Anti-aging composition and application thereof

Abstract

Provided herein are anti-aging agents for selectively killing aging cells associated with a variety of pathologies and diseases, including age-related pathologies and diseases. As disclosed herein, diseases and abnormalities associated with senescent cells may be treated or prevented by administering at least one anti-aging agent or a pharmaceutical composition thereof. The diseases or abnormalities associated with senescent cells treated or prevented by the methods described herein include, but are not limited to, cardiovascular diseases or abnormalities, cardiovascular diseases and abnormalities associated with arteriosclerosis (such as atherosclerosis), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory diseases or abnormalities, and the like. Autoimmune diseases or abnormalities, lung diseases or abnormalities, nervous system diseases or abnormalities, dermatological diseases or abnormalities, side effects of chemotherapy, side effects of radiotherapy, metastatic diseases and metabolic diseases.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority and benefit of U.S. Provisional Patent Application Serial No. 62 / 696,486, filed on July 11, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical field

[0003] The present disclosure provides anti - aging agents for selectively killing senescent cells associated with a variety of pathologies and diseases, including age - related pathologies and diseases. As disclosed herein, diseases and abnormalities associated with senescent cells can be treated or prevented by administering at least one anti - aging agent or a pharmaceutical composition thereof. Diseases or abnormalities associated with senescent cells that can be treated or prevented by the methods described herein include, but are not limited to, cardiovascular diseases or abnormalities, cardiovascular diseases and abnormalities associated with arteriosclerosis (such as atherosclerosis), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory diseases or abnormalities, autoimmune diseases or abnormalities, pulmonary diseases or abnormalities, neurological diseases or abnormalities, dermatological diseases or abnormalities, side effects of chemotherapy, side effects of radiotherapy, metastatic lesions, and metabolic diseases. Background art

[0004] Aging is a risk factor for most chronic diseases, disabilities, and poor health. Senescent cells, which are cells in a state of replicative arrest, accumulate in aging individuals and may contribute in part or significantly to cellular and tissue degeneration, which underlies aging and age - related diseases (see, e.g., Childs et al., Nat. Rev. Drug Discov. 16 (2017) 718 - 735). Cells can also become senescent after exposure to environmental, chemical, biological insults or due to diseases (see, e.g., Demaria et al., Cancer Discovery 7 (2017) 165 - 176; and Schafer et al., Nat. Commun. 8 (2017) doi:10.1038 / ncomms14532).

[0005] Anti-aging agents with multiple pharmacological mechanisms are known in the art. The anti-aging agent can be a specific inhibitor of one or more members of the Bcl-2 anti-apoptotic protein family, wherein the inhibitor at least inhibits Bcl-xL (e.g., Bcl-2 / Bcl-xL / Bcl-w inhibitor; a selective Bcl-xL inhibitor; Bcl-xL / Bcl-w inhibitor (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 inhibitor (e.g., MK-2206); receptor tyrosine kinase inhibitor (e.g., dasatinib, see, e.g., Zhu et al., Aging Cell 14(2015)654-658); CDK4 / 6 inhibitor (e.g., palbociclib (see, e.g., Whittaker et al., Pharmacol. Ther. 173(2017)83-105)); mTOR inhibitor (e.g., rapamycin, (see, e.g., Laberge et al., Nat. Cell Biol. 17(2015)1049-1061)); MDM2 inhibitor (e.g., Nutlin-3; and RG-7112, see, e.g., U.S. Patent Application 2016 / 0339019)); Hsp90 inhibitor (e.g., 17-DMAG; and ganetespib, see, e.g., Fuhrmann-Stroissnigg et al., Nat. Commun. 8(2017) doi:10.1038 / s41467-017-00314-z)); flavonoids (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 inhibitor (e.g., panobinostat (see, e.g., Samaraweera et al., Sci. Rep. 7(2017)1900.doi:10.1038 / s41598-017-01964-1)).

[0006] An important challenge is to identify anti-aging agents that selectively kill senescent cells while sparing non-senescent cells. Additionally, many known anti-aging agents were initially developed as cytotoxic anti-cancer agents and have subsequently been repurposed to 'selectively' eliminate senescent cell populations. Since proliferating cells are generally more sensitive to the cytotoxic or cytostatic effects of anti-tumor agents, dose-limiting toxicity in hematopoietic cells is a frequently observed side effect that limits the clinical utility of anti-aging 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 anti-aging drugs has been proposed as a mechanism to minimize exposure of non-senescent cells to these molecules and potentially limit off-target effects. Thus, what is needed are anti-aging agents with improved selectivity for killing senescent cells that have minimal toxicity to non-senescent cells. SUMMARY OF THE INVENTION

[0007] Disclosed herein are non-toxic prodrugs of anti-aging agents activated by hydrolases that preferentially accumulate within senescent cells, which meet these and other needs. In one aspect, the hydrolase is a glycosidase, and the elevated intracellular glycosidase activity associated with senescence is utilized to convert a non-toxic prodrug derivative (I) of a pro-apoptotic agent into a toxic pro-apoptotic parent compound (II), resulting in the specific killing of senescent cells.

[0008]

[0009] In some embodiments, compound (II) is capable of promoting apoptosis in non-proliferating cells.

[0010] In another aspect, non-toxic prodrugs of toxic anti-aging agents are provided that specifically cause the death of senescent cells when cleaved within senescent cells to the active anti-aging agent. In some embodiments, prodrugs of histone deacetylase inhibitors are provided. In other embodiments, prodrugs of Hsp90 inhibitors are provided. In still other embodiments, prodrugs of topoisomerase 1 inhibitors are provided. In still other embodiments, prodrugs of DNA alkylating agents are provided. In still other embodiments, prodrugs of Akt1 inhibitors are provided. In still other embodiments, prodrugs of proteasome inhibitors are provided. Derivatives of the prodrugs described herein are also provided, including salts, solvates, hydrates, metabolites. Compositions are also provided that comprise a prodrug provided herein and a carrier.

[0011] In another aspect, the present disclosure also provides methods for treating, preventing, or alleviating symptoms of a medical abnormality in a subject, such as, for example, cardiovascular diseases or abnormalities, cardiovascular diseases and abnormalities associated with arteriosclerosis (such as atherosclerosis), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory diseases or abnormalities, autoimmune diseases or abnormalities, pulmonary diseases or abnormalities, neurological diseases or abnormalities, dermatological diseases or abnormalities, side effects of chemotherapy, side effects of radiotherapy, metastatic lesions, and metabolic diseases. In practicing the methods, a therapeutically effective amount of an anti-aging agent or a pharmaceutical composition thereof is administered to the subject.

[0012] In yet another aspect, a method for treating an age-related disease or disorder is provided. The method includes administering to a subject a therapeutically effective amount of the anti-aging agent or a pharmaceutical composition thereof. In yet another aspect, a method for delaying at least one characteristic of aging in a subject is provided. The method includes administering to a subject a therapeutically effective amount of the anti-aging agent or a pharmaceutical composition thereof.

[0013] In yet another aspect, a method for killing therapy-induced senescent cells is provided. The method includes administering to a subject who has received DNA damage therapy a therapeutically effective amount of the anti-aging agent or a pharmaceutical composition thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A Shows the viability of proliferating mouse embryonic fibroblasts (MEFs) treated with 5-fluorouridine (FUR) (102) or 5-fluorouridine-5′-O-β-D-galactopyranoside (FURGal) (101) at various drug concentrations.

[0015] Figure 1B Shows the viability of senescent mouse embryonic fibroblasts (MEFs) treated with 5-fluorouridine (FUR) (102) or 5-fluorouridine-5′-O-β-D-galactopyranoside (FURGal) (101) at various drug concentrations.

[0016] Figure 2A Shows the quantification of blood cell counts from wild-type C57BL / 6 mice administered FUR (102) (100 mg / kg) or FURGal (101) (160 mg / kg) by single intraperitoneal injection after 6 days of treatment (N = 3 mice / group).

[0017] Figure 2BShows the quantification of the number of bone marrow cells in the femurs of wild-type C57BL / 6 mice administered FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) by single intraperitoneal injection 6 days after treatment (N = 3 mice / group).

[0018] Figure 2C Shows the quantification of the total spleen weight of wild-type C57BL / 6 mice administered FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) by single intraperitoneal injection 6 days after treatment (N = 3 mice / group).

[0019] Figure 3A Shows representative images of liver sections of C57BL / 6 mice injected with doxorubicin (25 mg / kg).

[0020] Figure 3B Shows representative images of liver sections of C57BL / 6 mice injected with doxorubicin (25 mg / kg) and FURGal (140 mg / kg).

[0021] Figure 3C Shows Figure 3A and Figure 3B Quantification of liver sections together with controls.

[0022] Figure 3D Shows the average body weight of C57BL / 6 mice on the day of analysis.

[0023] Figure 4A Shows the protocol for inducing hepatocyte senescence in C57BL / 6 mice and subsequent treatment with compound (113).

[0024] Figure 4B Compares representative images of liver sections of C57BL / 6 mice injected with doxorubicin (20 mg / kg) and then injected with vehicle or compound (113).

[0025] Figure 4C Shows from Figure 4B Quantification of SA-β-Gal in liver sections together with controls.

[0026] Figure 4D Shows the quantification of Cdkn2a expression in the livers of C57BL / 6 mice injected with doxorubicin (20 mg / kg) and then injected with vehicle or compound (113).

[0027] Figure 4E Shows the quantification of IL-6 expression in the livers of C57BL / 6 mice injected with doxorubicin (20 mg / kg) and then injected with vehicle or compound (113).

[0028] Figure 5A A protocol is shown for observing the anti-aging effect of compound (119) in the lung tissue of C57BL / 6 mice.

[0029] Figure 5B Representative images of lung sections of C57BL / 6 mice injected with doxorubicin (15 mg / kg) and then injected with 20 mg / kg of vehicle or compound (119) were compared.

[0030] Figure 5C Quantification of SA-β-Gal in lung sections along with controls is shown after intravenous administration of compound (119) at 10 mg / kg, 20 mg / kg, or 40 mg / kg.

[0031] Figure 5D Quantification of Cdkn2a expression in the lungs of C57BL / 6 mice injected with doxorubicin (15 mg / kg) and then injected with vehicle or compound (119) at 10 mg / kg, 20 mg / kg, or 40 mg / kg is shown. Detailed Description

[0032] Definitions

[0033] As used herein, "characteristics of aging" include, but are not limited to, a systemic decline in the immune system, muscle atrophy and reduced muscle strength, reduced skin elasticity, delayed wound healing, retinal atrophy, reduced lens transparency, reduced hearing, osteoporosis, sarcopenia, hair graying, skin wrinkling, poor vision, frailty, and cognitive impairment.

[0034] "Acyl" refers to an H-CO-, alkyl-CO-, alkenyl-CO-, or cycloalkyl-CO-group, where the alkyl, alkenyl, or cycloalkyl group is as described herein.

[0035] "Acylamino" is an acyl-NH-group, where acyl is as defined herein.

[0036] As used herein, "age-related disease or disorder" includes, but is not limited to, degenerative diseases or hypofunctional abnormalities 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 dysfunctions, coronary artery disease, cerebrovascular disease, periodontal disease, cancer treatment-related disabilities (such as atrophy and fibrosis in various tissues, brain and heart damage and therapy-related myelodysplastic syndromes and diseases associated with accelerated aging and / or defects in DNA damage repair and telomere maintenance, such as progeria syndromes (i.e., Hutchinson-Gilford progeria syndrome, Werner syndrome, Bloom syndrome, Rothmund-Thomson syndrome, Cockayne syndrome, xeroderma pigmentosum, trichothiodystrophy, combined xeroderma pigmentosum-Cockayne syndrome, restrictive dermopathy)), ataxia telangiectasia, Fanconi anemia, Friedrich's ataxia, dyskeratosis congenita, aplastic anemia, and others.

[0037] "Alkenyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond, and it can be straight-chain or branched-chain, having 2 to 20 carbon atoms in the chain. In some embodiments, the alkenyl group has 2 to 12 carbon atoms in the chain. In other embodiments, the alkenyl group has about 2 to 6 carbon atoms in the chain. In still other embodiments, the alkenyl group has 2 to 4 carbon atoms in the chain. As used herein and throughout the text, "branched-chain" refers to one or more lower alkyl groups (such as methyl, ethyl, or propyl) attached to a straight-chain; here it is a linear alkenyl chain. "Lower alkenyl" refers to about 2 to about 4 carbon atoms in the chain, which can be straight-chain or branched-chain. Exemplary alkenyl groups include, but are not limited to, vinyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl, and decenyl.

[0038] "Alkenylene" refers to an aliphatic divalent radical derived from a straight-chain or branched-chain alkenyl group, where the alkenyl group is as described herein. Exemplary alkenylene radicals include, but are not limited to, vinylene and propenylene.

[0039] "Alkoxy" refers to 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, isopropoxy, n-butoxy, and heptyloxy.

[0040] "Alkoxycarbonyl" refers to an alkyl-O-CO-group, where the alkyl group is as described herein. Exemplary alkoxycarbonyl groups include methoxy and ethoxycarbonyl.

[0041] Unless otherwise specified, "alkyl" refers to an aliphatic hydrocarbon group that can be straight-chain or branched and has 1 to 20 carbon atoms in the chain. In some embodiments, the alkyl group has 1 to 6 carbon atoms. Unless otherwise specified, "lower alkyl" as a group or moiety of a lower alkoxy, lower alkylthio, lower alkylsulfinyl, or lower alkylsulfonyl group refers to an aliphatic hydrocarbon group that can be straight-chain or branched and has 1 to 4 carbon atoms in the chain. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 3-pentyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0042] "Alkylene" refers to an aliphatic divalent radical derived from a straight-chain or branched alkyl group, where the alkyl group is as described herein. Exemplary alkylene radicals include, but are not limited to, methylene, ethylene, and trimethylene.

[0043] "Alkylenedioxy" refers to the -O-alkylene-O- group, where alkylene is defined as above. Exemplary alkylenedioxy groups include, but are not limited to, methylenedioxy and ethylenedioxy.

[0044] "Alkylsulfinyl" refers to the alkyl-SO- group, where the alkyl group is as described above. Exemplary alkylsulfinyl groups include, but are not limited to, those where the alkyl group is C 1-4 alkyl.

[0045] "Alkylsulfonyl" refers to the alkyl-SO 2 - group, where the alkyl group is as described above. In some embodiments, the alkylsulfonyl group is one where the alkyl group is C 1-4 alkyl.

[0046] "Alkylthio" refers to the alkyl-S- group, where the alkyl group is as described above. Exemplary alkylthio groups include, but are not limited to, methylthio, ethylthio, isopropylthio, and heptylthio.

[0047] "Alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, and it can be straight-chain or branched and has about 2 to about 20 carbon atoms in the chain. In some embodiments, the alkynyl group has 2 to 12 carbon atoms in the chain. In other embodiments, the alkynyl group has 2 to 6 carbon atoms in the chain. In still other embodiments, the alkynyl group has 2 to 4 carbon atoms in the chain. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, n-butynyl, isobutynyl, 3-methylbut-2-ynyl, and n-pentynyl.

[0048] "Alkynylene" refers to an aliphatic divalent radical derived from a straight-chain or branched alkynyl group, where the alkynyl group is as described herein. Exemplary alkynylene radicals include, but are not limited to, ethynylene and propynylene.

[0049] "Amino acid side chain" means a substituent found on the carbon between the amino and carboxyl groups of an α-amino acid. For examples of "corresponding protected derivatives" of amino acid side chains, see T.W. Greene and P.G.M. Wuts in "Protective Groups in Organic Chemistry", John Wiley and Sons, 1991.

[0050] "Aroyl" means an aryl-CO-group, where the aryl group is as described herein. Exemplary aroyl groups include benzoyl, 1-naphthoyl, and 2-naphthoyl.

[0051] "Aryl", as a group or part of a group, means: (i) an optionally substituted monocyclic or polycyclic aromatic carbocyclic moiety having from about 6 to about 14 carbon atoms, such as phenyl or naphthyl; or (ii) an optionally substituted partially saturated polycyclic aromatic carbocyclic moiety, where an aryl and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl, or indanyl ring.

[0052] "Aralkyl" means an aryl-alkyl-group, where the aryl and alkyl moieties are as described above. Exemplary aralkyl groups include, but are not limited to, benzyl, 2-phenylethyl, and naphthylmethyl.

[0053] "Arylene" means an optionally substituted divalent radical derived from an aryl group. Exemplary arylene groups include, but are not limited to, optionally substituted phenylene, naphthylene, and indanylene. Suitable substituents include one or more of the "aryl group substituents" defined above, particularly halogen, methyl, or methoxy.

[0054] "Aryloxy" means an aryl-O-group, where the aryl group is as described above. Exemplary aryloxy groups include, but are not limited to, optionally substituted phenoxy and naphthoxy.

[0055] "Aryloxycarbonyl" means an aryl-O-C(=O)-group, where the aryl group is as described above. Exemplary aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthoxycarbonyl.

[0056] "Arylsulfinyl" means an aryl-SO-group, where the aryl group is as described above.

[0057] "Arylsulfonyl" means an aryl-SO 2 -group, where the aryl group is as described above.

[0058] "Arylthio" means an aryl-S-group, where the aryl group is as described above. Exemplary arylthio groups include phenylthio and naphthylthio.

[0059] "Azaaryl" refers to an aromatic carbocyclic moiety of about 5 to about 10 ring members, wherein one of the ring members is nitrogen and the other ring members are selected from carbon, oxygen, sulfur or nitrogen. Examples of azaaryl groups include pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl, quinazolinyl, imidazolyl and benzimidazolyl.

[0060] "Compound" refers to a compound encompassed by the structural formulas disclosed herein and includes any particular compound of these structural formulas whose structure is disclosed herein. Compounds can be identified by their chemical structure and / or chemical name. When the chemical structure and chemical name conflict, the chemical structure determines the identity of the compound. The compounds described herein can contain one or more chiral centers and / or double bonds and can therefore exist as stereoisomers such as double bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein include the stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) depicted in the structure. The chemical structures described herein also include the enantiomers and stereoisomeric derivatives of the compounds, unless otherwise expressly stated. Mixtures of enantiomers and stereoisomers can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. These compounds can also exist in several tautomeric forms, including enol forms, keto forms and mixtures thereof. Accordingly, the chemical structures described herein include all possible tautomeric forms of the depicted compounds. The described compounds also include isotopically labeled compounds in which one or more atoms have an atomic mass different from the atomic mass found conventionally in nature. Examples of isotopes that can 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, etc. Compounds can exist in non-solvated forms as well as solvated forms, including hydrated forms. Generally, compounds can be hydrated or solvated. Certain compounds may exist in multiple crystalline forms or amorphous forms. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of this disclosure. In addition, it should be understood that when illustrating a partial structure of a compound, parentheses indicate the point of attachment of the partial structure to the remainder of the molecule.

[0061] "Cyclic amine" refers to a 3- to 8-membered monocyclic cycloalkyl ring system in which one of the ring carbon atoms is replaced by nitrogen and which may also contain a moiety selected from O, S, SO 2or an additional heteroatom of NY, where Y is hydrogen, alkyl, aryl, aralkyl, acyl, acyloxyalkyl, cycloalkyl, heteroaryl, heterocycloalkyl, or sulfonyl. Exemplary cycloamines include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, pyridoindolinyl, and tetrahydroquinolinyl.

[0062] "Cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic 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.

[0063] "Cycloalkyl" refers to a saturated monocyclic or bicyclic system of 3 to 10 carbon atoms optionally substituted with oxo. Exemplary monocyclic alkyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0064] "Cycloalkylene" refers to a divalent radical derived from a cycloalkyl group. Exemplary cycloalkylene radicals include, but are not limited to, cyclopentylene and cyclohexylene.

[0065] As used herein, "DNA damage therapy" includes, but is not limited to, γ-irradiation, alkylating agents (such as nitrogen mustards (e.g., chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (streptozocin, carmustine, lomustine), alkyl sulfonates (e.g., busulfan), triazines (dacarbazine, temozolomide), and ethyleneimines (e.g., thiotepa, altretamine)), platinum drugs (such as, for example, cisplatin, carboplatin, oxaliplatin), antimetabolites (such as, for example, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine), anthracyclines (such as, for example, 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)), and mitotic inhibitors (such as taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine), and estramustine).

[0066] "Halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0067] "Heteroaroyl" refers to a heteroaryl-C(=O)- group, where the heteroaryl group is as described herein. An exemplary group is pyridylcarbonyl.

[0068] "Heteroaryl", as a group or part of a group, means: (i) an aromatic monocyclic or polycyclic organic moiety of about 5 to about 10 ring members, wherein one or more ring members are elements other than carbon, such as nitrogen, oxygen or sulfur (examples of such groups include benzimidazolyl, benzothiazolyl, furyl, imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl and triazolyl groups, which are optionally substituted with one or more aryl group substituents as defined above); (ii) an optionally substituted partially saturated polycyclic heterocarbon ring moiety, wherein the heteroaryl and cycloalkyl or cycloalkenyl groups are fused together to form a cyclic structure (examples of such groups include pyrroloindanyl groups).

[0069] "Heteroarylene" refers to a divalent radical derived from a heteroaryl group.

[0070] "Heteroaryloxy" refers to a heteroaryl-O- group, wherein the heteroaryl group is as described above. An exemplary heteroaryloxy group is optionally substituted pyridyloxy.

[0071] "Heterocycle" means an optionally substituted saturated, partially saturated or fully unsaturated monocyclic organic moiety of 5 or 6 ring members, wherein one or more ring members are elements other than carbon, such as, for example, 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.

[0072] "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 heterocarbon ring moiety, wherein an aryl (or heteroaryl ring) and a heterocycloalkyl group are fused together to form a cyclic structure (examples of such groups include chromanyl, dihydrobenzofuranyl, dihydroindolyl and pyridoindolinyl groups).

[0073] As used herein, "histone deacetylase inhibitor" or "HDAC inhibitor" is a compound capable of inhibiting histone deacetylation in vivo, in vitro, or both in vivo and in vitro (see, e.g., 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. As a result of inhibiting the deacetylation of at least one histone, the amount of acetylated histone increases, and the accumulation of acetylated histone is a suitable biomarker for assessing the activity of an HDAC inhibitor. Thus, a procedure for measuring the accumulation of acetylated histone can be used to determine the HDAC inhibitory activity of a relevant compound. It should be understood that a compound capable of inhibiting histone deacetylase activity may also bind to other substrates and thus may inhibit other bioactive molecules, such as enzymes.

[0074] "Hydrate" refers to incorporation of water in stoichiometric proportion into the lattice of the compounds described herein, resulting in the formation of an adduct. Methods for preparing hydrates include, but are not limited to, storage in an atmosphere containing water vapor, dosage forms containing water, or conventional pharmaceutical processing steps such as, for example, crystallization (i.e., crystallization from water or a mixed aqueous solvent), lyophilization, wet granulation, aqueous film coating, or spray drying. In some cases, hydrates can also be formed from crystalline solvates upon exposure to water vapor or when an anhydrous material is suspended in water. Hydrates can also crystallize in more than one form, resulting in hydrate polymorphism. See, e.g., (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing hydrates are well within the capabilities of those skilled in the art, are entirely routine, and do not require any experimentation beyond typical methods 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 light optical microscopy, thermal microscopy, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, 1999). In addition, many commercial companies typically 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 ).

[0075] As used herein, "hydroxamic acid derivative histone deacetylase inhibitor" refers to a class of histone deacetylase inhibitors that are hydroxamic acid derivatives.

[0076] As used herein, "residue of a hydroxamic acid derivative histone deacetylase inhibitor" refers to the entire moiety of a hydroxamic acid derivative histone deacetylase inhibitor excluding the hydroxamic acid moiety.

[0077] As used herein, "pharmaceutical composition" refers to at least one compound and a pharmaceutically acceptable carrier, the compound being administered to a patient together with the carrier.

[0078] "Pharmaceutically acceptable salts" as used herein refers to salts of compounds that have the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxy-naphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or coordination with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc.

[0079] "Pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle administered together with a compound.

[0080] "Patient" includes humans. The terms "person" and "patient" are used interchangeably herein.

[0081] "Preventing" or "prevention" as used herein refers to reducing the risk of acquiring a disease or abnormality (i.e., causing at least one clinical symptom of the disease not to appear in a patient who may be exposed to the disease or is predisposed to the disease but has not yet experienced or manifested symptoms of the disease).

[0082] "Prodrug" as used herein refers to a derivative of a drug molecule that requires conversion in the body to release the active drug. Prodrugs are typically (although not necessarily) pharmacologically inactive until converted to the parent drug.

[0083] "Precursor moiety" as used herein refers to a form of protecting group that, when used to mask a functional group in a drug molecule, converts the drug into a prodrug. Typically, the precursor moiety will be linked to the drug by a bond that is cleaved in the body by enzymatic or non-enzymatic means.

[0084] As used herein, "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces, or prevents the reactivity of the functional group. Examples of protecting groups can be found in Green et al., "Protective Groups in Organic Chemistry", (Wiley, 2 nd nd ed. 1991) and Harrison et al., "Compendium of Synthetic Organic Methods", Vols. 1-8 (John Wiley and Sons, 1971-1996). Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("Cbz"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilylethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("Fmoc"), nitro-veratryloxycarbonyl ("Nvoc"), etc. 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 and alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, and allyl ethers.

[0085] As used herein, "senescence" or "senescent cell" refers to a state in which a cell acquires one or more senescence markers in response to certain cellular stresses. Such markers can typically include permanent exit from the cell cycle, expression of a bioactive secretome of inflammatory factors, altered methylation, senescence-associated heterochromatic foci (SAHF), expression markers of oxidative stress, DNA damage, expression of markers of protein and lipid modification, morphological features of senescence, altered lysosomes / vacuoles, and expression of senescence-associated β-galactosidase (see Lorenzo Galluzzi et al. (eds.), Cell Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 965, DOI 10.1007 / 978-1-62703-239-1_4, Springer Science+Business Media, LLC 2013).

[0086] As used herein, an "anti-aging agent" refers to an agent that "selectively" (preferably or to a greater extent) destroys, kills, eliminates senescent cells or promotes the selective destruction of senescent cells. In other words, compared to its ability to destroy or kill non-senescent cells, an anti-aging agent destroys or kills senescent cells in a biologically, clinically, and / or statistically significant manner. The amount and duration of use of the anti-aging agent are sufficient to selectively kill established senescent cells, but not sufficient to kill non-senescent cells in a clinically or biologically significant manner. In certain embodiments, the anti-aging agents described herein alter at least one signaling pathway in a manner that induces (i.e., initiates, stimulates, triggers, activates, promotes) and results in senescent cell death.

[0087] "Hydrate" refers to the incorporation of water into the crystal lattice of the compounds described herein in stoichiometric proportions, resulting in the formation of an adduct. Methods for preparing hydrates include, but are not limited to, storage in an atmosphere containing water vapor, dosage forms containing water, or conventional pharmaceutical processing steps such as, for example, crystallization (i.e., crystallization from water or a mixed aqueous solvent), lyophilization, wet granulation, aqueous film coating, or spray drying. In some cases, hydrates can also be formed from crystalline solvates upon exposure to water vapor or when an anhydrous material is suspended in water. Hydrates can also crystallize in more than one form, resulting in hydrate polymorphism. See, for example (Guillory, K., Chapter 5, pp. 202 - 205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above methods for preparing hydrates are well within the capabilities of those skilled in the art, are entirely conventional, and do not require any experimentation beyond typical methods 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 light optical microscopy, thermal microscopy, thermogravimetric analysis, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205 - 208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, 1999). In addition, many commercial companies typically 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 )

[0088] 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 independently replaced by the same or different substituents.

[0089] Substituent groups that can be used to substitute saturated carbon atoms in a specified group or radical group include, but are not limited to, -R a , halogen, -O - , =O, -OR b , -SR b , -S - , =S, -NR c R c , =NR b , =N-OR b , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N-OR b , -N-NR c R c , -NR b S(O) 2 R b , =N 2 , -N 3 , -S(O) 2 R b , -S(O) 2 NR b R b , -S(O) 2 O - , -S(O) 2 OR b , -OS(O) 2 R b , -OS(O) 2 O - , -OS(O) 2 OR b , -OS(O) 2 NR 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(NRb )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) 2 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) 2 OR b 、 -NR b S(O) 2 R b 、 -NR b C(NCN)NR c R c 、 -NR b C(NR b )R b and -NR bC(NR b )NR c R c , wherein R a is independently alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; each R b is independently hydrogen, R a , substituted alkyl, substituted heteroalkyl, substituted aryl, substituted arylalkyl, substituted heteroaryl, and substituted heteroarylalkyl; and each R c is independently R b , or alternatively, two R c together with the nitrogen atom to which they are attached form a 4-, 5-, 6-, or 7-membered heterocycloalkyl, substituted heterocycloalkyl, or heterocycloalkyl fused to an aryl group, which aryl group may optionally contain 1 to 4 additional heteroatoms, the same or different, selected from the group consisting of O, N, and S. As a specific example, -NR c R c means containing -NH 2 , NH-alkyl, N-pyrrolidinyl, and N-morpholinyl. Similarly, the substituent groups that can be used to substitute the unsaturated carbon atoms in the specified groups or radicals include, but are not limited to, -R a , halogen, -O - , -OR b , -SR b , -S - , -NR c R c , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -S(O) 2 R b , -S(O) 2 O - , -S(O) 2 OR b , -OS(O) 2 R b , -OS(O) 2 O - , -OS(O) 2 OR 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(NRb )R b 、-C(O)O - 、-C(O)OR b 、-C(S)OR b 、-C(O)NR c R c 、-C(NR b )NR c R c 、-OC(O)R b 、-OC(S)R b 、-OC(O)O - 、-OC(O)OR b 、-OC(S)OR b 、-OC(O)NR c R c 、-OS(O) 2 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) 2 OR a 、-NR b S(O) 2 R 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 As defined above. Substituent groups that can be used to replace the nitrogen atoms in heteroalkyl and cycloheteroalkyl include but are not limited to -R a , -O - 、-OR b 、-SR b , -S - 、-NR c R c, trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R b , -S(O) 2 O - , -S(O) 2 OR b , -OS(O) 2 R b , -OS(O) 2 O - , -OS(O) 2 OR 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 cAs defined above. Substituent groups from the above list that can be used to replace other specified groups or atoms will be apparent to those skilled in the art. In some embodiments, the substituents used to replace the specified groups can be further substituted, typically by one or more of the same or different groups selected from the various groups above.

[0090] As used herein, "treating" or "treatment" of any disease or disorder in some embodiments refers to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of its clinical symptoms). In other embodiments, "treating" or "treatment" refers to ameliorating at least one physical parameter that may not be discernible by a patient. In yet other embodiments, "treating" or "treatment" refers to inhibiting the disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In yet other embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

[0091] As used herein, "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 of the disease. A "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the patient to be treated.

[0092] Specific embodiments of the compounds and methods will now be referred to in detail. The disclosed embodiments are not intended to limit the claims. Instead, the claims are intended to cover all alternatives, modifications, and equivalents.

[0093] Detailed Description

[0094] Anti-aging agent

[0095] The hydroxamate derivative HDAC inhibitors that have been approved for clinical treatment of hematological cancers such as T-cell lymphoma, leukemia, and multiple myeloma include vorinostat (suberoylanilide hydroxamic acid or SAHA (1)), belinostat (2), and panobinostat (3). Many other hydroxamate derivative HDAC inhibitors (e.g., compounds (4)-(13)) are undergoing clinical studies for the treatment of hematological and solid tumors as single agents or in combination therapies with other oncolytic compounds. In addition to multiple inhibitory enzymes in classes I, II, and IV of HDAC, hydroxamate derivatives have been designed to simultaneously inhibit other therapeutic targets, such as CUDC-101 (12) (which effectively inhibits EGFR and HER-2 kinases) and CUDC-907 (13) (which additionally inhibits various PI3K isotypes). Many other hydroxamate derivative HDAC inhibitors have been disclosed, including the natural product trichostatin A (14) isolated from Streptomyces and many synthetically derived compounds, examples of which include compounds (15)-(21) and other compounds disclosed in Roche and Bertrand above; or any hydroxamic acid disclosed in U.S. Pat. Nos. 5,369,108, 5,932,616, 6,087,367, and 6,511,990.

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Previously, the anti-aging activity of the pan-HDAC inhibitor panobinostat (3) has been reported (Samaraweera et al. supra), and aging has been shown to be associated with a decrease in global histone acetylation (Li et al., Proteomics 13 (2013) 2585-2596). Several reports have documented the HDAC inhibitor-mediated decrease in Bcl-xL expression (e.g., see 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 being bound by any theory, one pharmacological basis for the anti-aging activity of HDAC inhibitors may be mediated by a decrease in the anti-apoptotic Bcl-xL protein level.

[0102] In some embodiments, the compounds effective as anti-aging agents are compounds of formula (IV) or (V):

[0103]

[0104] wherein: R is the residue of a hydroxamic acid derivative histone deacetylase inhibitor, R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are each independently hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII):

[0105]

[0106] R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are each independently hydrogen or -C(O)-R 2 ; each R 1 is independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 is one of the moieties of formula (VI) or formula (VII), then R 13 , R14 , R 15 and R 16 The remainder of is hydrogen or C(O)-R 1 ; and each R 2 is independently hydrogen, C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 is part of formula (VI) or formula (VII), then the remainder of R 23 , R 24 or R 25 is hydrogen or -C(O)-R 1 ; provided that when R 13 , R 14 , R 15 and R 16 are each hydrogen, R is not 7-heptanoylbenzamide.

[0107] In some embodiments of the compounds of formula (IV) or (V), the anomeric carbon (labeled *) of the pyranose ring is in the S configuration, and these compounds are β-D-galactoside and α-L-fucoside conjugates of hydroxamic acid derivative histone deacetylase inhibitors, respectively.

[0108] In some embodiments of the compounds 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, quinosinostat, vorinostat, dacinostat, givinostat, CUDC-907, CUDC-101, abexinostat, belinostat, pivanostat, reinostat, licinostat, trichostatin A, APHA, trichostatin A, oxamflatin and AR-42.

[0109] In some embodiments of the compounds of formula (IV), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (V), R 23 , R 24 and R 25 are each hydrogen.

[0110] In other embodiments of the compounds of formula (IV), R 13 , R 14 , R 15 and R 16 are each independently -C(O)-R 1 , wherein R 1 is C 1-4An alkyl group or a phenyl group. In other embodiments of the compound of formula (V), R 23 , R 24 and R 25 are each independently -C(O)-R 1 , where R 1 is C 1-4 alkyl or a phenyl group.

[0111] In still other embodiments of the compound of formula (IV), R 13 , R 14 , R 15 and R 16 are each independently -C(O)-R 1 , where R 1 is methyl. In still other embodiments of the compound of formula (V), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is methyl.

[0112] In still other embodiments of the compound of formula (IV), R 13 , R 14 , R 15 and R 16 are each independently -C(O)-R 1 , where R 1 is ethyl. In still other embodiments of the compound of formula (V), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl.

[0113] In a further embodiment, the compound of formula (IV) is a compound having any one of the following structures:

[0114]

[0115]

[0116] In a further embodiment, the compound of formula (V) is a compound having any one of the following structures:

[0117]

[0118]

[0119]

[0120] In yet a further embodiment, the compound of formula (IV) is a compound having any of the following structures;

[0121]

[0122]

[0123] In yet a further embodiment, the compound of formula (V) is a compound having any of the following structures:

[0124]

[0125]

[0126] In yet a further embodiment, the compound of formula (IV) is a compound having any of the following structures:

[0127]

[0128]

[0129]

[0130]

[0131] In yet a further embodiment, the compound of formula (V) is a compound having any of the following structures:

[0132]

[0133]

[0134]

[0135] The compound of formula (IV) or (V) can be synthesized by coupling the carboxylic acid precursor RCO 2 H (VIII) of the hydroxamic acid HDAC inhibitor with the glyoxime compounds (IX) and (X) respectively in the presence of an acyl coupling agent such as carbodiimide (e.g., EDC), or alternatively after pre-activation as an acyl chloride or mixed anhydride acylating agent, as shown below.

[0136]

[0137] Specifically, the O-β-galactoside derivative of SAHA, compound (22), can be prepared from the known bromogalactoside (23) according to the method of Thomas et al. Bioorg. Med. Chem. Lett. 17 (2007) 983-986:

[0138]

[0139]

[0140] 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 mainly depends on the zinc chelating activity of the free hydroxamic acid moiety (e.g., see Roche and Bertrand above). Thus, masking the hydroxamic acid functional group as a glycoside derivative in the compounds of formula (IV) or (V) ensures that these prodrugs are inactive as HDAC inhibitors but will become activated upon hydrolysis in the lysosomes of senescent cells.

[0141] Similarly, the O-β-galactoside derivative of panobinostat, compound (26), can be prepared by reductive amination of 4-formylcinnamic acid (27) with 2-(2-methyl-1H-indol-3-yl)ethylamine (28) (as described in International Application No. WO02 / 22577) and the basic nitrogen of the resulting protected amino acid, for example as a fluorenylmethoxycarbonyl (Fmoc) derivative (29). Coupling with compound (24) as described above, followed by successive deprotection, gives the panobinostat prodrug (26).

[0142]

[0143]

[0144] In an alternative route for compound (26), compound (24) is first coupled with (27) (e.g., using EDC, HOBt), and the resulting aldehyde is treated with the tryptamine derivative (28) in a reductive amination reaction, where removal of the acetyl protecting group mediated by sodium methoxide gives the prodrug (26).

[0145] Specific α-L-fucosyl conjugates of hydroxamic acid derivative histone deacetylase inhibitors can be prepared in a similar manner, starting from appropriately protected and activated fucose derivatives, which are prepared as described in Hou et al., Mater. Chem. Front. 1 (2017) 660 - 667 or U.S. Application No. 2015 / 0168374. For example, treatment of 1-fluoro-2,3,4-tri-O-acetyl-fucose (30) with N-hydroxyphthalimide gives a mixture of α- and β-L-fucosyl oxime derivatives, and the desired α-anomer (31) is the less polar product. Deprotection with hydrazine then gives α-L-fucosyl oxime (32), which can be further prepared into the α-L-fucoside prodrugs of SAHA and panobinostat, compounds (33) and (35), respectively:

[0146]

[0147]

[0148] Examples of Hsp90 inhibitors are the resorcinol compounds AT13387 (onalespib (36), NYP-AUY922 (luminespib (37)), ganetespib (38), VER-50589 (39), VER-49009 (40), CCT018159 (41) and KW-2478 (42), 2-(4-aminocyclohexanol)-benzamide derivatives exemplified by SNX-2112 (43) and (SNX-7081) (44). In some embodiments, the O-galactoside or O-fucoside conjugate of the Hsp90 inhibitor is an anti-aging compound. In other embodiments, the O-β-D-galactoside or O-α-L-fucoside conjugate of the Hsp90 inhibitor is an anti-aging compound.

[0149]

[0150]

[0151] In some embodiments, the compound of Formula (XI), (XII), (XIII) or (XIV) is an anti-aging agent:

[0152]

[0153] wherein Y is a carbonyl group or is 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 chlorine; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are independently hydrogen, C(O)-R 1 , a portion of formula (VI) or a portion of formula (VII);

[0154]

[0155] R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are independently hydrogen or -C(O)-R 2; each R 1 is independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 is part of formula (VI) or formula (VII), then the remaining portions of R 13 , R 14 , R 15 and R 16 are hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 is part of formula (VI) or formula (VII), then the remaining portions of R 23 , R 24 or R 25 are hydrogen or -C(O)-R 1 .

[0156] In some embodiments of the compounds of formula (XI), (XII), (XIII) or (XIV), the anomeric carbon (labeled *) of the pyranose ring is in the S configuration, and the compounds are β-D-galactoside and α-L-fucoside conjugates of resorcinol Hsp90 inhibitors, respectively.

[0157] In some embodiments of the compounds of formula (XI), (XII), (XIII) or (XIV), the moiety A-Y-C 6 H 2 (OH) 2 -B is an Hsp90 inhibitor selected from the group consisting of luminespib (NVP-AUY922), ganetespib, VER-50589, AT13387 and KW-2478.

[0158] In some embodiments of the compounds of formula (XI), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XII), R 23 , R 24 and R 25 are each hydrogen. In some embodiments of the compounds of formula (XIII), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XIV), R23 、R 24 and R 25 each is hydrogen.

[0159] In some embodiments of the compound of formula (XI), R 13 、R 14 、R 15 and R 16 each independently is -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XII), R 23 、R 24 and R 25 each independently is -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XIII), R 13 、R 14 、R 15 and R 16 each independently is -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XIV), R 23 、R 24 and R 25 each independently is -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl.

[0160] In some embodiments of the compound of formula (XI), R 13 、R 14 、R 15 and R 16 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compound of formula (XII), R 23 、R 24 and R 25 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compound of formula (XIII), R 13 、R 14 、R 15 and R 16 each is -C(O)-R 1 , where R 1is methyl. In some embodiments of the compound of formula (XIV), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is methyl.

[0161] In some embodiments of the compound of formula (XI), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compound of formula (XII), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compound of formula (XIII), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compound of formula (XIV), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl.

[0162] In a further embodiment, the compound of formula (XI) is a compound having any one of the following structures:

[0163]

[0164]

[0165] In a further embodiment, the compound of formula (XII) is a compound having any one of the following structures:

[0166]

[0167]

[0168] In a further embodiment, the compound of formula (XIII) is a compound having any one of the following structures:

[0169]

[0170]

[0171] In a further embodiment, the compound of formula (XIV) is a compound having any of the following structures:

[0172]

[0173]

[0174]

[0175] The compounds of formulae (XI) and (XIII) can be prepared by reacting a compound of formula (XV) with a protected D-galactosyl donor moiety under classical BF 3 -mediated glycosylation or Koenigs-Knorr coupling conditions, where the resulting regioisomers are separated by chromatographic means. Alternatively, the phenolic hydroxyl group of the resorcinol compound (XV) can first be selectively protected to allow regioselective glycosylation.

[0176]

[0177]

[0178] Specifically, the O-β-galactoside conjugates of AT13387 (36) (prepared as described in U.S. Patent No. 8,779,132), namely 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.

[0179]

[0180]

[0181]

[0182] Similarly, the O-β-galactoside conjugates of NVP-AUY922 (37) (prepared as described in Brough et al., J. Med. Chem. 51 (2008) 196-218), namely compounds (48) and (49), can be prepared by the reaction of (37) with (23):

[0183]

[0184] Compounds (50)-(53), specific α-L-fucosyl conjugates of the Hsp90 inhibitors (36) and (37), can be prepared in a similar manner starting from the protected fucose derivative (45):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)), and the O-α-fucosyl 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.

[0185]

[0186]

[0187]

[0188] There are other embodiments that relate to O-D-galactosyl and O-L-fucosyl conjugates of topoisomerase I (TOP1) inhibitory compounds as anti-aging compounds. Camptothecin (62), a cytotoxic pentacyclic quinoline alkaloid natural product, is a typical TOP1 inhibitor, and many synthetic analogs (including SN-38(63) and topotecan(64)) have been studied 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 dibenzo[h]naphthyridinones (exemplified by compounds (71)-(73)) (see, e.g., Sooryakumar et al., Mol.Cancer Ther. 10(2011)1490-1499).

[0189]

[0190] The O-β-D-galactoside conjugate of SN-38(74) (Chinese Patent No. CN1534046, 2004) and the O-β-L-fucosyl conjugate of SN-38(75) (Japanese Patent No. JP6328098, 1988) have been previously disclosed as antitumor agents.

[0191]

[0192] In some embodiments, the compounds of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV) or (XXV) are anti-aging agents:

[0193]

[0194]

[0195]

[0196]

[0197] wherein R 8 is a heteroaryl or heterocyclic group containing at least one nitrogen atom; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are each independently hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII):

[0198]

[0199] R 33 , R 34 , R 35 , R 36 , R 43 , R 44 and R 45 are each independently hydrogen or -C(O)-R 2 ; each R 1 is independently C 1-4 alkyl or phenyl, provided that if one of R 13 , R 14 , R 15 or R 16 is a moiety of formula (VI) or formula (VII), then the remaining portions of R 13 , R 14 , R 15 and R 16 are hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if one of 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 .

[0200] In some embodiments of the compounds of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV) or (XXV), the anomeric carbon (labeled *) of the pyranose ring has the S configuration, and these 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.

[0201] In some embodiments of the compounds of formula (XVI), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XVII), R 23 , R 24 and R 25 are each hydrogen. In some embodiments of the compounds of formula (XVIII), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XIX), R 23 , R 24 and R 25 are each hydrogen. In some embodiments of the compounds of formula (XX), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XXI), R 23 , R 24 and R 25 are each hydrogen. In some embodiments of the compounds of formula (XXII), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XXIII), R 23 , R 24 and R 25 are each hydrogen. In some embodiments of the compounds of formula (XXIV), R 13 , R 14 , R 15 and R 16Each is hydrogen. In some embodiments of the compound of formula (XXV), R 23 、R 24 and R 25 are each hydrogen.

[0202] In some embodiments of the compound of formula (XVI), R 13 、R 14 、R 15 and R 16 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XVII), R 23 、R 24 and R 25 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XVIII), R 13 、R 14 、R 15 and R 16 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XIX), R 23 、R 24 and R 25 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XX), R 13 、R 14 、R 15 and R 16 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XXI), R 23 、R 24 and R 25 are each independently -C(O)-R 1 wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compound of formula (XXII), R 13 、R 14 、R 15 and R 16 are each independently -C(O)-R1 , wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compounds of formula (XXIII), R 23 , R 24 and R 25 are each independently -C(O)-R 1 , wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compounds of formula (XXIV), R 13 , R 14 , R 15 and R 16 are each independently -C(O)-R 1 , wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compounds of formula (XXV), R 23 , R 24 and R 25 are each independently -C(O)-R 1 , wherein R 1 is C 1-4 alkyl or phenyl.

[0203] In some embodiments of the compounds of formula (XVI), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , wherein R 1 is methyl. In some embodiments of the compounds of formula (XVII), R 23 , R 24 and R 25 are each -C(O)-R 1 , wherein R 1 is methyl. In some embodiments of the compounds of formula (XVIII), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , wherein R 1 is methyl. In some embodiments of the compounds of formula (XIX), R 23 , R 24 and R 25 are each -C(O)-R 1 , wherein R 1 is methyl. In some embodiments of the compounds of formula (XX), R 13 , R 14 , R 15 and R16 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XXI), R 23 , R 24 and R 25 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XXII), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XIII), R 23 , R 24 and R 25 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XXIV), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XXV), R 23 , R 24 and R 25 each is -C(O)-R 1 , where R 1 is methyl.

[0204] In some embodiments of the compounds of formula (XVI), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XVII), R 23 , R 24 and R 25 each is -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XVIII), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , where R 1is ethyl. In some embodiments of the compounds of formula (XIX), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XX), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXI), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXII), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXIII), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXIV), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXV), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl.

[0205] In still other embodiments, O-D-galactosyl and O-L-fucosyl conjugates of DNA-alkylating agents based on cytotoxic calicheamicin-family antibiotics are anti-aging agents. Calicheamicin SA(76) isolated from Streptomyces DO-113 contains a highly reactive spirocyclopropyl cyclohexadienone moiety and has served as inspiration for the design of monosaccharide and disaccharide derivatives such as galactosyl compound (77) (e.g., see Tietze et al., Angew. Chem. Int. Ed. 45 (2006) 6574-6577; Tietze et al., J. Med. Chem. 52 (2009) 537-543). The cytotoxicity of compound (77) is more than 4000-fold lower than that of its hydrolyzed ring-opened product (78), which undergoes a so-called Winstein in situ cyclization to give the DNA-reactive spirocyclopropyl cyclohexadienone (79).

[0206]

[0207]

[0208] In some embodiments, the compounds of formula (XXVI) or (XXVII) are anti-aging agents:

[0209]

[0210]

[0211] wherein R 13 、R 14 、R 15 、R 16 、R 23 、R 24 and R 25 are each independently hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII):

[0212]

[0213] R 33 、R 34 、R 35 、R 36 、R 43 、R 44 and R 45 are each independently hydrogen or -C(O)-R 2 ; each R 1 is independently C 1-4 alkyl or phenyl, provided that if R 13 、R 14 、R 15 or R16 One is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 's remaining part is hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 one is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 's remaining part is hydrogen or -C(O)-R 1 ; provided that in the compound of formula (XXVI), R 13 , R 14 , R 15 and R 16 are not simultaneously hydrogen or acetyl.

[0214] In some embodiments of the compounds of formula (XXVI) or (XXVII), the anomeric carbon (labeled *) of the pyranose ring is in the S configuration, and these compounds are respectively the β-D-galactoside and α-L-fucoside conjugates of the doxorubicin analog.

[0215] In some embodiments of the compounds of formula (XXVI), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XXVII), R 23 , R 24 and R 25 are each hydrogen.

[0216] In some embodiments of the compounds of formula (XXVI), R 13 , R 14 , R 15 and R 16 are each independently -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compounds of formula (XXVII), R 23 , R 24 and R 25 are each independently -C(O)-R 1 , where R 1 is C 1-4 alkyl or phenyl.

[0217] In some embodiments of the compounds of formula (XXVI), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is methyl. In some embodiments of the compounds of formula (XXVII), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is methyl.

[0218] In some embodiments of the compounds of formula (XXVI), R 13 , R 14 , R 15 and R 16 are each -C(O)-R 1 , where R 1 is ethyl. In some embodiments of the compounds of formula (XXVII), R 23 , R 24 and R 25 are each -C(O)-R 1 , where R 1 is ethyl.

[0219] In some embodiments of the compounds of formula (XXVII), compound (80) is synthesized from compound (81) (prepared according to the method of Tietze et al. described above):

[0220]

[0221] There are other embodiments related to O-D-galactosyl and O-L-fucosyl conjugates of cytotoxic pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) as anti-aging agents. PBDs are a family of antitumor antibiotics that include the natural product anthramycin (85). These compounds covalently bind via their N10-C11 imine functional group to the exocyclic NH of guanine residues in the minor groove of DNA 2It exerts its cytotoxic effect on the group (see, for example, Antonow and Thurston, Chem. Rev. 111 (2011) 2815 - 2864; and Mantaj et al., Angew. Chem. Int. Ed. 56 (2017) 462 - 488). PBD monomers show significant cytotoxicity, and connecting two PBD monomers through a linker generates a PBD dimer that is capable of interstrand DNA cross - linking. SJG - 136(86) is such a dimer with high cytotoxic potency and has been used to construct antibody - drug conjugates with clinical utility.

[0222]

[0223] Kamal and colleagues have described β - galactoside analogs of both PBD monomers and dimers as anticancer agents (for example, see compounds (87) and (88) (Kamal et al., ChemMedChem 3 (2008) 794 - 802)).

[0224]

[0225]

[0226] In some embodiments, the compounds of formula (XXVIII) or (XXIX) are anti - aging agents:

[0227]

[0228] wherein R 9 is hydrogen, C 1-4 alkyl, CF 3 3, CN or NO 2 2; R 10 is hydrogen, C 1-4 alkyl or arylalkyl; R 13 1, R 14 2, R 15 3, R 16 4, R 23 5, R 24 6 and R 25 7 are each independently hydrogen, C(O) - R 1 8, a moiety of formula (VI) or a moiety of formula (VII):

[0229]

[0230] R 33 8, R 34 9, R 35 10, R 36 11, R 43 12, R44 and R 45 each independently is hydrogen or C(O)-R 2 and each R 1 is independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 is part of formula (VI) or formula (VII), then the remainder of R 13 , R 14 , R 15 and R 16 is hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 is part of formula (VI) or formula (VII), then the remainder of R 23 , R 24 or R 25 is hydrogen or C(O)-R 1 ; provided that in the compound of formula (XXVIII), when R 9 is NO 2 and R 10 is benzyl, R 13 , R 14 , R 15 , R 16 are not simultaneously hydrogen or acetyl.

[0231] In some embodiments of the compounds of formula (XXVIII) or (XXIX), the anomeric carbon (labeled *) of the pyranose ring is of the S configuration, and the compounds are β-D-galactoside and α-L-fucoside conjugates of pyrrolo[2,1-c][1,4]benzodiazepine analogs, respectively.

[0232] In some embodiments of the compounds of formula (XXVIII), R 13 , R 14 , R 15 and R 16 are each hydrogen. In some embodiments of the compounds of formula (XXIX), R 23 , R 24 and R 25 are each hydrogen.

[0233] In some embodiments of the compounds of formula (XXVIII), R 13 , R 14 , R 15 and R16 each independently is -C(O)-R 1 , wherein R 1 is C 1-4 alkyl or phenyl. In some embodiments of the compounds of formula (XXIX), R 23 , R 24 and R 25 each independently is -C(O)-R 1 , wherein R 1 is C 1-4 alkyl or phenyl.

[0234] In some embodiments of the compounds of formula (XXVIII), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , wherein R 1 is methyl. In some embodiments of the compounds of formula (XXIX), R 23 , R 24 and R 25 each is -C(O)-R 1 , wherein R 1 is methyl.

[0235] In some embodiments of the compounds of formula (XXVIII), R 13 , R 14 , R 15 and R 16 each is -C(O)-R 1 , wherein R 1 is ethyl. In some embodiments of the compounds of formula (XXIX), R 23 , R 24 and R 25 each is -C(O)-R 1 , wherein R 1 is ethyl.

[0236] In some embodiments of the compounds of formula (XXVIII), compound (89) is synthesized from compound (90) (prepared according to the method of Kamal et al. described above):

[0237]

[0238] Specifically, as an example of the compounds of formula (XXIX), compound (91) is prepared from compound (90) using a similar synthetic method.

[0239]

[0240] In still other embodiments, the O-galactoside or O-fucosidase conjugate of an Akt inhibitor is an anti-aging agent. Akt inhibitors useful for preparing such conjugates are exemplified by compounds such as ipatasertib (or GDC-0068) (92), AZD5363 (93), and triapine (94). In some embodiments, the O-β-D-galactoside or O-α-L-fucosidase conjugate of an Akt inhibitor is an anti-aging agent.

[0241]

[0242] Specific compounds are exemplified by compounds (95)-(100), where each R 46 is hydrogen, acetyl, or propionyl, and is prepared according to the methods previously disclosed herein:

[0243]

[0244]

[0245]

[0246] In some embodiments, a compound of formula (I), exemplified by 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101), is converted to an apoptotic compound of formula (II), specifically the cytotoxin 5-fluorouridine (FUR) (102) produced by the action of intracellular β-galactosidase (i.e., SA-β-Gal) enriched in senescent cells:

[0247]

[0248] In another embodiment, a compound of formula (I), exemplified by 5-fluorouridine-5'-O-α-L-fucopyranoside (FURFuc) (106), is converted to an apoptotic compound of formula (II), specifically the cytotoxin 5-fluorouridine (FUR) (102) produced by the action of intracellular α-fucosidase enriched in senescent cells:

[0249]

[0250] In additional embodiments, the O-galactoside or O-fucosidase conjugate of a proteasome inhibitor is an anti-aging agent. Proteasome inhibitors useful for preparing such conjugates are exemplified by compounds such as delanzomib (103). In some embodiments, the O-β-D-galactoside or O-α-L-fucosidase conjugates (104) and (105) are anti-aging agents.

[0251]

[0252] Methods for characterizing and identifying anti - aging agents

[0253] The characteristics of anti - aging agents can be determined by using one or more cell - based assays and one or more animal models described herein or in the art and familiar to those skilled in the art. Anti - aging agents can selectively kill one or more types of senescent cells (e.g., senescent preadipocytes, senescent endothelial cells, senescent fibroblasts, senescent neurons, senescent epithelial cells, senescent mesenchymal cells, senescent smooth muscle cells, senescent macrophages, or senescent chondrocytes). In certain embodiments, the anti - aging agent is capable of selectively killing at least senescent fibroblasts.

[0254] Characterizing a compound as an anti - aging agent can be accomplished using one or more cell - based assays and one or more animal models described herein or in the art. Those skilled in the art will readily understand that characterizing a compound as an anti - aging agent and determining the killing level of the compound can be done by comparing the activity of the test agent with appropriate negative controls (e.g., vehicle only or diluent and / or compositions or compounds known in the art that do not kill senescent cells) and appropriate positive controls. In vitro cell - based assays for characterizing anti - aging agents also include controls for determining the effect of the agent on non - senescent cells (e.g., quiescent cells or proliferating cells). Compared to one or more negative controls, the anti - aging agent reduces (i.e., decreases) the percentage of survival of multiple senescent cells (i.e., reduces the number of surviving senescent cells in an animal or cell - based assay in some manner). The conditions for a particular in vitro assay, including temperature, buffer (including salts, cations, medium), and other components that maintain the integrity of the test agents and reagents used in the assay, are familiar to those skilled in the art and / or can be readily determined by routine experimentation.

[0255] The source of senescent cells for assay can be primary cell cultures or cell lines adapted to 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 hybrid cell lines, differentiated or differentiable cell lines, transformed cell lines, etc. In some embodiments, senescent cells are isolated from biological samples obtained from a host or subject suffering from a disease or abnormality associated with senescent cells. In other embodiments, non-senescent cells (e.g., primary cells obtained from a subject or cell lines suitable for growth in culture) are used and senescence is induced by methods described herein and in the art, such as by exposure to radiation or chemotherapeutic agents (e.g., doxorubicin). Biological samples can be, for example, blood samples, biopsy samples, body fluids (e.g., bronchoalveolar lavage fluid, ascites, mucosal washings, synovial fluid, etc.), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation obtained from a subject. Biological samples can be tissue or cell preparations in which the morphological integrity or physical state has been disrupted, for example, by dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication, or any other means for processing samples from a subject or biological source. The subject can be a human or non-human animal. As an example, the anti-senescent effects of certain compounds of the invention on human fibroblasts in culture are characterized in Example 23 herein, and the anti-senescent effects on mouse embryonic fibroblasts are characterized in Example 24 herein. Examples 26 and 27 demonstrate that in vivo administration of the anti-senescent compounds of the invention results in a reduction of senescent hepatocytes in mice. Example 28 demonstrates that in vivo administration of the anti-senescent compounds of the invention results in a reduction of senescent lung cells in mice.

[0256] Transgenic animal models as described herein and in the art can be used to determine the killing or removal of senescent cells (see, for example, Baker et al. supra; Nature, 479 (2011) 232-236; International Application No. WO / 2012 / 177927; International Application No. WO 2013 / 090645). Exemplary transgenic animal models contain a transgene that includes a nucleic acid allowing for the controlled clearance of senescent cells (e.g., p16INK4a-positive senescent cells) as a positive control. The presence and level of senescent cells in transgenic animals can be determined by measuring the level of one or more detectable markers expressed in the senescent cells of the animal. The transgenic nucleotide sequence includes detectable markers, such as one or more red fluorescent proteins; green fluorescent proteins; and one or more luciferases to detect the clearance of senescent cells.

[0257] The animal models described herein or in the art include models accepted in the art for determining the effectiveness of anti-aging agents in treating or preventing specific age-related diseases or abnormalities (i.e., reducing the likelihood of occurrence of specific age-related diseases or abnormalities), such as atherosclerosis models, osteoarthritis models, COPD models, IPF models, etc. As described herein, murine models of lung diseases, such as bleomycin-induced pulmonary fibrosis models and chronic smoking models, are applicable to diseases such as COPD and can be routinely practiced by those skilled in the art. Animal models for determining the effectiveness of anti-aging agents in treating and / or preventing side effects of chemotherapy and radiotherapy (i.e., reducing the likelihood of occurrence of side effects of chemotherapy and radiotherapy) or for treating or preventing metastatic lesions (i.e., reducing the likelihood of occurrence of metastatic lesions) are described in International Applications Nos. WO 2013 / 090645 and WO 2014 / 205244. Animal models for determining the effectiveness of agents for treating eye 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).

[0258] As a non-limiting example and as described herein, an animal model of osteoarthritis has been developed. Osteoarthritis can be induced in animals, for example, by incomplete or total surgical transection of the anterior cruciate ligament, inducing damage to the joint, for example, in the knee. The osteoarthritis animal model can be used to evaluate the effectiveness of anti-aging agents in treating or preventing osteoarthritis (i.e., reducing the likelihood of occurrence of osteoarthritis), and results in a reduction in proteoglycan erosion, and induces (i.e., stimulates, enhances) the production of collagen (such as type II collagen), and alleviates pain in animals that have undergone 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 can also be performed using the methods and techniques described herein, such as assays for determining the levels of inflammatory molecules (e.g., IL-6) and assays for determining the levels of aging markers as described above, and these methods and techniques can be routinely practiced by those skilled in the art.

[0259] 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 the animals a high-fat diet or by using transgenic animals that are highly sensitive to developing atherosclerosis. The animal models can be used to determine the effectiveness of anti-aging agents in reducing the amount of plaque in atherosclerotic arteries or in inhibiting the formation of plaque in atherosclerotic arteries, in reducing the lipid content of atherosclerotic plaques (i.e., reducing the amount of lipids in the plaque), and in causing an increase in the thickness of the fibrous cap of the plaque or enhancing the thickness of the fibrous cap of the plaque. Sudan staining can be used to detect the lipid levels in atherosclerotic blood vessels. Immunohistology and immunochemistry and molecular biology assays (e.g., for determining the levels of inflammatory molecules (e.g., IL-6), and for determining the levels of the aging markers as described above) can all be performed according to the methods described herein, which are routine practices in the art.

[0260] In yet another non-limiting example and as described herein, a mouse model in which animals are treated with bleomycin has been described (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) for determining the effectiveness of agents for treating IPF. In animal models of lung diseases (e.g., bleomycin animal models, animal models exposed to smoke, etc.), respiratory measurements can be made to determine elasticity, compliance, static compliance, and peripheral capillary oxygen saturation (SpO 2 ). Immunohistology and immunochemistry and molecular biology assays (e.g., for determining the levels of inflammatory molecules (e.g., IL-6), and for determining the levels of the aging markers as described above) can all be performed according to the methods described herein, which are routine practices in the art.

[0261] One or more statistical analyses familiar to those skilled in the art can be used to determine the effectiveness of anti-aging agents in selectively killing senescent cells in animal models as described herein. By way of example, statistical analyses such as two-way analysis of variance (ANOVA) are used to determine the statistical significance of the differences between the group of animals treated with the agent and the group of animals not treated with the agent (i.e., the negative control group, which can contain only the vehicle and / or non-anti-aging agent). Statistical software packages such as SPSS, MINITAB, SAS, Statistika, Graphpad, GLIM, Genstat, and BMDP are readily available and are routinely used by those skilled in the field of animal models.

[0262] Those skilled in the art will readily understand that the characterization of anti - aging agents and determination of the killing level by such anti - aging agents can be accomplished by comparing the activity of the test agent with appropriate negative controls (e.g., carrier only and / or compositions, agents or compounds known in the art that do not kill senescent cells) and appropriate positive controls. In vitro cell - based assays for characterizing agents also include controls for determining the effect of the agent on non - senescent cells (e.g., quiescent cells or proliferating cells). Compared with one or more negative controls, useful anti - aging agents reduce (i.e., decrease) the percentage of viable senescent cells (i.e., reduce the amount of viable senescent cells in an animal or in a cell - based assay in some manner). Thus, compared with killing non - senescent cells, anti - aging agents selectively kill senescent cells (which may be referred to herein as selectively killing senescent cells relative to non - senescent cells).

[0263] In certain embodiments (in an in vitro assay or in an in vivo assay (in a human or non - human animal)), at least one anti - aging agent kills at least 20% of senescent cells and kills no more than 5% of non - senescent cells. In other embodiments (in an in vitro assay or in an in vivo assay (in a human or non - human animal)), at least one anti - aging agent kills at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% of senescent cells and kills no more than about 5% or 10% of non - senescent cells. In still other embodiments (in an in vitro assay or in an in vivo assay (in a human or non - human animal)), at least one anti - aging 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 (in an in vitro assay or in an in vivo assay (in a human or non - human animal)), at least one anti - aging 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 (in an in vitro assay or in an in vivo assay (in a human or non - human animal)), at least one anti - aging 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, compared with killing non - senescent cells, anti - aging agents are at least 5 - 25 times, 10 - 50 times, 10 - 100 times or 100 - 1000 times more selective for killing senescent cells.

[0264] For a specific embodiment of the method for treating an aging-related disease or disorder described herein, the percentage of senescent cells killed can refer to the percentage of senescent cells killed in a tissue or organ that includes senescent cells that contribute to the onset, progression, and / or exacerbation of the disease or disorder. As a non-limiting example, brain tissue, tissues and parts of the eye, lung tissue, heart tissue, arteries, joints, skin, and muscle can include senescent cells that can be reduced by an anti-aging agent as described herein by the percentages described above, and thereby provide a therapeutic effect. Additionally, selectively removing at least 20% or at least 25% of the senescent cells from an affected tissue or organ can have a clinically significant therapeutic effect.

[0265] For a specific embodiment of the method described herein, such as treating a cardiovascular disease or disorder (such as atherosclerosis) related to arteriosclerosis by administering an anti-aging agent (i.e., referring to the in vivo methods described above), the percentage of senescent cells killed can refer to the percentage of senescent cells killed in an affected artery containing plaque relative to non-senescent cells killed in the artery plaque. In certain embodiments, in a method for treating a cardiovascular disease such as atherosclerosis as described herein, at least one anti-aging agent kills at least 20% of the senescent cells in the artery and kills no more than 5% of the non-senescent cells. In other embodiments, the anti-aging agent selectively kills at least 25% of the senescent cells in atherosclerotic arteries.

[0266] In some embodiments, for a method for treating osteoarthritis by administering an anti-aging agent as described herein, the percentage of senescent cells killed can refer to the percentage of senescent cells killed in an osteoarthritic joint relative to non-senescent cells killed in the osteoarthritic joint. In certain embodiments, in a method for treating osteoarthritis as described herein, at least one anti-aging agent kills at least 20% of the senescent cells in the osteoarthritic joint and kills no more than 5% of the non-senescent cells. In other embodiments, the anti-aging agent selectively kills at least 25% of the senescent cells in the osteoarthritic joint.

[0267] In some embodiments, for a method for treating an aging-related lung disease or disorder (e.g., COPD, IPF) by administering at least one anti-aging agent as described herein, the percentage of senescent cells killed can refer to the percentage of senescent cells killed in affected lung tissue relative to non-senescent cells killed in the affected lung tissue. In certain embodiments, in a method for treating aging-related lung diseases and disorders as described herein, the anti-aging agent kills at least 20% of the senescent cells in the affected lung tissue and kills no more than 5% of the non-senescent cells. In other embodiments, the anti-aging agent selectively kills at least 25% of the senescent cells in the affected lung tissue.

[0268] In certain embodiments, methods are provided for identifying (i.e., screening) agents that are useful anti-aging agents for treating or preventing diseases or abnormalities associated with aging (i.e., reducing the likelihood of occurrence of diseases or abnormalities associated with aging). In some embodiments, a method for identifying an anti-aging agent for treating such diseases and abnormalities includes inducing cellular senescence to provide established senescent cells. Methods for inducing cellular senescence are described herein and in the art and include, for example, exposure to radiation (e.g., 10 Gy is typically sufficient) or chemotherapeutic agents (e.g., doxorubicin or other anthracyclines). After exposure to the agent, the cells are cultured for an appropriate time under appropriate conditions (e.g., a medium, temperature, CO 2 / O 2 level) suitable for the given cell type or cell line to allow senescence to be established. As discussed herein, senescence of the cells can be determined by determining a number of characteristics, such as morphological changes (e.g., as observed by microscopy); production of, for example, senescence-associated β-galactosidase (SA-β-gal), p16INK4a, p21, or any one or more SASP factors (e.g., IL-6, MMP3). A sample of the senescent cells is then contacted with a candidate agent (i.e., the cells and the agent are mixed, bound, or otherwise allowed to interact). Those skilled in the art will understand that the assay will include appropriate negative and positive controls, either historical or concurrent. For example, a sample of control non-senescent cells that have been cultured similarly to the senescent cells but not exposed to a senescence inducer is contacted with the candidate agent. The survival level of the senescent cells is determined and compared to the survival level of the non-senescent cells. When the survival level of the senescent cells is less than the survival level of the non-senescent cells, the anti-aging agent is identified.

[0269] In some embodiments, the above method for identifying an anti-aging agent may further include steps for identifying whether the anti-aging agent can be used for treating osteoarthritis. The method may further include contacting the identified anti-aging agent with cells capable of producing collagen; and determining the level of collagen produced by the cells. In an embodiment, the cells are chondrocytes and the collagen is type II collagen. The method may further include administering a candidate anti-aging agent to a non-human animal having an arthritic injury in a joint and determining one or more of the following: (a) the level of senescent cells in the joint; (b) the physical function of the animal; (c) the level of one or more inflammatory markers; (d) the histology of the joint; and (e) the level of type II collagen produced, thereby determining the therapeutic efficacy of the anti-aging agent, wherein compared to an animal not treated with the anti-aging agent, one or more of the following are observed in the treated animal: (i) a decrease in the level of senescent cells in the joint of the treated animal; (ii) improved physical function of the treated animal; (iii) a decrease in the level of one or more inflammatory markers in the treated animal; (iv) increased histological normality in the joint 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 for determining the sensitivity of the leg to an induced or natural osteoarthritis condition, e.g., by the tolerance of the animal to bear weight on the affected limb or the ability of the animal to move away from an uncomfortable stimulus such as heat or cold. One or more statistical analyses familiar to those skilled in the art can be used to determine the effectiveness of an agent as described herein for killing senescent cells in an animal model. Statistical analyses as described herein and as routinely practiced in the art can be used to analyze the data.

[0270] In other embodiments, the above method for identifying an anti-aging agent may further include steps for identifying whether the anti-aging agent can be used for treating cardiovascular diseases caused by or associated with atherosclerosis. Thus, the method may further include administering an anti-aging candidate agent in a non-human animal or an animal model for determining the effectiveness of the agent in reducing the amount of plaque, inhibiting the formation of plaque in atherosclerotic arteries, reducing the lipid content of atherosclerotic plaques (i.e., reducing the amount of lipid in the plaque) and / or causing an increase in the fibrous cap thickness of the plaque or enhancing the fibrous cap thickness of the plaque. Sudan staining can be used to detect the lipid level in atherosclerotic blood vessels. Immunohistology, assays for determining the level of inflammatory molecules (e.g., IL-6) and / or assays for determining the level of senescence markers as described above can all be performed according to the methods described herein and as routinely practiced in the art.

[0271] In a specific embodiment, the method for identifying an anti-aging agent described herein may further comprise administering a candidate anti-aging agent to a non-human animal having atherosclerotic plaques and determining one or more of the following: (a) the level of senescent cells in the artery; (b) the physical function of the animal; (c) the level of one or more inflammatory markers; (d) the histology of the affected blood vessel (e.g., artery); and thereby determining the therapeutic effect of the anti-aging agent, wherein compared to an animal not treated with the anti-aging agent, one or more of the following are observed in the treated animal: (i) a decrease in the level of senescent cells in the artery of the treated animal; (ii) improved physical function of the treated animal; (iii) a decrease in the level of one or more inflammatory markers in the treated animal; (iv) increased histological normality in the artery of the treated animal. As described herein and in the art, the physical function of an animal can be determined by measuring physical activity. Statistical analysis as described herein and in the conventional practice of the art can be used to analyze the data.

[0272] In some embodiments, the method for identifying an anti-aging agent described herein may comprise administering a candidate anti-aging agent to a non-human animal model of lung disease, such as a bleomycin model or an animal model exposed to smoke, and determining one or more of the following: (a) the level of senescent cells in the lung; (b) the lung function of the animal; (c) the level of one or more inflammatory markers; (d) the histology of the lung tissue, thereby determining the therapeutic effect of the anti-aging agent, wherein compared to an animal not treated with the anti-aging agent, one or more of the following are observed in the treated animal: (i) a decrease in the level of senescent cells in the lung 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 inflammatory markers in the treated animal; and (iv) increased histological normality in the lung tissue of the treated animal. Respiratory measurements can be made to determine elasticity, compliance, static compliance, and peripheral capillary oxygen saturation (SpO 2 )). Lung function can be evaluated by determining any one of a number of measurements, such as expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in one second, FEV1), FEV1 / FEV ratio, 25% to 75% forced expiratory flow, and maximum voluntary ventilation (MVV), peak expiratory flow (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 of carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO.sub.2) can also be measured. Statistical analysis as described herein and in the conventional practice of the art can be used to analyze the data.

[0273] Methods for treating and preventing diseases and abnormalities associated with aging

[0274] Provided herein are methods for treating in a subject in need thereof a disorder, disease or abnormality associated with, associated or caused by cellular senescence, including age-related diseases and abnormalities. Age-related diseases or abnormalities may also be referred to herein as diseases or abnormalities associated with senescent cells. Age-related diseases and abnormalities include, for example, age-related diseases and abnormalities induced by senescence; pulmonary diseases and abnormalities; neurological diseases and abnormalities (e.g., neurodegenerative diseases and abnormalities); ophthalmic diseases and abnormalities; metabolic diseases and abnormalities; cardiovascular diseases and abnormalities; inflammatory diseases and abnormalities; autoimmune diseases and abnormalities; dermatological diseases and abnormalities; skin disorders; age-related diseases; and transplantation-related diseases and abnormalities. A prominent feature of aging is the progressive loss or deterioration of function that occurs at the molecular, cellular, tissue, and organismal levels. Age-related degeneration gives rise to well-recognized pathologies such as sarcopenia, atherosclerosis and heart failure, osteoporosis, lung insufficiency, kidney failure, neurodegeneration (including macular degeneration, Alzheimer's disease, and Parkinson's disease), and many other diseases. Although different mammalian species vary in their susceptibility to specific age-related pathologies, generally, age-related pathologies typically begin to increase with approximately exponential kinetics at about the midpoint of the species-specific lifespan (e.g., 50-60 years of age for humans) (see, e.g., Campisi, Annu. Rev. Physiol. 75 (2013) 685-705; Naylor et al., Clin. Pharmacol. Ther. 93 (2013) 105-116).

[0275] Examples of aging-related conditions, abnormalities, or diseases that can be treated by administering any of the anti-aging agents described herein according to the methods described herein include aging-related diseases and abnormalities (e.g., kyphosis, abnormal kidney function, frailty, hair loss, hearing loss, muscle fatigue, skin conditions, sarcopenia, and herniated discs), as well as other aging-related diseases induced by aging (e.g., diseases / abnormalities caused by radiation, chemotherapy, smoking, high-fat / high-sugar diet, and environmental factors); lung diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiolitis obliterans, asthma); proliferative diseases, including cancer and metastatic lesions; side effects associated with chemotherapy side effects or radiotherapy; fibrotic diseases and abnormalities (e.g., cystic fibrosis, renal fibrosis, hepatic fibrosis, pulmonary fibrosis, submucous fibrosis of the oral cavity, cardiac fibrosis, and pancreatic fibrosis); cognitive diseases (e.g., mild cognitive impairment (MCI), Alzheimer's disease, and other dementias; Huntington's disease); motor function diseases and abnormalities (e.g., Parkinson's disease, motor neuron dysfunction (MND); Huntington's disease); cerebrovascular diseases; emphysema; osteoarthritis; benign prostatic hyperplasia; eye diseases or abnormalities (e.g., age-related macular degeneration, cataracts, glaucoma, vision loss, presbyopia); metabolic diseases and abnormalities (e.g., obesity, diabetes, metabolic syndrome); cardiovascular diseases (e.g., atherosclerosis, diastolic dysfunction of the heart, aortic aneurysm, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis); inflammatory / autoimmune diseases and abnormalities (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and abnormalities); dermatological diseases, such as diabetic ulcers, wound healing, and skin moles. In certain examples, any one or more of the above or other diseases or abnormalities described herein can be excluded.

[0276] In some embodiments, provided are methods for treating a disease or abnormality by killing senescent cells associated with the disease or abnormality (i.e., identified senescent cells) in a subject having an aging-related disease or abnormality by administering an anti-aging agent, wherein the disease or abnormality is an aging disease (e.g., frailty, muscle weakness, cognitive impairment); idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease (COPD); renal fibrosis or hepatic fibrosis; metastatic lesions or other proliferative abnormalities; osteoarthritis; or atherosclerosis.

[0277] Age-related diseases and abnormalities

[0278] The anti-aging agent described herein selectively kills senescent cells. In this way, targeting senescent cells during the aging process may be a preventive strategy. Accordingly, administering the anti-aging agent described herein to a subject can prevent comorbidities in elderly subjects and delay mortality. In addition, selectively killing senescent cells can enhance the immune system, extend healthspan, and improve the quality of life of the subject.

[0279] An anti-aging agent can also be used to treat or prevent age-related diseases or abnormalities (i.e., reduce the likelihood of occurrence of age-related diseases or abnormalities), which occur as part of the natural aging process or when a subject is exposed to aging-inducing agents or factors (e.g., radiation, chemotherapy, smoking, high-fat / high-sugar diet, other environmental factors). Age-related abnormalities or diseases or age-sensitive traits may be related to aging-inducing stimuli. The efficacy of the treatment methods described herein can be demonstrated by reducing the number of symptoms of age-related abnormalities or age-sensitive traits associated with aging-inducing stimuli, reducing the severity of one or more symptoms, or delaying the progression of age-related abnormalities or age-sensitive traits associated with aging-inducing stimuli. In other embodiments, preventing age-related abnormalities or age-sensitive traits associated with aging-inducing stimuli refers to preventing (i.e., reducing the likelihood of occurrence) or delaying the onset of age-related abnormalities or age-sensitive traits associated with aging-inducing stimuli, or the recurrence of one or more age-related abnormalities or age-sensitive traits associated with aging-inducing stimuli. Age-related diseases or disorders include, for example, abnormal kidney function, kyphosis, herniated disc, frailty, cognitive impairment, hair loss, hearing loss, vision loss (blindness or impaired vision), muscle fatigue, skin disorders, skin moles, diabetes, metabolic syndrome, and sarcopenia. Vision loss refers to the absence of vision when the subject previously had vision. Various scales have been developed to describe the degree of vision and vision loss based on visual acuity. Age-related diseases and disorders also include dermatological disorders, such as, but not limited to, treating one or more of the following conditions: wrinkles, including fine surface wrinkles; hyperpigmentation; scars; keloids; dermatitis; psoriasis; eczema (including seborrheic eczema); rosacea; vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis. Frailty has been defined as a clinically recognizable state of increased vulnerability due to age-related decline in reserve and function in multiple physiological systems, which impairs the subject's ability to cope with daily or acute stressors. The characteristics of frailty may be impaired energetic features, such as low grip strength, low energy, slow walking speed, low physical activity, and / or unintentional weight loss. Studies have shown that when three of the above five characteristics are observed, the patient may be diagnosed as frail (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 age-related diseases and abnormalities can be treated or prevented (i.e., the likelihood of their occurrence can be reduced) by administering an anti-aging agent. The anti-aging agent can inhibit the aging of adult stem cells, or inhibit the accumulation, kill, or promote the clearance of adult stem cells that have become senescent.The importance of preventing senescence in stem cells to maintain the regenerative capacity of tissues is discussed, for example, in Park et al., J. Clin. Invest. 113 (2004) 175-179; and Sousa-Victor, Nature 506 (2014) 316-321.

[0280] Methods of measuring aging are known in the art. For example, senescence in bone can be measured by, among other things, incident non-vertebral fractures, incident hip fractures, incident total fractures, incident vertebral fractures, incident repeated fractures, functional recovery after fracture, reduced bone mineral density in the lumbar spine and hip, knee flexion curvature, NSAID use, number of painful joints, and osteoarthritis. Senescence in muscle can also be measured by functional decline, fall rate, reaction time, and grip strength, decreased muscle mass in the upper and lower extremities, and dual-task 10-meter gait speed. In addition, senescence in the cardiovascular system can be measured by changes in systolic and diastolic blood pressure, incident hypertension, major cardiovascular events such as myocardial infarction, stroke, congestive heart disease, and cardiovascular mortality. In addition, senescence in the brain can be measured by cognitive decline, incident depression, and incident dementia. In addition, senescence in the immune system can be measured by infection rate, upper respiratory infection rate, influenza-like illness rate, incident severe infections leading to hospitalization, incident cancer, implant infection rate, and gastrointestinal infection rate. Other signs of aging may include, but are not limited to, decline in oral health, tooth loss, incidence of GI symptoms, changes in fasting glucose and / or insulin levels, body composition, decline in renal function, quality of life, accidental disability regarding activities of daily living, and accidental nursing home admission. Methods of measuring skin aging are known in the art and may include transepidermal water loss (TEWL), skin hydration, skin elasticity, area ratio analysis of crow's feet, sensitivity, gloss, roughness, spots, laxity, skin tone uniformity, softness, and undulation (depth change).

[0281] If a subject does not receive treatment, administration of the anti-aging agent described herein can prolong survival compared to expected survival. Subjects in need of treatment include those who already have the disease or disorder, as well as those who are predisposed to or at risk of developing the disease or disorder, and those in whom prophylactic treatment of the disease, condition, or disorder is needed. Subjects may have a genetic predisposition to developing a disease or disorder that would benefit from the clearance of senescent cells, or may not be young, in which case administration of the anti-aging agent would provide a clinical benefit in delaying the development or reducing the severity of the disease, including age-related diseases or disorders.

[0282] In other embodiments, methods for treating diseases or abnormalities associated with aging are provided, which further include identifying a subject who would benefit from treatment with an anti-aging agent as described herein (i.e., phenotype; personalized treatment). The method includes first detecting the level of senescent cells in a subject (such as in a specific organ or tissue of the subject). A biological sample can be obtained from the subject, such as a blood sample, serum or plasma sample, biopsy sample, body fluid (e.g., bronchoalveolar lavage fluid, ascites, mucosal washings, synovial fluid, vitreous humor, cerebrospinal fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation from the subject. The level of senescent cells can be determined according to any in vitro assay or technique described herein. For example, senescent cells can be detected by morphology (e.g., as observed by microscopy); 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 of a biological sample can also be used in in vitro cell assays, where the cells are exposed to any one of the anti-aging agents described herein to determine the ability of the anti-aging agent to kill the senescent cells of the subject without producing undesirable toxicity to non-senescent cells. In addition, these methods can be used to monitor the level of senescent cells in a subject before, during, and after treatment with an anti-aging agent. In certain embodiments, the presence of senescent cells can be detected (e.g., by determining the level of expression of senescent cell markers of mRNA), and the treatment course and / or non-treatment interval can be adjusted accordingly.

[0283] Lung diseases and abnormalities

[0284] In some embodiments, methods are provided for treating or preventing a disease or abnormality (i.e., reducing the likelihood of occurrence of the disease or abnormality) associated with aging, which is a lung disease or abnormality, by killing senescent cells (i.e., determined senescent cells) associated with the disease or abnormality in a subject suffering from the disease or abnormality associated with aging by administering an anti-aging agent as described herein. Aging-related lung diseases and abnormalities include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

[0285] COPD is a lung disease defined by persistent poor airflow due to breakdown of lung tissue (emphysema) and abnormal function of small airways (obstructive bronchiolitis). The main symptoms of COPD include shortness of breath, wheezing, chest tightness, chronic cough, and excessive sputum. Elastase from cigarette smoke-activated neutrophils and macrophages degrades the extracellular matrix of the alveolar structure, resulting in enlarged air spaces and loss of respiratory capacity (see, e.g., Shapiro et al., Am. J. Respir. Cell Mol. Biol. 32 (2005) 367-372). The most common causes of COPD are tobacco smoke (including cigarette smoke, cigar smoke, secondhand smoke, pipe smoke), occupational exposures (e.g., exposure to dust, fumes, or smoke), and pollution over decades, meaning that aging is a risk factor for developing COPD.

[0286] Processes causing lung injury include, for example, oxidative stress from high concentrations of free radicals in tobacco smoke; cytokine release due to inflammatory responses to irritants in the airways; and damage to anti-proteases by tobacco smoke and free radicals, allowing proteases to damage the lungs. Genetic susceptibility can also contribute to the disease. In about 1% of people with COPD, the disease is caused by a genetic abnormality that results in low levels of production of alpha-1-antitrypsin in the liver. This enzyme is normally secreted into the bloodstream to help protect the lungs.

[0287] Pulmonary fibrosis is a chronic and progressive lung disease characterized by hardening and scarring of the lungs, which can lead to respiratory failure, lung cancer, and heart failure. Fibrosis is associated with epithelial repair. Fibroblasts are activated, production of extracellular matrix proteins increases, and transdifferentiation into contractile myofibroblasts contributes to wound contraction. The provisional matrix plugs the damaged epithelium and provides a scaffold for epithelial cell migration (involving epithelial-mesenchymal transition (EMT)). Hemorrhage associated with epithelial injury induces platelet activation, production of growth factors, and an acute inflammatory response. Normally, the epithelial barrier heals and the inflammatory response subsides. However, in fibrotic diseases, the fibroblast response persists, leading to unresolved wound healing. The formation of fibroblast foci is a feature of the disease, reflecting the location of ongoing fibrogenesis. As the name implies, the etiology of IPF is unknown. The involvement of cellular senescence in IPF is shown by the observations that the incidence of the disease increases with age and that the lung tissue of IPF patients is rich in SA-β-Gal-positive cells and contains elevated 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 risk factor common to both IPF and cellular senescence (see, e.g., Alder et al., Proc. Natl. Acad. Sci. USA 105 (2008) 13051-13056). Without wishing to be bound by theory, the contribution of cellular senescence to IPF is shown by the report that the SASP components of senescent cells (such as IL-6, IL-8, and IL-1β) promote the differentiation of fibroblasts to myofibroblasts and epithelial-mesenchymal transition, resulting in extensive remodeling of the extracellular matrix in the alveolar and interstitial spaces (see, e.g., Minagawa et al. supra).

[0288] Subjects at risk of developing pulmonary fibrosis include subjects exposed to environmental or occupational pollutants such as asbestosis and silicosis; subjects who smoke; subjects with some typical connective tissue diseases (such as rheumatoid arthritis, SLE, and scleroderma); subjects with other diseases involving connective tissue (such as sarcoidosis and Wegener's granulomatosis); subjects with infectious diseases; subjects taking certain drugs (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin); subjects undergoing radiotherapy to the chest; and subjects whose family members have pulmonary fibrosis.

[0289] The symptoms of COPD can include any of the following: shortness of breath, especially during physical activity; wheezing; chest tightness; having to clear the throat first thing in the morning due to excess mucus in the lungs; a chronic cough that may produce clear, white, yellow, or green phlegm; bluish lips or nail beds (cyanosis); frequent respiratory infections; lack of energy; and unexpected weight loss (observed in the later stages of the disease). Subjects with COPD may also experience exacerbations, during which symptoms worsen and persist for days or longer. The symptoms of pulmonary fibrosis are known in the art and include shortness of breath, especially during exercise; frequent dry cough; rapid shallow breathing; gradual unexpected weight loss; fatigue; painful joints and muscles; and clubbing of the fingers (widening and rounding of the tips of the fingers or toes).

[0290] Subjects with COPD or pulmonary fibrosis can be identified using standard diagnostic methods practiced routinely in the art. Methods commonly used for diagnosis can be used to monitor the effects of one or more anti-aging agents administered to subjects with lung disease or at risk of developing lung disease. Generally, one or more of the following examinations or tests can be performed: physical examination, the patient's medical history, the patient's 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 test.

[0291] Other lung diseases or abnormalities that can be treated with anti-aging 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, pipe smoke), occupational exposure (e.g., exposure to dust, fumes, or smoke), infection, and / or pollutants that induce cellular senescence and thereby cause inflammation. Emphysema is sometimes considered a subgroup of COPD.

[0292] Bronchiectasis is caused by damage to the airways, which causes the airways to widen and become flabby and scarred. Bronchiectasis is usually caused by medical conditions that damage the airway walls or inhibit the airway's ability to clear mucus. Examples of such conditions include cystic fibrosis and primary ciliary dyskinesia (PCD). When only a part of the lungs is affected, the abnormality may be caused by an obstruction rather than a medical condition.

[0293] The methods described herein for treating or preventing age-related pulmonary diseases or abnormalities (i.e., reducing the likelihood or occurrence of age-related pulmonary diseases or abnormalities) can also be used to treat subjects who are aging and have a loss (or deterioration) of lung function (i.e., a decline or impairment of lung function compared to younger subjects) and / or degeneration of lung tissue. The respiratory system undergoes various anatomical, physiological, and immunological changes with aging. These structural changes include chest wall and thoracic spine deformities, which can compromise the compliance of the entire respiratory system, resulting in increased respiratory effort. The respiratory system undergoes structural, physiological, and immunological changes with aging. An increased proportion of neutrophils and a decreased percentage of macrophages are found in the bronchoalveolar lavage fluid (BAL) of the elderly compared to younger individuals. Persistent low-grade inflammation in the lower respiratory tract can lead to proteolytic and oxidant-mediated damage to the lung matrix, resulting in loss of alveolar units and impaired gas exchange across the alveolar membrane (which occurs with aging). Persistent lower respiratory tract inflammation can make the elderly more susceptible to toxic environmental exposures and accelerate the 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). Alterations in the redox balance and increased oxidative stress during aging promote the expression of cytokines, chemokines, adhesion molecules, and enzymes (see, e.g., Chung et al., Ageing Res. Rev. 8 (2009) 18-30). Constitutive activation and recruitment of macrophages, T cells, and mast cells promote the release of proteases, leading 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 an anti-aging agent to aging subjects (which includes asymptomatic middle-aged individuals), the decline in lung function can be slowed or inhibited by killing senescent cells and removing senescent cells from the respiratory tract.

[0294] The effectiveness of the anti-aging agent can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods (including physical examinations, assessment and monitoring of clinical symptoms, and performance of the analytical tests and methods described herein) can be used to monitor the health status of a subject. Using techniques known in the art, such as comparing the symptoms of patients with lung diseases who have received treatment or are at risk of developing lung diseases with those of patients who have not received such treatment or have been treated with a placebo, the therapeutic effect of the anti-aging agent or a pharmaceutical composition comprising the agent can be analyzed. In addition, methods and techniques for evaluating the mechanical function of the lungs can be performed, such as techniques for measuring lung volume, elasticity, and airway hyperresponsiveness. To determine lung function and monitor lung function throughout the treatment, any of a plurality of measurements can be obtained: expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in one second, FEV1), FEV1 / FEV ratio, forced expiratory flow at 25% to 75%, and maximum voluntary ventilation (MVV), peak expiratory flow (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 of carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO 2 ) can also be measured; normal oxygen levels are typically between 95% and 100%. An SpO 2 level below 90% indicates that the subject has hypoxemia. A value below 80% is considered a critical value and requires intervention to maintain brain and heart function and avoid cardiac or respiratory arrest.

[0295] Metastatic lesions

[0296] In some embodiments, methods are provided for treating or preventing a disease (or abnormality or disorder) associated with senescent cells (i.e., reducing the likelihood of its occurrence or development), said disease (or abnormality or disorder) being metastatic lesions. The anti-aging agents described herein can also be used, according to the methods described herein, for treating or preventing metastatic lesions (i.e., the spread and dissemination of cancer or tumor cells) from one organ or tissue to another in the body (i.e., reducing the likelihood of its occurrence).

[0297] Diseases or abnormalities associated with senescent cells include metastatic lesions, and a subject having cancer can benefit from administration of an anti-aging agent for inhibiting metastatic lesions as described herein. When administered to a subject having cancer according to the methods described herein, such an anti-aging agent can inhibit tumor proliferation. Metastatic lesions of cancer occur when cancer cells (i.e., tumor cells) spread beyond the anatomic site of origin and initial colonization to other regions throughout the subject's body. Tumor proliferation can be determined by tumor size, which can be measured by a variety of ways familiar to those skilled in the art, such as by PET scan, MRI, CAT scan, biopsy. The effect of a therapeutic agent on tumor proliferation can also be evaluated by examining the differentiation of tumor cells.

[0298] As used herein and in the art, the term cancer or tumor is a clinical descriptive term that includes diseases typically characterized by cells presenting abnormal cell proliferation. The term cancer is generally used to describe a malignant tumor or a disease state caused by a tumor. Alternatively, abnormal growth can be referred to as a tumor in the art. The term tumor, when referring to tissue, generally refers to any abnormal tissue growth, which is characterized at least in part by excessive and abnormal cell proliferation. A tumor can be metastatic and capable of spreading beyond the anatomic site of its origin and initial colonization to other regions throughout the subject's body. Cancer can include solid tumors or can include "liquid" tumors (e.g., leukemia and other blood cancers).

[0299] Cells are induced to senesce by cancer therapies such as radiation and certain chemotherapeutic drugs. The presence of senescent cells increases the secretion of inflammatory molecules, promoting tumor progression, which may include promoting tumor growth and increasing tumor size, promoting metastatic lesions, and altering differentiation. When senescent cells are destroyed, tumor progression is significantly inhibited, resulting in tumors of small size and little or no observed metastatic growth (see, e.g., International Publication No. WO 2013 / 090645).

[0300] In some embodiments, methods are provided for preventing (i.e., reducing the likelihood of occurrence of metastatic lesions), inhibiting, or delaying metastatic lesions in a subject having cancer by administration of an anti-aging agent as described herein. In other embodiments, the anti-aging agent is administered on one or more days within a treatment window (i.e., treatment course) of no more than 7 days or 14 days. In still other embodiments, the treatment course is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or no more than 21 days. In still other embodiments, the treatment course is one day. In still other embodiments, the anti-aging agent is administered on two or more days within a treatment window of no more than 7 days or 14 days.

[0301] Because cells can be induced to senesce by cancer therapies such as radiation and certain chemotherapeutic drugs (e.g., doxorubicin; paclitaxel; gemcitabine; pomalidomide; lenalidomide), the anti-aging agents described herein can be administered after chemotherapy or radiotherapy to kill (or promote the killing of) these senescent cells. As discussed herein and understood in the art, the establishment of senescence (such as indicated by the presence of the senescence-associated secretory phenotype (SASP)) occurs within days; thus, when senescence has been established, the anti-aging agent is administered to kill the senescent cells and thereby reduce the likelihood of occurrence or the extent of metastatic lesions. As discussed herein, the following regimens of administering an anti-aging agent can be used in the methods described herein for treating or preventing side effects of chemotherapy or radiotherapy (i.e., reducing the likelihood of their occurrence, or reducing their severity).

[0302] In certain embodiments, when chemotherapy or radiotherapy is administered during a treatment cycle of non-therapy (i.e., non-chemotherapy or non-radiotherapy) of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 (or about 2 weeks), 15, 16, 17, 18, 19, 20, 21 (or about 3 weeks) or about 4 weeks (about one month) after at least one day of therapy (i.e., chemotherapy or radiotherapy), the anti-aging agent is administered on one or more days during the non-therapy time interval (time period), which begins on the second day or later of the non-therapy time interval and ends on the last day or before of the non-therapy time interval. As an illustrative example, if n is the number of non-therapy days, then the anti-aging agent is administered on at least one day and no more than n - 1 days of the non-therapy time interval. In some embodiments, when chemotherapy or radiotherapy is administered during a treatment cycle of non-therapy of at least one week after at least one day of therapy (i.e., chemotherapy or radiotherapy), the anti-aging agent is administered on one or more days during the non-therapy time interval, which begins on the second day or later of the non-therapy time interval and ends on the last day or before of the non-therapy time interval.

[0303] Chemotherapy can be referred to as chemotherapy, chemotherapeutic agent or chemotherapeutic drug. Many chemotherapeutic agents are compounds known as organic small molecules. Chemotherapy is also a term used to describe a combination of chemotherapeutic drugs administered to treat a particular cancer. As understood by those skilled in the art, chemotherapy can also refer to a combination of two or more chemotherapeutic molecules administered in combination, and it can be referred to as combination chemotherapy. Many chemotherapeutic drugs are used in the field of oncology and include, but are not limited to, alkylating agents; antimetabolites; anthracyclines, plant alkaloids; and topoisomerase inhibitors.

[0304] The cancer that can metastasize can be a solid tumor or can be a liquid tumor (e.g., blood cancer such as leukemia). Cancers that are liquid tumors are classified in the art as those that occur in the blood, bone marrow, and lymph nodes and generally include leukemia (myeloid and lymphocytic), lymphoma (e.g., Hodgkin lymphoma), and melanoma (including multiple myeloma). Leukemia includes, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia. Cancers that are solid tumors and occur in humans with higher frequency include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, gastric cancer, lung cancer, ovarian cancer, Kaposi sarcoma, skin cancer (including squamous cell skin cancer), kidney cancer, head and neck cancer, laryngeal cancer, squamous carcinoma formed on the moist mucosal lining of the nose, mouth, larynx, etc., bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain specific 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 a metastatic lesion 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 sarcoma cells, skin cancer cells, kidney cancer cells, head or 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.

[0305] The methods described herein can also be used to inhibit, delay, or slow the progression of metastatic cancer of any type of tumor described in the medical field. The types of cancer (tumors) include the following: adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumors, childhood astrocytoma, childhood brainstem glioma, childhood central nervous system atypical teratoid / rhabdoid tumor, childhood central nervous system embryonal tumor, childhood central nervous system germ cell tumor, childhood craniopharyngioma brain tumor, childhood ependymoma brain tumor, breast cancer, childhood bronchial tumor, carcinoid tumor, childhood carcinoid tumor, gastrointestinal carcinoid tumor, cancer of unknown primary, childhood cancer of unknown primary, childhood heart (cardiac) tumor, cervical cancer, childhood cervical cancer, childhood chordoma, chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood olfactory neuroblastoma, eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric (stomach) cancer, childhood gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), childhood gastrointestinal stromal tumor (GIST), childhood extracranial germ cell tumor, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell renal carcinoma, Wilms' tumor, childhood kidney tumor, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving the NUT gene, oral cancer, childhood multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasms, myeloproliferative neoplasms, multiple myeloma, nasal cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhood oral cancer, oropharyngeal cancer, ovarian cancer, childhood ovarian cancer, epithelial ovarian cancer, ovarian cancer of low malignant potential, pancreatic cancer, childhood pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), childhood papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacytoma, childhood pleuropulmonary blastoma, prostate cancer, rectal cancer, transitional cell carcinoma of the renal pelvis, retinoblastoma, salivary gland cancer,Salivary gland carcinoma in children, Ewing sarcoma family of tumors, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, rhabdomyosarcoma in children, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer in children, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous cell carcinoma in children, testicular cancer, testicular cancer in children, laryngeal cancer, thymoma and thymic carcinoma, thymoma and thymic carcinoma in children, thyroid cancer, thyroid cancer in children, urothelial carcinoma of the ureter, urethral cancer, endometrial cancer, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia.

[0306] Side effects of chemotherapy and radiotherapy

[0307] In other embodiments, the abnormalities or disorders associated with senescent cells are side effects of chemotherapy or radiotherapy. Examples of chemotherapeutic agents that induce senescence in non-cancerous cells include anthracyclines (such as doxorubicin, daunorubicin); taxanes (e.g., paclitaxel); gemcitabine; pomalidomide; and lenalidomide. One or more anti-aging agents administered as described herein can be used to treat and / or prevent side effects of chemotherapy or radiotherapy (i.e., reduce their likelihood or occurrence). The removal or destruction of senescent cells can improve the acute toxicity of chemotherapy or radiotherapy, including acute toxicity including energy imbalance. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, malaise, hypoactivity, hematotoxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, dermatological toxicity (e.g., rash, dermatitis, hyperpigmentation, urticaria, photosensitivity, nail changes), mouth (e.g., oral mucositis), gum or throat problems, or any toxic side effect caused by chemotherapy or radiotherapy. For example, the toxic side effects caused by radiotherapy or chemotherapy can be improved by the methods described herein. Thus, in certain embodiments, provided herein are methods for improving the acute toxicity (reducing, inhibiting, or preventing its occurrence (i.e., reducing the likelihood of its occurrence)) of chemotherapy or radiotherapy or both or reducing the severity of its toxic side effects (i.e., harmful side effects) in a subject receiving therapy, wherein the method comprises administering to the subject an agent that selectively kills, removes, or destroys senescent cells or promotes the selective destruction of senescent cells. The administration of the anti-aging agents described herein for treating or reducing the likelihood of occurrence of side effects of chemotherapy or radiotherapy or reducing the severity of side effects of chemotherapy or radiotherapy can be accomplished by the same regimen as described above for treating / preventing metastatic lesions. As described for treating or preventing metastatic lesions (i.e., reducing the likelihood of occurrence of metastatic lesions), the anti-aging agent is administered during a non-therapy or non-therapy time interval or after the chemotherapy or radiotherapy treatment regimen has been completed.

[0308] In more specific embodiments, the acute toxicity is acute toxicity that includes an energy imbalance and can include one or more of weight loss, endocrine alterations (e.g., hormonal imbalances, altered hormonal signaling), and alterations in body composition. In certain embodiments, the acute toxicity that includes an energy imbalance involves a decrease or reduction in the physical activity ability of a subject, as indicated by a reduced or decreased energy expenditure as would be observed in a subject not receiving the pharmaceutical therapy. As a non-limiting example, such an acute toxic effect that includes an energy imbalance encompasses low physical activity. In other embodiments, the energy imbalance includes fatigue or discomfort.

[0309] In some embodiments, a chemotherapy side effect that is treated or prevented (i.e., the likelihood of occurrence is reduced) by an anti-aging agent described herein is cardiotoxicity. A subject having cancer being treated with an anthracycline (such as doxorubicin, daunorubicin) can be treated with one or more anti-aging agents described herein that reduce, ameliorate, or decrease the cardiotoxicity of the anthracycline. As is well understood in the medical field, due to the cardiotoxicity associated with anthracyclines, even if the cancer responds to the drug, the maximum lifetime dose that a subject can receive is limited. Administration of one or more anti-aging agents can reduce the cardiotoxicity such that an additional amount of the anthracycline can be administered to the subject, resulting in an improved prognosis associated with the cancer disease. In some embodiments, the cardiotoxicity is caused by the administration of an anthracycline such as doxorubicin. Doxorubicin is an anthracycline topoisomerase inhibitor that is approved for the treatment of patients having ovarian cancer after failure of platinum therapy; Kaposi sarcoma after failure of primary systemic chemotherapy or intolerance to therapy; or multiple myeloma in combination with bortezomib in patients who have not previously received bortezomib or who have received at least one therapy previously. If the total lifetime dose of the patient exceeds 550 mg / m 2 , doxorubicin may cause myocardial injury, which can lead to congestive heart failure. Cardiotoxicity may occur even at lower doses if the patient also receives mediastinal radiation or other cardiotoxic drugs.

[0310] In other embodiments, the anti-aging agents described herein can be used in methods for improving chronic or long-term side effects as provided herein. Chronic toxic side effects are typically caused by multiple exposures to chemotherapy or radiotherapy over a long period of time or the administration of chemotherapy or radiotherapy. Certain toxic effects occur a long time after treatment (also referred to as late toxic effects) and are caused by damage to organs or systems by the therapy. Abnormal organ function (e.g., neurological, pulmonary, cardiovascular, and endocrine dysfunction) has been observed in patients who received cancer treatment during childhood (see, e.g., Hudson et al., JAMA 309 92013 2371-2381). Without wishing to be bound by any particular theory, by destroying senescent cells, particularly normal cells that have been induced to senesce by chemotherapy or radiotherapy, the likelihood of chronic side effects can be reduced, or the severity of chronic side effects can be reduced or decreased, or the onset time of chronic side effects can be delayed. Chronic and / or late toxic side effects that occur in subjects receiving chemotherapy or radiotherapy include, as non-limiting examples, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancer, cataracts, and other vision problems, hearing loss, chronic fatigue, reduced lung capacity, and lung disease.

[0311] Furthermore, by killing or removing senescent cells in a subject with cancer via the administration of an anti-aging agent, the sensitivity to chemotherapy or radiotherapy can be enhanced in a clinically or statistically significant manner compared to not administering the anti-aging agent. In other words, the development of chemotherapy or radiotherapy resistance can be inhibited when an anti-aging agent is administered to a subject being treated with the corresponding chemotherapy or radiotherapy.

[0312] Neurological diseases and abnormalities

[0313] Aging-related diseases or abnormalities that can be treated by the administration of the anti-aging agents described herein include neurological diseases or abnormalities. Such aging-related diseases and abnormalities 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 abnormalities such as age-related macular degeneration. Other eye diseases associated with aging are glaucoma, vision loss, presbyopia, and cataracts.

[0314] Parkinson's disease (PD) is the second most common neurodegenerative disease. It is a disabling disorder of the brain characterized by slowness of movement (bradykinesia), tremors, stiffness, and in later stages, loss of balance. Many of these symptoms are due to the loss of certain nerves in the brain, which results in a deficiency of dopamine. The disease is characterized by neurodegeneration, such as the loss of about 50% to 70% of dopaminergic neurons in the substantia nigra pars compacta, a profound loss of dopamine in the striatum, and / or the presence of cytoplasmic inclusions (Lewy bodies), which consist mainly of α-synuclein and ubiquitin. Parkinson's disease also has motor function deficits, such as tremors, rigidity, bradykinesia, and / or postural instability. Subjects at risk of developing Parkinson's disease include those with a family history of Parkinson's disease and those exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., Agent Orange), or heavy metals. The aging of dopamine-producing neurons is thought to contribute to the cell death observed in PD by generating reactive oxygen species (see, e.g., Cohen et al., J. Neural Transm. Suppl. 19 (1983) 89-103); thus, the methods and anti-aging agents described herein can be used to treat and prevent Parkinson's disease.

[0315] Methods for detecting, monitoring, or quantifying neurodegenerative defects and / or motor defects associated with Parkinson's disease are known in the art, such as histological studies, biochemical studies, and behavioral assessments (see, e.g., U.S. Application Publication No. 2012 / 0005765). The symptoms of Parkinson's disease are known in the art and include, but are not limited to, difficulty starting or completing voluntary movements, jerks, rigid movements, muscle atrophy, tremors (trembling), and heart rate changes, but with normal reflexes, bradykinesia, and postural instability. It is increasingly recognized that individuals diagnosed with Parkinson's disease may also have cognitive impairments, including mild cognitive impairment, in addition to their physical symptoms.

[0316] Alzheimer's disease (AD) is a neurodegenerative disease characterized by a slowly progressive mental deterioration, accompanied by memory failure, sensory abnormalities, and confusion, leading to profound dementia. Age is the single greatest risk factor for developing AD, and AD is the leading cause of dementia in the elderly (see, e.g., Hebert, et al., Arch. Neural. 60 (2003) 1119-1122). Early clinical symptoms show significant similarities to mild cognitive impairment (see below). As the disease progresses, impaired judgment, confusion, behavioral changes, sensory abnormalities, and difficulty walking and swallowing occur.

[0317] Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid (senile) plaques in histological samples. The disease mainly involves the limbic and cortical regions of the brain. Argyrophilic plaques containing amyloidogenic Aβ fragments of amyloid precursor protein (APP) are scattered throughout the cerebral cortex and hippocampus. Neurofibrillary tangles are found in pyramidal neurons located mainly in the neocortex, hippocampus, and basal nucleus of Meynert. Other changes are observed, such as granular vacuolar degeneration in the pyramidal cells of the hippocampus and neuronal loss and gliosis in the cortex and hippocampus. Subjects at risk of 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 deterministic gene mutations (e.g., APP, PS1, or PS2), and subjects with a history of head trauma or cardiac / vascular disorders (e.g., hypertension, heart disease, stroke, diabetes, high cholesterol, etc.).

[0318] Many behavioral and histopathological assays are known in the art for assessing Alzheimer's disease phenotypes, for characterizing therapeutic agents, and for evaluating treatments. 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 deposition on sections of brain tissue (see, e.g., Holcomb et al., Nat. Med. 4 (1998) 97-100; Borchelt et al., Neuron 19 (1997) 939-945; Dickson et al., Am. J. Path. 132 (1998) 86-101). In vivo methods for visualizing Aβ deposition in transgenic mice have also been described. BSB ((trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene) and PET tracers 11 11C-labeled Pittsburgh compound-B (PIB) binds to Aβ 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). Containing 19The amyloidophilic Congo red-type compound of F, FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy) styrylbenzene), allows visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., Nature Neurosci. 8 (2005) 527-533). Radioactively labeled, putrescine-modified amyloid β-peptide labels amyloid deposits in a mouse model of Alzheimer's disease (see, e.g., Wengenack et al., Nat. Biotechnol. 18 (2000) 868-872).

[0319] Increased glial fibrillary acidic protein (GFAP) by astrocytes is a marker of astrocyte activation and gliosis during neurodegeneration. Aβ 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 performed with electron microscopy and axonal transport studies can be used to observe neuronal degeneration (see, e.g., Ishihara et al., Neuron 24 (1999) 751-762).

[0320] Subjects with Alzheimer's disease can be identified using standard diagnostic methods known in the art. Generally, the diagnosis of Alzheimer's disease is based on the patient's symptoms (e.g., progressive decline in memory function, gradual withdrawal and frustration from normal activities, apathy, agitation or irritability, aggression, anxiety, sleep disturbances, restlessness, abnormal motor behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia), medical history, neuropsychological tests, neurological and / or physical examinations. Various proteins associated with Alzheimer's pathology in cerebrospinal fluid can also be tested, including taurine, amyloid-β peptide, and AD7C-NTP. Genetic testing can also be used for early-onset familial Alzheimer's disease (eFAD), an autosomal dominant genetic disorder. Clinical genetic testing can be used for individuals with AD symptoms or at-risk family members of patients with early-onset disease. In the United States, mutations in PS2 and APP can be tested in clinical or federally approved laboratories under the Clinical Laboratory Improvement Amendments. Commercial tests for PS1 mutations are also available (Elan Pharmaceuticals).

[0321] Zhang et al. have reported that in the brains of patients with AD and in an AD mouse model, oligodendrocyte progenitor cells (OPCs) expressing Olig2 and NG2, rather than astrocytes, microglia, or oligodendrocytes, associated with Aβ plaques exhibit an aging-like phenotype characterized by upregulation of p21 / CDKN1A and p16 / INK4 / CDKN2A proteins and senescence-associated β-galactosidase activity (see Nature Neurosci. 22 (2019) 719-728). Molecular interrogation of the Aβ plaque microenvironment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Cultured OPCs directly exposed to aggregated Aβ trigger cellular senescence. Treatment of AD mice with an anti-aging cocktail including dasatinib and quercetin selectively removes senescent cells from the plaque microenvironment, reduces neuroinflammation, alleviates Aβ burden, and improves cognitive deficits. These findings suggest a role for Aβ-induced OPC cellular senescence in the neuroinflammation and cognitive deficits of AD, as well as the potential therapeutic benefits of anti-aging therapies.

[0322] The effectiveness of one or more anti-aging agents described herein and the monitoring of subjects receiving one or more anti-aging agents can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods (including physical examination, assessment and monitoring of clinical symptoms, and performance of the analytical tests and methods described herein) can be used to monitor the health status of a subject. The effects of administering one or more anti-aging agents can be analyzed using techniques known in the art, such as comparing the symptoms of patients with Alzheimer's disease or at risk of Alzheimer's disease who have received treatment with those of patients who have not received such treatment or who have received placebo treatment.

[0323] Mild cognitive impairment (MCI) is a brain function syndrome that involves the onset and evolution of cognitive impairment that exceeds what would be expected based on an individual's age and educational level, but is not yet sufficient to significantly interfere with an individual's daily activities. MCI is an aspect of cognitive aging and is considered to be a transitional state between normal aging and the dementia into which it may convert (see, Pepeu, Dialogues in Clinical Neuroscience 6(2004)369-377). MCI that primarily affects memory is called "amnestic MCI". Persons with amnestic MCI may begin to forget important information that he or she could previously recall easily, such as recent events. Amnestic MCI is often regarded as the prodromal phase of Alzheimer's disease. MCI that affects thinking abilities other than memory is called "non-amnestic MCI". This type of MCI affects thinking skills, such as the ability to make correct decisions, judge the time or sequence of steps required to complete a complex task, or visual perception ability. Individuals with non-amnestic MCI are considered more likely to convert to other types of dementia (e.g., dementia with Lewy bodies).

[0324] Persons in the medical field are increasingly recognizing that persons diagnosed with Parkinson's disease may also have MCI in addition to their physical symptoms. Recent studies have shown that 20-30% of persons with Parkinson's disease have MCI, and their MCI tends to be non-amnestic. Parkinson's disease patients with MCI sometimes develop into full-blown dementia (Parkinson's disease with dementia).

[0325] Methods for detecting, monitoring, quantifying or assessing neuropathological deficits associated with MCI are known in the art and include astrocyte morphology analysis, acetylcholine release, silver staining for assessing neurodegeneration, and PiB PET imaging for detecting amyloid-β deposition (see, e.g., U.S. Application Publication No. 2012 / 0071468; Pepeu, supra (2004)). Methods for detecting, monitoring, quantifying or assessing behavioral deficits associated with MCI are also known in the art and include the eight-arm radial maze paradigm, the non-match-to-sample task, the heterotopic location determination task in the water maze, the Morris maze test, the visuospatial task, the delayed response spatial memory task, and the olfactory novelty test.

[0326] Motor neuron dysfunction (MND) is a group of progressive neurological disorders that destroy motor neurons, the cells that control basic voluntary muscle activities such as speaking, walking, breathing, and swallowing. It is classified according to whether the degeneration affects upper motor neurons, lower motor neurons, or both. Examples of MND include, but are not limited to, amyotrophic lateral sclerosis (ALS) (also known as Lou Gehrig's disease), progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, progressive muscular atrophy, lower motor neuron disease, and spinal muscular atrophy (SMA) (e.g., SMA1 (also known as Werdnig-Hoffmann disease), SMA2, SMA3 (also known as Kugelberg-Welander disease), and Kennedy disease), postpoliomyelitis 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 the arm, leg, or facial muscles. Primary lateral sclerosis is a disease of the upper motor neurons, while progressive muscular atrophy affects only the lower motor neurons in the spinal cord. In progressive bulbar palsy, the motor neurons in the lowest part of the brainstem are most affected, resulting in slurred speech and difficulty chewing and swallowing. There are almost always mild abnormal signs in the arms and legs. Patients with MND exhibit a Parkinson's disease phenotype (e.g., with tremor, rigidity, bradykinesia, and / or postural instability). Methods for detecting, monitoring, or quantifying motor and / or other deficits associated with Parkinson's disease such as MND are known in the art (see, e.g., U.S. Application Publication No. 2012 / 0005765).

[0327] Methods for detecting, monitoring, quantifying or evaluating motor and histopathological deficits associated with MND are known in the art and include histopathological, biochemical and electrophysiological studies as well as motor activity analysis (see, e.g., Rich et al., J. Neurophysiol. 88 (2002) 3293-3304; Appel et al., Proc. Natl. Acad. Sci. USA 88 (1991) 647-651). Histopathologically, MND is characterized by the death of motor neurons, the progressive accumulation of detergent-resistant aggregates containing SOD1 and ubiquitin, and the abnormal accumulation of neurofilaments in degenerating motor neurons. In addition, reactive astrocytes and microglia are often detected in lesioned tissue. Patients with MND exhibit one or more motor deficits, including muscle weakness and wasting, uncontrollable twitching, spasticity, slow and labored movement, and hyperactive tendon reflexes.

[0328] Eye diseases and disorders

[0329] In certain embodiments, the aging-related disease or abnormality is an eye disease, disorder, or condition, such as presbyopia, macular degeneration, or cataract. In certain other embodiments, the aging-related disease or abnormality is glaucoma. Macular degeneration is a neurodegenerative disease that results in the loss of photoreceptor cells in the central part of the retina (called the macula). Macular degeneration is generally classified into two types: dry type and wet type. The dry form is more common than the wet form, with approximately 90% of patients with age-related macular degeneration (ARMD or AMD) being diagnosed with the dry form. The wet form of this disease typically results in more severe vision loss. Although the exact cause of age-related macular degeneration is unknown, the number of senescent retinal pigment epithelial (RPE) cells increases with age. Age and certain genetic factors, as well as 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 susceptibility factors include the intake of omega-3 fatty acids (see, e.g., Christen et al., Arch. Ophthalmol. 129 (2011) 921-929); estrogen exposure (see, e.g., Feshanich et al., Arch. Ophthalmol. 126(4) (2008) 519-524); and increased serum levels of vitamin D (see, e.g., Millen, et al., Arch. Ophthalmol. 129(4) (2011) 481-89). Genetic susceptibility risk factors include reduced levels of Dicer1 (an enzyme involved in microRNA maturation) in the eyes of patients with dry AMD, and reduced microRNAs result in a cellular senescence profile.

[0330] Dry ARMD is associated with atrophy of the RPE layer, which leads to the loss of photoreceptor cells. The dry form of ARMD may be caused by the aging and thinning of macular tissue and the deposition of pigments in the macula. Aging appears to simultaneously inhibit the replication and migration of RPE, resulting in the permanent depletion of RPE in the macula of patients with dry AMD (see, e.g., Iriyama et al., J. Biol. Chem. 283 (2008) 11947-11953). In the case of wet ARMD, new blood vessels grow under the retina and leak blood and fluid. This abnormally leaking choroidal neovascularization causes retinal cell death, creating a blind spot in central vision. Different forms of macular degeneration can also occur in younger patients. Age-independent etiologies may be related to genetics, diabetes, nutritional deficiencies, head injury, infection, or other factors.

[0331] A decrease in visual acuity noticed by a patient or an eye care professional during a routine eye examination may be the first indicator of macular degeneration. The formation of subretinal exudates or "drusen" in the Bruch's membrane of the macula is generally the first physical sign that macular degeneration may be developing. Symptoms include the perception of straight lines being distorted, and in some cases, the center of vision appears more distorted than the rest of the scene; dark, blurry areas or "holes" appear in the center of vision; and / or color perception is altered or diminished. The diagnosis and monitoring of a subject with macular degeneration can be accomplished by a technician in the field of ophthalmology based on the routine eye examination procedures accepted in the art and the subject's reported symptoms.

[0332] Presbyopia is an age-related condition in which the ability of the eye to focus on nearby objects gradually decreases as the normal eye's accommodation speed and amplitude decrease with age. The loss of lens elasticity and the loss of ciliary muscle contractility have been proposed as the causes (see, e.g., Heys et al., Mol. Vis. 10 (2004) 956-963; Petrash, Invest. Ophthalmol. Vis. Sci. 54 (2013) ORSF54-ORSF59). Age-related changes in the mechanical properties of the anterior and posterior lens capsules suggest that the mechanical strength of the posterior lens capsule decreases significantly with age (see, e.g., Kraget al., Invest. Ophthalmol. Vis. Sci. 44 (2003) 691-696; Krag et al., Invest. Ophthalmol. Vis. Sci. 38 (1997) 357-363).

[0333] The layered structure of the capsule also changes and may be caused at least in part by changes in tissue composition (see, e.g., Krag et al., 1997 above, and the references cited therein). The main structural component of the lens capsule is type IV collagen of the basement membrane, which is organized into a three-dimensional molecular network (see, e.g., Cummings et al., Connect. Tissue Res. 55 (2014) 8-12; Veis et al., Coll. Relat. Res. 1 (1981) 269-286). Type IV collagen consists of six homologous α-chains (α1-6), which combine into heterotrimeric type IV procollagen protomers, each protomer including a specific chain combination of α112, α345 or α556 (see, e.g., Khoshnoodi et al., Microsc. Res. Tech. 71 (2008) 357-370). The protomers share a structural similarity of the triple-helical collagen domain with the Gly-X-Y triplet peptide sequence (Timpl et al., Eur. J. Biochem. 95 (1979) 255-263), terminating in a globular C-terminal region called the non-collagen 1 (NC1) domain. The N-terminal consists of a helical domain called the 7S domain (see, e.g., Risteli et al., Eur. J. Biochem. 108 (1980) 239-250), which also participates in protomer-protomer interactions.

[0334] Studies have shown that collagen IV affects cell function, which is inferred from its location in the subepithelial basement membrane, and data support a role for collagen IV in tissue stability (see, e.g., Cummings et al. supra). Posterior capsule opacification (PCO) develops as a complication in approximately 20 - 40% of patients in the years following cataract surgery (see, e.g., Awasthi et al., Arch. Ophthalmol. 127(2009)555 - 562). PCO results from the proliferation and activity of residual lens epithelial cells along the posterior capsule in a response 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 discussed herein, the production of these factors and cytokines by senescent cells contributes to the SASP. In contrast, in vitro studies have shown that collagen IV promotes the adhesion of lens epithelial cells (see, e.g., Olivero et al., Invest. Ophthalmol. Vis. Sci. 34(1993)2825 - 2834). The adhesion of collagen IV, fibronectin, and laminin to the intraocular lens inhibits cell migration and may reduce the risk of PCO (see, e.g., Raj et al, Int. J. Biomed. Sci. 3(2007)237 - 250).

[0335] Without wishing to be bound by any particular theory, selective killing of senescent cells by the anti - aging agents described herein can slow or prevent (delay, inhibit, retard) the disruption of the type IV collagen network. Removal of senescent cells and thereby the inflammatory effects of the SASP can reduce or inhibit epithelial cell migration and can also delay (inhibit) the onset of presbyopia or reduce or slow the progressive severity of the condition (such as slowing the progression from mild to moderate or from moderate to severe). The anti - aging agents described herein can also be used in the post - cataract treatment to reduce the likelihood of PCO occurrence.

[0336] Although no direct evidence has been obtained from human studies that cellular senescence is associated with cataract development, BubR1 hypomorphic allele mice develop bilateral posterior subcapsular cataracts early in life, suggesting that senescence may play a role (see, e.g., Baker et al., Nat. Cell Biol. 10 (2008) 825-836). A cataract is an opacity of the eye lens that causes blurred vision and can lead to blindness if untreated. Surgery is effective and routinely performed to remove cataracts. Administration of one or more of the anti-aging agents described herein can result in a reduced likelihood of cataract development or can slow or inhibit the progression of cataracts. The presence and severity of cataracts can be monitored by an eye examination using methods routinely performed by those skilled in the art of ophthalmology.

[0337] In certain embodiments, at least one of the anti-aging agents described herein can be administered to a subject at risk of developing presbyopia, cataracts, or macular degeneration. Treatment with an anti-aging agent can be initiated when a human subject is at least 40 years old to delay or inhibit the onset or development of cataracts, presbyopia, and macular degeneration. Since almost all people develop presbyopia, in certain embodiments, an anti-aging agent can be administered to a human subject after the subject reaches 40 years of age in a manner as described herein to delay or inhibit the onset or development of presbyopia.

[0338] In certain embodiments, the age-related disease or abnormality is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause loss of the visual field, usually without any other major symptoms. The lack of symptoms often leads to a delayed diagnosis of glaucoma until the late stages of the disease. Even if a subject with glaucoma does not become blind, their vision is usually severely impaired. Normally, clear fluid flows into 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 an increase in pressure within the eye. If untreated, this high pressure subsequently damages the optic nerve and can lead to total blindness. The 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. A four-fold increase in senescence is observed in glaucoma patients when the network of cells required for fluid outflow undergoes SA-β-Gal staining (see, e.g., Liton et al., Exp. Gerontol. 40 (2005) 745-748).

[0339] To monitor the effectiveness of therapies for suppressing the progression of glaucoma, standard automated perimetry (perimetry) is the most widely used technique. In addition, several algorithms for progression detection have been developed (see, e.g., Wesselink et al., Arch. Ophthalmol. 127(3)(2009)270-274, and references therein). Additional methods include gonioscopy (examination of the trabecular meshwork and the angle through which fluid exits the eye); imaging techniques such as scanning laser tomography (e.g., HRT3), laser polarimetry (e.g., GDX), and optical coherence tomography of the eye); ophthalmoscopy; and pachymetry measurements to determine central corneal thickness.

[0340] Metabolic diseases or disorders

[0341] Age-related diseases or disorders treatable by administration of an anti-aging agent include metabolic diseases or disorders. Such diseases and disorders associated with senescent cells include diabetes, metabolic syndrome, diabetic ulcers, and obesity.

[0342] Diabetes is characterized by high blood glucose levels caused by defects in insulin production, insulin action, or both. Most (90% to 95%) of the diagnosed cases of adult diabetes are type 2 diabetes, which is characterized by a progressive loss of insulin secretion by the pancreas. In the United States, diabetes is the leading cause of new cases of kidney failure, non-traumatic lower limb amputations, and adult blindness. Diabetes is a major cause of heart disease and stroke and is 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 anti-aging agents described herein can be used to treat type 2 diabetes, particularly type 2 diabetes associated with age, diet, and obesity.

[0343] The involvement of senescent cells in metabolic diseases, such as obesity and type 2 diabetes, has been recognized 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 shows induction of the senescence markers SA-β-Gal, p53, and p21 (see, e.g., Tchkonia et al. supra; Minamino et al., Nat. Med. 15 (2009) 1082-1087). A concomitant upregulation of pro-inflammatory cytokines, such as tumor necrosis factor α and Ccl2 / MCP1, is observed in the same adipose tissue (see, e.g., Minamino et al. supra). The induction of senescent cells in obesity potentially has clinical significance, since pro-inflammatory SASP components are also thought to contribute to type 2 diabetes (see, e.g., Tchkonia et al. supra). A similar pattern of upregulation of senescence markers and SASP components is associated with diabetes, both in mice and in humans (see, e.g., Minamino et al. supra). Accordingly, the methods described herein, including the administration of anti-aging agents, can be used to treat or prevent type 2 diabetes as well as obesity and metabolic syndrome. Without wishing to be bound by theory, contacting pre-senescent adipocytes with an anti-aging agent to kill the pre-senescent adipocytes can provide clinical and health benefits to a person suffering from any one of diabetes, obesity, or metabolic syndrome.

[0344] Subjects with type 2 diabetes can be identified using standard diagnostic methods for type 2 diabetes known in the art. Generally, the diagnosis of type 2 diabetes is based on symptoms (e.g., increased thirst and frequent urination, increased hunger, weight loss, fatigue, blurred vision, slow wound healing or frequent infections and / or areas of darkened skin), medical history, and / or physical examination of the patient. Subjects at risk of developing type 2 diabetes include subjects with a family history of type 2 diabetes and subjects with other risk factors, such as overweight, fat distribution, inactivity, race, age, pre-diabetes, and / or gestational diabetes.

[0345] The effectiveness of anti-aging agents can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods (including physical examinations, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods such as those described herein) can be used to monitor the health status of a subject. For example, a subject receiving one or more anti-aging agents for the treatment or prevention of diabetes as described herein can be monitored by determining glucose and insulin tolerance, energy expenditure, body composition, adipose tissue, skeletal muscle, and liver inflammation and / or lipotoxicity (by in vivo imaging of muscle and liver lipids and by histological lipid accumulation and inflammation in muscle, liver, bone marrow, and pancreatic beta cells). Other characteristics or phenotypes of type 2 diabetes are known and can be determined as described herein and by using other methods and techniques known and routinely practiced in the art.

[0346] Obesity and obesity-related abnormalities are used to refer to the condition of a subject whose body weight is significantly greater than the ideal value for their height and build. Body mass index (BMI) is a measurement tool used to determine overweight and is calculated from a subject's height and weight. When a person has a BMI of 25 - 29, the person is considered overweight; when a person has a BMI of 30 - 39, the person is considered obese; and when a person has a BMI of >40, the person is considered severely obese. Thus, the terms obesity and obesity-related refer to human subjects with a body mass index value greater than 30, greater than 35, or greater than 40. A type of obesity not captured by BMI is known in the art as "abdominal obesity," which is related to excess fat found in a subject and is an important factor in health, even independent of BMI. The simplest and most commonly used measure of abdominal obesity is waist circumference. Generally, abdominal obesity in women is defined as a waist circumference of 35 inches or greater, and abdominal obesity in men is defined as a waist circumference 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.

[0347] A condition or abnormality associated with diabetes and aging is a diabetic ulcer (i.e., a diabetic wound). An ulcer is a break in the skin that can extend to involve subcutaneous tissue or even muscle or bone. These lesions occur particularly in the lower extremities. Patients with diabetic venous ulcers exhibit the presence of elevated cellular senescence at the site of the chronic wound (see, e.g., Stanley et al., J. Vas. Surg. 33 (2001) 1206 - 1211). Chronic inflammation has also been observed at the site of chronic wounds such as diabetic ulcers (see, e.g., Goren et al., Am. J. Pathol. 168 (2006) 65 - 77), suggesting that the pro-inflammatory cytokine phenotype of senescent cells has a role in pathology.

[0348] Subjects with type 2 diabetes or at risk of developing type 2 diabetes may have metabolic syndrome. Human metabolic syndrome is typically associated with obesity and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes, and insulin resistance. Subjects with metabolic syndrome may exhibit a range of metabolic abnormalities or disorders, which may for example include one or more of hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (such as hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis, and other metabolic abnormalities.

[0349] Abnormal renal function

[0350] Renal pathologies such as glomerular diseases occur in the elderly and can be treated by administration of the anti-aging compounds described herein. Glomerulonephritis is characterized by inflammation of the kidney 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 to be major regulators of the SASP (see, e.g., Coppe et al., PLoS Biol. 6 (2008) 2853-2868). Glomerular diseases are associated with an increased presence of senescent cells, particularly in fibrotic kidneys (see, e.g., Sis et al., Kidney Int. 71 (2007) 218-226).

[0351] Dermatological diseases or disorders

[0352] Aging-related diseases or disorders treatable by administering the anti-aging agents described herein include dermatological diseases or disorders. Such aging cell-related diseases and disorders include psoriasis and eczema, which are also inflammatory diseases and are discussed in more detail above. Other dermatological diseases and disorders related to aging include wrinkles (wrinkles caused by aging); pruritus (associated with diabetes and aging); paresthesia (a chemotherapy side effect associated with diabetes and multiple sclerosis); psoriasis (as described above) and other papular skin abnormalities such as erythroderma, lichen planus, and lichenoid dermatoses; atopic dermatitis (a form of eczema and associated with inflammation); eczematous rash (commonly observed in elderly patients and associated with side effects of certain medications). Other dermatological diseases and disorders related to aging include eosinophilic dermatoses (associated with certain types of hematological cancers); reactive neutrophilic dermatoses (associated with underlying diseases such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies against desmoglein are formed); pemphigoid and other immunobullous dermatoses (skin autoimmune blisters); cutaneous fibrohistiocytic hyperplasia, which is associated with aging; and cutaneous lymphoma, which is more common in the elderly population. Another dermatological disease treatable by the methods described herein includes cutaneous lupus, which is a symptom of lupus erythematosus. Late-onset lupus may be associated with a decline (i.e., reduction) in the function of T cells and B cells and cytokines associated with aging (immunosenescence).

[0353] Inflammatory and autoimmune diseases and disorders

[0354] In certain embodiments, the aging-related 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) by the methods described herein including administering an anti-aging agent. Other inflammatory or autoimmune diseases or disorders treatable by administering an anti-aging agent such as the inhibitors and antagonists described herein include osteoporosis, psoriasis, oral mucositis, rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis, herniated disc, and lung diseases, COPD, and idiopathic pulmonary fibrosis.

[0355] Osteoarthritis, a degenerative joint disease, is characterized by fibrillation of cartilage fibers, osteosclerosis, and thickening of the synovium and joint capsule at sites of high mechanical stress. Fibrillation is a local surface disintegration involving splitting of the superficial layer of cartilage. The early splits are tangential to the cartilage surface and follow the axis of the major collagen bundles. The collagen within the cartilage becomes disorganized, and proteoglycans are lost from the cartilage surface. In the absence of the protective and lubricating effects of proteoglycans in the joint, the collagen fibers become susceptible to degradation, and mechanical damage ensues. Risk factors predisposing to the development of osteoarthritis include aging, obesity, previous joint injury, joint overuse, thigh muscle weakness, and genetics. Symptoms of osteoarthritis include joint pain or stiffness, particularly in the hips, knees, and lower back after inactivity or overuse; stiffness that resolves after movement; and pain that is more severe after activity or near the end of the day. Osteoarthritis can also affect the neck, small finger joints, base of the thumb, ankles, and big toes. Chronic inflammation is considered a major age-related factor contributing to osteoarthritis. In combination with age, joint overuse and obesity appear to promote osteoarthritis.

[0356] By selectively killing senescent cells, senolytics prevent (i.e., reduce the likelihood of occurrence), 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) the production of collagen (e.g., type II collagen). Removal of senescent cells results in a decrease in the amount (i.e., level) of inflammatory cytokines such as IL-6 produced in joints, and inflammation is alleviated. Methods are provided herein for treating osteoarthritis, selectively killing senescent cells in the osteoarthritic joints of a subject, and / or inducing the production of collagen (such as type II collagen) in the joints of a subject 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. Senolytics can also be used to reduce (inhibit, decrease) the production of matrix metalloproteinase 13 (MMP-13), which degrades collagen in joints, and to restore the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Thus, treatment with senolytics can also prevent (i.e., reduce the likelihood of occurrence of) bone erosion, inhibit or reduce bone erosion, or slow bone erosion (i.e., reduce the rate of bone erosion). As described in detail herein, in certain embodiments, the senolytic agent is administered directly to the osteoarthritic joint (e.g., by intra-articular, topical, transdermal, intradermal, or subcutaneous delivery). Treatment with senolytics can also restore, improve, or inhibit the deterioration of joint strength. In addition, the methods comprising administering a senolytic agent can reduce joint pain and can thus be used for pain control in osteoarthritic joints.

[0357] The effectiveness of one or more anti-aging agents for treating or preventing osteoarthritis in a subject and the monitoring of a subject receiving one or more anti-aging agents can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination (such as determining tenderness, swelling, or redness of the affected joint), assessment and monitoring of clinical symptoms (such as pain, stiffness, range of motion), and performance of analytical tests and methods described herein and practiced in the art (e.g., determining the levels of inflammatory cytokines or chemokines; X-ray images to determine cartilage loss, as indicated by narrowing of the space between bones in the joint; magnetic resonance imaging (MRI), which provides detailed images of bone and soft tissue, including cartilage), can be used to monitor the health status of a subject. The therapeutic effect of one or more anti-aging agents can be analyzed by comparing the symptoms of patients with an inflammatory disease or disorder (such as osteoarthritis) or at risk of developing an inflammatory disease or disorder (who have been treated) with the symptoms of patients who have not received such treatment or have received placebo treatment.

[0358] In certain embodiments, the anti-aging agent can be used to treat and / or prevent rheumatoid arthritis (RA) (i.e., reduce or decrease the likelihood of developing rheumatoid arthritis). Aberrant regulation of innate and adaptive immune responses is characteristic of rheumatoid arthritis (RA), an autoimmune disease whose incidence increases with age. Rheumatoid arthritis is a chronic inflammatory disorder that typically affects the small joints of the hands and feet. Osteoarthritis is caused at least in part by wear and tear of the joints, while rheumatoid arthritis affects the lining of the joints, leading to painful swelling that can result in bone erosion and joint deformity. RA can sometimes also affect other organs of the body, such as the skin, eyes, lungs, and blood vessels. RA can occur in subjects of any age; however, RA typically begins to develop after the age of 40. This disorder is more common in women. In certain embodiments of the methods described herein, RA is excluded.

[0359] Chronic inflammation may also lead to other age-related or senescence-related diseases and abnormalities, such as kyphosis and osteoporosis. Kyphosis is a severe curvature in the spine and is often seen in normal and premature aging (see, e.g., Katzman et al., J. Orthop. Sports Phys. Ther. 40(2010)352 - 360). Age-related kyphosis typically occurs after osteoporosis weakens the spine to the point of fracture and compression. Several types of kyphosis are specific to infants or adolescents. Severe kyphosis can affect the lungs, nerves, and other tissues and organs, causing pain and other problems. Kyphosis is associated with cellular senescence. The ability of an anti-aging agent to treat kyphosis can be determined in preclinical animal models that can be 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 include Bub1 H / H mice, which are also known to develop kyphosis (see, e.g., Baker et al., Nature 479(2011)232 - 236). Over time, the formation of kyphosis is measured visually. The level of senescent cells reduced by treatment with an anti-aging agent can be determined by detecting the presence of one or more markers associated with senescent cells, such as by SA-β-Gal staining.

[0360] Osteoporosis is a progressive bone disease characterized by a reduction in bone mass and bone density, which may lead to an increased risk of fractures and can be treated or prevented by administering the anti-aging agents described herein. Bone mineral density (BMD) is reduced, the bone microarchitecture deteriorates, and the amount and type of proteins in the bone are altered. Osteoporosis is typically diagnosed and monitored by bone mineral density tests. Postmenopausal women or women with reduced estrogen are at greatest risk. Although both men and women over 75 years old are at risk, women are twice as likely to develop osteoporosis as men. The level of senescent cells reduced by treatment with an anti-aging agent can be determined by detecting the presence of one or more markers associated with senescent cells, such as by SA-β-Gal staining.

[0361] In still other embodiments, inflammatory / autoimmune disorders that can be treated or prevented (i.e., the likelihood of occurrence is reduced) with the anti-aging agents described herein include irritable bowel syndrome (IBS) and inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease. Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the digestive tract. In addition to life-threatening complications caused by IBD, the disease can also be painful and debilitating. Ulcerative colitis is an inflammatory bowel disease that causes long-term inflammation of part of the digestive tract. Symptoms usually develop over time rather than suddenly. Ulcerative colitis usually affects only the innermost lining of the large intestine (colon) and rectum. Crohn's disease is an inflammatory bowel disease that causes inflammation anywhere along the inner wall of your digestive tract and often penetrates into the affected tissues. This can lead to abdominal pain, severe diarrhea, and malnutrition. The inflammation caused by Crohn's disease can involve different areas of the digestive tract. Diagnosis and monitoring of the disease are carried out according to methods and diagnostic tests of conventional practice in the art, including blood tests, colonoscopy, flexible sigmoidoscopy, barium enema, CT scan, MRI, endoscopy, and small bowel imaging.

[0362] Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., the likelihood of occurrence is reduced) by using anti-aging agents include eczema, psoriasis, osteoporosis, and lung diseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma), inflammatory bowel disease, and mucositis (including oral mucositis, which is radiation-induced in some cases). Certain organ fibrosis or fibrotic disorders (such as renal fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis, skin wound healing, and oral submucous fibrosis) can be treated with the anti-aging agents described herein.

[0363] In certain embodiments, the abnormalities associated with senescent cells are inflammatory abnormalities of the skin, such as, by way of non-limiting example, psoriasis and 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 anti-aging agents. Psoriasis is characterized by abnormal excessive and rapid growth of the epidermal layer of the skin. The diagnosis of psoriasis is typically based on the appearance of the skin. The typical skin features of psoriasis are scaly red plaques, papules, or skin patches that may be painful and itchy. In psoriasis, cutaneous and systemic overexpression of various pro-inflammatory cytokines (such as IL-6, a key component of the SASP) is observed. Eczema is an inflammation of the skin characterized by redness, swelling of the skin, itching, and dryness, crusting, flaking, blistering, cracking, oozing, or bleeding. The effectiveness of anti-aging agents for treating psoriasis and eczema, as well as the monitoring of subjects receiving such anti-aging agents, can be readily determined by a person skilled in the medical or clinical arts. One or any combination of diagnostic methods, including physical examination (such as the appearance of the skin), monitoring and assessment of clinical symptoms (such as itching, swelling, and pain), and performance of analytical tests and methods described herein and practiced in the art (i.e., determination of the levels of pro-inflammatory cytokines).

[0364] Other immune abnormalities or disorders that can be treated or prevented (i.e., the likelihood of occurrence is reduced) with the anti-aging agents described herein include disorders caused by the host immune response to an organ transplant (e.g., kidney, bone marrow, liver, lung, or heart transplant), such as rejection of the transplanted organ. The anti-aging agents described herein can also be used to treat or reduce the likelihood of occurrence of graft-versus-host disease.

[0365] Cardiovascular diseases and abnormalities

[0366] In other embodiments, the age-related diseases or abnormalities treated by the methods described herein are cardiovascular diseases. Cardiovascular diseases can be any one or more of angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease (CAD), carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction [MI]), high blood pressure / hypertension, aortic aneurysm, brain aneurysm, cardiac fibrosis, diastolic dysfunction of the heart, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral artery disease (PAD)), cardiac stress resistance, and stroke.

[0367] In certain embodiments, methods are provided for treating age-related cardiovascular diseases associated with or caused by arteriosclerosis (i.e., hardening of the arteries). The cardiovascular diseases can be atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease); any one or more of angina, congestive heart failure, and peripheral vascular diseases (e.g., peripheral artery disease (PAD)). The methods for treating cardiovascular diseases associated with or caused by arteriosclerosis can reduce the likelihood of occurrence of high blood pressure / hypertension, angina, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In certain examples, methods are provided for stabilizing atherosclerotic plaques in a subject's blood vessels (e.g., arteries), thereby reducing the likelihood of occurrence of thrombotic events (such as stroke or myocardial infarction) or delaying the occurrence of thrombotic events. In certain embodiments, these methods include administering an anti-aging agent, reducing (i.e., causing a reduction in) the lipid content of atherosclerotic plaques in the subject's blood vessels (e.g., arteries) and / or increasing the fibrous cap thickness (i.e., causing an increase, enhancement, or promotion of the thickening of the fibrous cap).

[0368] Atherosclerosis is characterized by patchy intimal plaques (atheromas) that invade the lumens of medium and large arteries; the plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect medium and large arteries, including coronary arteries, carotid arteries, and cerebral arteries, the aorta and its branches, and the major arteries of the extremities. In some embodiments, methods are provided for inhibiting the formation of atherosclerotic plaques (or reducing, decreasing the formation of atherosclerotic plaques, causing a reduction in the formation of atherosclerotic plaques) by administering an anti-aging agent. In other embodiments, methods are provided for reducing (decreasing, lessening) the amount (i.e., level) of plaques. A reduction in the amount of plaques in a blood vessel (e.g., artery) can be determined, for example, by a reduction in the plaque surface area, or by a reduction in the extent or degree (e.g., percentage) of occlusion of the blood vessel (e.g., artery), which can be determined by angiography or other visualization methods used in the cardiovascular field. Also provided herein are methods for increasing the stability (or improving, promoting, enhancing the stability) of atherosclerotic plaques present in one or more blood vessels (e.g., one or more arteries) of a subject, the method comprising administering to the subject any one of the anti-aging agents described herein.

[0369] Subjects with cardiovascular diseases can be identified using standard diagnostic methods known in the field of cardiovascular diseases. Generally, the diagnosis of atherosclerosis and other cardiovascular diseases is based on symptoms (e.g., chest pain or pressure (angina), numbness or weakness in the arm or leg, difficulty speaking or slurring of speech, drooping of facial muscles, leg pain, high blood pressure, kidney failure, and / or erectile dysfunction), medical history, and / or physical examination of the patient. The diagnosis can be confirmed by angiography, ultrasound, or other imaging tests. Subjects at risk of developing cardiovascular diseases include those with any one or more susceptibility factors (such as a family history of cardiovascular diseases) and those with other risk factors (i.e., susceptibility factors) (such as high blood pressure, dyslipidemia, high cholesterol, diabetes, obesity, and smoking, sedentary lifestyle, and hypertension). In certain embodiments, the cardiovascular disease that is a disease / abnormality associated with senescent cells is atherosclerosis.

[0370] The effectiveness of one or more anti-aging agents in treating or preventing cardiovascular diseases (e.g., atherosclerosis) (i.e., reducing or decreasing the likelihood of its development or occurrence) can be readily determined by those skilled in the medical and clinical arts. One or any combination of diagnostic methods, including physical examination, assessment and monitoring of clinical symptoms, and performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiogram, stress test, non-stress test), can be used to monitor the health status of the subject. Techniques known in the art can be used to analyze the therapeutic effect of the anti-aging agent or a pharmaceutical composition comprising the anti-aging agent, such as comparing the symptoms of patients with cardiovascular diseases or at risk of cardiovascular diseases who have received treatment with those of patients who have not received such treatment or have received placebo treatment.

[0371] Combination therapy

[0372] The anti-aging agents and compositions disclosed herein can also be used in combination with one or more other active ingredients. In certain embodiments, the compounds can be administered in combination with or sequentially with another therapeutic agent. Such other therapeutic agents include those known to be used for treating, preventing, or ameliorating one or more of the symptoms or abnormalities described herein.

[0373] For example, the anti-aging agent can be administered in combination with or after a chemotherapeutic agent. In one embodiment, a tumor is treated with a chemotherapeutic agent that induces a senescent state in tumor cells, and the co-administered anti-aging agent kills the senescent tumor cells. Examples of chemotherapeutic drugs that can be combined with anti-aging compounds for tumor treatment include topoisomerase inhibitors (such as doxorubicin), CDK4 / 6 inhibitors (such as palbociclib), and PARP inhibitors such as olaparib (e.g., see Fleury et al, Nature Communications, 10 (2019) 2556).

[0374] It should be understood that any suitable combination of the compounds and pharmaceutical compositions provided herein with one or more of the foregoing therapeutic agents and optionally one or more other 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.

[0375] Pharmaceutical Compositions and Methods of Administration

[0376] The present disclosure also provides pharmaceutical compositions comprising an anti-aging agent as described herein and at least one pharmaceutically acceptable excipient, which may also be 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 compositions may be sterile aqueous or non-aqueous solutions, suspensions, or emulsions (e.g., microemulsions). The excipients described herein are exemplary and in no way limiting. An effective amount or therapeutically effective amount refers to the amount of one or more anti-aging agents that, when administered to a subject as a single dose or as part of a series of doses, effectively produces the desired therapeutic effect.

[0377] When two or more anti-aging agents are administered to a subject for the treatment of a disease or disorder described herein, each anti-aging agent may be formulated into a separate pharmaceutical composition. A pharmaceutical preparation may be prepared comprising each separate pharmaceutical composition (which, for convenience, may be referred to as, for example, a first pharmaceutical composition and a second pharmaceutical composition comprising a first anti-aging agent and a second anti-aging agent, respectively). Each pharmaceutical composition in the preparation may be administered at the same time (i.e., simultaneously) and by the same route of administration, or may be administered at different times by the same or different routes of administration. Alternatively, two or more anti-aging agents may be formulated together into a single pharmaceutical composition.

[0378] The pharmacokinetics of the anti-aging agent (or one or more of its metabolites) administered to a subject may be monitored by determining the level of the anti-aging agent in a biological fluid, such as the subject's blood, a blood fraction (e.g., serum) and / or urine and / or other biological samples or biological tissues. Any method of detecting pharmaceutical agents practiced in the art and described herein may be used to measure the level of the anti-aging agent during the course of treatment.

[0379] The dosage of the anti - aging agent for treating diseases or abnormalities associated with senescent cells as described herein can depend on the condition of the subject, namely the stage of the disease, the severity of the symptoms caused by the disease, general health status, and age, sex, and weight, and other factors that are obvious to those skilled in the medical art. The pharmaceutical composition can be administered in a manner suitable for the disease to be treated as determined by those skilled in the medical art. In addition to the factors described herein and above related to the use of anti - aging agents for treating diseases or abnormalities associated with aging, the appropriate duration and frequency of administration of the anti - aging agent can also be determined or adjusted by factors such as the condition of the patient, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. The optimal dosage of the agent can generally be determined using experimental models and / or clinical trials. The optimal dosage may depend on the body mass, weight, or blood volume of the subject. It is generally preferred to use the minimum dosage sufficient to provide an effective therapy. The design and conduct of pre - clinical and clinical studies of the anti - aging agents described herein (including when administered for prophylactic benefits) are entirely within the skills of those skilled in the relevant art. When two or more anti - aging agents are administered to treat diseases or abnormalities associated with aging, the optimal dosage of each anti - aging agent can be different from when any one of the agents is administered alone as a single - agent therapy, such as less. In certain embodiments, two anti - aging agents are combined to act synergistically or additively, and either agent can be used in an amount less than if administered alone. The amount of anti - aging agent that can be administered daily can be, for example, between about 0.01 mg / kg and 100 mg / kg (e.g., between about 0.1 mg / kg and 1 mg / kg, between about 1 mg / kg and 10 mg / kg, between about 10 - 50 mg / kg, between about 50 - 100 mg / kg body weight). In other embodiments, the amount of anti - aging agent that can be administered daily is between about 0.01 mg / kg and 1000 mg / kg, between about 100 - 500 mg / kg, or between about 500 - 1000 mg / kg body weight. The optimal dosage (per day or per course of treatment) may be different for the aging - related diseases or abnormalities to be treated and may also vary with the route of administration and treatment regimen.

[0380] By using techniques conventional in the art, a pharmaceutical composition comprising an anti - aging agent can be formulated in a manner suitable for the delivery method. The composition can be in the form of a solid (e.g., tablets, capsules), semi - solid (e.g., gels), liquid, or gas (aerosol). In certain other specific embodiments, the anti - aging agent (or the pharmaceutical composition comprising the anti - aging agent) is administered by bolus infusion. In certain embodiments, when the anti - aging agent is delivered by infusion, the anti - aging agent is delivered via a blood vessel to an organ or tissue comprising senescent cells to be killed according to techniques routinely performed by those skilled in the medical art.

[0381] Pharmaceutically acceptable excipients are well known in the medical art and are described, for example, in Rowe et al., Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 th th Ed., 2006 and Remington: The Science and Practice of Pharmacy (Gennaro, 21 st th 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. In addition, antioxidants and suspending agents may also be used. Generally, the type of excipient is selected according to the mode of administration and the chemical composition of the active ingredient. Alternatively, the compositions described herein may be formulated as lyophilized products. The compositions described herein may be lyophilized or otherwise formulated as a lyophilized product and solubilized and / or diluted with one or more suitable excipient solutions upon administration. In other embodiments, the agent may be encapsulated within liposomes using techniques known and practiced in the art. The pharmaceutical compositions may be formulated for any suitable mode of administration described herein and in the art.

[0382] The pharmaceutical compositions may be delivered to a subject in need thereof by any one of several routes known to those of skill in the art. By way of non-limiting example, the compositions may be delivered orally, intravenously, intraperitoneally, by infusion (e.g., bolus infusion), subcutaneously, enterally, rectally, intranasally, by inhalation, buccally, sublingually, intramuscularly, transdermally, intradermally, topically, intravitreally, vaginally, rectally, or by intracranial injection or any combination thereof. In certain embodiments, as described above, the dose is administered by intravenous, intraperitoneal, direct into the target tissue or organ, or subcutaneous routes. In certain embodiments, the delivery method comprises a drug-coated or permeable stent where the drug is an anti-aging agent. Formulations suitable for such delivery methods are described in more detail herein.

[0383] In certain embodiments, an anti-aging 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 that includes senescent cells contributing to a disease or abnormal manifestation. In a specific embodiment in treating osteoarthritis, the at least one anti-aging agent is administered directly into the osteoarthritic joint (i.e., intra-articularly) of a subject in need thereof. In other specific embodiments, the anti-aging agent may be administered to the joint by topical, transdermal, intradermal, or subcutaneous routes. In certain other embodiments, methods are provided herein for treating cardiovascular diseases or abnormalities associated with arteriosclerosis, such as atherosclerosis, by direct administration into an artery. In other embodiments, an anti-aging agent (which may be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) for treating a lung disease or abnormality associated with aging may be administered by inhalation, intranasally, by intubation, or intrathecally, for example, to provide the anti-aging agent more directly to the affected lung tissue. As another non-limiting example, an anti-aging agent (or a pharmaceutical composition comprising an anti-aging agent) may be delivered directly to the eye by injection (e.g., intravitreally or subretinally) or by applying a cream, ointment, gel, or eye drops under the eyelid conjunctiva. In more specific embodiments, the anti-aging agent or a pharmaceutical composition comprising an anti-aging agent may be formulated as a timed-release (also known as sustained-release, controlled-release) composition or may be administered as a bolus infusion.

[0384] A pharmaceutical composition (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery, or other methods) may be in liquid form. The liquid pharmaceutical composition may contain, for example, one or more of the following: a sterile diluent, such as water, a salt solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a fixed oil that may be used as a solvent or suspension medium, polyethylene glycol, glycerol, propylene glycol, or other solvents; an antibacterial agent; an antioxidant; a chelating agent; a buffering agent; and an agent for adjusting tonicity, such as sodium chloride or glucose. The parenteral composition may be enclosed in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic. Use of physiological saline is preferred, and the injectable pharmaceutical composition is preferably sterile. In other embodiments, for treating an ocular disorder or disease, the liquid pharmaceutical composition may be administered to the eye in the form of eye drops. The liquid pharmaceutical composition may be delivered orally.

[0385] For oral preparations, at least one of the anti-aging agents described herein can be used alone or in combination with suitable additives to prepare tablets, powders, granules or capsules, and if desired, can be used in combination with diluents, buffers, wetting agents, preservatives, colorants and flavoring agents. The compound can be formulated with a buffer (to protect the compound from the low pH of the gastric environment) and / or with an enteric coating. The anti-aging agent contained in the pharmaceutical composition can be formulated with a flavoring agent for oral delivery, for example, in liquid, solid or semi-solid preparations and / or with an enteric coating.

[0386] A pharmaceutical composition comprising any one of the anti-aging agents described herein can be formulated for sustained or slow release (also known as timed release or controlled release). Such compositions can generally be prepared using well-known techniques and administered, for example, orally, rectally, intradermally or subcutaneously by implantation, or by implantation at the desired target site. Sustained release formulations can contain the compound dispersed in a carrier matrix and / or contained within a reservoir surrounded by a rate-controlling membrane. The excipients used in such formulations are biocompatible and can also be biodegradable; preferably, the formulation provides a relatively constant level of release of the active ingredient. The amount of the active agent contained in the sustained release formulation depends on the site of implantation, the rate of release and the expected duration, as well as the nature of the condition, disease or disorder to be treated or prevented.

[0387] In certain embodiments, a pharmaceutical composition comprising an anti-aging agent is formulated for transdermal, intradermal or topical administration. The composition can be administered in the form of a powder / talc or other solid, liquid, spray, aerosol, ointment, foam, cream, gel, paste using a syringe, bandage, transdermal patch, insert or syringe-like applicator. This is preferably in the form of a controlled release formulation or a sustained release formulation, which is topically applied or directly injected into the skin near or within the area to be treated (intradermally or subcutaneously). The active composition can also be delivered by iontophoresis. Preservatives can be used to prevent the growth of fungi and other microorganisms. Suitable preservatives include, but are not limited to, benzoic acid, butyl paraben, ethyl paraben, methyl paraben, propyl paraben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenethyl alcohol, thimerosal and combinations thereof.

[0388] A pharmaceutical composition comprising an anti-aging agent can be formulated as an emulsion for topical application. The emulsion contains a liquid that is distributed in the bulk of a second liquid. The emulsion can be an oil-in-water emulsion or a water-in-oil emulsion. Either or both of the oil phase and the water phase can contain one or more surfactants, emulsifiers, emulsion stabilizers, buffering agents, and other excipients. The oil phase can contain other oil-based pharmaceutically approved excipients. Suitable surfactants include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. The composition for topical application can also contain at least one suitable suspending agent, antioxidant, chelating agent, emollient, or humectant.

[0389] Ointments and creams can be formulated, for example, with an aqueous or oily base in the presence of a suitable thickening agent and / or gelling agent. Lotions can be formulated with an aqueous or oily base and generally also will contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents, or colorants. Liquid sprays can be delivered from a pressurized package, for example, through a specially shaped closure. Oil-in-water emulsions can also be used in compositions, patches, bandages, and articles. These systems are semi-solid emulsion, microemulsion, or foam emulsion systems.

[0390] Controlled-release or sustained-release transdermal or topical formulations can be achieved by adding timed-release additives (such as polymeric structures, matrices) available in the art. For example, the composition can be administered by using a hot-melt extrusion article (such as a bioadhesive hot-melt extrusion 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 adhesion to allow the system to remain attached to the target epithelial or endothelial cell surface for a sufficient length of time to allow the desired release of the compound.

[0391] Inserts, transdermal patches, bandages, or articles can include a mixture or coating of polymers that provides a constant rate of release of the active agent over a long period of time. In some embodiments, the article, transdermal patch, or insert includes a water-soluble pore former, such as polyethylene glycol (PEG), which can be mixed with a water-insoluble polymer to increase the durability of the insert and extend the release of the active ingredient.

[0392] Polymeric formulations can also be used to provide controlled or sustained release. Bioadhesive polymers described in the art can be used. For example, a sustained release gel and a compound can be incorporated into a polymeric matrix, such as a hydrophobic polymeric matrix. Examples of polymeric matrices include microparticles. The microparticles can be microspheres, and the core can be a material different from the polymeric shell. Alternatively, the polymer can be cast into thin sheets or films, powders or gels (such as hydrogels) produced by milling or other standard techniques. The polymer can also be in the form of a coating or part of a bandage, stent, catheter, vascular graft or other device to facilitate delivery of the anti-aging agent. The matrix can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.

[0393] A kit is provided having a unit dose of one or more of the agents described herein, typically in an oral or injectable dosage. Such a kit can include a container containing the unit dose, an information insert describing the use of the drug in treating diseases associated with senescent cells and the attendant benefits, and optionally an appliance or device for delivering the composition.

[0394] For all purposes, all references and patent documents are hereby incorporated by reference in their entirety.

[0395] Examples

[0396] 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)

[0397]

[0398] Dissolve 2-(2-methyl-1H-indol-3-yl)ethyl-1-amine (28) (400 mg, 2.2 mmol) in a mixture of THF:dichloroethane:methanol (5:5:0.5). To this solution, add (E)-3-(4-formylphenyl)acrylic acid (27) (367 mg, 2.09 mmol, 0.95 eq), triacetoxyborohydride (2320 mg, 11 mmol, 5.0 eq) and 3 drops of acetic acid. Let the mixture stir overnight and remove volatiles in vacuo. LC / MS shows the presence of the desired product. Add water and the solid precipitates out of solution. After washing with dilute NaHCO 3After the pH was adjusted to 7, 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 light yellow solid. (620 mg, 81% yield). LC / MS: RT=2.41 min; m / z=335.4 [M+H] + .

[0399] The aforementioned 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 (230 mg, 0.89 mmol, 1.15 equiv) was added portionwise at 0°C. The reaction mixture was allowed to warm to room temperature. Analysis by LC / MS showed the desired product. Dilute HCl was added to pH 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 10-100% 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] + .

[0400] 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)

[0401]

[0402] 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) was added. 2Acetone (440 mg, 0.48 mmol, 0.075 eq). The solution was purged with nitrogen (3x). Then xanthene (558 mg, 0.96 mmol, 0.15 eq) was added in one portion. Within a few minutes, the suspension changed from dark red to yellow-green. Then it was heated at 70 °C for 30 min, at which point LC / MS analysis showed 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, 3x). The solvent was concentrated to dryness and the residue was subjected to normal phase purification, eluting with hexanes:ethyl acetate (40 - 100%). The product fractions were collected, combined and concentrated to give methyl 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-carboxylate as an off-white solid. (1650 mg). LC / MS: RT = 3.23 min; m / z = 351.6 [M+H] + .

[0403] Methyl 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-5-carboxylate (1.65 g, 4.7 mmol) was dissolved in THF (10 mL). 4N HCl / dioxane (9.4 mL, 37.6 mmol, 8.0 eq) was added and the solution was heated at 60 °C for 2 h, during which time a solid precipitated. The hydrochloride precipitate was filtered, washed with ether / hexanes (3x) and dried to give methyl 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylate hydrochloride as a white solid. (1.08 g). LC / MS: RT = 1.88 min; m / z = 251.4 [M+H] + .

[0404] To a solution of methyl 2-(4-(aminomethyl)piperidin-1-yl)pyrimidine-5-carboxylate (1080 mg, 3.77 mmol, 1.0 equiv) and triethylamine (1.5 mL, 10.5 mmol, 2.5 equiv) in THF:DCE (1:1) 5% methanol 18 mL 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) was added along with 4 drops of acetic acid. NMP (1.8 mL) was added and the mixture was stirred at room temperature for 2 days. LC / MS analysis showed 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 afford methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (1300 mg) as a white solid. This material was used in the next step without further purification. LC / MS: RT = 2.67 min; m / z = 394.5 [M+H] + .

[0405] The 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 showed completion of the reaction. The solvent was concentrated to remove the excess alcohol and the mixture was acidified to pH 5 and washed with water then hexanes. The grey solid was dried under high vacuum to afford 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] + .

[0406] Suspend 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) in dioxane:water (3:1) (1.4 mL). Add Fmoc chloride (136 mg, 0.5 mmol, 1.0 equiv) in portions until the solution is clear. LC / MS analysis shows the desired product. Adjust the pH of the solution to 2 and add ethyl acetate. Extract the mixture with water (3x) and wash with brine. Dry the combined organic layers over sodium sulfate, concentrate to dryness to afford a white foam which is triturated with dichloromethane-methanol-hexanes (1:5, 3x). Dry thoroughly to give 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) (200 mg). LC / MS: RT = 3.96 min; m / z = 602.3 [M+H] + 。

[0407] Example 3: Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24)

[0408]

[0409] (2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (23) (6600 mg, 16.05 mmol, 1.0 eq) was dissolved in dichloromethane (80 mL). 2-Hydroxyisoindoline-1,3-dione (2600 mg, 16.05 mmol, 1.0 eq) was added to this solution. At the ice bath temperature, tetrabutylammonium hydrogensulfate (1090 mg, 3.21 mmol, 0.2 eq) in 1 M sodium carbonate (32.0 mL, 32 mmol, 2.0 eq) was added slowly. The mixture was stirred overnight at room temperature. TLC analysis showed the appearance of a new spot at a lower rf compared to the starting material stained with PMA. Water was added and the mixture was extracted with dichloromethane (3x). 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:hexane. 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] + 。

[0410] (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 eq) was dissolved in methanol (35 mL, 0.1 M). Hydrazine hydrate (0.20 mL, 4.25 mmol, 1.05 eq) was added slowly. After five minutes, LC / MS analysis showed the desired product. Dichloromethane (125 mL) was added and the solution was washed with saturated NaHCO 3 (3x). 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:hexane. 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] + 。

[0411] Example 4: Preparation of (2S,3S,4R,5R,6S)-2-(Aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (32)

[0412]

[0413] Dissolve (3S,4R,5R,6S)-2-Hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (5000 mg, 17 mmol) in THF (100.0 mL). Add DAST (18.0 mL, 137 mmol, 8.0 eq) at -30 °C and cool with a dry ice - methanol bath. Allow the reaction mixture to warm to room temperature and stir for 1 h at this temperature. Add another portion of DAST (4 mL) at -30 °C and allow to warm to room temperature again and stir for 1 h. LC / MS analysis indicates completion of the reaction. Add methanol at -20 °C and evaporate the solvent. Add NaHCO 3 , and extract the solution with dichloromethane (3x). Wash the combined organic layers with water. Add dilute HCl to decompose the remaining DAST, and the solution is extracted with ethyl acetate (2x). The combined organic layers are washed with brine, dried over sodium sulfate and concentrated to give the crude product (4000 mg) as an oil. Subject the brown oil to normal phase purification, eluting with hexane - ethyl acetate (0 to 40%). Collect the product fractions, combine and concentrate 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 + H 2 O] + .

[0414] Dissolve (3S,4R,5R,6S)-2-Fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (30) (1800 mg, 6.16 mmol, 1.0 eq) in acetonitrile (30 mL). Add N-Hydroxyphthalimide (1100 mg, 6.78 mmol, 1.1 eq), followed by TEA (1.15 mL, 6.78 mmol, 1.05 eq). Add BF 3 :Et 2 O (1.1 mL, 6.78 mmol, 1.05 eq) dropwise and stir the reaction product for 1 h, at which point LC / MS analysis shows the presence of the desired product. Pour the mixture into 10% NaHCO 3In / EtOAc. The layers were shaken and separated, and the organic layer was washed with sodium bicarbonate (2x), 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 to 50%). The product fractions were collected and combined (low-polarity product - fractions 55 - 64, 1.0 g; high-polarity product - fractions 91 - 105, 500 mg). The low-polarity product was the α-anomer of the desired (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (31). The high-polarity product was the undesired β-anomer. LC / MS: RT = 3.02 min and 3.28 min; m / z = 436.5 [M+H] + .

[0415] (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 showed completion of the reaction within a few minutes. The white precipitate was filtered off. The reaction product was diluted with dichloromethane and filtered a second time. The filtered solution was washed with NaHCO 3 (3x). 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) (500 mg) as a white foam solid. LC / MS: RT = 2.2 min; m / z = 306.6 [M+H] + .

[0416] 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)

[0417]

[0418] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (114 mg, 0.59 mmol, 1.33 eq) and 1-hydroxybenzotriazole (HOBt) (91 mg, 0.59 mmol, 1.33 eq) were added to a solution of (E)-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 eq) in N,N-dimethylformamide (DMF) (1.4 mL) and the mixture was stirred at room temperature for 30 min. Then, at ice bath temperature, (2R,3S,4S,5R,6S)-2-(acetyloxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) (244 mg, 0.67 mmol, 1.5 eq) and DIPEA (0.078 mL, 0.449 mmol, 1.4 eq) were added to the mixture. The mixture was stirred at room temperature overnight. LC / MS analysis showed the desired product. The mixture was quenched with cold saturated NH 4 Cl solution. The white precipitate thus formed was filtered and washed with water (2x). The white solid was redissolved in ethyl acetate and washed with water, NaHCO 3 and brine. The combined organic layers were dried over sodium sulfate and concentrated to afford 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-(acetyloxymethyl)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] + .

[0419]

[0420] (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 eq) was dissolved in methanol (3.0 mL), and 25% sodium methoxide in methanol (0.1 mL, 0.49 mmol, 3.0 eq) was added slowly at the ice bath temperature. After 20 min, LC / MS showed the desired product. The reaction was quenched by adding 10% acetic acid. The water was removed and replaced with methanol. The insoluble salts were removed by filtration twice, 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 (500 MHz, methanol-d 4 ): δ 7.59 - 7.45 (m, 3H), 7.40 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 8.1 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.50 (J = 15.8 Hz, 1H), 4.56 (d, J = 8.0 Hz, 1H), 3.85 (m, 4H), 3.78 - 3.67 (m, 2H), 3.65 - 3.60 (m, 1H), 3.57 (dd, J = 9.6, 3.4 Hz, 1H), 2.97 (dd, J = 7.9, 5.8 Hz, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.36 (s, 3H).

[0421] Example 6: Preparation of (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)

[0422]

[0423] (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 eq) was dissolved in dichloromethane (5 mL). Piperidine (10% in DCM) (7.0 mL, 8.2 mmol, 10.0 eq) was added and the mixture was stirred at room temperature for 3 h. LC / MS analysis showed completion of the reaction. EtOAc (200 mL) was added and the solution was washed with NaHCO 3 (2x). The organic layer was washed with brine, dried over sodium sulfate and concentrated to dryness to afford 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 (550 mg, 80% yield) after purification by HPLC using a HCl-containing buffer. LC / MS: RT = 4.38 min; m / z = 680.7 [M+H] + . 1 HNMR (500 MHz, methanol-d 4 ): δ 7.61 - 7.48 (m, 3H), 7.40 (dt, J = 7.8 Hz, 1.0 Hz, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 6.48 (d, J = 16.1 Hz, 1H), 5.43 (d, J = 3.3 Hz, 1H), 5.32 - 5.19 (m, 2H), 5.02 (d, J = 8.0 Hz, 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).

[0424] 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)

[0425]

[0426] 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, and then DIEA (418 μL, 1.5 equiv) was added at the ice bath temperature. The reaction product was stirred overnight at room temperature. LC / MS analysis showed the desired product. Saturated ammonium chloride solution (20 mL) was added, and a white solid precipitated. The solid was filtered, washed with water (2x), 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) (650 mg) as an off-white solid. LC / MS: RT = 3.89 min; m / z = 844.5 [M+H] + 。

[0427]

[0428] (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%) in methanol (1.0 mL) (0.085 mL, 2.6 equiv) were mixed at ice-bath temperature. The mixture was stirred at room temperature for 2 h. 1N HCl (1.0 equiv) was added and the solution was concentrated to dryness, and the residue was subjected to HPLC purification, where 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) was isolated as a white solid (70 mg). LC / MS: RT = 1.35 min; m / z = 496.5 [M+H] + 。 1 H NMR (500 MHz, methanol-d 4 ): δ 7.69 (d, J = 8.5 Hz, 3H), 7.53 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H) 7.08 - 6.97 (m, 2H), 6.59 (d, J = 15.8 Hz, 1H), 4.57 (d, J = 7.9 Hz, 1H), 4.28 (s, 2H), 3.74 (q, J = 6.4 Hz, 1H), 3.68 - 3.61 (m, 2H), 3.57 (dd, J = 9.7, 3.3 Hz, 1H), 3.30 - 3.22 (m, 2H), 3.14 (dd, J = 9.3, 6.6 Hz, 2H), 2.42 (s, 3H), 1.33 (d, J = 6.4 Hz, 3H).

[0429] 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)

[0430]

[0431] To a solution of compound (117) (640 mg, 0.75 mmol, 1.0 eq) in DCM:DMF (1:1) (10 mL) was added 50% triethylamine / DCM (10.0 mL). The mixture was stirred overnight at room temperature. LC / MS analysis showed the presence of the desired product and starting material. The solvent was concentrated. The residue was dissolved in 50% TEA / DCM (5 mL) and DMF (2.0 mL), and the mixture was stirred for 4 h. Now LC / MS showed that the starting material had been consumed. The solvent was removed under reduced pressure. The residue was triturated with hexane to remove most of the 9-methylene-9H-fluorene. Saturated ammonium chloride solution and ethyl acetate were added, and the organic layer was washed with water (3x), then with brine, and dried over sodium sulfate. The solvent was removed, and the residue 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-triyl triacetate (119) was isolated as its hydrochloride as a white solid. LC / MS: RT = 1.89 min; m / z = 622.4 [M+H] + . 1 H NMR (500 MHz, methanol-d 4 ): δ 7.73 - 7.61 (m, 3H), 7.54 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.55 (d, J = 15.9 Hz, 1H), 5.48 - 5.31 (m, 3H), 5.16 (dd, J = 11.3, 3.9 Hz, 1H), 4.63 (s, 1H), 4.28 (s, 2H), 3.26 (dd, J = 9.5, 6.5 Hz, 2H), 3.14 (dd, J = 9.3, 6.5 Hz, 2H), 2.42 (s, 3H), 2.18 (s, 3H), 2.16 (s, 3H), 2.01 (s, 3H), 1.20 (d, J = 6.5 Hz, 3H).

[0432] 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)

[0433]

[0434] 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 min, after which compound (24) (148 mg, 0.45 mmol, 1.5 equiv) was added, followed by DIEA (80 μL, 1.5 equiv) at the ice bath temperature. The solution was stirred overnight at room temperature. LC / MS analysis showed 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 (2x). The solid was filtered and dried to afford (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) (260 mg) as a white solid, which was used in the subsequent step without further purification. LC / MS: RT = 7.07 min; m / z = 947.9 [M+H] + 。

[0435] 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)

[0436]

[0437] 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 the ice-bath temperature. After stirring at room temperature for 2 h, the Fmoc protecting group was not completely removed. Another 0.2 equiv of sodium methoxide was added at the ice-bath temperature, and the reaction was completed within 20 min. 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 afford 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-d 4 ): 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, 4.6 Hz, 1H), 3.71 (dd, J = 9.6, 8.0 Hz, 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).

[0438] 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)

[0439]

[0440] 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 h. LC / MS analysis showed completion of the reaction. 1N HCl / dioxane (1.0 equiv) was added. The solution was concentrated to dryness and the crude residue was subjected to HPLC purification to afford 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 HNMR (500 MHz, methanol-d 4 ): 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 = 3.5 Hz, 1H), 5.34 (m, 1H), 5.21 (m, 1H), 5.07 (d, J = 8.3 Hz, 1H), 4.81 (d, J = 13.3 Hz, 2H), 4.26 - 4.13 (m, 5H), 3.82 (s, 3H), 2.94 (t, J = 12.8 Hz, 2H), 2.77 (d, J = 6.8 Hz, 2H), 2.13 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.85 (d, J = 11.6 Hz, 2H), 1.19 (m, 2H).

[0441] 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)

[0442]

[0443] 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 min, at which point a portion of compound (32) (96 mg, 0.37 mmol, 1.4 equiv) was added. DIEA (1.5 equiv, 0.073 mL) was added at ice-bath temperature, and the mixture was stirred at room temperature for 1 h. LC / MS analysis showed the presence of the desired product. Saturated ammonium chloride solution was added to the reaction product, and the yellow solid was filtered off and washed with water. Ethyl acetate was added to the solution, and the solution was washed with brine (2x). 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) (200 mg) as a foamy solid, which was used in the next reaction without purification. LC / MS: RT = 6.83 min; m / z = 889.9 [M+H] + 。

[0444] 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)

[0445]

[0446] At ice-bath temperature, compound (115) (70 mg, 0.079 mmol, 1.0 equiv) and sodium methoxide (25%) (2.0 equiv) were dissolved in methanol (0.8 mL). The mixture was stirred at room temperature for 2 h. 1N HCl (1.0 equiv) was added, and 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 4): δ 8.71 (s, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.51 - 7.43 (m, 2H), 7.30 (m, 1H), 7.21 (m, 1H), 4.93 (d, J = 3.9 Hz, 1H), 4.59 (d, J = 7.9 Hz, 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 = 12.4 Hz, 2H), 1.33 (d, J = 6.5 Hz, 3H), 1.26 (m, 2H).

[0447] 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)

[0448]

[0449] 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 time more propionic anhydride (5.0 mL) was added and stirring was continued for an additional day. The mixture was concentrated to dryness, ethyl acetate (80 mL) and water (30 mL) were added, and the aqueous layer was further extracted twice. The combined organic layers were washed with dilute 1N HCl and then with brine. The solution was dried over sodium sulfate and concentrated to dryness to afford (2R,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetra(propionate) (10 g) as a brown oil. LC / MS: RT = 3.3 min; m / z = 467.5 [M + 79] + .

[0450] 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-tetrayl tetra(propionate) (8 g, 20.66 mmol, 1.0 equiv) was added in one portion. The reaction mixture was stirred overnight at room temperature, at which time LC / MS analysis showed mainly unreacted starting material. The reaction product was reprocessed and again subjected to the same reaction conditions, stirred overnight at room temperature. A second batch was repeated on the same scale. LC / MS analysis showed completion of the reaction. 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 and then with brine. The solution was dried over sodium sulfate and the solvent was removed to afford (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate as a white viscous solid. (5.8 g). LC / MS: RT = 2.6 min; m / z = 350.1 [M+18].

[0451] (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 min and the solution was cooled to -50 °C. DAST (5.0 mL) was added dropwise via a plastic pipette. The solution was allowed to warm to 0 °C. TLC analysis showed mainly starting material. Another portion of DAST (2.0 mL) (1.5 equiv) was added at -50 °C and the temperature was raised to 0 °C. TLC analysis showed low polarity spots and a small amount of starting material. As described previously, two additional portions of DAST (3.0 mL each) were added and the reaction product was stirred until the starting material was completely consumed (total of about 8 h). The solution was cooled to -30 °C, methanol (15 mL) was added slowly, and the mixture was allowed to warm to room temperature. Cold NaHCO 3The solution, and the mixture was diluted with dichloromethane. The organic layer was separated and washed with 1 N HCl (2 x), then with brine. The solution was dried over sodium sulfate and the solvent removed to give a yellow oil (4.1 g). TLC analysis showed the product to be impure. The crude product was subjected to normal phase purification, eluting with a gradient of 100% hexane to 15% ethyl acetate in hexane for 60 min. The product fractions were collected and the solvent 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] + 。

[0452] (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 dark red. BF 3 -etherate (1.03 mL, 8.08 mmol, 1.8 equiv) was added and the solution became clear. Stirred at room temperature for 1 h. LC / MS showed the desired product. A cold solution of NaHCO 3 was added and the mixture was extracted twice with ethyl acetate. The solution turned dark red. The combined organic layers were washed twice with NaHCO 3 until no color was detected. The organic solvents were 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, eluting with a gradient of 100% hexane to 25% ethyl acetate in hexane for 40 min. The product fractions were collected as 2 peaks. Peak #1 (normal phase, low polarity) LC / MS (reverse phase): RT = 3.56 min; m / z = 478.4 [M+H]+. Peak #2 (normal phase, high polarity) LC / MS (reverse phase): RT = 3.31 Min; m / z = 478.4 [M+H]+. By comparing its 1 1H NMR spectrum with that of the literature-characterized triacetoxy analogue (31), peak #1 was identified as 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. It was isolated as a white solid (500 mg).

[0453] Dissolve (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) in methanol (10 mL). Hydrazine hydrate (65%, 0.090 mL, 1.04 mmol, 1.0 equiv) was added dropwise at the ice bath temperature. The mixture was stirred for 30 minutes and the solution changed from clear to turbid. LC / MS showed the reaction was complete. Dichloromethane (15 mL) was added and the mixture was extracted twice with cold saturated aqueous NaHCO 3 Extract twice. 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 (280 mg) as a white solid. LC / MS: RT = 2.34 min; m / z = 348.4 [M+H] + .

[0454] Dissolve (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) 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 minutes. (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 h, LC / MS showed the desired product. Saturated NH 4 Cl solution was added, then ethyl acetate was added, and the organic phase was washed with NaHCO 3Extract twice. Wash the combined organic layer with brine and concentrate the solvent to afford a pale yellow foam. (490 mg). Purify the product using normal phase purification on a 40 g silica gel column, eluting with a gradient of 100% hexane to 50% ethyl acetate in hexane for 45 minutes. Collect the product fractions and concentrate the solvent to afford (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] + .

[0455] Dissolve (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 (200 mg, 0.225 mmol, 1.0 equiv) in 50% NEt 3 in DCM / DMF. Stir the solution at room temperature for 3 days to remove the Fmoc protecting group. LC / MS shows the reaction is complete. Concentrate the solvent, add saturated NH 4 Cl solution, then extract the mixture twice with ethyl acetate. Dry the combined organic layers over sodium sulfate. Grind the crude mixture twice with hexane. Concentrate the final product to afford a white foamy solid. (138 mg). Purify the solid 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-triyl tripropionate (121) is isolated as its hydrochloride as a white solid. LC / MS: RT = 4.98 min; m / z = 664.7 [M+H] + . 1 HNMR (500 MHz, methanol-d 4): δ 7.70 - 7.68 (m, 3H), 7.53 (d, J = 7.9 Hz, 2H), 7.47 - 7.40 (m, 1H), 7.28 (dt, J = 8.0, 1.0 Hz, 1H), 7.06 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.03 - 6.97 (m, 1H), 6.55 (d, J = 15.8 Hz, 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 = 7.6 Hz, 3H).

[0456] 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-triyl tris(2-methylpropanoate) (122)

[0457]

[0458] Dissolve L-fucose (3 g, 18.4 mmol, 1.0 eq) in pyridine (13 mL) and chloroform (19.5 mL). Under a nitrogen atmosphere, slowly add isobutyryl chloride (13.14 mL, 123.8 mmol, 6.7 eq) via syringe at ice bath temperature. Allow the reaction mixture to warm to room temperature and then stir for 48 h. Concentrate the solution to dryness under high vacuum and then quench with 2 M HCl (15 mL). Add ethyl acetate (200 mL) and wash the solution twice with water. Wash the organic layer again with 2 M HCl (20 mL), then with saturated sodium bicarbonate solution (30 mL) and then water (30 mL). Dry the organic layer over sodium sulfate solution. Remove the solvent to obtain (2S,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetrakis(2-methylpropanoate) (9.8 g) as a pale yellow oil. LC / MS: RT = 3.93 min; m / z = 462.8 [M + 18].

[0459] 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). (2S,3S,4R,5R,6S)-6-Methyltetrahydro-2H-pyran-2,3,4,5-tetrayl tetrakis(2-methylpropanoate) (8 g, 20.66 mmol, 1.0 equiv) was added in one portion to the suspension of the precipitated solid. The mixture was stirred overnight at room temperature. The next day, LC / MS analysis showed mainly unreacted starting material. The reaction product was reworked and subjected to the same reaction conditions again, this time stirring for a total of five 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 afford (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate) as a yellow oil. The crude product was dried and loaded onto an 80 g silica gel column and purified using a gradient of 100% hexane to 30% ethyl acetate in hexane for 1 h. The product fractions were collected and concentrated to afford (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate) as a colorless oil. (2.89 g). LC / MS: RT = 3.19 min; m / z = 392.5 [M+18].

[0460] At 0 °C, (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate) (2.4 g, 6.4 mmol, 1.0 equiv) in dichloromethane (60 mL) was added in 3 portions over 30 min

[0461] DAST (3.69 mL, 27.5 mmol, 4.3 equiv) was added dropwise. The mixture was stirred for an additional 30 min at this temperature. TLC analysis showed no starting material. The reaction mixture was cooled to -20 °C and methanol (5.0 mL) was added slowly. The mixture was stirred for an additional 15 min, 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 1N HCl, then with saturated sodium bicarbonate solution, and finally with brine. After drying over sodium sulfate, the solvent was removed to afford 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 of 100% hexanes to 25% ethyl acetate in hexanes. The product fractions were collected and the solvent was removed to afford (2RS,3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate) as a white foam (2.0 g). LC / MS: RT = 3.64, 3.78 min; m / z = 377.5 [M+H] + .

[0462] To a solution of (2RS,3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(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). BF 3 .Et 2 O (2.1 mL, 15.9 mmol, 3.0 equiv) was added slowly via syringe. The solution changed from red to clear pale yellow. After 30 min, LC / MS analysis showed the reaction was complete, giving two isomeric products. Dichloromethane was added and the solution was poured into cold sodium bicarbonate solution. 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 afford 2500 mg of crude (2RS,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate). The product was purified by normal-phase column chromatography (on a 40 g silica gel column) using a gradient of 100% hexanes to 25% ethyl acetate in hexanes over 40 min. The product fractions were collected as 2 peaks. Peak #1 (normal-phase, low polarity) LC / MS (reverse-phase): RT = 4.04 min; m / z = 520.6 [M+H]+. Peak #2 (normal-phase, high polarity) LC / MS (reverse-phase): RT = 3.74 min; m / z = 520.6 [M+H]+.

[0463] By comparing its 1 1H NMR spectrum with that of the triacetoxy analogue (31) characterized in the literature, peak #1 was identified as the desired α-isomer, (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate). It was isolated as a white solid (1400 mg). Peak #2 was identified as the β-isomer, (2R,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate). It was isolated as a white solid (500 mg).

[0464] (2R,3S,4R,5R,6S)-2-((1,3-Dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(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 min. The solution changed from clear to turbid. LC / MS analysis showed completion of the reaction. Dichloromethane (25 mL) was added, and the separated organic layer was washed twice with cold saturated NaHCO 3 solution. The organic layer was washed with brine and dried over sodium sulfate. Removal of the solvent gave (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropanoate) (750 mg) as a white solid. 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 min, after which (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(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 h. LC / MS analysis showed the formation of the desired product. Ethyl acetate was added and the mixture was washed twice with saturated NH 4 Cl solution. The ethyl acetate layer was washed twice with aqueous NaHCO 3 and finally with brine. The solvent was removed to afford a pale yellow foam (590 mg). The product was purified using a 10 g silica gel column with normal phase purification, eluting with a gradient of 100% hexane to 30% ethyl acetate in hexane for 45 min. The product fractions were collected and the solvent was removed to afford (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 tris(2-methylpropanoate) (250 mg) as a white foam. LC / MS: RT = 8.13 min; m / z = 829.1 [M+H] + .

[0465] . (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 tris(2-methylpropanoate) (250 mg, 0.26 mmol, 1.0 equiv) was dissolved in DMF (1.0 mL). Triethylamine (1.0 mL) was added and the mixture was stirred overnight at room temperature 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 washed with saturated NH 4The Cl was extracted twice. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was concentrated to afford 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). The 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-triyl tris(2-methylpropanoate) (122) was isolated as its hydrochloride as a white solid. LC / MS: RT = 5.67 min; m / z = 706.9 [M+H] + . 1 H NMR (500 MHz, methanol-d 4 ): δ 7.72 - 7.61 (m, 3H), 7.53 (d, J = 8.2 Hz, 2H), 7.43 (m, 1H), 7.28 (dt, J = 8.0, 1.0 Hz, 1H), 7.06 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.00 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.55 (d, J = 15.9 Hz, 1H), 5.48 (dd, J = 11.1, 3.3 Hz, 1H), 5.40 (dd, J = 3.4, 1.4 Hz, 1H), 5.33 (d, J = 3.9 Hz, 1H), 5.24 (d, J = 10.3 Hz, 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 = 7.0 Hz, 6H).

[0466] 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)

[0467]

[0468] 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) was added LiOH (340 mg, 14.2 mmol), and the resulting mixture was stirred at 50 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with water (10 mL), and acidified to pH 5 with aqueous 2 M HCl. The reaction mixture was then concentrated in vacuo to remove dioxane. The precipitated product was filtered, washed with water (15 mL), and dried under high vacuum to afford 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylic acid (1.5 g, 85%) as an off-white solid. LC / MS: RT = 2.52 min; m / z = 494.4 [M+H] + 。

[0469] 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), then 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 Et 3 N (0.3 mL, 2.1 mmol) was added. The reaction mixture was gradually warmed to room temperature and stirred for 12 h. Saturated aqueous NH 4 Cl (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, and dried over anhydrous Na 2 SO 4Dry, filter, and concentrate to obtain the crude product as a white foamy solid (1 g, 80% yield, 90% purity). Purify 350 mg of this crude material by reverse-phase HPLC to obtain 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 (500 MHz, methanol-d 4 ): δ 9.08 (d, J = 2.6 Hz, 1H), 8.79 (s, 2H), 8.51 (dd, J = 8.8, 2.6 Hz, 1H), 7.56 (s, 1H), 7.04 (d, J = 8.7 Hz, 1H), 5.45 (d, J = 3.3 Hz, 1H), 5.33 (s, 2H), 5.30 (dd, J = 10.4, 8.1 Hz, 1H), 5.24 (dd, J = 10.4, 3.4 Hz, 1H), 5.06 (d, J = 8.1 Hz, 1H), 4.29 - 4.16 (m, 7H), 4.06 (s, 3H), 3.91 (m, 4H), 3.38 (s, 3H), 2.18 (s, 3H). 2.16 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H).

[0470] 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)

[0471]

[0472] In an ice / water bath, a 25% solution of NaOMe in MeOH (0.21 mL, 1 mmol) was added dropwise to a solution of compound (123) (650 mg, 0.77 mmol) in MeOH (7.7 mL). The reaction mixture was then warmed to room temperature and stirred for 1 h. The reaction mixture was quenched by addition of aqueous 1 N HCl to adjust the pH to 7. The solvent was removed under reduced pressure and the residue was purified by reverse phase HPLC to afford 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 (500 MHz, methanol-d 4 ): δ 9.08 (dd, J = 2.7, 0.7 Hz, 1H), 8.85 (s, 2H), 8.51 (dd, J = 8.9, 2.6 Hz, 1H), 7.58 (d, J = 1.0 Hz, 1H), 7.05 (dd, J = 8.9, 0.7 Hz, 1H), 5.36 - 5.34 (m, 2H), 4.63 (d, J = 7.9 Hz, 1H), 4.27 (m, 4H), 4.07 (s, 3H), 3.94 - 3.82 (m, 6H), 3.78 (dd, J = 11.4, 4.6 Hz, 1H), 3.71 (dd, J = 9.7, 7.9 Hz, 1H), 3.65 (ddd, J = 7.6, 4.6, 1.1 Hz, 1H), 3.58 (dd, J = 9.6, 3.4 Hz, 1H), 3.38 (s, 3H).

[0473] 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)

[0474]

[0475] At room temperature, EDC·HCl (377 mg, 1.9 mmol) was added in one portion 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), followed by the addition of HOBt (290 mg, 1.9 mmol). 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 placed at room temperature and stirred for 2 h. Cold saturated NH 4 Cl aqueous solution (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 Na 2 SO 4 , filtered and concentrated to give the crude product (125) (1.1 g, quantitative, 90% purity) as a white foamy solid. 350 mg of this crude material was purified by 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] + . 1 H NMR (500 MHz, methanol-d 4 ): δ 9.08 (dd, J = 2.6, 0.8 Hz, 1H), 8.80 (s, 2H), 8.52 (dd, J = 8.8, 2.6 Hz, 1H), 7.51 (d, J = 0.9 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 5.46 - 5.36 (m, 3H), 5.32 (s, 2H), 5.17 (dd, J = 11.1, 4.0 Hz, 1H), 4.70 (d, J = 6.7 Hz, 1H), 4.20 (m, 4H), 4.05 (s, 3H), 3.90 (m, 4H), 3.37 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 2.01 (s, 3H), 1.19 (d, J = 6.5 Hz, 3H).

[0476] 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))

[0477]

[0478] In an ice / water bath, a 25% solution of NaOMe in MeOH (0.22 mL, 1.06 mmol) was added dropwise to a solution of compound (125) (750 mg, 0.96 mmol) in MeOH (9.6 mL). The reaction mixture was then warmed to room temperature and stirred for 1 h. The reaction mixture was quenched by adding aqueous 1N HCl to adjust the pH to 7. The solvent was removed under reduced pressure, and the residue was purified by reverse-phase HPLC to afford 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] + 。 1 H NMR (500 MHz, methanol-d 4 ): δ 9.08 (dd, J = 2.7, 0.7 Hz, 1H), 8.81 (s, 2H), 8.51 (dd, J = 8.8, 2.6 Hz, 1H), 7.57 (d, J = 0.9 Hz, 1H), 7.05 (dd, J = 8.8, 0.7 Hz, 1H), 5.34 (m, 2H), 5.15 (d, J = 3.7 Hz, 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, 1.3 Hz, 1H), 3.38 (s, 3H), 1.26 (d, J = 6.5 Hz, 3H).

[0479] 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)

[0480]

[0481] The β-D-galactoside 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-phenyglycosylation in aqueous media. Starting from the 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 gave a mixture of regioisomers of the β-D-galactoside separated by HPLC.

[0482] α-D-galactopyranosyl fluoride was prepared as follows: A cold solution of 70% HF·pyridine (17 mL, 1.5 M) was slowly added via syringe to β-D-galactose pentaacetate (10 g, 25.7 mmol, 1 equiv) in a plastic bottle cooled in an ice / water bath. The reaction vessel was capped and 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 NaHCO 3 aqueous solution (150 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and dried under high vacuum to give the crude residue.

[0483] To the above residue was added anhydrous MeOH (130 mL), and the resulting mixture was stirred for 15 min. The reaction mixture was then cooled in ice / water for 10 min, NaOMe (139 mg, 2.57 mmol) was added, and after 10 min, the ice / water bath was removed and the reaction mixture was allowed to warm gradually to room temperature over 3 h. The reaction mixture was cooled again in the 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 well for 5 min and filtered. The residue was washed with 7:3 EtOAc:MeOH (60 mL), and the combined filtrates were 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 any further purification.

[0484] 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) 2 (488 mg, 6.6 mmol), and the resulting mixture was stirred at room temperature for 8 h. LC / MS analysis showed a clear conversion to the desired compounds (46) and (47). The reaction mixture was quenched by adding aqueous 1 M HCl to adjust the pH to 8, and the resulting mixture was concentrated in vacuo to remove water. The DMSO solution of the residue was purified directly by reverse-phase HPLC, and two regioisomeric products were isolated. The regiochemistry of the products was deduced by correlating the retention times on reverse-phase LC / MS with the calculated LogP values (the isomer with the lower calculated lipophilicity was assigned to the earlier eluting peak on HPLC).

[0485] The earlier 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) (cLogP = 1.8) as a white solid. LC / MS: RT = 1.13 min; m / z = 572.5 [M+H] + . 1 H NMR (500 MHz, methanol-d 4): 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 = 6.9 Hz, 3H) 1.24 (d, J = 6.9 Hz, 3H).

[0486] The subsequent elution 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) (cLogP = 2.0) as a white solid. LC / MS: RT = 1.20 min; m / z = 572.5 [M+H] + . 1 1H NMR (500 MHz, methanol-d 4 ): δ 7.42 - 7.35 (m, 1H), 7.35 - 7.27 (m, 1H), 7.25 - 7.18 (m, 1H), 7.12 (d, J = 1.7 Hz, 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 = 8.8 Hz, 3H), 1.24 (d, J = 6.8 Hz, 3H), 1.22 (d, J = 6.8 Hz, 3H).

[0487] 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)

[0488]

[0489] 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-phenyglycosylation 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) 2 afforded a regioisomeric mixture of β-D-galactosides, which was separated by HPLC.

[0490] 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) 2 (47 mg, 0.64 mmol) in water (0.4 mL) were suspended in a 4 mL vial. The reaction vial was capped, and the resulting mixture was stirred at room temperature for 3 h. LC / MS indicated a distinct conversion to the desired compounds (48) and (49). The reaction mixture was quenched by addition of aqueous 1 M HCl to adjust to pH 8, and the resulting mixture was purified directly by reverse-phase HPLC to afford the two regioisomeric products. The regiochemistry of the products was deduced by correlating the retention times on reverse-phase LC / MS with the calculated LogP values (the isomer with the lower calculated lipophilicity was assigned to the earlier eluting peak on HPLC).

[0491] The earlier 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) (cLogP = 0.88) as a white solid. LC / MS: RT = 1.29 min; m / z = 628.4 [M+H] + . 1 1H NMR (500 MHz, methanol-d 4): 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 = 7.3 Hz, 3H), 1.06 (d, J = 6.9 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H).

[0492] The subsequent elution 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) (cLogP = 1.02) as a white solid. LC / MS: RT = 1.43 min; m / z = 628.4 [M + H] + . 1 H NMR (500 MHz, methanol - d 4 ): δ 7.45 (s, 4H), 7.05 (s, 1H), 6.72 (s, 1H), 4.71 (d, J = 7.7 Hz, 1H), 4.35 (q, J = 7.3 Hz, 2H), 4.06 (br.s, 1H), 3.86 (m, 1H), 3.77 - 3.70 (m, 3H), 3.60 (m, 1H), 3.48 - 3.20 (m, 10H), 3.16 (m, 1H), 1.23 (t, J = 7.3 Hz, 3H), 1.10 (d, J = 6.9 Hz, 3H), 1.09 (d, J = 6.9 Hz, 3H).

[0493] 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)

[0494]

[0495] 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 Cs 2 CO 3 (3.2 g, 9.8 mmol), and the resulting mixture was stirred at room temperature for 15 h. Water (100 mL) was added, and the resulting mixture was extracted with EtOAc (3 X 50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was dried under high vacuum overnight.

[0496] To the above residue was added MeOH (5 mL) and NaOMe (25% solution in MeOH, 25 μL, 0.11 mmol), and the resulting mixture was stirred at room temperature for 1 h. LC / MS indicated significant conversion to the desired compounds (127) and (128). The reaction mixture was quenched by addition of 4 M HCl in dioxane solution to adjust to pH 8. The reaction mixture was then concentrated in vacuo and purified by reverse-phase HPLC to afford two regioisomer products. The regiochemistry of the products was deduced by correlating the retention times on reverse-phase LC / MS with the calculated LogP values (the isomer with the lower calculated lipophilicity was assigned to the earlier eluting peak on HPLC).

[0497] The earlier 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) (cLogP = 1.96) as a white solid. LC / MS: RT = 1.30 min; m / z = 527.2 [M+H] + 。 1 H NMR (500 MHz, methanol-d 4): δ 7.53 - 7.46 (m, 2H), 7.29 (d, J = 3.2 Hz, 1H), 7.08 (dd, J = 8.6, 2.1 Hz, 1H), 6.71 (s, 1H), 6.67 (s, 1H), 6.49 (dd, J = 3.1, 0.8 Hz, 1H), 4.80 (d, J = 7.8 Hz, 1H), 3.90 (d, J = 2.9 Hz, 1H), 3.86 (s, 3H), 3.82 - 3.71 (m, 3H), 3.67 (m, 1H), 3.55 (dd, J = 9.7, 3.4 Hz, 1H), 3.14 (m, 1H), 0.73 (dd, J = 6.9, 1.7 Hz, 6H). The subsequent elution 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) (cLogP = 2.09) as a white solid. LC / MS: RT = 1.54 min; m / z = 527.2 [M + H] + 。 1 H NMR (500 MHz, methanol-d 4 ): δ 7.55 - 7.49 (m, 2H), 7.30 (d, J = 3.1 Hz, 1H), 7.09 (dd, J = 8.6, 2.0 Hz, 1H), 6.59 (s, 1H), 6.51 (d, J = 3.0 Hz, 1H), 6.29 (s, 1H), 5.27 (d, J = 9.1 Hz, 1H), 4.40 (m, 1H), 3.98 (d, J = 3.3 Hz, 1H), 3.87 (s, 3H), 3.85 - 3.75 (m, 3H), 3.72 (dd, J = 9.6, 3.3 Hz, 1H), 2.91 (m, 1H), 0.67 (d, J = 6.9 Hz, 3H), 0.65 (d, J = 6.9 Hz, 3H).

[0498] Example 23: Toxicity of the compound to normal proliferating, senescent or quiescent fibroblasts

[0499] Normal human fibroblasts (IMR90) and patient-derived cells deficient in β-galactosidase (GLB1) or α-fucosidase (FUCA1) were used to determine changes in cell viability after exposure to compounds. Cells were cultured in DMEM and 10% heat-inactivated FBS under a controlled atmosphere of 5% carbon dioxide and 5% oxygen. Senescence was induced by contact inhibition of cells and confirmed by the absence of staining for senescence-associated β-galactosidase (SA-β-Gal) and the ability to re-enter the cell cycle. Senescence was induced by treatment with doxorubicin and confirmed by SA-β-Gal positive staining and the lack of DNA replication. Cells were treated with compounds for three days. Cell viability was determined by mitochondrial dehydrogenase activity (XTT assay, Cayman Chemical) at ten concentrations of the test compound. To generate dose-response curves, the data were fitted to a four-parameter Hill function, and the IC 50 was determined at a viability of Y = 0.5. The senolytic index (S.I.) for each compound was determined by dividing the IC 50 for normal proliferating cells by the IC 50 for senescent cells. The senolytic index data are shown in Table 1, where the compounds have S.I. < 1 (−), S.I. > 1 (+), S.I. > 5 (++), and S.I. > 10 (+++).

[0500]

[0501]

[0502] Example 24: Senolytic effect of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) on mouse embryonic fibroblasts (MEF)

[0503] Mouse embryonic fibroblasts were incubated with 300 nM doxorubicin for 24 h to induce senescence. Cells were washed and, after incubation in medium for 7 days, senescence was confirmed by SA-β-Gal staining (Itahana et al., Methods Mol. Biol. 371 (2007) 21-31) and the lack of incorporation of 5-ethynyl-2'-deoxyuridine (EdU) (see Yu et al., J. Immunol. Methods 350 (2009) 29-35).

[0504] Proliferating or senescent MEF were treated with increasing concentrations of 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), and the proportion of live cells was evaluated by the ratio of PI-positive to PI-negative nuclei. Figure 1AThe results shown in Figure 1B indicate that the active drug (102) significantly reduces cell viability, while the prodrug (101) is completely non-toxic to proliferating cells at concentrations up to 4 mM. In contrast, compounds (101) and (102) have equal toxicity to senescent MEFs, as

[0505] Example 25: Comparative toxicity of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) and 5-fluorouridine (FUR) (102) in C57BL / 6 mice

[0506] Equimolar doses of (101) (160 mg / kg) and (102) (100 mg / kg) were administered by single-dose intraperitoneal injection to two groups of mice (N = 3 per group). Six days after treatment, blood cell counts were determined by standard methods ( Figure 2A ), bone marrow cells from the femurs were counted ( Figure 2B ), and spleen weights were determined ( Figure 2C ) to compare the toxicity of the two drugs. The prodrug (101) showed negligible toxicity to platelets, neutrophils, lymphocytes, and bone marrow cells, while FUR (102) induced a significant reduction in all cell populations. The prodrug (101) also showed a minimal effect on spleen weight compared to FUR (102).

[0507] Example 26: Effect of 5-fluorouridine-5'-O-β-D-galactopyranoside (FURGal) (101) on hepatocytes of senescent C57BL / 6 mice after in vivo administration

[0508] Two groups of C57BL / 6 mice (N = 5 per group) were intraperitoneally injected with doxorubicin (25 mg / kg) to induce hepatocyte senescence. Four and six days later, each group was injected with PBS or FURGal (140 mg / kg) (101). Five animals in a third group were used as controls. Four days later, before SA-β-Gal staining, the livers were removed and sectioned (30 μm) (six fields of view / animal were evaluated) ( Figure 3A and 3B ). As Figure 3C shown, quantification revealed a numerical trend of reduced senescent hepatocytes after FURGal treatment. The average body weights of the mice on the day of analysis are shown in Figure 3D .

[0509] Example 27: Effect of (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) (also known as RBO-013) on senescent hepatocytes in aged C57BL / 6 mice

[0510] Two groups of C57BL / 6 mice (N = 9 mice / group) were intraperitoneally injected with doxorubicin (20 mg / kg) to induce hepatocyte senescence. Four and six days later, each group was intraperitoneally injected with PBS or compound (113) (also known as RBO-013) (2 x 20 mg / kg). A third group of animals was used as a control. After an additional 2 days, as described previously, the livers were removed and sectioned (30 μm) before SA-β-Gal staining ( Figure 4A and 4B ). As Figure 4C shown, quantification revealed a decrease in senescent hepatocytes after treatment with (113) (visualized by SA-β-Gal staining). This anti-aging effect was also confirmed by detecting the mRNA levels of Cdkn2a (p16 INK4a ) and IL-6 (note that the expression of Cdkn2a was only detected in animals receiving doxorubicin) using quantitative PCR. The expression levels were reported relative to Actb as a reference gene. Figure 4D and 4E respectively showed a significant decrease in the gene expression of Cdkn2a and IL-6.

[0511] Example 28: Anti-aging 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) (also known as RBO-019) on senescent lung cells in C57BL / 6 mice

[0512] Four groups of C57BL / 6 mice (N = 9 mice / group) were intravenously injected with doxorubicin (15 mg / kg) to induce senescence of lung tissue. Five days later, each group was injected with vehicle or compound (119) (also known as RBO-019) (10 mg / kg, 20 mg / kg or 40 mg / kg intravenously). After an additional 3 days, as described previously, the lungs were removed and the left lobe was sectioned ( Figure 5A and 5B ). As Figure 5CAs shown, quantification revealed a dose-dependent decrease in SA-β-Gal staining after treatment with (119). This anti-aging effect was also confirmed by using quantitative PCR to detect the mRNA levels of Cdkn2a (p16 INK4a ). The expression levels relative to Actb, which was used as a reference gene, were reported. Figure 5D A dose-dependent decrease in the expression of the Cdkn2a gene in the lung was shown.

Claims

1. A compound of formula (IV) or (V) or a pharmaceutically acceptable salt, hydrate or solvate thereof: Wherein, R is a residue of a hydroxamic acid derivative histone deacetylase inhibitor, R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 are each independently hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): R 33 、R 34 、R 35 、R 36 、R 43 、R 44 and R 45 each independently is hydrogen or -C(O)-R 2 ; each R 1 independently is C 1-4 alkyl or phenyl, provided that if R 13 、R 14 、R 15 or R 16 is part of formula (VI) or formula (VII), then the remaining portions of R 13 、R 14 、R 15 and R 16 are hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if R 23 、R 24 or R 25 is part of formula (VI) or formula (VII), then the remaining portions of R 23 、R 24 or R 25 are hydrogen or -C(O)-R 1 ; provided that when R 13 、R 14 、R 15 and R 16 are each hydrogen, then R is not 7-heptanoylbenzamide.

2. The compound according to claim 1, wherein the anomeric carbon of the pyranose ring (marked with *) is in the S configuration, and the compound is a β-D-galactoside and an α-L-fucoside conjugate of a hydroxamic acid derivative histone deacetylase inhibitor, respectively.

3. The compound according to claim 2, wherein the hydroxamic acid derivative histone deacetylase inhibitor is selected from the group consisting of: panobinostat, quisinostat, vorinostat, danostat, givinostat, CUDC-907, CUDC-101, abexinostat, belinostat, punostat, reinostat, licinostat, pyroglutamide, APHA, trichostatin A, oxaladine and AR-42.

4. The compound according to claim 3, having any one of the following structures:

5. The compound according to claim 3, having any one of the following structures:

6. The compound according to claim 3, having any one of the following structures:

7. A compound of formula (XI), (XII), (XIII) or (XIV) or a pharmaceutically acceptable salt, hydrate or solvate thereof: wherein Y is a carbonyl group 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 chlorine; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 each independently is hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): R 33 、R 34 、R 35 、R 36 、R 43 、R 44 and R 45 Each is independently hydrogen or -C(O)-R 2 ; each R 1 is independently C 1-4 alkyl or phenyl, provided that if one of R 13 、R 14 、R 15 or R 16 is part of formula (VI) or formula (VII), then the remaining portions of R 13 、R 14 、R 15 and R 16 are hydrogen or C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if one of R 23 、R 24 or R 25 is part of formula (VI) or formula (VII), then the remaining portions of R 23 、R 24 or R 25 are hydrogen or -C(O)-R 1 .

8. The compound according to claim 7, wherein the anomeric carbon of the pyranose ring (marked with *) is in the S configuration, and the compound is a β-D-galactoside and an α-L-fucoside conjugate of an Hsp90 inhibitor, respectively.

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

10. The compound according to claim 9, having any one of the following structures:

11. A compound of formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV) or (XXV) or a pharmaceutically acceptable salt, hydrate or solvate thereof: wherein R 8 is 4-morpholinyl or 1-imidazolyl; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 , and R 25 each independently is hydrogen, -C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): R 33 、R 34 、R 35 、R 36 、R 43 、R 44 and R 45 Each is independently hydrogen or -C(O)-R 2 ; each R 1 is independently C 1-4 alkyl or phenyl, provided that if one of R 13 、R 14 、R 15 or R 16 is part of formula (VI) or formula (VII), then the remaining portions of R 13 、R 14 、R 15 and R 16 are hydrogen or -C(O)-R 1 ; and each R 2 is independently C 1-4 alkyl or phenyl, provided that if one of R 23 、R 24 or R 25 is part of formula (VI) or formula (VII), then the remaining portions of R 23 、R 24 or R 25 are hydrogen or -C(O)-R 1 .

12. The compound according to claim 11, wherein the anomeric carbon of the pyranose ring (marked with *) has the S configuration, and the compound is a β-D-galactoside and an α-L-fucoside conjugate of a TOP1 inhibitor, respectively.

13. The compound according to claim 12, having any one of the following structures:

14. The compound according to claim 12, having any one of the following structures:

15. A compound of formula (XXVIII) or (XXIX) or a pharmaceutically acceptable salt, hydrate or solvate thereof: wherein R 9 is hydrogen, C 1-4 alkyl, CF 3 , CN or NO 2 ; R 10 is hydrogen, C 1-4 alkyl or arylalkyl; R 13 , R 14 , R 15 , R 16 , R 23 , R 24 and R 25 each independently is hydrogen, C(O)-R 1 , a moiety of formula (VI) or a moiety of formula (VII): R 33 、R 34 、R 35 、R 36 、R 43 、R 44 and R 45 Each independently is hydrogen or C(O)-R 2 ,each R 1 is independently a C 1-4 alkyl or phenyl, provided that if R 13 、R 14 、R 15 or R 16 is part of formula (VI) or formula (VII), then the remainder of R 13 、R 14 、R 15 and R 16 is hydrogen or C(O)-R 1 ; and each R 2 is independently a C 1-4 alkyl or phenyl, provided that if R 23 、R 24 or R 25 is part of formula (VI) or formula (VII), then the remainder of R 23 、R 24 or R 25 is hydrogen or C(O)-R 1 ; provided that in the compounds of formula (XXVIII), when R 9 is NO 2 and R 10 is benzyl, R 13 、R 14 、R 15 、R 16 are not simultaneously hydrogen or acetyl.

16. The compound according to claim 15, wherein the anomeric carbon of the pyranose ring (marked with *) is in the S configuration, and the compound is a β-D-galactoside and an α-L-fucoside conjugate of a pyrrolo[2,1-c][1,4]benzodiazepine analogue, respectively.

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

18. A method for treating a disease or disorder associated with aging, comprising administering to a subject in need thereof an effective therapeutically amount of a compound of the following chemical formula or a pharmaceutically acceptable salt, pharmaceutical composition, hydrate or solvate thereof: wherein R 46 is hydrogen, an acetyl group or a propionyl group.

19. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is selected from the group of age-related disorders consisting of: kidney disease, kidney failure, frailty, cognitive impairment, hearing loss, muscle fatigue, skin disorders, skin wound healing, liver fibrosis, pancreatic fibrosis, submucosal fibrosis of the oral cavity, and sarcopenia.

20. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is selected from the group of lung diseases consisting of: pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, and age-related loss of lung function.

21. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is a metastatic lesion.

22. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is a side effect of chemotherapy or radiotherapy.

23. The method according to claim 22, wherein the side effect of chemotherapy or radiotherapy is gastrointestinal toxicity, peripheral neuropathy, fatigue, malaise, low physical activity, frailty, hematotoxicity, hepatotoxicity, alopecia (hair loss), pain, infection, mucositis, fluid retention, dermatological toxicity, mouth, gum or throat problems, cardiomyopathy, congestive heart disease, inflammation, premature menopause, osteoporosis, infertility, impaired cognitive function, secondary cancer, cataracts and other vision problems, hearing loss, reduced lung volume, and lung diseases.

24. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is selected from the group of neurodegenerative diseases consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, and abnormal motor neuron function.

25. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is selected from the group of eye diseases or disorders consisting of: macular degeneration, glaucoma, cataracts, presbyopia, and vision loss.

26. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is selected from the group of metabolic diseases consisting of: diabetes, diabetic ulcers, metabolic syndrome, and obesity.

27. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is renal dysfunction.

28. The method according to claim 17 or 18, wherein the disease or disorder associated with aging is a dermatological disease or disorder selected from the group consisting of: eczema, psoriasis, hyperpigmentation, moles, rashes, atopic dermatitis, urticaria, diseases and disorders associated with photosensitivity or photoaging, wrinkles; pruritus; paresthesia ； eczematoid rash; eosinophilic dermatoses; reactive neutrophilic dermatoses; pemphigus; pemphigoid; immunobullous dermatoses; Dermal fibrohistiocytic hyperplasia; cutaneous lymphoma; and cutaneous lupus.

29. The method according to 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. The method according to 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 thrombosis, myocardial infarction, hypertension, aortic aneurysm, diastolic dysfunction of the heart, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, cerebral aneurysm, and stroke.

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

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

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

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

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