhood cardiac (heart) tumor, 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 nasal neuroblastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric (stomach) cancer, childhood gastric (stomach) cancer, gastrointestinal stromal tumor (GIST) ), pediatric gastrointestinal stromal tumor (GIST), pediatric extracranial germ cell tumor, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, pediatric head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell kidney cancer, Wilms tumor, pediatric kidney tumor, Langerhans cell histiocytosis, laryngeal cancer, pediatric 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 lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous cell neck cancer of unknown primary, midline tract carcinoma involving the NUT genecarcinoma), oral cancer, childhood multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor, myeloproliferative neoplasm, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, pediatric nasopharyngeal cancer, neuroblastoma, oral cancer, pediatric oral cancer, oropharyngeal cancer, ovarian cancer, pediatric ovarian cancer, epithelial ovarian cancer, low malignant potential ovarian cancer, pancreatic cancer, pediatric pancreatic cancer, pancreatic neuroendocrine tumor (insulin cell tumor), pediatric papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacytoma, pediatric pleuropulmonary blastoma, prostate cancer, rectal Cancer, renal pelvis transitional cell carcinoma, retinoblastoma, salivary gland cancer, pediatric salivary gland cancer, Ewing's sarcoma family of tumors, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, pediatric rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, pediatric skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, pediatric squamous cell carcinoma, testicular cancer, pediatric testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, pediatric thymoma and thymic carcinoma, thyroid cancer, pediatric thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, uterine endometrial cancer, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0191] 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 radiation therapy. Examples of chemotherapeutic agents that induce senescence in non-cancerous cells include anthracyclines (doxorubicin, daunorubicin, etc.); taxol (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more of the senolytic agents administered as described herein can be used to treat and / or prevent (i.e., reduce the likelihood of) the side effects of chemotherapy or radiation therapy. Removal or destruction of senescent cells can ameliorate the acute toxicity of chemotherapy or radiation therapy, including acute toxicity involving energy imbalance. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, loss of appetite, diarrhea), peripheral neuropathy, fatigue, 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), oral problems (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 can 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) the occurrence of) the acute toxicity of chemotherapy or radiation therapy, or both, or reducing the severity of its toxic side effects (i.e., adverse side effects) in a treated subject, 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 or reduce the severity of chemotherapy or radiation therapy side effects can be carried out via the same course of treatment as described above for the treatment / prevention of metastasis.As described for treating or preventing metastasis (i.e., reducing the likelihood of its development), the senolytic agent is administered during a chemotherapy-free or radiotherapy-free interval or after the chemotherapy or radiotherapy treatment regimen is completed.

[0192] In more particular embodiments, the acute toxicity is an acute toxicity involving energy imbalance, which may include one or more of weight loss, endocrine changes (e.g., hormone imbalance, changes in hormone signaling), and changes in body composition. In particular embodiments, the acute toxicity involving energy imbalance is associated with a decreased or diminished ability of the subject to be physically active, as indicated by decreased or diminished energy expenditure compared to that observed in subjects not receiving the medical therapy. By way of non-limiting example, such acute toxic effects involving energy imbalance include reduced physical activity. In other embodiments, the energy imbalance includes fatigue or lethargy.

[0193] In certain embodiments, a chemotherapy side effect treated or prevented (i.e., the likelihood of occurrence is reduced) by the senolytic agents described herein is cardiotoxicity. A subject suffering from cancer who is being treated with an anthracycline (e.g., doxorubicin, daunorubicin), can be treated with one or more senolytic agents described herein that alleviate, 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 a subject can receive, even if the cancer responds to the drug. Administration of one or more senolytic agents can reduce cardiotoxicity, allowing additional amounts of the anthracycline to be administered to the subject, resulting in an improved prognosis associated with the cancer disease. In certain embodiments, the cardiotoxicity results from the administration of an anthracycline, such as doxorubicin. Doxorubicin is an anthracycline topoisomerase inhibitor approved for the treatment of patients with ovarian cancer after failure of platinum-based therapy; Kaposi's sarcoma after failure of or intolerance to neoadjuvant 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. Doxorubicin is administered to patients at a total lifetime dose of 550 mg / m 2 Above this dose, it can cause myocardial damage that can lead to congestive heart failure. Cardiotoxicity can occur even at lower doses if the patient also receives mediastinal radiation or another cardiotoxic drug.

[0194] In other embodiments, the senolytic agents described herein can be used in the methods provided herein to ameliorate chronic or long-term side effects. Chronic toxic side effects typically result from multiple exposures or administrations of chemotherapy or radiation therapy over an extended 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 caused by the treatment. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients treated for cancer in childhood (see, e.g., Hudson et al., JAMA 309 92013) 2371-2381). Without wishing to be bound by any particular theory, destroying senescent cells, certain normal cells whose senescence has been induced by chemotherapy or radiation therapy, may reduce the likelihood of chronic side effects occurring, or may alleviate or reduce the severity of chronic side effects, or may delay the time of onset of chronic side effects. Chronic and / or late toxic side effects occurring in subjects receiving chemotherapy or radiation therapy include, by way of non-limiting example, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancers, cataracts and other visual impairment, hearing loss, chronic fatigue, decreased lung capacity, and lung disease.

[0195] Furthermore, by administering a senolytic agent to kill or remove senescent cells in a subject suffering from cancer, sensitivity to chemotherapy or radiotherapy may be clinically or statistically significantly increased compared to when the senolytic agent is not administered. In other words, the development of resistance to chemotherapy or radiotherapy may be inhibited when a senolytic agent is administered to a subject treated with the respective chemotherapy or radiotherapy.

[0196] Neurological Diseases and Disorders Age-related diseases or disorders that can be treated by administering the senolytic agents described herein include neurological 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 include glaucoma, vision loss, presbyopia, and cataracts.

[0197] Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is a disabling condition of the brain characterized by slow movement (bradykinesia), tremors, rigidity, and, in later stages, impaired balance. Many of these symptoms result from the loss of specific neurons in the brain, which leads to a lack of dopamine. The disease is characterized by neurodegeneration, such as the loss of approximately 50% to 70% of dopaminergic neurons in the substantia nigra pars compacta, severe loss of dopamine in the striatum, and / or the presence of intracytoplasmic inclusions (Lewy bodies) composed primarily of α-synuclein and ubiquitin. Parkinson's disease is also characterized by impaired locomotor activity, such as tremors, rigidity, bradykinesia, and / or postural instability. Subjects at risk for 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. Because senescence of dopamine-producing neurons is thought to contribute to the cell death observed in PD through the production of reactive oxygen species (see, e.g., Cohen et al., J. Neural Transm. Suppl. 19 (1983) 89-103), the methods and senolytic agents described herein are useful for treating and preventing Parkinson's disease.

[0198] Methods for detecting, monitoring, or quantifying neurodegenerative defects and / or impaired locomotor activity associated with Parkinson's disease, such as histological studies, biochemical tests, and behavioral assessments, are known in the art (see, for example, U.S. Patent Application Publication No. 2012 / 0005765). Symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty initiating or ending voluntary movements, jerky and stiff movements, muscle atrophy, tremors, and changes in heart rate, normal reflexes, bradykinesia, and postural instability. There is increasing recognition that individuals diagnosed with Parkinson's disease may have cognitive impairment, including mild cognitive impairment, in addition to their physical symptoms.

[0199] Alzheimer's disease (AD) is a neurodegenerative disease that presents with a slowly progressive mental decline accompanied by memory impairment, disorientation, and confusion, leading to severe dementia. Aging is the single most significant predisposing risk factor for developing AD, 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.

[0200] Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid (senile) plaques in histological samples. The disease primarily involves the limbic and cortical regions of the brain. Argyrophilic plaques containing amyloidogenic Aβ fragments of the 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, such as granulovacuolar degeneration in hippocampal pyramidal cells, as well as neuronal loss and gliosis in the cortex and hippocampus, are observed. Subjects at risk for developing Alzheimer's disease include elderly subjects, subjects with a family history of Alzheimer's disease, subjects with genetic risk genes (e.g., ApoE4) or definitive gene mutations (e.g., APP, PS1, or PS2), and subjects with a history of head trauma or cardiovascular conditions (e.g., hypertension, heart disease, stroke, diabetes, hypercholesterolemia, etc.).

[0201] Numerous behavioral and histopathological assays for assessing Alzheimer's disease phenotypes, characterizing therapeutic agents, and evaluating treatments are known in the art. Histological analysis is typically performed postmortem. Histological analysis of Aβ levels can be performed using thioflavin-S, Congo red, or anti-Aβ staining (e.g., 4G8, 10D5, or 6E10 antibodies) to visualize Aβ deposits on sectioned 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). An in vivo method for visualizing Aβ deposits in transgenic mice has 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 amyloidophilic Congo red-type compound FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) enables visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., Nature Neurosci. 8 (2005) 527-533). Radiolabeled, putrescine-modified amyloid-β peptide labels amyloid deposits in vivo in a mouse model of Alzheimer's disease (see, e.g., Wengenack et al., Nat. Biotechnol. 18 (2000) 868-872).

[0202] Increased glial fibrillary acidic protein (GFAP) expression 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 Gallyas silver staining (see, e.g., Gotz et al., J. Biol. Chem. 276 (2001) 529-534; U.S. Patent No. 6,664,443). Axonal staining using electron microscopy and axonal transport studies can be used to visualize neurodegeneration (see, for example, Ishihara et al., Neuron 24 (1999) 751-762).

[0203] Subjects suffering from Alzheimer's disease can be identified using standard diagnostic methods known in the field of Alzheimer's disease. Generally, the diagnosis of Alzheimer's disease is based on the patient's symptoms (e.g., progressive decline in memory function, gradual withdrawal from daily activities and resulting frustration, apathy, agitation or irritability, aggression, anxiety, sleep disorders, dysphoria, abnormal motor behavior, disinhibition, social withdrawal, anorexia, hallucinations, dementia), medical history, neuropsychological testing, neurological testing, and / or physical examination. Cerebrospinal fluid can also be tested for various proteins associated with Alzheimer's pathology, including tau, amyloid beta peptide, and AD7C-NTP. Genetic testing is also available for early-onset familial Alzheimer's disease (eFAD), an autosomal dominant genetic disease. Clinical genetic testing is available for individuals with AD symptoms or family members of patients at risk of developing early-onset disease. In the United States, mutations to PS2 and APP can be tested clinically or in federally approved laboratories under the Clinical Laboratory Improvement Amendments. Commercial tests for PS1 mutations are also available (Elan Pharmaceuticals).

[0204] Zhang et al. reported that in the brains of patients with AD and in AD mouse models, Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs) associated with Aβ plaques exhibited a senescence-like phenotype characterized by upregulation of p21 / CDKN1A and p16 / INK4 / CDKN2A proteins and senescence-associated β-galactosidase activity, whereas astrocytes, microglia, or oligodendrocytes did not (see Nature Neurosci. 22 (2019) 719-728). Molecular interrogation of the Aβ plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregated Aβ induced cellular senescence. Treatment of AD mice with a senolytic cocktail containing dasatinib and quercetin selectively removed senescent cells from the plaque environment, attenuated neuroinflammation, reduced Aβ burden, and improved cognitive impairment. These findings suggest a role for Aβ-induced OPC senescence in AD neuroinflammation and cognitive impairment, and the potential therapeutic benefit of senolytic treatment.

[0205] The effectiveness of one or more senolytic agents described herein and monitoring of subjects receiving one or more senolytic agents can be readily determined by those skilled in the medical and clinical arts. One or any combination of the diagnostic methods described herein, including physical examination, evaluation and monitoring of clinical symptoms, and performance of analytical tests and methods, can be used to monitor a subject's health status. The effect of administering one or more senolytic agents can be analyzed using techniques known in the art, such as comparing the symptoms of patients with or at risk for Alzheimer's disease who received the treatment with the symptoms of patients who did not receive such treatment or who received a placebo.

[0206] Mild cognitive impairment (MCI) is a brain function syndrome involving the onset and progression of cognitive impairment beyond that expected based on the individual's age and education, but not significant enough to interfere with an individual's daily activities. MCI is a form of cognitive aging that is considered a transitional state between normal aging and possible conversion to dementia (see Pepeu, Dialogues in Clinical Neuroscience 6 (2004) 369-377). MCI that primarily affects memory is known as "amnestic MCI." Individuals with amnestic MCI may begin to forget recent events or important information that was previously easily recalled. Amnestic MCI is often considered a precursor to Alzheimer's disease. MCI that affects thinking skills other than memory is known as "non-amnestic MCI." This type of MCI affects thinking skills such as the ability to make correct decisions, determine the timing or sequence of steps required to complete a complex task, or perform visual recognition. Individuals with non-amnestic MCI are thought to be more likely to convert to other types of dementia (e.g., Lewy body dementia).

[0207] Those skilled in the medical arts are increasingly aware that individuals diagnosed with Parkinson's disease may have MCI in addition to physical symptoms. Recent studies indicate that 20-30% of individuals with Parkinson's disease have MCI, and that MCI tends to be non-amnestic. Parkinson's disease patients with MCI may occasionally progress to fully developed dementia (Parkinson's disease with dementia).

[0208] Methods for detecting, monitoring, quantifying, or assessing neuropathological deficits associated with MCI are known in the art, including morphological analysis of astrocytes, acetylcholine release, 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 arm maze paradigm, non-matching-to-sample task, allocentric place determination task in a water maze, Morris maze test, visuospatial task, delayed-response spatial memory task, and olfactory novelty test.

[0209] Motor neuron dysfunction (MND) is a group of progressive neurological disorders that destroy motor neurons, the cells that control essential voluntary muscle activities such as speech, 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, SMA3, also known as Kugelberg-Welander disease, and 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. While primary lateral sclerosis is a disease of the upper motor neurons, 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, causing slurred speech and difficulty chewing and swallowing. There are almost always mildly abnormal signs in the arms and legs. Patients with MND exhibit a Parkinson's disease phenotype (e.g., tremor, rigidity, bradykinesia, and / or postural instability). Methods for detecting, monitoring, or quantifying locomotor disorders and / or other deficits associated with Parkinson's disease, such as MND, are known in the art (see, for example, U.S. Patent Application Publication No. 2012 / 0005765).

[0210] Methods for detecting, monitoring, quantifying, or evaluating motor disorders and histopathological defects associated with MND, including histopathological, biochemical, and electrophysiological tests and motor activity analyses, are known in the art (see, e.g., Rich et al., J. Neurophysiol. 88 (2002) 3293-3304; Appel et al., Proc. Natl. Acad. Sci. USA 88 (1991) 647-651). Histopathologically, MND is characterized by the death of motor neurons, the gradual accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin, and the accumulation of abnormal neurofilaments in degenerating motor neurons. In addition, reactive astroglia and microglia are often detected in diseased tissue. Patients with MND exhibit one or more motor disorders, including muscle weakness and wasting, uncontrollable twitching, spasticity, slow and effortful movements, and hyperactive tendon reflexes.

[0211] Eye Diseases and Disorders In certain embodiments, the aging-related disease or disorder is an ocular disease, disorder, or condition, such as presbyopia, macular degeneration, or cataract. In other specific embodiments, the aging-related disease or disorder is glaucoma. Macular degeneration is a neurodegenerative disease that causes 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 approximately 90% of patients with age-related macular degeneration (ARMD or AMD) are diagnosed with the dry form. The wet form of the disease usually leads to more severe vision loss. Although the exact cause of age-related macular degeneration is still unknown, the number of senescent retinal pigment epithelial (RPE) cells increases with age. Aging 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 blood levels of vitamin D (see, e.g., Millen, et al., Arch. Ophthalmol. 129 (4) (2011) 481-89). Genetic predisposing risk factors include decreased levels of Dicer 1 (an enzyme involved in the maturation of microRNAs) in the eyes of patients with dry AMD, and the reduction in microRNAs contributes to a senescent cell profile.

[0212] Dry ARMD is associated with atrophy of the RPE layer, which leads to the loss of photoreceptor cells. Dry forms of ARMD may be due to aging and thinning of macular tissue and pigmentation in the macula. Aging appears to inhibit both RPE replication and migration, resulting in permanent RPE deficiency in the macula of dry AMD patients (see, e.g., 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 causes retinal cell death and blind spots in central vision. Different forms of macular degeneration may also occur in younger patients. Non-aging etiologies may be associated with genetics, diabetes, nutritional deficiencies, head trauma, infection, or other factors.

[0213] Decreased vision noticed by a patient or ophthalmologist during a routine eye examination may be the first indicator of macular degeneration. The formation of exudates, or "drusen," under the Bruch's membrane in the macula is often the first physical sign that macular degeneration may develop. Symptoms include perceived distortion of straight lines, and in some cases, the center of the visual field appears more distorted than the rest of the vision; a dark, blurred area or "whiteout" appears in the center of the visual field; and / or color perception is altered or diminished. Diagnosis and monitoring of subjects with macular degeneration can be performed by those skilled in the art of ophthalmology, following art-recognized procedures for routine eye examinations and subject reporting of symptoms.

[0214] Presbyopia is an age-related condition in which the speed and amplitude of normal eye accommodation decrease with age, resulting in a gradual decline in the eye's ability to focus on near objects. Loss of lens elasticity and loss of ciliary muscle contractility are hypothesized to be the cause (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 capsules 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).

[0215] The lamination structure of the ocular capsule also changes, which may be at least partially attributable to changes in tissue composition (see, e.g., Krag et al., 1997, supra, and references cited therein). The primary structural component of the lens capsule is basement membrane type IV collagen, 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 (α1-6) that assemble into heterotrimeric collagen IV protomers, each containing a specific combination of α112, α345, or α556 chains (see, e.g., Khoshnoodi et al., Microsc. Res. Tech. 71 (2008) 357-370). The protomers share structural similarities with triple-helical collagen domains, with a Gly-XY triplet peptide sequence terminating in a globular C-terminal region called the non-collagenous 1 (NC1) domain (Timpl et al., Eur. J. Biochem. 95 (1979) 255-263). The N-terminus consists of a helical domain called the 7S domain (see, e.g., Risteli et al., Eur. J. Biochem. 108 (1980) 239-250), which is also involved in interactions between protomers.

[0216] Studies suggest that collagen IV influences cellular functions inferred from the positioning of the basement membrane beneath the epithelial layer, and data support the role of collagen IV in tissue stabilization (see, e.g., Cummings et al., supra). Posterior capsule opacification (PCO) develops as a complication in approximately 20–40% of patients several years after cataract surgery (see, e.g., Awasthi et al., Arch. Ophthalmol. 127 (2009) 555–562). PCO results from the proliferation and activity of remaining lens epithelial cells along the posterior capsule in a response similar to wound healing. Growth factors such as fibroblast growth factor, transforming growth factor-β, 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 explained herein, the production of these factors and cytokines by senescent cells contributes to SASP. In contrast, in vitro studies show that collagen IV promotes the attachment of lens epithelial cells (see, e.g., Olivero et al., Invest. Ophthalmol. Vis. Sci. 34 (1993) 2825-2834). Attachment of collagen IV, fibronectin, and laminin to intraocular lenses may inhibit cell migration and reduce the risk of PCO (see, e.g., Raj et al., Int. J. Biomed. Sci. 3 (2007) 237-250).

[0217] Without wishing to be bound by any particular theory, selectively killing senescent cells with the senolytic agents described herein may slow or prevent (delay, inhibit, delay) the division of type IV collagen network. Removal of senescent cells, and thereby elimination of the inflammatory effects of SASP, may reduce or inhibit epithelial cell migration, further delaying (inhibiting) the onset of presbyopia or reducing or slowing the progressive severity of the condition (e.g., slowing progression from mild to moderate or moderate to severe). The senolytic agents described herein may also be useful after cataract surgery to reduce the likelihood of developing PCO.

[0218] Although direct evidence of the involvement of cellular senescence in the development of cataracts has not been obtained from clinical studies, BubR1 hypomorphic mice develop bilateral posterior subcapsular cataracts early in life, suggesting that aging may play a role (see, e.g., Baker et al., Nat. Cell Biol. 10 (2008) 825-836). Cataracts are opacities of the eye's lens that cause blurred vision and, if left untreated, can lead to blindness. Surgery to remove cataracts is effective and routinely performed. Administration of one or more of the senolytic agents described herein may reduce the likelihood of cataract development or slow or inhibit cataract progression. The presence and severity of cataracts can be monitored by ophthalmic examination using methods routinely performed by those skilled in the art of ophthalmology.

[0219] In certain embodiments, at least one senolytic agent described herein can be administered to a subject at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with the senolytic agent can be initiated when the human subject is at least 40 years of age to delay or inhibit the onset or occurrence of cataracts, presbyopia, and macular degeneration. Because nearly all humans develop presbyopia, in certain embodiments, a senolytic agent can be administered in a manner as described herein to a human subject after the subject reaches 40 years of age to delay or inhibit the onset or occurrence of presbyopia.

[0220] 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 cause visual field loss without other current symptoms. The lack of symptoms often leads to a delay in diagnosing glaucoma until the late stages of the disease. Subjects affected by glaucoma often have severely impaired vision, even if they do not become blind. Normally, clear fluid flows in and out of the front part of the eye, known as the anterior chamber. In individuals with open-angle / wide-angle glaucoma, this fluid drains too slowly, causing increased intraocular pressure. If left untreated, this high pressure can subsequently damage the optic nerve and cause complete blindness. Loss of peripheral vision is caused by 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 outflow was subjected to SA-β-Gal staining, a four-fold increase in senescence was observed in glaucoma patients (see, for example, Liton et al., Exp. Gerontol. 40 (2005) 745-748).

[0221] To monitor the effectiveness of treatment in inhibiting the progression of glaucoma, standard automated perimetry (visual field testing) is the most widely used technique. Furthermore, several algorithms for progression detection have been developed (see, for example, Wesselink et al., Arch. Ophthalmol. 127(3)(2009)270-274, and references therein). Additional methods include gonioscopy (examining the trabecular meshwork and angle where fluid leaves the eye); imaging techniques such as scanning laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and ocular coherence tomography); fundus examination; and pachymetry to measure central corneal thickness.

[0222] metabolic disease or disorder Senescence-related diseases or disorders that can be treated by administering senolytic agents include metabolic diseases or disorders, such as diabetes, metabolic syndrome, diabetic ulcers, and obesity.

[0223] Diabetes is characterized by high blood glucose levels due to defects in insulin production, insulin action, or both. The majority (90-95%) of all diagnosed cases of diabetes in adults are type 2 diabetes, which is characterized by the gradual loss of insulin production by the pancreas. Diabetes is the leading cause of new cases of kidney failure, non-traumatic lower limb amputations, and blindness among 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 can be used to treat type 2 diabetes, particularly type 2 diabetes associated with aging, diet, and obesity.

[0224] 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-β-Gal, p53, and p21 (see, e.g., Tchkonia et al., supra; Minamino et al., Nat. Med. 15 (2009) 1082-1087). Concurrent upregulation of inflammatory cytokines such as tumor necrosis factor-α and Ccl2 / MCP1 was observed in the same adipose tissue (see, e.g., Minamino et al., supra). The induction of senescent cells in obesity potentially has clinical implications, as inflammatory SASP components have also been 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 the senolytic agent, thereby killing the senescent preadipocytes, may provide clinical and health benefits to individuals with any one of diabetes, obesity, or metabolic syndrome.

[0225] Subjects suffering from type 2 diabetes can be identified using standard diagnostic methods known in the field of type 2 diabetes.Generally, the 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 dark 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 and those with other risk factors such as excess weight, fat distribution, lack of exercise, race, age, prediabetes, and / or gestational diabetes.

[0226] The effectiveness of senolytic agents can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of analytical tests and methods, such as those described herein, can be used to monitor a subject's health status. 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 tolerance, energy expenditure, body composition, adipose tissue, skeletal muscle, and liver inflammation, and / or lipotoxicity (muscle and liver lipids by in vivo imaging and muscle, liver, bone marrow, and pancreatic beta cell lipid accumulation and inflammation by histology). Other characteristics or phenotypes of type 2 diabetes are known and can be assayed using other methods and techniques known and routinely practiced in the art, as described herein.

[0227] Obesity and obesity-related disorders are used to refer to the condition of a subject who weighs significantly more than 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 a subject's height and weight. A person is considered overweight if they have a BMI between 25 and 29; obese if they have a BMI between 30 and 39; and severely obese if they have a BMI >40. Thus, the terms obesity and obesity-related refer to human subjects with a body mass index greater than 30, greater than 35, or greater than 40. A category of obesity not captured by BMI is referred to in the art as "abdominal obesity," which refers to excess fat around a subject's waist and is an important health factor independent of BMI. The simplest and most commonly used measure of abdominal obesity is waist size. Generally, abdominal obesity in women is defined as a waist size of 35 inches or greater, and in men as a waist size of 40 inches or greater. More complex methods for determining obesity require specialized equipment such as magnetic resonance imaging or dual energy X-ray absorptiometry machines.

[0228] A diabetes- and aging-related pathology or disorder is diabetic ulcers (i.e., diabetic wounds). Ulcers are breakdowns in the skin that can extend to involve subcutaneous tissue or even muscle or bone. These lesions occur particularly on the lower extremities. Patients with diabetic venous ulcers show 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, such as diabetic ulcers, is also observed at the site of chronic wounds (see, e.g., Goren et al., Am. J. Pathol. 168 (2006) 65-77), suggesting that the inflammatory cytokine phenotype of senescent cells plays a role in the pathology.

[0229] Subjects who suffer from type 2 diabetes or are 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.Subjects with metabolic syndrome may exhibit a group of metabolic disorders or metabolic abnormalities, which may include 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.

[0230] Renal dysfunction Renal pathologies, such as glomerular disease, occur in elderly individuals and can be treated by administering the senolytic compounds described herein. Glomerulonephritis is characterized by renal inflammation and the expression of two proteins, IL1α and IL1β (see, e.g., Niemir et al., Kidney Int. 52 (1997) 393-403). IL1α and IL1β are considered key regulators of SASP (see, e.g., Coppe et al., PLoS. Biol. 6 (2008) 2853-2868). Glomerular disease is associated with an increased presence of senescent cells, particularly in fibrotic kidneys (see, e.g., Sis et al., Kidney Int. 71 (2007) 218-226).

[0231] Skin diseases or disorders Senescence-related diseases or disorders treatable by administering the senolytic agents described herein include skin diseases or disorders. Such senescent cell-associated diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and are described in more detail above. Other senescence-related skin diseases and disorders include wrinkles (age wrinkles); pruritus (associated with diabetes and aging); paresthesia (a chemotherapy side effect associated with diabetes and multiple sclerosis); psoriasis (as indicated) and other papulosquamous disorders such as erythroderma, lichen planus, and lichenoid skin diseases; atopic dermatitis (a form of eczema associated with inflammation); and eczematous rash (often observed in elderly patients and associated with the side effects of certain medications). Other aging-related skin diseases and disorders include eosinophilic skin disease (associated with certain types of blood cancer); reactive neutrophilic dermatosis (associated with underlying conditions such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies form against desmoglein); pemphigoid and other immune bullous skin diseases (autoimmune blistering of the skin); aging-associated fibrous histiocytic proliferation of the skin; and cutaneous lymphoma, which is more common in older populations. Another skin disease treatable according to the methods described herein is cutaneous lupus, a symptom of lupus erythematosus. Late-onset lupus may be associated with decreased (i.e., impaired) function of T cells and B cells and aging-related cytokines (immunosenescence).

[0232] Inflammatory and autoimmune diseases and disorders In certain embodiments, the senescence-associated disease or disorder is an inflammatory disease or disorder, such as, by way of non-limiting example, osteoarthritis, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) according to the methods described herein, including the administration of a senolytic agent. Other inflammatory or autoimmune diseases or disorders that can 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 lung diseases COPD and idiopathic pulmonary fibrosis.

[0233] Osteoarthritis, a degenerative joint disease, is characterized by cartilage fibrillation at sites of high mechanical stress, osteosclerosis, and thickening of the synovium and articular cavity. Fibrillation is a localized surface breakdown associated with splitting of the superficial cartilage layer. Initial splitting occurs along the axis of the major collagen bundles toward the cartilage. Collagen within the cartilage breaks down, and proteoglycans are lost from the cartilage surface. Without the protective and lubricating effects of proteoglycans in the joint, collagen fibers become susceptible to degradation, leading to subsequent mechanical breakdown. Risk factors that predispose to the development of osteoarthritis include aging, obesity, previous joint injury, joint overuse, weak thigh muscles, and genetic predisposition. Symptoms of osteoarthritis include pain or stiffness in the joints, especially the hips, knees, and lower back, after inactivity or overuse; stiffness after rest that resolves after movement; and pain that worsens after activity or toward the end of the day. Osteoarthritis can also affect the neck, little knuckles, base of the thumb, ankle, and big toe. Chronic inflammation is thought to be the main age-related factor contributing to osteoarthritis. In combination with aging, joint overuse and obesity appear to promote osteoarthritis.

[0234] By selectively killing senescent cells, senolytic agents prevent (i.e., reduce the likelihood of onset), reduce, or inhibit the loss or erosion of the proteoglycan layer in joints, reduce inflammation in affected joints, and promote (i.e., stimulate, enhance, induce) collagen (e.g., type II collagen) production. Removal of senescent cells causes a decrease in the amount (i.e., level) of inflammatory cytokines, such as IL-6, produced in joints, thereby reducing inflammation. Provided herein are methods for selectively killing senescent cells in osteoarthritic joints of a subject and / or inducing collagen (such as type II collagen) production in the joints of a subject to treat osteoarthritis by administering to the subject at least one senolytic agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition). The senolytic agent may also be used to reduce (inhibit, alleviate) the production of metalloproteinase 13 (MMP-13), which degrades collagen in joints, and to restore the proteoglycan layer or inhibit its loss and / or degradation. Treatment with the senolytic agent also thereby prevents (i.e., reduces the likelihood of its occurrence), inhibits, or reduces bone erosion, or slows (i.e., reduces 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, transdermal, intradermal, or subcutaneous delivery). Treatment with the senolytic agent can also restore, improve, or inhibit the deterioration of joint strength. Furthermore, methods comprising administering the senolytic agent can alleviate joint pain and are therefore useful for pain management in osteoarthritic joints.

[0235] The effectiveness of one or more senolytic agents for treating or preventing osteoarthritis in a subject and monitoring a subject receiving one or more senolytic agents can be easily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods can be used to monitor a subject's health status, including physical examination (e.g., measuring the flexibility, swelling, or redness of the affected joint), assessing and monitoring clinical symptoms (e.g., pain, stiffness, mobility), and performing analytical tests and methods described herein and practiced in the art (e.g., measuring levels of inflammatory cytokines or chemokines; X-ray imaging to measure cartilage loss, as indicated by narrowing of the spaces between bones in joints; magnetic resonance imaging (MRI), which provides detailed images of bones and soft tissues, including cartilage). The effectiveness of treatment with one or more senolytic agents can be analyzed by comparing the symptoms of patients suffering from or at risk of an inflammatory disease or disorder, such as osteoarthritis, who received the treatment with those of patients who did not receive such treatment or who received a placebo treatment.

[0236] In certain embodiments, senolytic agents can be used to treat and / or prevent (i.e., reduce or decrease the likelihood of developing) 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 disease that typically affects the small joints of the hands and feet. While osteoarthritis may be caused, at least in part, by wear and tear on joints, rheumatoid arthritis affects the lining of joints, resulting in painful swelling that can lead to bone erosion and joint deformity. RA can also occasionally affect other organs of the body, such as the skin, eyes, lungs, and blood vessels. While RA can occur in subjects of any age; RA usually develops after the age of 40. This disorder is significantly more prevalent in women. Certain embodiments of the methods described herein exclude RA.

[0237] Chronic inflammation can also contribute to other age-related or aging-related diseases and disorders, such as kyphosis and osteoporosis. Kyphosis is a severe curvature of the spine that is common in normal and premature aging (see, e.g., Katzman et al., J. Orthop. Sports Phys. Ther. 40 (2010) 352-360). Age-related kyphosis often occurs after osteoporosis weakens the vertebrae to the point of cracking and compression. Some types of kyphosis affect young children or teenagers. Severe kyphosis can affect the lungs, nerves, and other tissues and organs, causing pain and other problems. Kyphosis is associated with cellular senescence. Characterization of the ability of senolytic agents to treat kyphosis can be determined in preclinical animal models used in the art. For example, TTD mice develop kyphosis (see, e.g., de Boer et al., Science 296 (2002) 1276-1279); other mice that can be used are BubR1 H / H These include 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 senescent cell-associated markers, such as SA-β-Gal staining.

[0238] Osteoporosis is a progressive bone disease characterized by a decrease in bone mass and density, which can lead to an increased risk of fracture, and can be treated or prevented by administration of the senolytic agents described herein. Bone mineral density (BMD) is reduced, bone microarchitecture deteriorates, and the amount and type of protein in bone is altered. Osteoporosis is typically diagnosed and monitored by bone mineral density testing. Postmenopausal women or women with reduced estrogen are at greatest risk. Both men and women over 75 years of age are at risk, although 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 senescent cell-associated markers, such as SA-β-Gal staining.

[0239] In yet other embodiments, inflammatory / autoimmune diseases that can be treated or prevented (i.e., the likelihood of developing 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 part of the digestive tract. In addition to life-threatening complications resulting from IBD, the disease can be painful and debilitating. Ulcerative colitis is an inflammatory bowel disease that causes long-term inflammation in parts of the digestive tract. Symptoms usually progress over time rather than suddenly. Ulcerative colitis usually affects only the innermost parts of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that causes inflammation anywhere along the membrane of the digestive tract, often extending deep into the affected tissue. This can lead to abdominal pain, severe diarrhea, and malnutrition. The inflammation caused by Crohn's disease can involve various areas of the digestive tract. Diagnosis and monitoring of this disease is performed according to methods and diagnostic tests routinely practiced in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small bowel imaging.

[0240] Other inflammatory or autoimmune diseases that can 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, oral mucositis caused by radiation). Specific fibrosis or fibrotic diseases of organs, such as renal fibrosis, liver fibrosis, pancreatic fibrosis, myocardial fibrosis, skin wound healing, and oral submucous fibrosis, can be treated with the senolytic agents described herein.

[0241] In certain embodiments, the senescent cell-associated disorder is, by way of non-limiting example, an inflammatory skin disease, such as psoriasis or eczema, which can be treated or prevented (i.e., the likelihood of occurrence is reduced) according to the methods described herein, including the administration of a senolytic agent. 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, red plaques, papules, and patches of skin that may be painful and itchy. In psoriasis, cutaneous and systemic overexpression of various inflammatory cytokines, such as IL-6, a major component of the SASP, is observed. Eczema is a skin inflammation characterized by redness, swelling, itching, dryness, crusting, peeling, blistering, cracking, weeping, or bleeding. The effectiveness of senolytic agents for the treatment of psoriasis and eczema and the monitoring of subjects receiving such senolytic agents can be readily determined by those skilled in the medical or clinical arts. One or any combination of diagnostic methods, including physical examination (such as skin appearance), evaluation of monitoring clinical symptoms (such as itching, swelling, and pain), and performance of analytical tests and methods described herein and practiced in the art (i.e., determining the level of inflammatory cytokines), can be used. Other immune disorders or diseases that can be treated or prevented (i.e., reduced likelihood of occurrence) with the senolytic agents described herein include conditions resulting from the host immune response to organ transplants (e.g., kidney, bone marrow, liver, lung, or heart transplants), such as rejection of transplanted organs. The senolytic agents described herein can also be used to treat or reduce the likelihood of occurrence of graft-versus-host disease.

[0242] Cardiovascular Diseases and Disorders In another embodiment, the aging-related disease or disorder treated by the methods described herein is a cardiovascular disease, which can be any one or more of angina pectoris, 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, myocardial fibrosis, cardiac diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral arterial disease (PAD)), cardiac stress tolerance, and stroke.

[0243] In certain embodiments, methods are provided for treating aging-related cardiovascular diseases associated with or caused by arteriosclerosis (i.e., hardening of the arteries). Cardiovascular diseases can 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)). Methods for treating cardiovascular diseases associated with or caused by arteriosclerosis can reduce the likelihood of developing high blood pressure / hypertension, angina pectoris, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In certain embodiments, methods are provided for stabilizing atherosclerotic plaques in a subject's blood vessels (e.g., arteries), thereby reducing the likelihood of developing or delaying the development of thrombotic events such as stroke or myocardial infarction. In certain embodiments, these methods involving administration of a senolytic agent reduce (i.e., cause a decrease in) the lipid content of atherosclerotic plaques in a subject's blood vessel (e.g., artery) and / or increase (i.e., cause an increase in, enhance or promote the thickening of) the fibrous cap.

[0244] Atherosclerosis is characterized by patchy intimal plaques (atheromas) that invade the lumen of medium-sized and large arteries; plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect medium-sized and large arteries, including the coronary, carotid, and cerebral arteries, the aorta and its branches, and the major arteries of the limbs. In certain embodiments, methods are provided for inhibiting atherosclerotic plaque formation (or reducing, decreasing, or causing a decrease in atherosclerotic plaque formation) by administering a senolytic agent. In other embodiments, methods are provided for decreasing (lowering, reducing) the amount (i.e., level) of plaque. A decrease in the amount of plaque in a blood vessel (e.g., an artery) can be determined, for example, by a decrease in plaque surface area or by a decrease in the extent or degree (e.g., percent) of blockage of the blood vessel (e.g., an artery), which can be determined by angiography or other visualization methods used in the cardiovascular field. Also provided herein are methods for increasing the stability (or improving, promoting, or enhancing the stability) of atherosclerotic plaques present in one or more blood vessels (e.g., one or more arteries) in a subject, the methods comprising administering to the subject any one of the senolytic agents described herein.

[0245] Subjects suffering from cardiovascular disease can be identified using standard diagnostic methods known in the field of cardiovascular disease. Generally, the diagnosis of atherosclerosis and other cardiovascular diseases is based on the patient's symptoms (e.g., chest pain or tightness (angina pectoris), 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 can 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 smoking, a sedentary lifestyle, and hypertension. In certain embodiments, the cardiovascular disease that is a disease / disorder associated with senescent cells is atherosclerosis.

[0246] The effectiveness of one or more senolytic agents for treating or preventing (i.e., reducing or decreasing the likelihood of occurrence or development of) cardiovascular disease (e.g., atherosclerosis) can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, evaluation and monitoring of clinical symptoms, and performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiography, stress tests, non-stress tests), can be used to monitor a subject's health status. The therapeutic effect of 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 treated patients suffering from or at risk of cardiovascular disease with the symptoms of patients who have not received such treatment or who have received a placebo treatment.

[0247] 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 with or sequentially with another therapeutic agent, including those known to treat, prevent, or ameliorate one or more of the symptoms or disorders described herein.

[0248] For example, the senolytic agent may be administered in combination with or following the administration of a chemotherapeutic agent. In one embodiment, a tumor is treated with a chemotherapeutic agent that induces a state of senescence in tumor cells, and the co-administered senolytic agent kills the senescent tumor cells. Examples of chemotherapeutic agents useful for treating tumors in combination with senolytic compounds include topoisomerase inhibitors such as doxorubicin, CDK4 / 6 inhibitors such as parbocyclib, and PARP inhibitors such as olaparib (see, e.g., Fleury et al., Nature Communications, 10 (2019) 2556).

[0249] 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 before or after one or more additional active ingredients.

[0250] Pharmaceutical compositions and methods of administration Also provided herein are pharmaceutical compositions comprising a senolytic agent described herein and at least one pharmaceutically acceptable excipient, also referred to as a pharmaceutically suitable excipient 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 excipients described herein are examples and are in no way limiting. An effective amount or therapeutically effective amount refers to the amount of one or more senolytic agents administered to a subject, either in a single dose or as part of a series, that is effective to produce the desired therapeutic effect.

[0251] 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 formulation containing each of the separate pharmaceutical compositions (which may be conveniently referred to as, for example, a first pharmaceutical composition and a second pharmaceutical composition containing the first and second senolytic agents, respectively) may be prepared. Each of the pharmaceutical compositions in the preparation may be administered simultaneously (i.e., concurrently) and via the same administration route, or may be administered at different times by the same or different administration routes. Alternatively, two or more senolytic agents may be formulated together in a single pharmaceutical composition.

[0252] The pharmacokinetics of a senolytic agent (or one or more metabolites thereof) administered to a subject can 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 tissue from the subject. Any method practiced in the art and described herein for detecting agents can be used to measure the level of the senolytic agent during the course of treatment.

[0253] The dose of the senolytic agent described herein for treating a senescent cell-associated disease or disorder may depend on the subject's condition, i.e., the stage of the disease, the severity of symptoms caused by the disease, overall health, as well as the age, sex, and weight of the subject, as well as other factors apparent to those skilled in the medical art. Pharmaceutical compositions may be administered in a manner appropriate to the disease being treated, as determined by those skilled in the medical art. In addition to factors related to the use of the senolytic agent described herein for treating a senescence-associated disease or disorder, the appropriate duration and frequency of administration of the senolytic agent may also be determined or adjusted depending on factors such as the patient's condition, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. The optimal dose of the agent may generally be determined using experimental models and / or clinical trials. The optimal dose may depend on the subject's size, weight, or blood volume. The use of the minimum amount sufficient to provide effective treatment is generally preferred. Designing and conducting preclinical and clinical trials for the senolytic agents described herein (including when administered for prophylactic effects) is well within the skill of those skilled in the relevant art. When two or more senolytic agents are administered to treat a senescence-related disease or disorder, the optimal dose of each senolytic agent may be different, such as being lower than when either agent is administered alone as a single drug treatment. In certain embodiments, the combination of two senolytic agents acts synergistically or additively, allowing either agent to be used in a lower amount than when administered alone. The amount of the senolytic agent that can be administered per day can 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, or about 50 to 100 mg / kg of body weight). In other embodiments, the amount of the senolytic agent that can 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 dose (per day or per course of treatment) can vary depending on the aging-related disease or disorder being treated, and can also vary depending on the route of administration and treatment regimen.

[0254] Pharmaceutical compositions containing senolytic agents can be formulated in a manner appropriate for the delivery method using techniques routinely practiced in the art. The compositions can be in the form of a solid (e.g., tablet, capsule), semisolid (e.g., gel), liquid, or gas (aerosol). In certain other embodiments, the senolytic agent (or pharmaceutical composition containing it) is administered as a bolus injection. In certain embodiments, when the senolytic agent is delivered by injection, the senolytic agent is delivered via blood vessels to the organ or tissue containing the senescent cells to be killed, according to techniques routinely practiced by those skilled in the medical arts.

[0255] Pharmaceutically acceptable excipients are well known in the pharmaceutical arts 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 st Ed. Mack Pub. Co., Easton, Pa. (2005). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate-buffered saline at physiological pH. Preservatives, stabilizers, dyes, buffers, etc. may be provided in the pharmaceutical composition. Additionally, antioxidants and suspending agents may also be used. Generally, the type of excipient is selected based on the method 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 formulated as a lyophilized product using one or more appropriate excipient solutions to solubilize and / or dilute the agent of the composition after administration. In other embodiments, the agent may be encapsulated in liposomes using techniques known and practiced in the art. The pharmaceutical compositions may be formulated for any suitable method of administration described herein and in the art.

[0256] The pharmaceutical composition can be delivered to a subject in need thereof by any one of several routes known to those skilled in the art. By way of non-limiting example, the composition can be delivered orally, intravenously, intraperitoneally, by injection (e.g., bolus injection), subcutaneously, intestinal, rectal, intranasal, inhalation, buccal, sublingual, intramuscular, transdermal, intradermal, topical, intraocular, vaginal, rectal, or intracranial injection, or any combination thereof. In certain embodiments, administration of the dose as described above is via intravenous, intraperitoneal, direct to the target tissue or organ, or subcutaneous route. In certain embodiments, the delivery method includes a stent coated or impregnated with a drug that is a senolytic agent. Suitable formulations for such delivery methods are described in further detail herein.

[0257] In certain embodiments, the senolytic agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) is administered directly to a target tissue or organ containing senescent cells that contribute to the development 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 via a topical, transdermal, intradermal, or subcutaneous route. In other certain embodiments, provided herein are methods for treating cardiovascular diseases or disorders associated with arteriosclerosis, such as atherosclerosis, by administering directly to an artery. In other embodiments, the senolytic agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) for treating a senescence-associated lung disease or disorder may be administered by inhalation, intranasal administration, intubation, or intratracheal administration, for example, to provide the senolytic agent more directly to the affected lung tissue. As another non-limiting example, the senolytic agent (or pharmaceutical composition comprising the senolytic agent) may be delivered directly to the eye by injection (e.g., intraocular or intravitreal) or by application of a cream, ointment, gel, or eye drops to the conjunctiva under the eyelid. In more specific embodiments, the senolytic agent or 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.

[0258] Pharmaceutical compositions (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery, or other methods) can be in liquid form. Liquid pharmaceutical compositions can contain, for example, one or more of the following: a sterile diluent, such as water, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other solvents that can serve as a solvent or suspending medium; antibacterial agents; antioxidants; chelating agents; buffers and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral compositions can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. The use of saline is preferred, and pharmaceutical compositions for injection are preferably sterilized. In other embodiments, for the treatment of ophthalmic diseases or disorders, liquid pharmaceutical compositions can be applied to the eye in the form of eye drops. Liquid pharmaceutical compositions can be delivered orally.

[0259] For oral formulations, at least one of the senolytic agents described herein may be used alone or in combination with suitable additives for producing tablets, powders, granules, or capsules, as needed, including diluents, buffers, wetting agents, preservatives, coloring agents, and flavoring agents. The compound may be formulated with a buffer to protect the compound from the low pH of the gastric environment and / or to provide an enteric coating. The senolytic agent included in the pharmaceutical composition may be formulated for oral delivery, for example, with a flavoring agent and / or with an enteric coating in a liquid, solid, or semisolid formulation.

[0260] Pharmaceutical compositions comprising any one of the senolytic agents described herein may be formulated for sustained or delayed release (also referred to as extended 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 a 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. Excipients for use in such formulations may be biocompatible and biodegradable; preferably, the formulation provides a relatively constant level of release of the active ingredient. The amount of active agent contained within a sustained-release formulation depends on the site of implantation, the rate and expected duration of release, and the nature of the condition, disease, or disorder being treated or prevented.

[0261] In certain embodiments, pharmaceutical compositions containing senolytic agents are formulated for transdermal, intradermal, or topical administration. The compositions may be administered as powders / talc or other solids, liquids, sprays, aerosols, ointments, foams, creams, gels, or pastes using syringes, bandages, transdermal patches, inserts, or syringe-like applicators. This is preferably in the form of a controlled-release or sustained-release formulation administered topically or injected directly into the skin (intradermally or subcutaneously) adjacent to or within the area to be treated. The active composition may also be delivered by iontophoresis. Preservatives may be used to prevent the growth of fungi and other microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

[0262] Pharmaceutical compositions containing senolytic agents can be formulated as emulsions for topical application. Emulsions contain one liquid distributed throughout the body of a second liquid. The emulsions can be oil-in-water or water-in-oil emulsions. Either or both of the oil and aqueous phases can contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. The oil phase can contain other oily pharmaceutically acceptable excipients. Suitable surfactants include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. Compositions for topical application can also contain at least one suitable suspending agent, antioxidant, chelating agent, emollient, or humectant.

[0263] Ointments and creams can be formulated with an aqueous or oily base, for example, to which suitable thickening and / or gelling agents are added.Lotions can be formulated with an aqueous or oily base, and generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners, or colorants.Liquid sprays can be delivered from pressurized packs, for example, through specially shaped closures.Oil-in-water emulsions can also be used in compositions, patches, bandages, and articles.These systems are semisolid emulsions, microemulsions, or foam emulsion systems.

[0264] Controlled-release or sustained-release transdermal or topical formulations can be achieved by adding sustained-release additives, such as polymeric structures and matrices, available in the art. For example, the composition can be administered by the use of a hot-melt extrudate, such as a bioadhesive hot-melt extruded film. The formulation can include a cross-linked polycarboxylic acid polymer formulation. The cross-linking agent can be present in an amount that provides sufficient adhesiveness to keep the system attached to the target epithelial or endothelial cell surface for a sufficient period of time to allow the desired release of the compound.

[0265] The insert, transdermal patch, bandage, or article may include a polymer blend or coating that provides a constant rate of release of the active agent over an extended period of time. In certain embodiments, the article, transdermal patch, or insert includes a water-soluble pore-generating agent, such as polyethylene glycol (PEG), that may be blended with a water-insoluble polymer to increase the durability of the insert and prolong the release of the active ingredient.

[0266] Polymer formulations can also be utilized to provide controlled or sustained release. Bioadhesive polymers described in the art can be used. For example, sustained-release gels and compounds can be incorporated into polymer matrices, such as hydrophobic polymer matrices. Examples of polymer matrices include microparticles. Microparticles can be microspheres, where the core can be composed of a different material than the polymer shell. Alternatively, the polymer can be cast as a thin slab or film, a powder produced by milling or other standard techniques, or a gel, such as 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.

[0267] Kits containing one or more unit doses of the agents described herein are provided, typically in oral or injectable dosage forms. Such kits may include a container containing the unit dose, an informational package insert describing the use and associated benefits of the agent in treating senescence-related diseases, and, optionally, an implement or device for delivery of the composition.

[0268] All references and patents are incorporated herein in their entirety for all purposes. [Example]

[0269] Example 1: Preparation of (E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (29) [ka] 2-(2-Methyl-1H-indol-3-yl)ethan-1-amine (28) (400 mg, 2.2 mmol) was dissolved in a mixture of THF:dichloroethane:methanol (5:5:0.5). To this solution was added (E)-3-(4-formylphenyl)acrylic acid (27) (367 mg, 2.09 mmol, 0.95 equiv.), triacetoxyborohydride (2320 mg, 11 mmol, 5.0 equiv.), and three drops of acetic acid. The mixture was stirred overnight, and the volatiles were removed under reduced pressure. LC / MS indicated the presence of the desired product. Upon addition of water, a solid precipitated from the solution. The pH was adjusted to 7 with dilute NaHCO3, and the aqueous solution was washed with ethyl acetate. The white solid was filtered and washed with water, ether, and hexane to give (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid as a pale yellow solid. (620 mg, 81% yield). LC / MS: RT=2.41 min; m / z=335.4 [M+H] + .

[0270] The previous product, (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (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 warmed to room temperature. Analysis by LC / MS showed the desired product. Dilute HCl was added until the pH was approximately 2. The aqueous solution was extracted twice with ethyl acetate. 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 to 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 (29) as an off-white solid. (240 mg, 56% yield). LC / MS: RT=3.91 min; m / z=557.6 [M+H] + .

[0271] Example 2: Preparation 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 (110) [ka] tert-Butyl (piperidin-4-ylmethyl)carbamate (1400 mg, 6.5 mmol, 1.0 equiv.) and methyl 2-chloropyrimidine-5-carboxylate (1180 mg, 6.8 mmol, 1.05 equiv.) in dioxane (28.0 mL, 0.23 M) were treated with cesium carbonate (5.27 g, 16.2 mmol, 2.5 equiv.) and Pd(dba)2 acetone (440 mg, 0.48 mmol, 0.075 equiv.) was added. The solution was purged with nitrogen (three times). Xantphos (558 mg, 0.96 mmol, 0.15 equiv.) was then added in one portion. The suspension changed from deep red to yellow-green within minutes. It was then heated at 70 °C for 30 min, at which point LC / MS 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 (20 mL x 3). The solvent was concentrated to dryness, 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-carboxylic acid methyl ester as an off-white solid (1650 mg). LC / MS: RT = 3.23 min; m / z = 351.6 [M+H]. + .

[0272] 2-[4-(tert-Butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-5-carboxylic acid methyl ester (1.65 g, 4.7 mmol) was dissolved in THF (10 mL). 4N HCl / dioxane (9.4 mL, 37.6 mmol, 8.0 equiv.) was added and the solution was heated at 60° C. for 2 hours, during which time a solid precipitated. The hydrochloride precipitate was filtered, washed with ether / hexane (3 times), and dried to give 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester hydrochloride as a white solid (1.08 g). LC / MS: RT=1.88 min; m / z=251.4 [M+H] + .

[0273] To 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester (1080 mg, 3.77 mmol, 1.0 equiv.) and triethylamine (1.5 mL, 10.5 mmol, 2.5 equiv.) in 18 mL of 5% THF:DCE (1:1) methanol was added 1-methyl-1H-indole-3-carbaldehyde (600 mg, 3.77 mmol, 0.95 equiv.) in one portion. Sodium triacetoxyborohydride (6300 mg, 30.6 mmol, 8.0 equiv.) and 4 drops of acetic acid were added. NMP (1.8 mL) was added, and the mixture was stirred at room temperature for 2 days. LC / MS analysis indicated 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 and ethyl acetate. 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 as a white solid (1300 mg). This material was used in the next step without further purification. LC / MS: RT=2.67 min; m / z=394.5 [M+H] + .

[0274] Crude methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (1300 mg, 3.3 mmol, 1.0 equiv) and sodium hydroxide (1058 mg, 26.4 mmol, 8.0 equiv) were suspended in dioxane:water (3:1) (10.0 mL). The solution was heated at 70° C. for 2 h. LC / MS analysis indicated complete reaction. The solvent was concentrated to remove excess alcohol, and the mixture was acidified to pH ∼5 and washed with water followed by hexanes. The gray 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 (220 mg). LC / MS: RT=2.41 min; m / z=380.6[M+H] + .

[0275] 2-(4-((((1-Methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (200 mg, 0.52 mmol, 1.0 equiv) and sodium bicarbonate (165 mg, 1.9 mmol, 3.8 equiv) were suspended in dioxane:water (3:1) (1.4 mL). Fmoc chloride (136 mg, 0.5 mmol, 1.0 equiv) was added portionwise until the solution became clear. LC / MS analysis showed the desired product. The pH of the solution was adjusted to 2, and ethyl acetate was added. The mixture was extracted with water (3 times) and washed with brine. The combined organic layers were dried over sodium sulfate and concentrated to dryness to give a white foam, which was triturated with dichloromethane-methanol-hexane (1:5, 3 times). Thorough drying afforded the title compound 2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)-methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (110) as a white foam (200 mg). LC / MS: RT=3.96 min; m / z=602.3 [M+H] + .

[0276] Example 3: Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) [ka] (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (23) (6600 mg, 16.05 mmol, 1.0 equiv.) was dissolved in dichloromethane (80 mL). To this solution was added 2-hydroxyisoindoline-1,3-dione (2600 mg, 16.05 mmol, 1.0 equiv.). Tetrabutylammonium hydrogen sulfate (1090 mg, 3.21 mmol, 0.2 equiv.) in 1 M sodium carbonate (32.0 mL, 32 mmol, 2.0 equiv.) was added slowly at ice bath temperature. The mixture was stirred overnight at room temperature. TLC analysis by PMA staining showed a new spot with a lower rf compared to the starting material. Water was added, and the mixture was extracted with dichloromethane (3×). The combined organic layers were washed with water and brine, then dried over sodium sulfate and concentrated to give a red solid. The crude product was subjected to normal phase purification eluting with 20-70% ethyl acetate:hexanes. The product fractions were collected, combined, and concentrated to give pure (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((1,3-dioxoisoindolin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white foam (2000 mg, 55%). LC / MS: RT=2.96 min; m / z=494.4 [M+H]. + .

[0277] (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((1,3-dioxoisoindolin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2000 mg, 4.05 mmol, 1.0 equiv) was dissolved in methanol (35 mL, 0.1 M). Hydrazine hydrate (0.20 mL, 4.25 mmol, 1.05 equiv) was added slowly. After 5 min, LC / MS analysis showed the desired product. Dichloromethane (125 mL) was added, and the solution was washed with saturated NaHCO3 (3 times). The combined organic layers were washed with water and brine, dried over sodium sulfate, and concentrated to give a solid (2000 mg). The product was subjected to normal-phase purification, eluting with 30-90% ethyl acetate:hexanes. The product fractions were collected, combined, and concentrated to give the title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) as a white foam (1170 mg, 79% yield). LC / MS: RT=1.98 min; m / z=364.3 [M+H] + .

[0278] Example 4: Preparation of (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (32) [ka] (3S,4R,5R,6S)-2-Hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (5000 mg, 17 mmol) was dissolved in THF (100 mL). DAST (18 mL, 137 mmol, 8 equiv.) was added at −30° C. with cooling in a dry ice-methanol bath. The reaction mixture was warmed to room temperature and stirred at this temperature for 1 h. Another portion of DAST (4 mL) was added at −30° C., warmed to room temperature again, and stirred for 1 h. LC / MS analysis indicated the reaction was complete. Methanol was added at −20° C., and the solvent was evaporated. NaHCO3 was added, and the solution was extracted with dichloromethane (3 times). The combined organic layers were washed with water. Dilute HCl was added to decompose residual DAST, and the solution was extracted with ethyl acetate (2 times). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated to give the crude product as an oil (4000 mg). This brown oil was subjected to normal-phase purification eluting with hexane-ethyl acetate (0-40%). The product fractions were collected, combined, and concentrated to give (3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (30) as a white foam solid (2000 mg). LC / MS: RT = 2.45 min; m / z = 310 [M + HO]. + .

[0279] (3S,4R,5R,6S)-2-Fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (30) (1800 mg, 6.16 mmol, 1.0 equiv.) was dissolved in acetonitrile (30 mL). N-Hydroxyphthalimide (1100 mg, 6.78 mmol, 1.1 equiv.) was added, followed by TEA (1.15 mL, 6.78 mmol, 1.05 equiv.). BF:EtO (1.1 mL, 6.78 mmol, 1.05 equiv.) was added dropwise, and the reaction was stirred for 1 h, at which point LC / MS analysis indicated the presence of the desired product. The mixture was poured into 10% NaHCO / EtOAc. The layers were shaken, separated, and the organic layer was washed with sodium bicarbonate (twice), water, and brine. The organic layer was dried over sodium sulfate and concentrated to give a dark oil. The crude product was subjected to normal-phase purification eluting with hexane:ethyl acetate (0-50%). Product fractions were collected and combined (less polar product—fractions 55-64, 1.0 g; more polar product—fractions 91-105, 500 mg). The less polar product is the desired α-anomer of (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (31). The more polar product is the undesired β-anomer. LC / MS: RT = 3.02 min and 3.28 min; m / z = 436.5 [M+H]. + .

[0280] (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (31) (350 mg, 0.8 mmol, 1.0 equiv.) was dissolved in methanol (8.0 mL). Hydrazine hydrate (65%, 0.066 mL, 0.8 mmol, 1.0 equiv.) was added slowly at ice bath temperature. LC / MS analysis indicated complete reaction within minutes. The white precipitate was filtered off. The reaction was diluted with dichloromethane and filtered a second time. The filtered solution was washed with NaHCO3 (3 times). The organic layer was washed with brine, dried over sodium sulfate, and concentrated to give the desired (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (32) as a white foam solid (500 mg). LC / MS: RT=2.2 min; m / z=306.6 [M+H]. + .

[0281] Example 5 Preparation of (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)acrylamide (26) [ka] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (114 mg, 0.59 mmol, 1.33 equiv.) and 1-hydroxybenzotriazole (HOBt) (91 mg, 0.59 mmol, 1.33 equiv.) were added to a solution of (£)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (29) (250 mg, 0.449 mmol, 1.0 equiv.) in N,N-dimethylformamide (DMF) (1.4 mL) and stirred at room temperature for 30 min. Next, (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) (244 mg, 0.67 mmol, 1.5 equiv.) and DIPEA (0.078 mL, 0.449 mmol, 1.4 equiv.) were added to the mixture at ice bath temperature. The mixture was stirred at room temperature overnight. LC / MS analysis showed the desired product. The mixture was quenched with cold saturated NH4Cl solution. The white precipitate thus formed was filtered and washed with water (twice). The white solid was redissolved in ethyl acetate and washed with water, NaHCO3, and brine. The combined organic layers were dried over sodium sulfate and concentrated to give pure (2S,3R,4S,5S,6R)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (116) as a pale yellow foamy solid. (320 mg, 79% yield). LC / MS: RT=6.95 min; m / z=902.7 [M+H] + .

[0282] [ka] (2S,3R,4S,5S,6R)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (116) (150 mg, 0.166 mmol, 1.0 equiv) was dissolved in methanol (3.0 mL) and 25% sodium methoxide in methanol (0.1 mL, 0.49 mmol, 3.0 equiv) was added slowly at ice bath temperature. LC / MS showed the desired product after 20 min. The reaction was quenched by the addition of 10% acetic acid. The water was removed and replaced with methanol. Insoluble salts were removed by two filtrations, and the crude product after solvent removal was subjected to HPLC purification for final analysis. The title compound (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)acrylamide (26) was isolated as a white solid. LC / MS: RT=1.28 min; m / z=512.5 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.59-7.45(m, 3H), 7.40(d, J=7.8Hz, 1H), 7.31(d, J=7.9Hz, 2H), 7.25(d, J=8.1H) z, 1H), 7.02(t, J=7.5Hz, 1H), 6.94(t, J=7.2Hz, 1H), 6.50(J=15.8Hz, 1H), 4.56( d, J=8.0Hz, 1H), 3.85(m, 4H), 3.78-3.67(m, 2H), 3.65-3.60(m, 1H), 3.57(dd, J= 9.6, 3.4Hz, 1H), 2.97(dd, J=7.9, 5.8Hz, 2H), 2.90(t, J=7.3Hz, 2H), 2.36(s, 3H).

[0283] Example 6 Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (113) [ka] (2S,3R,4S,5S,6R)-2-(((E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (116) (745 mg, 0.825 mmol, 1.0 equiv) was dissolved in dichloromethane (5 mL). Piperidine (10% in DCM) (7.0 mL, 8.2 mmol, 10.0 equiv) was added and the mixture was stirred at room temperature for 3 h. LC / MS analysis showed the reaction was complete. EtOAc (200 mL) was added and the solution was washed with NaHCO3 (twice). The organic layer was washed with brine, dried over sodium sulfate, and concentrated to dryness to give the crude product. The title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (113) was isolated as its white hydrochloride salt after HPLC purification using HCl-containing buffer (550 mg, 80% yield). LC / MS: RT=4.38 min; m / z=680.7 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 7.61-7.48(m, 3H), 7.40(dt, J=7.8Hz, 1.0Hz, 1H), 7.33(d, J=7.9Hz, 2H), 7.25(d, J=8.0Hz, 1 H), 7.02(t, J=7.5Hz, 1H), 6.95(t, J=7.4Hz, 1H), 6.48(d, J=16.1Hz, 1H), 5.43(d, J=3.3Hz, 1 H), 5.32-5.19(m, 2H), 5.02(d, J=8.0Hz, 1H), 4.27-4.13(m, 3H), 3.89(s, 2H), 3.11(t, J=5.7 Hz, 1H), 3.02-2.87(m, 4H), 2.37(s, 3H), 2.15(s, 3H), 2.13(s, 3H), 2.03(s, 3H), 1.98(s, 3H).

[0284] Example 7: Preparation of (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)acrylamide (35) [ka] EDC (430 mg, 2.2 mmol, 1.4 equiv) and HOBt (344 mg, 2.2 mmol, 1.4 equiv) were added to 29 (900 mg, 1.6 mmol, 1.0 equiv) in DMF (8.0 mL). The mixture was stirred for 10 min. Compound 32 (500 mg, 1.63 mmol, 1.05 equiv) was added, followed by DIEA (418 μL, 1.5 equiv) at ice bath temperature. The reaction was stirred overnight at room temperature. LC / MS analysis showed the desired product. Saturated ammonium chloride solution was added (20 mL), and a white solid precipitated. The solid was filtered, washed with water (twice), and dried to give a white solid (1.30 g). The solid was purified by normal phase chromatography eluting with hexane-ethyl acetate (20-75%). The product fractions were collected, combined, and the solvent was concentrated to give (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (117) as an off-white solid (650 mg). LC / MS: RT=3.89 min; m / z=844.5 [M+H] + .

[0285] [ka] (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (117) (150 mg, 0.17 mmol, 1.0 equiv.) and sodium methoxide (25%) (0.085 mL, 2.6 equiv.) in methanol (1.0 mL) were mixed at ice bath temperature. The mixture was stirred at room temperature for 2 hours. 1N HCl (1.0 equiv.) was added, the solution was concentrated to dryness, and the residue was subjected to HPLC purification to isolate the title compound (E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)acrylamide (35) as a white solid (70 mg). LC / MS: RT=1.35 min; m / z=496.5 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.69(d, J=8.5Hz, 3H), 7.53(d, J=8.2Hz, 2H), 7.43(d, J=7.8Hz, 1H), 7.28(d, J=8 .0Hz, 1H)7.08-6.97(m, 2H), 6.59(d, J=15.8Hz, 1H), 4.57(d, J=7.9Hz, 1H), 4.28( s, 2H), 3.74(q, J=6.4Hz, 1H), 3.68-3.61(m, 2H), 3.57(dd, J=9.7, 3.3Hz, 1H), 3. 30-3.22(m, 2H), 3.14(dd, J=9.3, 6.6Hz, 2H), 2.42(s, 3H), 1.33(d, J=6.4Hz, 3H).

[0286] Example 8: Preparation of (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (119) [ka] To compound (117) (640 mg, 0.75 mmol, 1.0 equiv.) in DCM:DMF (1:1) (10 mL) was added 50% triethylamine / DCM (10.0 mL). The mixture was stirred at room temperature overnight. LC / MS analysis indicated the presence of the desired product and starting material. The solvent was concentrated. The residue was taken up in 50% TEA / DCM (5 mL) and DMF (2.0 mL), and the mixture was stirred for 4 h. LC / MS indicated that the starting material had been consumed at this point. The solvent was removed under reduced pressure. The residue was triturated with hexane to remove any remaining 9-methylene-9H-fluorene. Saturated ammonium chloride solution and ethyl acetate were added, and the organic layer was washed with water (three times), followed by brine, and dried over sodium sulfate. The solvent was removed and the residue was purified by HPLC using a buffer containing HCl to isolate the title compound (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (119) as its hydrochloride salt as a white solid. LC / MS: RT=1.89 min; m / z=622.4 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.73-7.61(m, 3H), 7.54(d, J=8.1Hz, 2H), 7.44(d, J=7.8Hz, 1H), 7.28(d, J=8.0Hz, 1H), 7.0 6(t, J=7.5Hz, 1H), 7.00(t, J=7.4Hz, 1H), 6.55(d, J=15.9Hz, 1H), 5.48-5.31(m, 3H), 5.16( dd, J=11.3, 3.9Hz, 1H), 4.63(s, 1H), 4.28(s, 2H), 3.26(dd, J=9.5, 6.5Hz, 2H), 3.14(dd, J= 9.3, 6.5Hz, 2H), 2.42(s, 3H), 2.18(s, 3H), 2.16(s, 3H), 2.01(s, 3H), 1.20(d, J=6.5Hz, 3H).

[0287] Example 9 Preparation of (2S,3R,4S,5S,6R)-2-((2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (111) [ka] EDC (76 mg, 0.40 mmol, 1.4 equiv.) and HOBt (61 mg, 0.4 mmol, 1.4 equiv.) were added to 2-(4-{[(9H-Fluoren-9-ylmethoxycarbonyl)-(1-methyl-1H-indol-3-ylmethyl)-amino]-methyl}-piperidin-1-yl)-pyrimidine-5-carboxylic acid (110) (170 mg, 0.28 mmol, 1.0 equiv.) in DMF (0.5 mL). The solution was stirred for 10 minutes, after which compound (24) (148 mg, 0.45 mmol, 1.5 equiv.) was added, followed by DIEA (80 μL, 1.5 equiv.) at ice bath temperature. The solution was stirred overnight at room temperature. LC / MS analysis indicated the formation of the desired product. Saturated ammonium chloride solution (2.0 mL) was added, and a white solid precipitated. The precipitate was washed with water (twice). The solid was filtered and dried to give (2S,3R,4S,5S,6R)-2-((2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (111) as a white solid (260 mg), which was used in the next step without further purification. LC / MS: RT=7.07 min; m / z=947.9 [M+H] + .

[0288] Example 10 Preparation of 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (112) [ka] Compound (111) (170 mg, 0.18 mmol) was dissolved in methanol (1.79 mL) and 25% sodium methoxide in methanol (0.040 mL, 1.0 equiv.) was added slowly at ice bath temperature. After stirring at room temperature for 2 hours, the Fmoc protecting group was not completely removed. An additional 0.2 equiv. of sodium methoxide was added at ice bath temperature, and the reaction was complete within 20 minutes. HCl / dioxane (1 N, 1.2 equiv.) was added to adjust the pH to 5-6. The solvent was concentrated to dryness and the residue was subjected to HPLC purification to give the title compound 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (112) (80 mg). LC / MS: RT=1.33 min; m / z=557.3 [M+H] + . 1 H NMR (500 MHz, methanol-d4): δ 8.72 (s, 2H), 7.75 (m, 1H), 7.47 (m, 2H), 7.29 (m, 1H), 7.20 (m, 1H), 4.95-4.91 (m, 2H), 4.60 (d, J = 7.8 Hz, 1H), 4.45 (s 2H), 3.88-3.81(m, 5H), 3.77(dd, J=11.3, 4.6Hz, 1H), 3.71(dd, J=9.6, 8.0Hz, 1H), 3.64(m, 1H) ), 3.57(m, 1H), 3.05-2.96(m, 4H), 2.10(m, 1H), 2.06(s, 2H), 1.92-1.85(m, 2H), 1.26(m, 2H).

[0289] Example 11: Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (114) [ka] Compound 111 (88 mg, 0.092 mmol) was dissolved in dichloromethane (0.1 mL). Piperidine (20% in DCM) (0.47 mmol, 1.1 mmol, 12.0 equiv.) was added. The mixture was stirred at room temperature for 6 hours. LC / MS analysis indicated the reaction was complete. 1 N HCl / dioxane (1.0 equiv.) was added. The solution was concentrated to dryness, and the crude residue was subjected to HPLC purification to give 60 mg of the title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (114). LC / MS: RT=1.89 min; m / z=725.3[M+H] + . 1 H NMR (500MHz, methanol-d4):δ 8.64(s, 2H), 7.69(m, 1H), 7.40(m, 1H), 7.31(s, 1H), 7.23(m, 1H), 7.13(m, 1H), 5.42( d, J=3.5Hz, 1H), 5.34(m, 1H), 5.21(m, 1H), 5.07(d, J=8.3Hz, 1H), 4.81(d, J=13.3Hz, 2 H), 4.26-4.13(m, 5H), 3.82(s, 3H), 2.94(t, J=12.8Hz, 2H), 2.77(d, J=6.8Hz, 2H), 2.1 3(s, 3H), 2.11(s, 3H), 2.02(s, 3H), 1.97(s, 3H), 1.85(d, J=11.6Hz, 2H), 1.19(m, 2H).

[0290] Example 12: Preparation of (2S,3R,4R,5S,6S)-2-methyl-6-((2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (115) [ka] EDC (54 mg, 0.28 mmol) and HOBt (42 mg, 0.28 mmol, 1.05 equiv.) were added to a solution of compound 110 (96 mg, 0.31 mmol, 1.4 equiv.) in DMF (0.5 mL). The solution was stirred at room temperature for 15 minutes, at which point compound 32 (96 mg, 0.37 mmol, 1.4 equiv.) was added in one portion. DIEA (1.5 equiv., 0.073 mL) was added at ice bath temperature, and the mixture was stirred at room temperature for 1 hour. LC / MS analysis indicated the presence of the desired product. Saturated ammonium chloride solution was added to the reaction, and the yellow solid was filtered off and washed with water. Ethyl acetate was added to the solution, which was then washed with brine (twice). The combined organic layers were dried over sodium sulfate and concentrated to give (2S,3R,4R,5S,6S)-2-methyl-6-((2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (115) as a foamy solid (200 mg), which was used directly in the next reaction without purification. LC / MS: RT=6.83 min; m / z=889.9 [M+H] + .

[0291] Example 13 Preparation of 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (120) [ka] Compound 115 (70 mg, 0.079 mmol, 1.0 equiv.) and sodium methoxide (25%) (2.0 equiv.) were dissolved in methanol (0.8 mL) at ice bath temperature. The mixture was stirred at room temperature for 2 hours. 1 N HCl (1.0 equiv.) was added, the solution was concentrated to dryness, and the residue was subjected to HPLC purification to give the title compound 120. LC / MS: RT=3.05 min; m / z=541.3 [M+H] + . 1 H NMR (500 MHz, methanol-d): δ 8.71(s, 2H), 7.74(d, J=8.0Hz, 1H), 7.51-7.43(m, 2H), 7.30(m, 1H), 7.21( m, 1H), 4.93(d, J=3.9Hz, 1H), 4.59(d, J=7.9Hz, 1H), 4.45(s, 2H), 3.87(s, 3H), 3.77-3.71(m, 1H), 3.68-3.61(m, 2H), 3.57(m, 1H), 3.05-2.95(m, 4H) , 2.09(m, 1H), 1.88(d, J=12.4Hz, 2H), 1.33(d, J=6.5Hz, 3H), 1.26(m, 2H).

[0292] Example 14: Preparation of (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl tripropionate (121) [ka] L-Fucose (3000 mg, 18.2 mmol, 1.0 equiv) was stirred in a mixture of pyridine (9.0 mL) and propionic anhydride (27.0 mL). The solution was heated at 80° C. for 2 days, at which point additional propionic anhydride (5.0 mL) was added and stirring continued for another day. The mixture was concentrated to dryness, and ethyl acetate (80 mL) and water (30 mL) were added, and the aqueous layer was extracted two more times. The combined organic layers were washed with 1N dilute HCl, followed by brine. The solution was dried over sodium sulfate, and the solution was concentrated to dryness to give (2R,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(propionate) as a brown oil (10 g). LC / MS: RT=3.3 min; m / z=467.5 [M+79]. + .

[0293] Acetic acid (1.8 mL, 28.8 mmol, 1.4 equiv.) was added to a solution of ethylenediamine (1.78 mL, 24.7 mmol, 1.2 equiv.) in THF (200 mL). A solid precipitated, and (2R,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(propionate) (8 g, 20.66 mmol, 1.0 equiv.) was added in one portion. The reaction mixture was stirred at room temperature overnight, at which point LC / MS analysis indicated primarily unreacted starting material. The reaction was worked up and subjected again to the same reaction conditions, stirring at room temperature overnight. A second lot was repeated on the same scale. LC / MS analysis indicated the reaction was complete. Water was added, and the two layers were separated. Dilute HCl (2%) and ethyl acetate were added, and the organic layer was washed twice with water, followed by brine. The solution was dried over sodium sulfate and the solvent was removed to give (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate as a white sticky solid (5.8 g). LC / MS: RT = 2.6 min; m / z = 350.1 [M+18].

[0294] (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (3.0 g, 9.03 mmol, 1.0 equiv.) was dissolved in THF (150 mL). The solution was purged with nitrogen for 5 minutes and cooled to -50°C. DAST (5.0 mL) was added dropwise via a plastic pipette. The solution was warmed to 0°C. TLC analysis showed mostly starting material. Another portion of DAST (2.0 mL) was added at -50°C (1.5 equiv.), and the temperature was warmed to 0°C. TLC analysis showed a less polar spot and a small amount of starting material. Two additional portions of DAST were added as before (3.0 mL, 3.0 mL, respectively), and the reaction was allowed to stir until the starting material was completely consumed (approximately 8 h total). The solution was cooled to -30°C, methanol (15 mL) was added slowly, and the mixture was allowed to warm to room temperature. Cold NaHCO3 solution was added, and the mixture was diluted with dichloromethane. The organic layer was separated and washed with 1N HCl (twice), followed by brine. The solution was dried over sodium sulfate, and the solvent was removed to give a yellow oil (4.1 g). TLC analysis showed the product was impure. The crude product was subjected to normal-phase purification, eluting with a gradient from 100% hexane to 15% ethyl acetate in hexane over 60 minutes. The product fractions were collected, and the solvent was removed to give pure (2S,3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate as a colorless oil (2.4 g). LC / MS: RT = 3.2 min; m / z = 413.1 [M+79] + .

[0295] (2S,3S,4R,5R,6S)-2-Fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (1500 mg, 4.49 mmol, 1.0 equiv.) was dissolved in acetonitrile (15 mL). N-Hydroxyphthalimide (915 mg, 5.61 mmol, 1.25 equiv.) was added, followed by triethylamine (0.81 mL, 5.61 mmol, 1.25 equiv.). The solution turned deep red. BF3-etherate (1.03 mL, 8.08 mmol, 1.8 equiv.) was added, and the solution turned clear. It was stirred at room temperature for 1 hour. LC / MS showed the desired product. A cold solution of NaHCO3 was added, and it was extracted twice with ethyl acetate. The solution turned deep red. The combined organic layers were washed twice with NaHCO3 until no color was detected. The organic solvent was dried over sodium sulfate. The solvent was concentrated to give a yellow oil (2000 mg). The crude mixture was subjected to normal phase purification using a 25 g silica gel column eluted with a gradient of 100% hexane to 25% ethyl acetate in hexane over 40 minutes. The product fractions were collected as two peaks. Peak #1 (normal phase, less polar) LC / MS (reverse phase): RT = 3.56 minutes; m / z = 478.4 [M+H] + Peak #2 (normal phase, more polar) LC / MS (reverse phase): RT = 3.31 min; m / z = 478.4 [M+H] + Peak #1 is the 1 Comparison of the H NMR spectrum with the triacetoxy analogue (31) characterized in the literature revealed the desired α-isomer, (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate, which was isolated as a white solid (500 mg).

[0296] (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (500 mg, 1.04 mmol, 1.0 equiv) was dissolved in methanol (10 mL). Hydrazine hydrate (65%, 0.090 mL, 1.04 mmol, 1.0 equiv) was added dropwise at ice bath temperature. The mixture was stirred for 30 minutes, at which point the solution changed from clear to cloudy. LC / MS showed the reaction was complete. Dichloromethane (15 mL) was added, and the mixture was extracted twice with cold saturated aqueous NaHCO3. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was concentrated to give (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate as a white solid (280 mg). LC / MS: RT=2.34 min; m / z=348.4 [M+H] + .

[0297] (E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (29) (281 mg, 0.503 mmol, 1.0 equiv.) was dissolved in DMF (1.5 mL). EDC (124 mg, 0.653 mmol, 1.3 equiv.) and 1-hydroxybenzotriazole (100 mg, 0.65 mmol, 1.3 equiv.) were added, and the mixture was stirred for 10 min. (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (226 mg, 0.503 mmol, 1.0 equiv.) was added, followed by DIPEA (1.4 equiv.). After stirring at room temperature for 2 hours, LC / MS showed the desired product. Saturated NH4Cl solution was added, followed by ethyl acetate, and the organic phase was extracted twice with NaHCO3. The combined organic layers were washed with brine, and the solvent was concentrated to give a pale yellow foam (490 mg). The product was purified using normal-phase purification on a 40 g silica gel column eluting with a gradient from 100% hexane to 50% ethyl acetate in hexane over 45 minutes. The product fractions were collected and the solvent was concentrated to give (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)-oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate as a white foamy solid (200 mg). LC / MS: RT=7.48 min; m / z=886.7 [M+H] + .

[0298] (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltripropionate (200 mg, 0.225 mmol, 1.0 equiv) was dissolved in 50% NEt in DCM / DMF. The solution was stirred at room temperature for 3 days to remove the Fmoc protecting group. LC / MS showed the reaction was complete. The solvent was concentrated, saturated MLCl solution was added, and the mixture was then extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate. The crude mixture was triturated twice with hexane. The final product was concentrated to give a white foamy solid (138 mg). The solid was purified by HPLC using a buffer containing HCl to isolate the title compound (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl tripropionate (121) as its hydrochloride salt as a white solid. LC / MS: RT=4.98 min; m / z=664.7 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.70-7.68(m, 3H), 7.53(d, J=7.9Hz, 2H), 7.47-7.40(m, 1H), 7.28(dt, J=8.0, 1.0Hz, 1H), 7.06(ddd, J=8.2, 7.1, 1.2Hz, 1H), 7.03-6.97(m, 1H), 6.55(d, J=15.8Hz, 1H), 5.49-5.43(m, 1H), 5.43-5.39(m, 1H), 5.38-5.31(m, 1H), 5.24-5.17(m, 1H), 4.66(s, 1H), 4.28(s, 2H), 3.29-3.23(m, 2H), 3.17-3.10( m, 2H), 2.55-2.46(m, 3H), 2.42(s, 3H), 2.34-2.20(m, 2H), 1.25-1.14(m, 9H), 1.10(t, J=7.6Hz, 3H).

[0299] Example 15: Preparation of (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (122) [ka] L-Fucose (3 g, 18.4 mmol, 1.0 equiv.) was dissolved in pyridine (13 mL) and chloroform (19.5 mL). Under a nitrogen atmosphere, isobutyryl chloride (13.14 mL, 123.8 mmol, 6.7 equiv.) was added slowly via syringe at ice bath temperature. The reaction mixture was allowed to warm to room temperature and then stirred for 48 hours. The solution was concentrated to dryness under high vacuum and then quenched with 2 M HCl (15 mL). Ethyl acetate (200 mL) was added, and the solution was washed twice with water. The organic layer was washed once more with 2 M HCl (20 mL), followed by saturated sodium bicarbonate solution (30 mL) and then water (30 mL). The organic layer was dried over sodium sulfate solution. The solvent was removed to give (2S,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(2-methylpropanoate) as a pale yellow oil (9.8 g). LC / MS: RT = 3.93 min; m / z = 462.8 [M+18].

[0300] Acetic acid (1.5 mL, 24 mmol, 1.4 equiv.) was added to a solution of ethylenediamine (1.6 mL, 20.5 mmol, 1.2 equiv.) in THF (200 mL). To the suspension of the precipitated solid, (2S,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayltetrakis(2-methylpropanoate) (8 g, 20.66 mmol, 1.0 equiv.) was added in one portion. The mixture was stirred at room temperature overnight. The next day, LC / MS analysis indicated mostly unreacted starting material. The reaction was worked up and subjected again to the same reaction conditions, this time stirring for a total of 5 days, at which point water was added. The layers were separated, and dilute HCl (2%) was added, followed by ethyl acetate (200 mL). After shaking, the layers were separated, and the organic layer was washed twice with water. The organic layer was further washed with brine and dried over sodium sulfate. The solvent was removed to give (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) as a yellow oil. The crude product was dry-loaded onto an 80 g silica gel column and purified using a gradient of 100% hexane to 30% ethyl acetate in hexane over 1 hour. The product fractions were collected and concentrated to give (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) as a colorless oil (2.89 g). LC / MS: RT = 3.19 min; m / z = 392.5 [M+18].

[0301] To a solution of (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (2.4 g, 6.4 mmol, 1.0 equiv.) in dichloromethane (60 mL) was added DAST (3.69 mL, 27.5 mmol, 4.3 equiv.) dropwise in three portions over 30 minutes at 0° C. The mixture was stirred at this temperature for an additional 30 minutes. TLC analysis indicated the absence of starting material. The reaction mixture was cooled to −20° C., and methanol (5.0 mL) was slowly added. The mixture was stirred for an additional 15 minutes, at which point dichloromethane was added and the mixture was poured into a cold solution of sodium bicarbonate. The separated organic layer was washed twice with 1 N HCl, followed by saturated sodium bicarbonate solution, and finally with brine. After drying over sodium sulfate, the solvent was removed to give a yellow oil (2.5 g). The crude product was subjected to normal phase purification using a 25 g silica gel column, eluting with a gradient from 100% hexane to 25% ethyl acetate in hexane. The product fractions were collected and the solvent was removed to give (2RS,3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) as a white foam (2.0 g). LC / MS: RT=3.64, 3.78 min; m / z=377.5 [M+H] + .

[0302] To a solution of (2RS,3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (2000 mg, 5.3 mmol, 1.0 equiv.) and N-hydroxyphthalimide (1100 mg, 6.89 mmol, 1.3 equiv.) in acetonitrile (4.0 mL) was added triethylamine (0.98 mL, 6.8 mmol, 1.3 equiv.). BF3.Et2O (2.1 mL, 15.9 mmol, 3.0 equiv.) was added slowly via syringe. The solution changed from red to a clear, pale yellow. After 30 min, LC / MS analysis indicated the reaction was complete, yielding two isomeric products. Dichloromethane was added, and the solution was poured into a cold solution of sodium bicarbonate. The organic layer was separated and washed twice with aqueous sodium bicarbonate until colorless. The solution was then washed with brine and dried over sodium sulfate. The solution was concentrated to dryness to give 2500 mg of crude (2RS,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate). The product was purified by normal-phase column chromatography (on a 40 g silica gel column) using a gradient of 100% hexane to 25% ethyl acetate in hexane over 40 minutes. The product fractions were collected as two peaks. Peak #1 (normal phase, less polar) LC / MS (reverse phase): RT = 4.04 min; m / z = 520.6 [M+H] + Peak #2 (normal phase, more polar) LC / MS (reverse phase): RT = 3.74 min; m / z = 520.6 [M+H] + .

[0303] Peak #1 is the 1Comparison of the H NMR spectrum with the literature characterized triacetoxy analog (31) revealed the desired α-isomer, (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate). It was isolated as a white solid (1400 mg). Peak #2 was found to be the β-isomer, (2R,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate). It was isolated as a white solid (500 mg).

[0304] (2R,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (900 mg, 2.3 mmol, 1.0 equiv.) was dissolved in methanol (20 mL). Hydrazine hydrate (65%, 2.3 mL, 2.3 mmol, 1.0 equiv.) was added dropwise at ice bath temperature, and the reaction mixture was stirred for 30 minutes. The solution changed from clear to cloudy. LC / MS analysis indicated the reaction was complete. Dichloromethane (25 mL) was added, and the separated organic layer was washed twice with cold saturated NaHCO3 solution. The organic layer was washed with brine and dried over sodium sulfate. The solvent was removed to give (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) as a white solid (750 mg). LC / MS: RT=2.87 min; m / z=390.5 [M+H] +(E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (29) (300 mg, 0.53 mmol, 1.0 equiv.) was dissolved in DMF (1.5 mL). EDC (124 mg, 0.653 mmol, 1.3 equiv.) and 1-hydroxybenzotriazole (100 mg, 0.65 mmol, 1.3 equiv.) were added. The reaction mixture was stirred for 10 minutes, after which time (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (247 mg, 0.53 mmol, 1.0 equiv.) was added, followed by DIPEA (1.4 equiv.). The mixture was stirred at room temperature for 2 hours. LC / MS analysis indicated the formation of the desired product. Ethyl acetate was added, and the mixture was washed twice with saturated NH4Cl solution. The ethyl acetate layer was washed twice with aqueous NaHCO3, and finally with brine. The solvent was removed to give a pale yellow foam (590 mg). The product was purified using normal-phase purification on a 10 g silica gel column eluting with a gradient from 100% hexane to 30% ethyl acetate in hexane over 45 minutes. The product fractions were collected and the solvent removed to give (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) as a white foam (250 mg). LC / MS: RT=8.13 min; m / z=829.1 [M+H] + .

[0305] (2S,3S,4R,5R,6S)-2-(((E)-3-(4-(((((9H-Fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (250 mg, 0.26 mmol, 1.0 equiv) was dissolved in DMF (1.0 mL). Triethylamine (1.0 mL) was added, and it was stirred at room temperature overnight to remove the Fmoc protecting group. LC / MS showed the desired product. The mixture was triturated with hexane, ethyl acetate was added, and the mixture was extracted twice with saturated NH4Cl. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was concentrated to give a yellow foam (300 mg). The residue was triturated with hexane / DCM (10%). The crude product was concentrated and dried to give an orange solid (180 mg). This solid was purified by HPLC using a buffer containing HCl, and the title compound (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropanoate) (122) was isolated as its hydrochloride salt as a white solid. LC / MS: RT=5.67 min; m / z=706.9 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 7.72-7.61(m, 3H), 7.53(d, J=8.2Hz, 2H), 7.43(m, 1H), 7.28(dt, J=8.0, 1.0Hz, 1H), 7.06(ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.00(ddd, J=8.0, 7.1, 1.1Hz, 1H), 6.55(d, J=15.9Hz, 1H), 5.48(dd, J=11.1, 3.3Hz, 1H), 5.40(dd, J=3 .4, 1.4Hz, 1H), 5.33(d, J=3.9Hz, 1H), 5.24(d, 7=10.3Hz, 1H), 4.70(br.s, 1H), 4.28(s, 2H), 3.26(m, 2H), 3.17 -3.10(m, 2H), 2.73(m, 1H), 2.67(s, 2H), 2.47(m, 1H), 2.42(s, 3H), 1.31-1.15(m, 15H), 1.12(d, J=7.0Hz, 6H).

[0306] Example 16: Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (123) [ka] To a solution of methyl 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylate (prepared as described in WO2018085342A1) (1.8 g, 3.5 mmol) in dioxane (60 mL) and water (15 mL), LiOH (340 mg, 14.2 mmol) was added and the resulting mixture was stirred at 50° C. for 16 hours. The reaction mixture was cooled to room temperature, diluted with water (10 mL), and acidified to approximately pH 5 with 2 M aqueous HCl. The reaction mixture was then concentrated under reduced pressure to remove dioxane. The precipitated product was filtered, washed with water (15 mL), and dried under high vacuum to give 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylic acid as an off-white solid (1.5 g, 85%). LC / MS: RT=2.52 min; m / z=494.4 [M+H] + .

[0307] To a suspension of 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylic acid (740 mg, 1.5 mmol) in DMF (4.2 mL) was added EDC.HCl (401 mg, 2.1 mmol), followed by HOBt (321 mg, 2.1 mmol), and the resulting mixture was stirred at room temperature for 5 min. Compound 24 (700 mg, 1.92 mmol) was added in one portion. The reaction mixture was cooled in an ice / water bath, and EtN (0.3 mL, 2.1 mmol) was added. The reaction mixture was gradually warmed to room temperature and stirred for 12 h. Saturated aqueous NHCl (6 mL) was added, and the resulting precipitate was filtered and washed with water (10 mL). The residue was dissolved in EtOAc (20 mL), washed with brine, dried over anhydrous NaSO, filtered, and concentrated to give the crude product (1 g, 80% yield, 90% purity) as a white foamy solid. 350 mg of this crude material was purified on reverse-phase HPLC to give 146 mg of the title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (123). LC / MS: RT=1.92 min; m / z=839.4 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 9.08(d, J=2.6Hz, 1H), 8.79(s, 2H), 8.51(dd, J=8.8, 2.6Hz, 1H), 7.56(s, 1H), 7.04 (d, J=8.7Hz, 1H), 5.45(d, J=3.3Hz, 1H), 5.33(s, 2H), 5.30(dd, J=10.4, 8.1Hz, 1H) , 5.24(dd, J=10.4, 3.4Hz, 1H), 5.06(d, J=8.1Hz, 1H), 4.29-4.16(m, 7H), 4.06(s, 3 H), 3.91(m, 4H), 3.38(s, 3H), 2.18(s, 3H), 2.16(s, 3H), 2.04(s, 3H), 1.98(s, 3H).

[0308] Example 17 Preparation of 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (124) [ka] To a solution of compound (123) (650 mg, 0.77 mmol) in MeOH (7.7 mL) was added dropwise a 25% NaOMe solution in MeOH (0.21 mL, 1 mmol) in an ice / water bath. The reaction mixture was then warmed to room temperature and stirred for 1 hour. The reaction mixture was quenched by the addition of 1 N aqueous HCl to adjust the pH to 7. The solvent was removed under reduced pressure and the residue was purified by reverse-phase HPLC to give the title compound 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (124) (267 mg) as a white powder. LC / MS: RT=1.34 min; m / z=671.6 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 9.08(dd, J=2.7, 0.7Hz, 1H), 8.85(s, 2H), 8.51(dd, J=8.9, 2.6Hz, 1H), 7.58(d, J=1.0H) z, 1H), 7.05(dd, J=8.9, 0.7Hz, 1H), 5.36-5.34(m, 2H), 4.63(d, J=7.9Hz, 1H), 4.27(m, 4H), 4.07(s, 3H), 3.94-3.82(m, 6H), 3.78(dd, J=11.4, 4.6Hz, 1H), 3.71(dd, J=9.7, 7. 9Hz, 1H), 3.65 (ddd, J=7.6, 4.6, 1.1Hz, 1H), 3.58 (dd, J=9.6, 3.4Hz, 1H), 3.38 (s, 3H).

[0309] Example 18: Preparation of (2S,3S,4R,5R,6S)-2-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (125) [ka] To a solution of 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylic acid (700 mg, 1.4 mmol) in DMF (4.6 mL) was added EDC.HCl (377 mg, 1.9 mmol) followed by HOBt (290 mg, 1.9 mmol) in one portion at room temperature. After 20 min, the reaction mixture was cooled in an ice / water bath, and compound 32 (491 mg, 1.6 mmol) and DIPEA (0.34 mL, 1.4 equiv.) were added at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 2 h. A cold saturated aqueous solution of NH4Cl (10 mL) was added, and the precipitated product was filtered and washed with water (5 mL). The residue was dissolved in EtOAc (20 mL), washed with brine, dried over anhydrous NaSO, filtered, and concentrated to give crude product (125) (1.1 g, quantitative, 90% purity) as a white foamy solid. 350 mg of this crude material was purified on reverse-phase HPLC to give 153 mg of the title compound (2S,3S,4R,5R,6S)-2-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)pyrimidine-5-carboxamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (125). LC / MS: RT=1.96 min; m / z=781.5 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 9.08(dd, J=2.6, 0.8Hz, 1H), 8.80(s, 2H), 8.52(dd, J=8.8, 2.6Hz, 1H), 7.51(d, J=0.9Hz, 1H), 7.00(d, J=8.8Hz, 1H), 5.46-5.36(m, 3H), 5.32(s, 2H), 5.17(dd, J=11.1, 4.0Hz, 1H), 4.70(d, J=6.7Hz, 1H), 4.20(m, 4H), 4.05(s, 3H), 3.90(m, 4) H), 3.37(s, 3H), 2.19(s, 3H), 2.17(s, 3H), 2.01(s, 3H), 1.19(d, J=6.5Hz, 3H).

[0310] Example 19 Preparation of 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (126) [ka] To a solution of compound (125) (750 mg, 0.96 mmol) in MeOH (9.6 mL) was added dropwise a 25% NaOMe solution in MeOH (0.22 mL, 1.06 mmol) in an ice / water bath. The reaction mixture was then warmed to room temperature and stirred for 1 hour. The reaction mixture was quenched by the addition of 1 N aqueous HCl to adjust the pH to 7. The solvent was removed under reduced pressure and the residue was purified by reverse-phase HPLC to give the title compound 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)(methyl)amino)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (126) (359 mg) as a white powder. LC / MS: RT=1.40 min; m / z=655.4 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 9.08(dd, J=2.7, 0.7Hz, 1H), 8.81(s, 2H), 8.51(dd, J=8.8, 2.6Hz, 1H), 7.57(d, J=0.9Hz, 1H), 7.05(dd, J=8.8, 0.7Hz, 1H), 5.34(m, 2H), 5.15 (d, J=3.7Hz, 1H), 4.42(m, 1H), 4.27(m, 4H), 4.07(s, 3H), 3.95-3.84(m, 6H), 3.76(dd, J=3.0, 1.3Hz, 1H), 3.38(s, 3H), 1.26(d, J=6.5Hz, 3H).

[0311] Example 20 Preparation of (2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (46) and (4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (47) [ka] The β-D-galactosyl conjugates 46 and 47 were synthesized by adapting the synthetic protocol of Wadzinski et al. (Nature Chem. Biol., 10 (2018) 644–652) for rapid O-phenol glycosylation in aqueous media. Starting from commercially available (2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone, AT13387 (36), treatment with α-D-galactopyranosyl fluoride in the presence of Ca(OH)2 afforded a regioisomeric mixture of β-D-galactosides, which were separated by HPLC.

[0312] α-D-Galactopyranosyl fluoride was prepared as follows: To β-D-galactose pentaacetate (10 g, 25.7 mmol, 1 equiv.) in a plastic bottle cooled in an ice / water bath, a cold solution of 70% HF and pyridine (17 mL, 1.5 M) was slowly added via syringe. The reaction vessel was capped, gradually warmed to room temperature, and stirred for 16 h. The reaction mixture was quenched by the addition of cold water (50 mL). DCM (50 mL) was added, and the resulting mixture was stirred for 30 min. The organic layer was then separated, and the aqueous layer was extracted with DCM (2 × 50 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (150 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under high vacuum to give a crude residue.

[0313] To the above residue, anhydrous MeOH (130 mL) was added, and the resulting mixture was stirred for 15 minutes. The reaction mixture was then cooled in ice / water for 10 minutes, and NaOMe (139 mg, 2.57 mmol) was added. After 10 minutes, the ice / water bath was removed, and the reaction mixture was allowed to warm gradually to room temperature over 3 hours. The reaction mixture was cooled again in an ice / water bath, and silica gel (11 g) was added. The resulting suspension was concentrated to a thick paste. A solution of 7:3 EtOAc:MeOH (50 mL) was added, stirred thoroughly for 5 minutes, and filtered. The residue was washed with 7:3 EtOAc:MeOH (60 mL), and the combined filtrate was concentrated to dryness using a rotary evaporator. The residue was dried under high vacuum overnight to give the desired product, α-D-galactopyranosyl fluoride (4.2 g, 91%), as a foamy solid, which was used in the next step without further purification.

[0314] To a solution of AT13387 (36) (90 mg, 0.22 mmol) and α-D-galactopyranosyl fluoride (1.2 g, 6.6 mmol) in water (7 mL) and DMSO (7 mL) was added Ca(OH) (488 mg, 6.6 mmol), and the resulting mixture was stirred at room temperature for 8 h. LC / MS showed substantial conversion to the desired compounds 46 and 47. The reaction mixture was quenched by the addition of 1 M aqueous HCl to adjust the pH to 8, and the resulting mixture was concentrated under reduced pressure to remove water. The DMSO solution of the residue was directly purified by reverse-phase HPLC, and the two regioisomeric products were isolated. The regiochemical identities of the products were inferred by correlation of their retention times in reverse-phase LC / MS with their calculated LogP values ​​(the calculated less lipophilic isomer was assigned to the earlier-eluting peak in HPLC).

[0315] The early eluting peak gave 34 mg of (2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (46) as a white solid (cLogP=1.8). LC / MS: RT=1.13 min; m / z=572.5 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ 7.41-7.35(br s, 2H), 7.33(s, 1H), 7.30(s, 2H), 5.01-4.90(m, 6H), 4.10-4.00(m, 2H), 3.83-3. 63(m, 9H), 3.29(m, 4H), 2.88(s, 3H), 1.25(d, J=6.9Hz, 3H) 1.24(d, J=6.9Hz, 3H).

[0316] The later eluting peak gave 22 mg of (4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (47) as a white solid (cLogP=2.0). LC / MS: RT=1.20 min; m / z=572.5 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.42-7.35(m, 1H), 7.35-7.27(m, 1H), 7.25-7.18(m, 1H), 7.12(d, J=1.7Hz, 1H), 6.78(s, 1H), 4.98-4.78(m, 6H), 4.71(m, 1H), 3.92- 3.86(m, 1H), 3.85-3.62(m, 6H), 3.55(m, 1H), 3.30-3.22(m, 7H), 2.88(d, J=8.8Hz, 3H), 1.24(d, J=6.8Hz, 3H), 1.22(d, J=6.8Hz, 3H).

[0317] Example 21: Preparation of N-ethyl-5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (48) and N-ethyl-5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (49) [ka] [ka] The β-D-galactosyl conjugates 48 and 49 were synthesized by adapting the synthetic protocol of Wadzinski et al. (Nature Chem. Biol., 10 (2018) 644–652) for rapid O-phenol glycosylation in aqueous media. Starting from the commercially available resorcinol compound NVP-AUY922 37, treatment with α-D-galactopyranosyl fluoride in the presence of Ca(OH) afforded a regioisomeric mixture of β-D-galactosides, which were separated by HPLC.

[0318] In a 4 mL vial, 5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide, NVP-AUY922 (37), (100 mg, 0.21 mmol), α-D-galactopyranosyl fluoride (352 mg, 1.9 mmol), and Ca(OH) (47 mg, 0.64 mmol) were suspended in water (0.4 mL). The reaction vial was capped, and the resulting mixture was stirred at room temperature for 3 h. LC / MS showed substantial conversion to the desired compounds (48) and (49). The reaction mixture was quenched by the addition of 1 M aqueous HCl to adjust the pH to 8, and the resulting mixture was directly purified by reverse-phase HPLC to give the two regioisomeric products. The regiochemical identity of the products was inferred by correlation of retention times in reversed-phase LC / MS with calculated LogP values ​​(the calculated less lipophilic isomer was assigned to the earlier-eluting peak in HPLC).

[0319] The early eluting peak gave 7 mg of N-ethyl-5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (48) as a white solid (cLogP=0.88). LC / MS: RT=1.29 min; m / z=628.4 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 7.44(m, 4H), 6.98(s, 1H), 6.73(s, 1H), 4.83(m, 1H), 4.31(s, 1H), 3.94(s, 1H), 3.87-3.76(m, 4H), 3.71(m, 1H), 3.59(m, 1H), 3.39-3.15(m, 11H), 1.23(t, J=7.3Hz, 3H), 1.06(d, J=6.9Hz, 3H), 1.05(d, J=6.9Hz, 3H).

[0320] The later eluting peak gave 22 mg of N-ethyl-5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (49) as a white solid (cLogP=1.02). LC / MS: RT=1.43 min; m / z=628.4 [M+H] + . 1 H NMR (500MHz, methanol-d4):δ 7.45(s, 4H), 7.05(s, 1H), 6.72(s, 1H), 4.71(d, J=7.7Hz, 1H), 4.35(q, J=7.3Hz, 2H), 4.06(br.s, 1H), 3.86(m, 1H), 3.77-3.7 0(m, 3H), 3.60(m, 1H), 3.48-3.20(m, 10H), 3.16(m, 1H), 1.23(t, J=7.3Hz, 3H), 1.10(d, J=6.9Hz, 3H), 1.09(d, J=6.9Hz, 3H).

[0321] Example 22 Preparation of 5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (127) and 5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (128) [ka] [ka] To a mixture of 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one, ganetespib (38) (200 mg, 0.55 mmol), and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (1.35 g, 3.3 mmol) in anhydrous DMF (10 mL) was added CsCO (3.2 g, 9.8 mmol), and the resulting mixture was stirred at room temperature for 15 hours. Water (100 mL) was added, and the resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dried under high vacuum overnight.

[0322] To the above residue, MeOH (5 mL) and NaOMe (25% solution in MeOH, 25 μL, 0.11 mmol) were added, and the resulting mixture was stirred at room temperature for 1 h. LC / MS showed substantial conversion to the desired compounds 127 and 128. The reaction mixture was quenched by the addition of 4 M HCl in dioxane solution and adjusted to pH 8. The reaction mixture was then concentrated under reduced pressure and purified by reverse-phase HPLC to give two regioisomeric products. The regiochemical identities of the products were inferred by correlation of their retention times in reverse-phase LC / MS with the calculated LogP values ​​(the calculated less lipophilic isomer was assigned to the earlier-eluting peak in HPLC).

[0323] The early eluting peak gave 25 mg of 5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (127) as a white solid (cLogP=1.96). LC / MS: RT=1.30 min; m / z=527.2 [M+H] + . 1H NMR (500MHz, methanol-d4):δ 7.53-7.46(m, 2H), 7.29(d, J=3.2Hz, 1H), 7.08(dd, J=8.6, 2.1Hz, 1H), 6.71(s, 1H), 6.67(s, 1H), 6.49(dd, J=3.1, 0.8Hz, 1H), 4.80(d, J=7.8Hz, 1H), 3.90(d, J=2.9Hz, 1H), 3.86(s, 3H), 3.82-3.71(m, 3H), 3.67(m, 1H), 3.55(dd, J=9.7, 3.4Hz, 1H), 3.14(m, 1H), 0.73(dd, J=6.9, 1.7Hz, 6H). The later eluting peak gave 35 mg of 5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (128) as a white solid (cLogP=2.09). LC / MS: RT=1.54 min; m / z=527.2 [M+H] + . 1 H NMR (500 MHz, methanol-d): δ 7.55-7.49(m, 2H), 7.30(d, J=3.1Hz, 1H), 7.09(dd, J=8.6, 2.0Hz, 1H), 6.5 9(s, 1H), 6.51(d, J=3.0Hz, 1H), 6.29(s, 1H), 5.27(d, J=9.1Hz, 1H), 4.40( m, 1H), 3.98(d, J=3.3Hz, 1H), 3.87(s, 3H), 3.85-3.75(m, 3H), 3.72(dd, J= 9.6, 3.3Hz, 1H), 2.91(m, 1H), 0.67(d, J=6.9Hz, 3H), 0.65(d, J=6.9Hz, 3H).

[0324] Example 23: Toxicity of compounds to normal proliferating, senescent or quiescent fibroblasts Changes in cell viability after exposure to compounds were determined using normal human fibroblasts (IMR90) and patient-derived β-galactosidase (GLB1)- or α-fucosidase (FUCA1)-deficient cells. Cells were cultured with DMEM and 10% heat-inactivated FBS under a controlled atmosphere of 5% carbon dioxide and 5% oxygen. Senescence was induced by cell contact inhibition and confirmed by the absence of senescence-associated β-galactosidase (SA-β-Gal) staining and the ability to re-enter the cell cycle. Senescence was induced by treatment with doxorubicin and confirmed by positive SA-β-Gal staining and the absence of DNA replication. Cells were treated with compounds for 3 days. Cell viability was determined by mitochondrial dehydrogenase activity (XTT assay, Cayman Chemical) at 10 concentrations of the tested compound. To generate dose-response curves, data were fitted to a four-parameter Hill function, and IC 50 The senolytic index (SI) for each compound was calculated as the IC400 / 10000 relative to normal proliferating cells. 50 IC against senescent cells 50 The senolytic index was determined by dividing by 0. The senolytic index data are shown in Table 1, with compounds having an SI<1(-), SI>1(+), SI>5(++), and SI>10(+++).

[0325] [Table 1]

[0326] Example 24: Senescent cell-eliminating effect of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) on mouse embryonic fibroblasts (MEFs) Mouse embryonic fibroblasts were incubated with 300 nM doxorubicin for 24 hours to induce senescence. After washing the cells and incubating them in culture medium for 7 days, senescence was confirmed by staining for SA-β-Gal (Itahana et al., Methods Mol. Biol. 371 (2007) 21-31) and the lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation (Yu et al., J. Immunol. Methods 350 (2009) 29-35).

[0327] Proliferating or senescent MEFs were treated with increasing concentrations of either FURGal (101) or 5-fluorouridine (FUR) (102) for 4 days. Cells were washed and stained with 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). The fraction of viable cells was assessed by the ratio of PI-positive to PI-negative nuclei. The results, shown in Figure 1A, demonstrate that the active drug (102) significantly reduces cell viability, while the prodrug (101) is completely non-toxic to proliferating cells at concentrations as low as 4 mM. In contrast, both compounds (101) and (102) are equitoxic to senescent MEFs, as shown in Figure 1B, indicating that the prodrug (101) is efficiently converted to the cytotoxic FUR (102) by hydrolase enzymes in the senescent cell population.

[0328] Example 25: Comparison of the toxicity of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) and 5-fluorouridine (FUR) (102) in C57BL / 6 mice Equimolar doses of 101 (160 mg / kg) and 102 (100 mg / kg) were administered by single intraperitoneal injection to two groups of mice (n=3 each). Six days after treatment, blood counts were determined by standard methods (Figure 2A), bone marrow cells from femurs were counted (Figure 2B), and spleen weights (Figure 2C) were determined to compare the toxicity of the two drugs. Prodrug 101 exhibited minimal toxicity to platelets, neutrophils, lymphocytes, and myeloid cells, while FUR 102 induced a significant reduction in all cell populations. Prodrug 101 also showed minimal effect on spleen weight compared to FUR 102.

[0329] Example 26: Effect of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) on senescent C57BL / 6 mouse hepatocytes after in vivo administration Two groups of C57BL / 6 mice (N = 5 / group) were intraperitoneally injected with doxorubicin (25 mg / kg) to induce hepatocyte senescence. Four and six days later, the groups were injected with PBS or FURGal (140 mg / kg) (101). A third group of five animals served as a control. After an additional four days, livers were removed, sectioned (30 μm), and stained for SA-β-Gal (six fields per animal were evaluated) (Figures 3A and 3B). As shown in Figure 3C, quantification revealed a numerical trend toward a reduction in senescent hepatocytes after FURGal treatment. The average body weight of the mice on the day of analysis is shown in Figure 3D.

[0330] Example 27: Effect of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (113) (aka RBO-013) on senescent C57BL / 6 mouse hepatocytes after in vivo administration Two groups of C57BL / 6 mice (N = 9 / group) were intraperitoneally injected with doxorubicin (20 mg / kg) to induce hepatocyte senescence. Four and six days later, the groups were intraperitoneally (ip) injected with PBS or compound 113 (also known as RBO-013) (2 × 20 mg / kg). A third group of animals served as a control. After an additional two days, livers were removed, sectioned (30 μm), and stained for SA-β-Gal as described above (Figures 4A and 4B). As shown in Figure 4C, quantification demonstrated a decrease in senescent hepatocytes (visualized by SA-β-Gal staining) after treatment with 113. This senolytic effect was also associated with the upregulation of Cdkn2a (p16 INK4a This finding was corroborated by using quantitative PCR to detect mRNA levels of Cdkn2a and IL-6 (NBCdkn2a expression was detected only in animals treated with doxorubicin). Expression levels were reported relative to Actb as a reference gene. Figures 4D and 4E show significant decreases in Cdkn2a and IL-6 gene expression, respectively.

[0331] Example 28: Senescent cell elimination effect of (2S,3R,4R,5S,6S)-2-methyl-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (119) (aka RBO-019) on senescent lung cells of C57BL / 6 mice after in vivo administration Four groups of C57BL / 6 mice (N = 9 / group) were intravenously injected with doxorubicin (15 mg / kg) to induce senescence in lung tissue. Five days later, the groups were injected with vehicle or compound 119 (also known as RBO-019) (10, 20, or 40 mg / kg intravenously (iv)). After an additional 3 days, the lungs were removed, and the left lobes were sectioned and stained for SA-β-Gal as described above (Figures 5A and 5B). As shown in Figure 5C, quantification revealed a dose-dependent decrease in SA-β-Gal staining after treatment with 119. This senolytic effect was also associated with an increase in Cdkn2a (p16 INK4aThis was confirmed by using quantitative PCR to detect mRNA levels of Cdkn2a. Expression levels were reported relative to Actb as a reference gene. Figure 5D shows a dose-dependent decrease in Cdkn2a gene expression in the lung.

Claims

1. Formula (IV) or (V): 【Chemical 1】 wherein R is a residue of a hydroxamic acid derivative histone deacetylase inhibitor; １３ , R １４ , R １５ , R １６ , R ２３ , R ２４ and R ２５ each independently represents hydrogen, C(O)—R １ , a moiety of formula (VI) or a moiety of formula (VII): 【Chemistry 2】 and each R ３３ , R ３４ , R ３５ , R ３６ , R ４３ , R ４４ and R ４５ are independently hydrogen or —C(O)—R ２ and each R １ But independently, C １～４ alkyl or phenyl, provided that R １３ , R １４ , R １５ or R １６ is a moiety of formula (VI) or formula (VII), then R １３ , R １４ , R １５ and R １６ The remainder is hydrogen or C(O)—R １ and each R ２ But independently, C １～４ alkyl or phenyl, provided that R ２３ , R ２４ or R ２５ is a moiety of formula (VI) or formula (VII), then R ２３ , R ２４ or R ２５ The remainder is hydrogen or —C(O)—R １ where R １３ , R １４ , R １５ and R １６ is hydrogen, then R is not 7-heptanoylphenylamido. or a pharmaceutically acceptable salt, hydrate or solvate thereof.

2. The anomeric carbon of the pyranose ring (labeled ＊ 2. The compound of claim 1, wherein: R is an integer from 1 to 10; and R is an integer from 1 to 20; and R is an integer from 1 to 20; and R is an integer from 1 to 20; and R is an integer from 1 to 20; and R is an integer from 1 to 20; and R is an integer from 1 to 20; and

3. 3. The compound of claim 2, wherein the hydroxamic acid derivative histone deacetylase inhibitor is selected from the group consisting of panobinostat, xinostat, vorinostat, dacinostat, gibinostat, CUDC-907, CUDC-101, abexinostat, belinostat, pracinostat, resminostat, licorinostat, piroxamide, APHA, trichostatin A, oxamflatin, and AR-42.

4. structure: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 【Chemistry 3-4】 The compound of claim 3, having any one of:

5. structure: 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】 【Chemistry 4-4】 The compound of claim 3, having any one of:

6. structure: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 【Chemistry 5-5】 The compound of claim 3, having any one of:

7. Formula (XI), (XII), (XIII) or (XIV): 【Chemistry 6】 wherein Y is carbonyl or absent; A is a substituted or benzo-fused 5-membered heteroaryl or heterocyclic group containing at least one nitrogen atom; B is selected from the group consisting of ethyl, isopropyl, or chloro; and R １３ , R １４ , R １５ , R １６ , R ２３ , R ２４ and R ２５ each independently represents hydrogen, C(O)—R １ , a moiety of formula (VI) or a moiety of formula (VII): 【Chemistry 7】 and each R ３３ , R ３４ , R ３５ , R ３６ , R ４３ , R ４４ and R ４５ are independently hydrogen or —C(O)—R ２ and each R １ But independently, C １～４ alkyl or phenyl, provided that R １３ , R １４ , R １５ or R １６ is a moiety of formula (VI) or formula (VII), then R １３ , R １４ , R １５ and R １６ The remainder is hydrogen or C(O)—R １ and each R ２ But independently, C １～４ alkyl or phenyl, provided that R ２３ , R ２４ or R ２５ is a moiety of formula (VI) or formula (VII), then R ２３ , R ２４ or R ２５ The remainder is hydrogen or —C(O)—R １ is) or a pharmaceutically acceptable salt, hydrate or solvate thereof.

8. The anomeric carbon of the pyranose ring (labeled ＊ 8. The compound of claim 7, wherein:

9. Part A-Y-C ６ H ２ (OH) ２ The compound of claim 8, wherein -B is an Hsp90 inhibitor selected from the group consisting of luminespib (NVP-AUY922), ganetespib, VER-50589, AT13387, and KW-2478.

10. structure: 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 【Chemistry 8-5】 【Chemistry 8-6】 10. The compound of claim 9, wherein

11. Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV) or (XXV): 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 【Chemistry 9-4】 【Chemistry 9-5】 (In the formula, R ８ is 4-morpholinyl or 1-imidazolyl; each R １３ , R １４ , R １５ , R １６ , R ２３ , R ２４ and R ２５ are independently hydrogen, C(O)—R １ , a moiety of formula (VI) or a moiety of formula (VII): 【Chemistry 10】 and each R ３３ , R ３４ , R ３５ , R ３６ , R ４３ , R ４４ and R ４５ are independently hydrogen or —C(O)—R ２ and each R １ But independently, C １～４ alkyl or phenyl, provided that R １３ , R １４ , R １５ or R １６ is a moiety of formula (VI) or formula (VII), then R １３ , R １４ , R １５ and R １６ The remainder is hydrogen or C(O)—R １ and each R ２ But independently, C １～４ alkyl or phenyl, provided that R ２３ , R ２４ or R ２５ is a moiety of formula (VI) or formula (VII), then R ２３ , R ２４ or R ２５ The remainder is hydrogen or —C(O)—R １ is) or a pharmaceutically acceptable salt, hydrate or solvate thereof.

12. The anomeric carbon of the pyranose ring (labeled ＊ 12. The compound of claim 11, wherein: R is in the S configuration; and the compounds are β-D-galactoside and α-L-fucoside conjugates of a TOP1 inhibitor, respectively.

13. structure: 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 13. The compound of claim 12, having any one of:

14. structure: 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 13. The compound of claim 12, having any one of:

15. Formula (XXVIII) or (XXIX): 【Chemistry 13】 (In the formula, R ９ But hydrogen, C １～４ Alkyl, CF ３ , CN or NO ２ and R １０ But hydrogen, C １～４ alkyl or arylalkyl; R １３ , R １４ , R １５ , R １６ , R ２３ , R ２４ and R ２５ each independently represents hydrogen, C(O)—R １ , a moiety of formula (VI) or a moiety of formula (VII): 【Chemistry 14】 and each R ３３ , R ３４ , R ３５ , R ３６ , R ４３ , R ４４ and R ４５ are independently hydrogen or C(O)—R ２ and each R １ But independently, C １～４ alkyl or phenyl, provided that R １３ , R １４ , R １５ , or R １６ is a moiety of formula (VI) or formula (VII), then R １３ , R １４ , R １５ and R １６ The remainder is hydrogen or C(O)—R １ and each R ２ But independently, C １～４ alkyl or phenyl, provided that R ２３ , R ２４ or R ２５ is a moiety of formula (VI) or formula (VII), then R ２３ , R ２４ or R ２５ The remainder is hydrogen or C(O)—R １ with the proviso that in the compound of formula (XXVIII), R ９ But NO ２ and R １０ is benzyl, R １３ , R １４ , R １５ , R １６ cannot be hydrogen or acetyl at the same time. or a pharmaceutically acceptable salt, hydrate or solvate thereof.

16. The anomeric carbon of the pyranose ring (labeled ＊ 16. The compound of claim 15, wherein:

17. 20. A method for treating an aging-related disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 16 or a pharmaceutical composition thereof.

18. 1. A method for treating an aging-related disease or disorder, comprising administering to a subject in need thereof a compound of the formula: 【Chemistry 15】 (In the formula, R ４６ is either hydrogen, acetyl or propionyl) or a pharmaceutically acceptable salt, pharmaceutical composition, hydrate or solvate thereof in a therapeutically effective amount.

19. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is an aging-related disease selected from the group consisting of kidney disease, renal failure, frailty, cognitive impairment, hearing loss, muscle wasting, skin disease, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucous fibrosis, and sarcopenia.

20. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a pulmonary disease selected from the group consisting of pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and age-related decline in lung function.

21. 19. The method of claim 17 or 18, wherein the aging-associated disease or disorder is metastasis.

22. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a chemotherapy side effect or a radiotherapy side effect.

23. 23. The method of claim 22, wherein the chemotherapy side effects or radiation therapy side effects are gastrointestinal toxicity, peripheral neuropathy, fatigue, lethargy, low physical activity, frailty, hematotoxicity, hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, skin toxicity, mouth, gum or throat problems, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, impaired cognitive function, secondary cancers, cataracts and other visual impairment, hearing loss, decreased lung capacity and lung disease.

24. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a neurodegenerative disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, and motor neuron dysfunction.

25. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is an ocular disease or disorder selected from the group consisting of macular degeneration, glaucoma, cataracts, presbyopia, and vision loss.

26. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a metabolic disease selected from the group consisting of diabetes, diabetic ulcers, metabolic syndrome, and obesity.

27. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is renal dysfunction.

28. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a skin disease or disorder selected from the group consisting of eczema, psoriasis, hyperpigmentation, nevi, rash, atopic dermatitis, hives, diseases and disorders related to photosensitivity or photoaging, wrinkles; pruritus; paresthesia; eczematous rash; eosinophilic skin diseases; reactive neutrophilic dermatoses; pemphigus; pemphigoid; immune bullous skin diseases; cutaneous fibrous histiocytosis; cutaneous lymphoma; and cutaneous lupus.

29. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is an inflammatory or autoimmune disease or disorder selected from osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, kyphosis, and herniated disc.

30. 19. The method of claim 17 or 18, wherein the aging-related disease or disorder is a cardiovascular disease selected from atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary artery thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, myocardial fibrosis, cerebral aneurysm, and stroke.

31. The method of claim 17 or 18, wherein the senescent cells associated with an aging-related disease or disorder are senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes.

32. 19. The method of claim 17 or 18, wherein the compound kills at least 20% of senescent cells and kills no more than 5% of non-senescent cells in an organ or tissue containing senescent cells associated with the aging-associated disease or disorder.

33. 19. The method of claim 17 or 18, wherein the compound is administered in combination with one or more other therapeutic agents.

34. 34. The method of claim 33, wherein the compound is administered in combination with a chemotherapeutic agent.

35. A pharmaceutical composition comprising a compound according to any one of claims 1 to 16 and a pharmaceutically acceptable vehicle.

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