Anti-aging composition and use thereof

Abstract

Anti-aging agents for selectively killing senescent cells associated with a variety of pathologies and diseases, including age-related pathologies and diseases, are provided herein. 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 are 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, nervous system diseases or abnormalities, dermatological diseases or abnormalities, chemotherapy side effects, radiation therapy side effects, metastatic lesions, and metabolic diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the 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] Anti-aging agents for selectively killing senescent cells associated with a variety of pathologies and diseases, including age-related pathologies and diseases, are provided herein. 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 are 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, nervous system diseases or abnormalities, dermatological diseases or abnormalities, chemotherapy side effects, radiation therapy side effects, metastatic lesions, and metabolic diseases. Background Art

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

[0005] Anti-aging agents with multiple pharmacological mechanisms are known in the art. Anti-aging agents can be specific inhibitors of one or more members of the Bcl-2 anti-apoptotic protein family, wherein the inhibitor inhibits at least 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 inhibitors (e.g., MK-2206); receptor tyrosine kinase inhibitors (e.g., dasatinib, see, e.g., Zhu et al., Aging Cell 14 (2015) 654-658); CDK4 / 6 inhibitors (e.g., palbociclib (see, e.g., Whittaker et al., Pharmacol. Ther. 173 (2017) 83-105)); mTOR inhibitors (e.g., rapamycin, (see, e.g., Laberge et al., Nat. Cell Biol. 17 (2015) 1049-1061)); MDM2 inhibitors (e.g., Nutlin-3; and RG-7112, see, e.g., U.S. Patent Application 2016 / 0339019)); Hsp90 inhibitors (e.g., 17-DMAG; and ganetespib, see, e.g., Fuhrmann-Stroissnigg et al., al., Nat. Commun. 8 (2017) doi: 10.1038 / s41467-017-00314-z)); 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 inhibitors (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 retaining non-senescent cells. In addition, many known anti-aging agents were originally developed as cytotoxic anticancer agents and were subsequently reused to 'selectively' remove 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 the exposure of non-senescent cells to these molecules and potentially limit off-target effects. Therefore, what is needed is an anti-aging agent with improved selectivity for killing senescent cells, which has minimal toxicity to non-senescent cells. Summary of the invention

[0007] Disclosed herein are non-toxic prodrugs of anti-senescence agents activated by hydrolases that preferentially accumulate in 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 of a pro-apoptotic agent (I) into a toxic pro-apoptotic parent compound II), resulting in specific killing of senescent cells.

[0008]

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

[0010] In another aspect, non-toxic prodrugs of toxic anti-aging agents are provided, which, when cleaved into active anti-aging agents in senescent cells, specifically cause senescent cell death. In some embodiments, prodrugs of histone deacetylase inhibitors are provided. In other embodiments, prodrugs of Hsp90 inhibitors are provided. In yet other embodiments, prodrugs of topoisomerase 1 inhibitors are provided. In yet other embodiments, prodrugs of DNA alkylating agents are provided. In yet other embodiments, prodrugs of Akt1 inhibitors are provided. In yet other embodiments, prodrugs of proteasome inhibitors are provided. Derivatives of the prodrugs described herein are also provided, including salts, solvates, hydrates, and metabolites. Compositions are also provided, which include the prodrugs provided herein and carriers.

[0011] In another aspect, provided herein are methods for treating, preventing or alleviating symptoms of a medical abnormality in a subject, such as, for example, cardiovascular disease or abnormality, cardiovascular disease and abnormality associated with arteriosclerosis (such as atherosclerosis), idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, inflammatory disease or abnormality, autoimmune disease or abnormality, pulmonary disease or abnormality, nervous system disease or abnormality, dermatological disease or abnormality, chemotherapy side effects, radiation therapy side effects, metastatic lesions, and metabolic diseases. In practicing the method, 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 condition is provided. The method comprises 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 comprises 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, comprising administering a therapeutically effective amount of the anti-aging agent or a pharmaceutical composition thereof to a subject who has received DNA damaging therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A Shown is 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 Shown is the viability of aged 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 Shown are quantifications of blood cell counts from wild-type C57BL / 6 mice dosed with FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) by a single intraperitoneal injection after 6 days of treatment (N=3 mice / group).

[0017] Figure 2BShown are quantifications of bone marrow cell numbers in femurs from wild-type C57BL / 6 mice dosed with FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) by a single intraperitoneal injection 6 days after treatment (N=3 mice / group).

[0018] Figure 2C Shown are quantifications of total spleen weights from wild-type C57BL / 6 mice dosed with FUR(102) (100 mg / kg) or FURGal(101) (160 mg / kg) by a single intraperitoneal injection after 6 days of treatment (N=3 mice / group).

[0019] Figure 3A Representative images of liver sections from C57BL / 6 mice injected with doxorubicin (25 mg / kg) are shown.

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

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

[0022] Figure 3D Shown are the mean body weights of C57BL / 6 mice on the day of analysis.

[0023] Figure 4A A protocol for induction of hepatocyte senescence in C57BL / 6 mice and subsequent treatment with compound (113) is shown.

[0024] Figure 4B Representative images of liver sections from C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113) are compared.

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

[0026] Figure 4D Shown is the quantification of Cdkn2a expression in the liver of C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113).

[0027] Figure 4E Shown is the quantification of IL-6 expression in the liver of C57BL / 6 mice injected with doxorubicin (20 mg / kg) followed by vehicle or compound (113).

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

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

[0030] Figure 5C Shown are the quantification of SA-β-Gal in lung sections following intravenous administration of compound (119) at 10 mg / kg, 20 mg / kg or 40 mg / kg along with controls.

[0031] Figure 5D Shown is the quantification of Cdkn2a expression in the lungs of C57BL / 6 mice injected with doxorubicin (15 mg / kg) followed by vehicle or compound (119) at 10 mg / kg, 20 mg / kg or 40 mg / kg. DETAILED DESCRIPTION

[0032] definition

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

[0034] "Acyl" means a H-CO-, alkyl-CO-, alkenyl-CO- or cycloalkyl-CO- group, wherein the alkyl, alkenyl or cycloalkyl group is as described herein.

[0035] "Acylamino" is an acyl-NH- group in which acyl is as defined herein.

[0036] As used herein, "age-related diseases or conditions" include, but are not limited to, degenerative diseases or functional impairments such as Alzheimer's disease, Parkinson's disease, cataracts, macular degeneration, glaucoma, frailty, muscle weakness, cognitive impairment, atherosclerosis, acute coronary syndrome, myocardial infarction, stroke, hypertension, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), osteoarthritis, type 2 diabetes, obesity, adipose dysfunction, coronary artery disease, cerebrovascular disease, periodontal disease, cancer treatment-related disabilities (such as atrophy and loss of function in various tissues), and other diseases. fibrosis, brain and cardiac damage and therapy-associated 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, Roth-Thompson Syndrome, Cockaine Syndrome, Xeroderma Pigmentosum, Trichothiodystrophy, Combined Xeroderma Pigmentosum-Cockaine Syndrome, Restrictive Dermatosis)), ataxia-telangiectasia, Fanconi anemia, Friedreich'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 or branched, with 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 some other embodiments, the alkenyl group has 2 to 4 carbon atoms in the chain. "Branched" as used herein and throughout the text refers to one or more lower alkyl groups (such as methyl, ethyl or propyl) connected to a straight chain; Here is a linear alkenyl chain. "Lower alkenyl" refers to about 2 to about 4 carbon atoms in the chain, which can be straight or branched. 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 or branched alkenyl group, wherein the alkenyl group is as described herein. Exemplary alkenylene radicals include, but are not limited to, ethenylene and propenylene.

[0039] "Alkoxy" refers to an alkyl-O- group, wherein the alkyl group is as described herein. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and heptoxy.

[0040] "Alkoxycarbonyl" means an alkyl-O-CO- group in which the alkyl group is as described herein. Exemplary alkoxycarbonyl groups include methoxy and ethoxycarbonyl.

[0041] Unless otherwise stated, "alkyl" refers to an aliphatic hydrocarbon group having 1 to 20 carbon atoms in the chain that can be straight or branched. In certain embodiments, the alkyl group has 1 to 6 carbon atoms. Unless otherwise stated, "lower alkyl" as a group or part of a lower alkoxy, lower alkylthio, lower alkyl sulfinyl or lower alkyl sulfonyl group refers to an aliphatic hydrocarbon group having 1 to 4 carbon atoms in the chain that can be straight or branched. 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 or branched chain alkyl group, wherein the alkyl group is as described herein. Exemplary alkylene radicals include, but are not limited to, methylene, ethylene, and trimethylene.

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

[0044] "Alkylsulfinyl" means an alkyl-SO- group in which the alkyl group is as previously described. Exemplary alkylsulfinyl groups include, but are not limited to, 1-4 Those of alkyl groups.

[0045] "Alkylsulfonyl" refers to an alkyl-SO2- group, wherein the alkyl group is as previously described. In some embodiments, the alkylsulfonyl group is 1-4 Those of alkyl groups.

[0046] "Alkylthio" means an alkyl-S- group in which the alkyl group is as previously described. Exemplary alkylthio groups include, but are not limited to, methylthio, ethylthio, isopropylthio, and heptylthio.

[0047] "Alkynyl" refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond, and it can be straight or branched, with 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 yet 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 or branched chain alkynyl group, wherein the alkynyl group is as described herein. Exemplary alkynylene radicals include, but are not limited to, ethynylene and propynylene.

[0049] "Amino acid side chain" refers to the 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 TW Greene and PGM Wuts in "Protective Groups in Organic Chemistry" John Wiley and Sons, 1991.

[0050] "Aroyl" means an aryl-CO- group wherein the aryl group is as described herein. Exemplary aroyl groups include benzoyl and 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 about 6 to about 14 carbon atoms, such as phenyl or naphthyl; or (ii) an optionally substituted partially saturated polycyclic aromatic carbocyclic moiety in which the aryl and cycloalkyl or cycloalkenyl groups are fused together to form a cyclic structure, such as a tetrahydronaphthyl, indenyl or indanyl ring.

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

[0053] "Aryldiyl" refers to an optionally substituted divalent free radical derived from an aryl group. Exemplary aryldiyl groups include, but are not limited to, optionally substituted phenylene, naphthylene and indanylene. Suitable substituents include one or more "aryl group substituents" as defined above, particularly halogen, methyl or methoxy.

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

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

[0056] "Arylsulfinyl" means an aryl-SO- group in which the aryl group is as previously described.

[0057] "Arylsulfonyl" means an aryl-SO2- group in which the aryl group is as previously described.

[0058] "Arylthio" means an aryl-S- group in which the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

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

[0060] "Compound" refers to a compound contained in the structural formula disclosed herein, and includes any specific compound in these structural formulas whose structures are 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 may contain one or more chiral centers and / or double bonds, and therefore may exist as stereoisomers such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Therefore, the chemical structures depicted herein include stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) depicted in the structure. The chemical structures described herein also include enantiomers and stereoisomeric derivatives of the compounds, unless otherwise explicitly stated. Enantiomers and stereoisomer mixtures can be split into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. These compounds may also exist in several tautomeric forms (including enol forms, keto forms, and mixtures thereof). Therefore, the chemical structures described herein include all possible tautomeric forms of the compounds shown. The compounds described also include isotopically labeled compounds in which one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that 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 unsolvated 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 intended uses herein and are intended to be within the scope of the present disclosure. In addition, it should be understood that when illustrating a partial structure of a compound, the brackets represent 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 additional heteroatoms selected from O, S, SO2 or NY (wherein Y is hydrogen, alkyl, aryl, aralkyl, acyl, acyloxyalkyl, cycloalkyl, heteroaryl, heterocycloalkyl or sulfonyl). Exemplary cyclic amines include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, indolyl, pyridoindolinyl, tetrahydroquinolinyl.

[0062] "Cycloalkenyl" refers to a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having 3 to 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include, but are not limited to, cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0063] "Cycloalkyl" refers to a saturated monocyclic or bicyclic ring 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 damaging therapy" includes, but is not limited to, gamma-irradiation, alkylating agents (such as nitrogen mustards (e.g., chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (streptozotocin, carmustine, lomustine), alkyl sulfonates (e.g., busulfan), triazines (dacarbazine, temozolomide), and ethyleneimines (e.g., thiotepa, altretamine)), platinum drugs (such as, for example, cisplatin, carboplatin, oxalaplatin), antimetabolites (such as, for example, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, guanine, 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)), 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" means a heteroaryl-C(=O)- group in which 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 of the ring members is an element other than carbon, such as nitrogen, oxygen or sulfur (examples of such groups include benzimidazolyl, benzothiazolyl, furanyl, imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, 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 heterocarbocyclic moiety in which a heteroaryl and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure (examples of such groups include pyrrolodihydroindanyl groups).

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

[0070] "Heteroaryloxy" means a heteroaryl-O- group in which the heteroaryl group is as previously described. An exemplary heteroaryloxy group is optionally substituted pyridinyloxy.

[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 is an element other than carbon, e.g., nitrogen, oxygen or sulfur. Exemplary 5- or 6-membered heterocycles include furanyl, 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" refers to: (i) a cycloalkyl radical of about 3 to 7 ring members containing one or more heteroatoms selected from O, S or N and optionally substituted with oxo; (ii) a partially saturated polycyclic heterocarbocyclic moiety in which an aryl (or heteroaryl ring) and a heterocycloalkyl radical are fused together to form a cyclic structure (examples of such radicals include chromanyl, dihydrobenzofuranyl, dihydroindolinyl and pyridoindolinyl radicals).

[0073] As used herein, "histone deacetylase inhibitor" or "HDAC inhibitor" is a compound that can inhibit the deacetylation of histones in vivo, in vitro or in vivo and in vitro (e.g., see Mottamal et al., Molecules 20 (2015) 3898-3941; Roche and Bertrand, Eur. J. Med. Chem. 121 (2016) 451-483). Therefore, HDAC inhibitors inhibit the activity of at least one histone deacetylase. As a result of inhibiting the deacetylation of at least one histone, acetylated histones increase, and the accumulation of acetylated histones is a suitable biomarker for assessing the activity of HDAC inhibitors. Therefore, the program for the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of related compounds. It should be understood that compounds that can inhibit the activity of histone deacetylases can also be combined with other substrates, and therefore other biologically active molecules, such as enzymes, can be inhibited.

[0074] "Hydrate" refers to the incorporation of water into the crystal lattice of the compound described herein in a stoichiometric ratio, resulting in the formation of an adduct. The method for preparing hydrates includes, but is not limited to, storage in an atmosphere containing water vapor, a dosage form containing water, or a conventional pharmaceutical processing step, such as, for example, crystallization (i.e., crystallization from water or a mixed aqueous solvent), freeze drying, wet granulation, aqueous film coating, or spray drying. In some cases, hydrates may also be formed by crystalline solvates when exposed to water vapor or when anhydrous materials are suspended in water. Hydrates may also crystallize in more than one form, resulting in hydrate polymorphism. See, for example (Guillory, K., Chapter 5, pp. 202205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above method for preparing hydrates is completely within the capabilities of those skilled in the art, is completely conventional, and does not require any experimentation except for 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 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 generally provide services involving 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, "the residue of a hydroxamic acid-derivative histone deacetylase inhibitor" refers to the entire portion of the hydroxamic acid-derivative histone deacetylase inhibitor excluding the hydroxamic acid portion.

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

[0078] As used herein, "pharmaceutically acceptable salt" refers to a salt of a compound that has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, 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-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid or (2) salts formed when the 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 coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc.

[0079] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered.

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

[0081] As used herein, "preventing" or "prevention" refers to reducing the risk of acquiring a disease or disorder (i.e., preventing a patient who may be exposed to or susceptible to the disease but does not yet experience or show symptoms of the disease from developing at least one clinical symptom of the disease).

[0082] As used herein, "prodrug" refers to a derivative of a drug molecule that requires transformation in vivo to release the active drug. A prodrug is generally (although not necessarily) pharmacologically inactive until converted to the parent drug.

[0083] As used herein, "promoiety" 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 promoiety will be attached to the drug via a bond that is cleaved in vivo by enzymatic or non-enzymatic means.

[0084] As used herein, a "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 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-butyloxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethylsulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("Fmoc"), nitro-veratryloxycarbonyl ("Nvoc"), and the like. Representative hydroxy protecting groups include, but are not limited to, those in which the hydroxy group is acylated or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers, and allyl ethers.

[0085] As used herein, "senescence" or "senescent cells" refers to a state in which a cell acquires one or more senescence markers in response to certain cellular stresses. Such markers may generally include permanent withdrawal from the cell cycle, expression of a biologically active secretome of inflammatory factors, altered methylation, expression of heterochromatin foci associated with senescence (SAHF), markers of oxidative stress, DNA damage, expression of markers of protein and lipid modification, morphological features of senescence, altered lysosomes / vacuoles, and expression of β-galactosidase associated with senescence (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, "anti-aging agent" refers to an agent that "selectively" (preferentially or to a greater extent) destroys, kills, removes, or promotes the selective destruction of senescent cells. In other words, the anti-aging agent destroys or kills senescent cells in a biologically, clinically, and / or statistically significant manner compared to its ability to destroy or kill non-senescent cells. The amount and duration of the anti-aging agent are sufficient to selectively kill established senescent cells, but are not sufficient to kill non-senescent cells in a clinically significant 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 leads to the death of senescent cells.

[0087] "Hydrate" refers to the incorporation of water into the crystal lattice of the compound described herein in a stoichiometric ratio, resulting in the formation of an adduct. The method for preparing hydrates includes, but is not limited to, storage in an atmosphere containing water vapor, a dosage form containing water, or a conventional pharmaceutical processing step, such as, for example, crystallization (i.e., crystallization from water or a mixed aqueous solvent), freeze drying, wet granulation, aqueous film coating, or spray drying. In some cases, hydrates may also be formed by crystalline solvates when exposed to water vapor or when anhydrous materials are suspended in water. Hydrates may also crystallize in more than one form, resulting in hydrate polymorphism. See, for example (Guillory, K., Chapter 5, pp. 202205 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999). The above method for preparing hydrates is completely within the capabilities of those skilled in the art, is completely conventional, and does not require any experimentation except for 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 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 generally provide services involving 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 with the same or different substituents.

[0089] Substituent groups that can be used to replace saturated carbon atoms in the specified group or free radical 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, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N-OR b 、-N-NR c R c 、-NR b S(O)2R b , =N2, -N3, -S(O)2R b 、-S(O)2NR b R b 、-S(O)2O - 、-S(O)2OR b 、-OS(O)2R b 、-OS(O)2O - 、-OS(O)2OR b 、-OS(O)2NR c NR c 、-P(O)(O - )2、-P(O)(OR b )(O - )、-P(O)(OR b )(OR b )、-C(O)R b 、-C(O)NR b -OR b -C(S)R b 、-C(NR b )R b 、-C(O)O - 、-C(O)OR b 、-C(S)OR b 、-C(O)NR c R c 、-C(NR b )NR c R c、-OC(O)R b 、-OC(S)R b 、-OC(O)O - 、-OC(O)OR b 、-OC(O)NR c R c 、-OC(NCN)NR c R c -OC(S)OR b 、-NR b C(O)R b 、-NR b C(S)R b 、-NR b C(O)O - 、-NR b C(O)OR b 、-NR b C(NCN)OR b 、-NR b S(O)2NR c R c 、-NR b C(S)OR b 、-NR b C(O)NR c R c 、-NR b C(S)NR c R c 、-NR b C(S)NR b C(O)R a 、-NR b S(O)2OR b 、-NR b S(O)2R b 、-NR b C(NCN)NR c R c 、-NR b C(NR b )R b and-NR b C(NR b )NR c R c , where R a are independently alkyl, heteroalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; each R b are independently hydrogen, R a , substituted alkyl, substituted heteroalkyl, substituted aryl, substituted arylalkyl, substituted heteroaryl, and substituted heteroarylalkyl; and each R c Independently R b, or alternatively, two R c Together with the nitrogen atom to which they are bound, they form a 4-membered, 5-membered, 6-membered or 7-membered cycloheteroalkyl, a substituted cycloheteroalkyl or a cycloheteroalkyl fused to an aryl group, which aryl group may optionally contain 1 to 4 additional heteroatoms that are the same or different and are selected from the group consisting of O, N and S. As a specific example, -NR c R c Means include -NH2, NH-alkyl, N-pyrrolidinyl and N-morpholinyl. Similarly, the substituent groups that can be used to replace the unsaturated carbon atoms in the specified group or free radical include but are not limited to -R a , halogen, -O - 、-OR b 、-SR b , -S - 、-NR c R c , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -S(O)2R b 、-S(O)2O - 、-S(O)2OR b 、-OS(O)2R b 、-OS(O)2O - 、-OS(O)2OR b 、-P(O)(O - )2、-P(O)(OR b )(O - )、-P(O)(OR b )(OR b )、-C(O)R b 、-C(S)R b 、-C(NR b )R b 、-C(O)O - 、-C(O)OR b 、-C(S)OR b 、-C(O)NR c R c 、-C(NR b )NR c R c 、-OC(O)R b 、-OC(S)R b 、-OC(O)O - 、-OC(O)OR b 、-OC(S)OR b 、-OC(O)NR c R c 、-OS(O)2NR c NRc 、-NR b C(O)R b 、-NR b C(S)R b 、-NR b C(O)O - 、-NR b C(O)OR b 、-NR b S(O)2OR a 、-NR b S(O)2R a 、-NR b C(S)OR b 、-NR b C(O)NR c R c 、-NR b C(NR b )R b and-NR b C(NR b )NR c R c , where R a , R b and R c 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, -CF3, -CN, -NO, -NO2, -S(O)2R b 、-S(O)2O - 、-S(O)2OR b 、-OS(O)2R b 、-OS(O)2O - 、-OS(O)2OR b 、-P(O)(O - )2、-P(O)(OR b )(O - )、-P(O)(OR b )(OR b )、-C(O)R b 、-C(S)R b 、-C(NR b )R b 、-C(O)OR b 、-C(S)OR b 、-C(O)NRc R c 、-C(NR b )NR c R c 、-OC(O)R b 、-OC(S)R b 、-OC(O)OR b 、-OC(S)OR b 、-NR b C(O)R b 、-NR b C(S)R b 、-NR b C(O)OR b 、-NR b C(S)OR b 、-NR b C(O)NR c R c 、-NR b C(NR b )R b and-NR b C(NR b )NR c R c , where R a , R b and R c As defined above. Substituent groups from the above list that can be used to replace other specified groups or atoms will be obvious to those skilled in the art. In some embodiments, the substituents used to replace the specified groups can be further substituted, usually with one or more of the same or different groups selected from the various groups described above.

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

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

[0093] Detailed Description

[0094] Anti-aging agents

[0095] Hydroxamic acid 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 hydroxamic acid derivative HDAC inhibitors (e.g., compounds (4)-(13)) have been clinically studied as single agents or in combination with other oncolytic compounds for the treatment of hematological tumors and solid tumors. In addition to the various inhibitory enzymes in HDAC classes I, II and IV, hydroxamic acid derivatives have also 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 isoforms). Many other hydroxamic acid derivative HDAC inhibitors have been disclosed, including the natural product trichostatin A isolated from Streptomyces (14) and a number of synthetically derived compounds, examples of which include compounds (15)-(21) and others disclosed in Roche and Bertrand, supra; or any of the hydroxamic acids disclosed in U.S. Pat. Nos. 5,369,108, 5,932,616, 6,087,367, and 6,511,990.

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] The anti-aging activity of the pan-HDAC inhibitor panobinostat (3) has been reported previously (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 HDAC inhibitor-mediated reductions 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 wishing to be bound by any theory, one pharmacological basis for the anti-aging activity of HDAC inhibitors may be mediated by reducing anti-apoptotic Bcl-xL protein levels.

[0102] In some embodiments, the compound effective as an anti-aging agent is a compound of formula (IV) or (V):

[0103]

[0104] Where: 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 independently hydrogen, -C(O)-R 1 , a portion of formula (VI) or a portion 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 Independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 One of them is a moiety of formula (VI) or formula (VII), then R 13 , R14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 ; and each R 2 are independently hydrogen, C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 One of them 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 ; The condition is that when R 13 , R 14 , R 15 and R 16 When each is hydrogen, R is not 7-heptanoylbenzamide.

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

[0108] In some embodiments of compounds of Formula (IV) or (V), R is the residue of a hydroxamic acid derivative histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected from the group consisting of panobinostat, quinostat, vorinostat, danostat, givista, CUDC-907, CUDC-101, abestat, belinostat, prenostat, rinotostat, liconostat, pyroxacillin, APHA, trichostatin A, oxalectin and AR-42.

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

[0110] In other embodiments of the compound of formula (IV), R 13 , R 14 , R 15 and R 16 Each is independently -C(O)-R 1 , where R 1 It is C 1-4In other embodiments of the compound of formula (V), R 23 , R 24 and R 25 Each is independently -C(O)-R 1 , where R 1 It is C 1-4 Alkyl or phenyl.

[0111] In some further embodiments of the compound of formula (IV), R 13 , R 14 , R 15 and R 16 Each is independently -C(O)-R 1 , where R 1 In some other embodiments of the compound of formula (V), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 It's methyl.

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

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

[0114]

[0115]

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

[0117]

[0118]

[0119]

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

[0121]

[0122]

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

[0124]

[0125]

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

[0127]

[0128]

[0129]

[0130]

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

[0132]

[0133]

[0134]

[0135] Compounds of formula (IV) or (V) can be synthesized by coupling the carboxylic acid precursor of the hydroxamic acid HDAC inhibitor RCO2H (VIII) with the glycoxime compounds (IX) and (X), respectively, in the presence of an acyl coupling agent such as a carbodiimide (e.g. EDC), or alternatively after prior 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 brominated galactoside (23) according to the method of Thomas et al. Bioorg. Med. Chem. Lett. 17 (2007) 983-986:

[0138]

[0139]

[0140] Compound (22) was incubated with β-galactosidase and showed quantitative conversion to (1). It is well known that the HDAC inhibitory activity of hydroxamic acid derivative compounds is mainly dependent on the zinc chelating activity of the free hydroxamic acid moiety (e.g., see Roche and Bertrand above). Therefore, masking the hydroxamic acid functional group as a glycoside derivative in compounds of formula (IV) or (V) ensures that these prodrugs are inactive as HDAC inhibitors, but will become activated when hydrolyzed 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 amino acid protected, for example as a fluorenylmethoxycarbamate (Fmoc) derivative (29). Coupling with compound (24) as described above followed by sequential deprotection affords the panobinostat prodrug (26).

[0142]

[0143]

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

[0145] Specific α-L-fucoside conjugates of hydroxamic acid derivative histone deacetylase inhibitors can be prepared in a similar manner, starting from appropriately protected and activated fucose derivatives, 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, 1-fluoro-2,3,4-tri-O-acetyl-fucose (30) is treated with N-hydroxyphthalimide to obtain a mixture of α- and β-L-fucosyl oxime derivatives, the desired α anomer (31) being the less polar product. Deprotection with hydrazine then affords α-L-fucosyl oxime (32), which can be further prepared into 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 each independently hydrogen or -C(O)-R 2 ; Each R1 Independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 One of them is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 ; and each R 2 Independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 One of them 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 .

[0156] In some embodiments of compounds of Formula (XI), (XII), (XIII) or (XIV), the anomeric carbon of the pyranose ring (marked with *) 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 compounds of Formula (XI), (XII), (XIII) or (XIV), the moiety AY-C6H2(OH)2-B is an Hsp90 inhibitor selected from the group consisting of luminespib (NVP-AUY922), ganetespib, VER-50589, AT13387 and KW-2478.

[0158] In some embodiments of compounds of Formula (XI), R 13 , R 14 , R 15 and R 16 In some embodiments of compounds of Formula (XII), R 23 , R 24 and R 25 In some embodiments of compounds of Formula (XIII), R 13 , R 14 , R 15 and R 16 In some embodiments of compounds of Formula (XIV), R 23 , R 24 and R 25Each is hydrogen.

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

[0160] In some embodiments of compounds of Formula (XI), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 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 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 1 In some embodiments of the compound of formula (XIV), R 23 , R 24 and R25 Each is -C(O)-R 1 , where R 1 It's methyl.

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

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

[0163]

[0164]

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

[0166]

[0167]

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

[0169]

[0170]

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

[0172]

[0173]

[0174]

[0175] Compounds of formula (XI) and (XIII) can be prepared by reacting a compound of formula (XV) with a protected D-galactosyl donor moiety under classical BF3-mediated glycosylation or Koenigs-Knorr coupling conditions, wherein 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 for regioselective glycosylation.

[0176]

[0177]

[0178] Specifically, the O-β-galactoside conjugates of AT13387 (36) (prepared as described in U.S. Pat. No. 8,779,132), i.e., compounds (46) and (47), can be prepared by reacting (36) with (23) according to the method of Shie et al., Carbohydrate Res. 341 (2006) 443-456.

[0179]

[0180] Similarly, O-β-galactoside conjugates of NVP-AUY922 (37) (prepared as described in Brough et al., J. Med. Chem. 51 (2008) 196-218), i.e., compounds (48) and (49), can be prepared by reaction of (37) with (23):

[0181]

[0182] Compounds (50)-(53), specific α-L-fucoside conjugates of Hsp90 inhibitors (36) and (37), can be prepared in a similar manner starting from the protected fucose derivative (45):

[0183]

[0184] 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-fucoside conjugates of KW-2478 (42) (i.e., compounds (58) and (59)), SNX-2112 (43) (i.e., compound (60)) and SNX-7081 (44) (i.e., compound (61)) can be prepared in a similar manner.

[0185]

[0186]

[0187]

[0188] Still other embodiments relate to OD-galactosyl and OL-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, for example, Cinelli et al., J. Med. Chem. 55 (2012) 10844-10862; and Lv et al., J. Med. Chem. 59 (2016) 4890-4899) and dibenzonaphthyridinones (exemplified by compounds (71)-(73)) (see, for example, 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-fucoside conjugate of SN-38 (75) (Japanese Patent No. JP6328098, 1988) have been previously disclosed as antitumor agents.

[0191]

[0192] In some embodiments, the compound of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), or (XXV) is an anti-aging agent:

[0193]

[0194]

[0195]

[0196]

[0197] Where 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 independently hydrogen, -C(O)-R 1 , a portion of formula (VI) or a portion 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 Independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 One of them is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 ; and each R 2 Independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 One of them 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 compounds of Formula (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV) or (XXV), the anomeric carbon of the pyranose ring (marked with *) has an S configuration, and these compounds are β-D-galactoside and α-L-fucoside conjugates of TOP1 inhibitors, respectively. In other embodiments of compounds of Formula (XVIII), (XIX), (XX) or (XXI), R 8 It is 4-morpholinyl or 1-imidazolyl.

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

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

[0203] In some embodiments of compounds of Formula (XVI), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of the compound of formula (XVII), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of Formula (XVIII), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of Formula (XIX), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of formula (XX), R 13 , R 14 , R 15 and R16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of formula (XXI), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 In some embodiments of the compound of formula (XXII), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of the compound of formula (XXIII), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 In some embodiments of the compound of formula (XXIV), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of Formula (XXV), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 It's methyl.

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

[0205] In yet other embodiments, OD-galactosyl and OL-fucosyl conjugates of DNA alkylating agents based on cytotoxic duocarmycin family antibiotics are anti-aging agents. Duocarmycin SA (76) isolated from Streptomyces DO-113 contains a highly active spirocyclopropylcyclohexadienone moiety and has been used as inspiration for the design of mono- and disaccharide derivatives such as galactosyl compounds (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 times lower than that of its hydrolyzed ring-opening product (78), which undergoes the so-called Winstein in situ cyclization to give the DNA-reactive spirocyclopropylcyclohexadienone (79).

[0206]

[0207]

[0208] In some embodiments, the compound of formula (XXVI) or (XXVII) is an anti-aging agent:

[0209]

[0210]

[0211] Where 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):

[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 Independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R16 One of them is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 ; and each R 2 Independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 One of them is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or -C(O)-R 1 ; Provided that in the compound of formula (XXVI), R 13 , R 14 , R 15 and R 16 Each is not hydrogen or acetyl at the same time.

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

[0215] In some embodiments of compounds of Formula (XXVI), R 13 , R 14 , R 15 and R 16 In some embodiments of compounds of Formula (XXVII), R 23 , R 24 and R 25 Each is hydrogen.

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

[0217] In some embodiments of compounds of Formula (XXVI), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of formula (XXVII), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 It's methyl.

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

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

[0220]

[0221] Still other embodiments relate to OD-galactosyl and OL-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 exert their cytotoxic effects by covalently binding to the exocyclic NH2 groups of guanine residues in the minor groove of DNA via their N10-C11 imine functional groups (see, e.g., Antonow and Thurston, Chem. Rev. 111 (2011) 2815-2864; and Mantaj et al., Angew. Chem. Int. Ed. 56 (2017) 462-488). PBD monomers exhibit significant cytotoxicity, and connecting two PBD monomers via a linker produces a PBD dimer that is capable of interchain DNA crosslinking. SJG-136 (86) is one such dimer with high cytotoxic potency and has been used to construct antibody-drug conjugates with clinical utility.

[0222]

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

[0224]

[0225]

[0226] In some embodiments, the compound of formula (XXVIII) or (XXIX) is an anti-aging agent:

[0227]

[0228] Where R 9 It is hydrogen, C 1-4 Alkyl, CF3, CN or NO2; R 10 It is hydrogen, C 1-4 Alkyl or arylalkyl; 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):

[0229]

[0230] 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 Independently C 1-4 alkyl or phenyl, provided that if R 13 , R 14 , R 15 or R 16 One of them is a moiety of formula (VI) or formula (VII), then R 13 , R 14 , R 15 and R 16 The remainder is hydrogen or C(O)-R 1 ; and each R 2 Independently C 1-4 alkyl or phenyl, provided that if R 23 , R 24 or R 25 One of them is a moiety of formula (VI) or formula (VII), then R 23 , R 24 or R 25 The remainder is hydrogen or C(O)-R 1 ; Provided that in the compound of formula (XXVIII), when R 9 is NO2 and R 10 When it is benzyl, R 13 , R 14 , R 15 , R 16 Each is not hydrogen or acetyl at the same time.

[0231] In some embodiments of compounds of formula (XXVIII) or (XXIX), the anomeric carbon of the pyranose ring (marked with *) is in 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 compounds of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 In some embodiments of compounds of Formula (XXIX), R 23 , R 24 and R 25Each is hydrogen.

[0233] In some embodiments of compounds of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 Each is independently -C(O)-R 1 , where R 1 It is C 1-4 In some embodiments of the compound of formula (XXIX), R 23 , R 24 and R 25 Each is independently -C(O)-R 1 , where R 1 It is C 1-4 Alkyl or phenyl.

[0234] In some embodiments of compounds of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of formula (XXIX), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 It's methyl.

[0235] In some embodiments of compounds of Formula (XXVIII), R 13 , R 14 , R 15 and R 16 Each is -C(O)-R 1 , where R 1 In some embodiments of compounds of Formula (XXIX), R 23 , R 24 and R 25 Each is -C(O)-R 1 , where R 1 It is ethyl.

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

[0237]

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

[0239]

[0240] In yet other embodiments, the O-galactoside or O-fucoside conjugate of the Akt inhibitor is an anti-aging agent. Akt inhibitors that can be used to prepare such conjugates are exemplified by compounds such as ipatasertib (or GDC-0068) (92), AZD5363 (93), and triciribine (94). In some embodiments, the O-β-D-galactoside or O-α-L-fucoside conjugate of the Akt inhibitor is an anti-aging agent.

[0241]

[0242] Specific compounds are exemplified by compounds (95)-(100) wherein each R 46 is hydrogen, acetyl or propionyl, 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 a pro-apoptotic compound of formula (II), specifically 5-fluorouridine (FUR) (102), a cytotoxin 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 a pro-apoptotic compound of formula (II), specifically 5-fluorouridine (FUR) (102), a cytotoxin produced by the action of intracellular α-fucosidase enriched in senescent cells:

[0249]

[0250] In further embodiments, the O-galactoside or O-fucoside conjugate of a proteasome inhibitor is an anti-aging agent. Proteasome inhibitors that can be used to prepare such conjugates are exemplified by compounds such as delanzomib (103). In some embodiments, O-β-D-galactoside or O-α-L-fucoside conjugates (104) and (105) are anti-aging agents.

[0251]

[0252] Methods for Characterization and Identification of Anti-Aging Agents

[0253] Anti-aging agents can be characterized by using one or more cell-based assays and one or more animal models described herein or in the art and that will be 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, anti-aging agents are 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 appreciate that characterizing a compound as an anti-aging agent and determining the level of killing of the compound can be accomplished by comparing the activity of the test agent with an appropriate negative control (e.g., only a carrier or diluent and / or a composition or compound known in the art that does not kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing anti-aging agents also include controls for determining the effects of the agent on non-senescent cells (e.g., resting cells or proliferating cells). Compared to one or more negative controls, anti-aging agents reduce (i.e., reduce) the survival percentage of a variety of senescent cells (i.e., reduce the number of surviving senescent cells in an animal or cell-based assay in some way). The conditions for specific in vitro assays include temperature, buffers (including salts, cations, media), 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 easily determined by routine experiments.

[0255] The source of senescent cells for determination can be a primary cell culture or a cell line adapted to culture, including but not limited to a genetically engineered cell line, an immortalized or immortalizable cell line, a somatic hybrid cell line, a differentiated or differentiable cell line, a transformed cell line, etc., which may contain a chromosomal integration or an episomal recombinant nucleic acid sequence. In some embodiments, senescent cells are isolated from a biological sample 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 a cell line 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 a chemotherapeutic agent (e.g., doxorubicin). The biological sample can be, for example, a blood sample, a biopsy sample, a body fluid (e.g., lung 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. A biological sample can be a tissue or cell preparation in which the morphological integrity or physical state has been destroyed, for example, by dissection, dissociation, solubilization, fractionation, homogenization, biochemical or chemical extraction, pulverization, lyophilization, sonication, or any other means for processing a sample from a subject or biological source. The subject can be a human or non-human animal. As an example, the anti-aging effects of certain compounds of the present invention on human fibroblasts in culture medium are characterized in Example 23 herein, and the anti-aging effects on mouse embryonic fibroblasts are characterized in Example 24 herein. Examples 26 and 27 demonstrate that in vivo administration of the anti-aging compounds of the present invention results in a reduction in senescent liver cells in mice. Example 28 demonstrates that in vivo administration of the anti-aging compounds of the present invention results in a reduction in 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, e.g., 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 transgenes that contain nucleic acids that allow controlled removal of senescent cells (e.g., p16INK4a positive senescent cells) as positive controls. 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 contains detectable markers, such as one or more red fluorescent proteins; green fluorescent proteins; and one or more luciferases to detect the removal 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 aging-related diseases or abnormalities (i.e., reducing the likelihood of specific aging-related diseases or abnormalities occurring), such as atherosclerosis models, osteoarthritis models, COPD models, IPF models, etc. As described herein, lung disease mouse models, such as the bleomycin pulmonary fibrosis model and the chronic smoking model are suitable for 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 chemotherapy and radiotherapy side effects (i.e., reducing the likelihood of chemotherapy and radiotherapy side effects occurring) or treating or preventing metastatic lesions (i.e., reducing the likelihood of metastatic lesions occurring) are described in International Application Nos. WO 2013 / 090645 and WO 2014 / 205244. Animal models for determining the effectiveness of agents for treating ocular diseases, particularly age-related macular degeneration, are also routinely used in the art (see, e.g., Pennesi et al., Mol. Aspects Med. 33 (2012) 487-509; Zeiss et al., Vet. Pathol. 47 (2010) 396-413; and Chavala et al., J. Clin. Invest. 123 (2013) 4170-4181).

[0258] As a non-limiting example and as described herein, an osteoarthritis animal model has been developed. Osteoarthritis can be induced in animals, for example, by incomplete or complete surgical cutting of the anterior cruciate ligament, inducing damage to the joint in, for example, 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 possibility of the occurrence of osteoarthritis), and leading to a reduction in proteoglycan erosion, and inducing (i.e., stimulating, enhancing) the production of collagen (such as type 2 collagen), and alleviating the pain of animals undergoing ACL surgery. Immunohistology can be performed to examine the integrity and composition of tissues and cells in the joints. Immunochemistry and / or molecular biology techniques can also be performed using the methods and techniques described herein, such as for determining the level of inflammatory molecules (e.g., IL-6) and for determining the level 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, an atherosclerosis animal model has been developed. Atherosclerosis can be induced in animals, for example, by feeding animals with high-fat foods or by using transgenic animals that are highly sensitive to developing atherosclerosis. The animal model can be used to determine that anti-aging agents reduce the amount of plaques in atherosclerotic arteries or suppress the formation of plaques in atherosclerotic arteries, reduce the lipid content of atherosclerotic plaques (i.e., reduce, reduce the amount of lipids in plaques) and cause the increase of the fibrous cap thickness of plaques or improve the effectiveness of the fibrous cap thickness of plaques. Sudan staining can be used to detect the lipid levels in atherosclerotic blood vessels. Immunohistology and immunochemistry and molecular biology assays (for example, for determining the level of inflammatory molecules (for example, IL-6), and for determining the level of aging markers as described above) can all be carried out according to the methods described herein, and these methods are routine practices in the art.

[0260] In 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 a medicament for treating IPF. In animal models of lung disease (e.g., bleomycin animal models, animal models exposed to smoke, etc.), respiratory measurements can be performed to determine elasticity, compliance, static compliance, and peripheral capillary oxygen saturation (SpO2). Immunohistology and immunochemistry and molecular biology assays (e.g., for determining the level of inflammatory molecules (e.g., IL-6), and for determining the level of aging markers as described above) can all be performed according to the methods described herein, which are routinely practiced in the art.

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

[0262] Those skilled in the art will readily appreciate that characterizing anti-aging agents and determining the level of killing by the anti-aging agent can be accomplished by comparing the activity of the test agent to an appropriate negative control (e.g., a carrier alone and / or a composition, agent, or compound known in the art that does not kill senescent cells) and an appropriate positive control. In vitro cell-based assays for characterizing agents also include controls for determining the effects of agents on non-senescent cells (e.g., quiescent cells or proliferating cells). Useful anti-aging agents reduce (i.e., decrease) the percentage of surviving senescent cells (i.e., reduce the amount of surviving senescent cells in an animal or in a cell-based assay in some manner) compared to one or more negative controls. Thus, anti-aging agents selectively kill senescent cells (which may be referred to herein as selectively killing senescent cells relative to non-senescent cells) compared to killing non-senescent cells.

[0263] In certain embodiments (in an in vitro assay or 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 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 yet other embodiments (in an in vitro assay or 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 yet other embodiments (in an in vitro assay or 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 yet other embodiments (in an in vitro assay or 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, the anti-aging agent is at least 5-25 times, 10-50 times, 10-100 times or 100-1000 times more selective for killing senescent cells than for killing non-senescent cells.

[0264] With respect to specific embodiments of the methods described herein for treating a disease or disorder associated with aging, the percentage of senescent cells killed may 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 non-limiting examples, brain tissue, tissue and portions of the eye, lung tissue, heart tissue, arteries, joints, skin, and muscle may include senescent cells that may be reduced by the anti-aging agents described herein at percentages as described above and thereby provide a therapeutic effect. In addition, selectively removing at least 20% or at least 25% of senescent cells from the affected tissue or organ may have a clinically significant therapeutic effect.

[0265] With respect to specific embodiments of the methods described herein, such as, for example, treating a cardiovascular disease or disorder associated with arteriosclerosis (such as atherosclerosis) by administering an anti-senescent agent (i.e., with reference to the in vivo methods described above), the percentage of senescent cells killed may refer to the percentage of senescent cells killed in an affected artery containing plaque relative to the percentage of non-senescent cells killed in an arterial plaque. In certain embodiments, in a method for treating a cardiovascular disease such as atherosclerosis, as described herein, at least one anti-senescent agent kills at least 20% of senescent cells in an artery and kills no more than 5% of non-senescent cells. In other embodiments, the anti-senescent agent selectively kills at least 25% of senescent cells in an arteriosclerotic artery.

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

[0267] In some embodiments, with respect to the methods described herein for treating aging-related lung diseases or disorders (e.g., COPD, IPF) by administering at least one anti-aging agent, the percentage of senescent cells killed may refer to the percentage of senescent cells killed in the affected lung tissue relative to the percentage of non-senescent cells killed in the affected lung tissue. In certain embodiments, in the methods for treating aging-related lung diseases and disorders as described herein, the anti-aging agent kills at least 20% of senescent cells in the affected lung tissue and kills no more than 5% of non-senescent cells. In other embodiments, the anti-aging agent selectively kills at least 25% of 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 anti-aging agents for treating such diseases and abnormalities comprises 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 generally sufficient) or chemotherapeutic agents (e.g., doxorubicin or other anthracyclines). After exposure to the agent, the cells are cultured for an appropriate period of time under appropriate conditions (e.g., culture medium, temperature, CO2 / O2 levels suitable for a given cell type or cell line) to allow senescence to be established. As discussed herein, senescence of cells can be determined by determining a number of features, such as changes in morphology (e.g., as observed by microscopy); production of, for example, β-galactosidase (SA-β-gal), p16INK4a, p21, or any one or more SASP factors (e.g., IL-6, MMP3) associated with senescence. The sample of senescent cells is then contacted with the candidate agent (i.e., the cells and agent are mixed, combined, or in some way allowed to interact). One skilled in the art will appreciate that the assay will include appropriate controls, both historical and concurrent, both negative and positive. For example, a sample of control non-senescent cells that have been cultured similarly to senescent cells but not exposed to a senescence-inducing agent is contacted with the candidate agent. The level of survival of senescent cells is determined and compared to that of non-senescent cells. When the level of survival of senescent cells is less than that of non-senescent cells, an anti-senescent agent is identified.

[0269] In some embodiments, the above method for identifying an anti-aging agent may further include a step for identifying whether the anti-aging agent can be used to treat osteoarthritis. The method may further include contacting the identified anti-aging agent with a cell capable of producing collagen; and determining the level of collagen produced by the cell. In an embodiment, the cell is a chondrocyte and the collagen is type 2 collagen. The method may further include administering a candidate anti-aging agent to a non-human animal having an arthritic lesion in a joint, and determining one or more of: (a) the level of senescent cells in the joint; (b) the animal's physical function; (c) the level of one or more inflammatory markers; (d) the histology of the joint; and (e) the level of type 2 collagen produced, thereby determining the therapeutic efficacy of the anti-aging agent, wherein one or more of the following is observed in the treated animal as compared to an animal not treated with the anti-aging agent: (i) a reduction in the level of senescent cells in the joint of the treated animal; (ii) improved physical function of the treated animal; (iii) a reduction 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 2 collagen produced in the treated animal. As described herein and in the art, the animal's physical function can be determined by techniques that determine the sensitivity of the leg to an induced or natural osteoarthritic condition, for example, by the animal's tolerance to bearing weight on the affected limb or the animal's ability to stay away from uncomfortable stimuli (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 in killing senescent cells in an animal model. Statistical analyses as described herein and routinely practiced in the art can be used to analyze the data.

[0270] In other embodiments, the above-mentioned method for identifying anti-aging agents may further include a step for identifying whether the anti-aging agent can be used to treat cardiovascular diseases caused by or associated with atherosclerosis. Therefore, the method may further include administering an anti-aging candidate agent in a non-human animal or animal model to determine the effectiveness of the agent in reducing the amount of plaque, inhibiting the formation of plaques in atherosclerotic arteries, reducing the lipid content of atherosclerotic plaques (i.e., reducing, reducing the amount of lipids in plaques) and / or causing an increase in the thickness of the fibrous cap of the plaque or improving the thickness of the fibrous cap of the plaque. Sudan staining can be used to detect lipid levels 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 aging markers as described above can all be performed according to the methods described herein and are routinely practiced in the art.

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

[0272] In some embodiments, the methods described herein for identifying anti-aging agents may include administering a candidate anti-aging agent to a non-human animal lung disease model, 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 one or more of the following is observed in the treated animal compared to an animal not treated with the anti-aging agent: (i) a reduction in the level of senescent cells in the lungs and lung tissue of the treated animal; (ii) improved lung function of the treated animal; (iii) a reduction 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 may be performed to determine elasticity, compliance, static compliance, and peripheral capillary oxygen saturation (SpO2). Lung function can be assessed by determining any of a number of measurements, such as expiratory reserve volume (ERV), forced vital capacity (FVC), forced expiratory volume (FEV) (e.g., FEV in one second, FEV1), FEV1 / FEV ratio, 25% to 75% forced expiratory flow and maximum spontaneous 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 diffusion capacity of carbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO.sub.2) can also be measured. Statistical analysis as described herein and routinely practiced in the art can be used to analyze the data.

[0273] Methods of treating and preventing diseases and disorders associated with aging

[0274] Provided herein are methods for treating conditions, diseases or abnormalities (including age-related diseases and abnormalities) associated with, associated with or caused by cellular senescence in a subject in need thereof. Diseases or abnormalities associated with aging may also be referred to herein as diseases or abnormalities associated with senescent cells. Diseases and abnormalities associated with aging include, for example, age-related diseases and abnormalities induced by aging; pulmonary diseases and abnormalities; nervous system diseases and abnormalities (e.g., neurodegenerative diseases and abnormalities); ocular 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 diseases and abnormalities associated with transplantation. A notable feature of aging is the gradual loss or degeneration of function occurring at the molecular, cellular, tissue and organismal levels. Age-related degeneration causes well-recognized pathologies such as sarcopenia, atherosclerosis and heart failure, osteoporosis, pulmonary insufficiency, renal failure, neurodegeneration (including macular degeneration, Alzheimer's disease and Parkinson's disease) and many other diseases. Although different mammalian species differ in their susceptibility to specific age-related pathologies, in general, age-related pathologies typically begin to rise with approximately exponential kinetics at about the midpoint of the species-specific lifespan (e.g., 50-60 years for humans) (see, e.g., Campisi, Annu. Rev. Physiol. 75 (2013) 685-705; Naylor et al., Clin. Pharmacol. Ther. 93 (2013) 105-116).

[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, renal dysfunction, frailty, hair loss, hearing loss, muscle fatigue, skin disorders, sarcopenia, and herniated discs) and other aging-related diseases induced by aging (e.g., diseases / abnormalities caused by radiation, chemotherapy, smoking, eating a high-fat / high-sugar diet, and environmental factors); pulmonary diseases (e.g., idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), emphysema, bronchiolitis obstructive, asthma); proliferative diseases, including cancer and metastatic lesions; side effects associated with chemotherapy or radiation therapy; fibrotic diseases and abnormalities (e.g., cystic fibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oral submucous fibrosis, 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 disorders (e.g., Parkinson's disease, motor neuron dysfunction (MND); Huntington's disease); cerebrovascular disease; emphysema; osteoarthritis; benign prostatic hypertrophy; ocular diseases or disorders (e.g., age-related macular degeneration, cataracts, glaucoma, vision loss, presbyopia); metabolic diseases and disorders (e.g., obesity, diabetes, metabolic syndrome); cardiovascular diseases (e.g., atherosclerosis, diastolic dysfunction, aortic aneurysm, angina, arrhythmias, cardiomyopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid artery disease, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis); inflammatory / autoimmune diseases and disorders (e.g., osteoarthritis, eczema, psoriasis, osteoporosis, mucositis, transplant-related diseases and disorders); dermatological diseases, such as diabetic ulcers, wound healing, and skin nevi. In certain examples, any one or more of the diseases or disorders described above or herein may be excluded.

[0276] In some embodiments, methods are provided for treating a disease or disorder associated with aging by killing senescent cells associated with the disease or disorder (i.e., established senescent cells) in a subject suffering from the disease or disorder via administration of an anti-senescent agent, wherein the disease or disorder is a disease of aging (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 disorders; osteoarthritis; or atherosclerosis.

[0277] Age-related diseases and disorders

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

[0279] Anti-aging agents 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) that 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 diets, other environmental factors). Age-related abnormalities or diseases or age-sensitive traits may be associated with 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 conditions include, for example, abnormal renal function, kyphosis, herniated disc, weakness, 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 loss 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 conditions also include dermatological conditions, such as but not limited to the treatment of one or more of the following conditions: wrinkles, including fine wrinkles on the surface; 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 a decline in reserves and functions associated with aging in a variety of physiological systems, which impairs the ability of the subject to cope with daily or acute stressors. Frailty may be characterized by impaired energetics, 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 five characteristics are observed, a patient may be diagnosed with frailty (see, e.g., Fried et al., J. Gerontol. A Biol. Sci. Med, Sci. 56 (3) (2001) M146-M156; Xue, Clin. Geriatr. Med. 27 (1) (2001) 1-15). In certain embodiments, aging and aging-related diseases and abnormalities can be treated or prevented (i.e., reduce the likelihood of their occurrence) by administering anti-aging agents. Anti-aging agents can inhibit the aging of adult stem cells, or inhibit the accumulation of, kill, or promote the clearance of adult stem cells that have become aged.The importance of preventing senescence in stem cells to maintain the regenerative capacity of tissues is discussed in, for example, Park et al., J. Clin. Invest. 113 (2004) 175-179; and Sousa-Victor, Nature 506 (2014) 316-321.

[0280] The method for measuring aging is known in the art. For example, aging in bone can be measured by: occasional non-vertebral fracture, occasional hip fracture, occasional total fracture, occasional vertebral fracture, occasional repeated fracture, functional recovery after fracture, bone mineral density reduction of lumbar vertebra and hip, knee flexion rate, use of NSAID, number of painful joints and osteoarthritis. Aging in muscle can also be measured by functional decline, fall rate, reaction time and grip strength, upper limb and lower limb muscle mass decline and dual-task 10 meters gait speed. In addition, aging in cardiovascular system can be measured by systolic and diastolic blood pressure changes, occasional hypertension, major cardiovascular events (such as myocardial infarction, stroke, congestive heart disease and cardiovascular mortality). In addition, aging in brain can be measured by cognitive decline, occasional depression and occasional dementia. In addition, aging in immune system can be measured by infection rate, upper respiratory tract infection rate, influenza-like illness rate, occasional serious infection caused by hospitalization, occasional cancer, implant infection rate and gastrointestinal infection rate. Other signs of aging may include, but are not limited to, decreased oral health, tooth loss, incidence of GI symptoms, changes in fasting blood glucose and / or insulin levels, body composition, decreased renal function, quality of life, unexpected disability with respect to activities of daily living, and unexpected nursing home admissions. 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, radiance, roughness, spots, laxity, skin tone uniformity, softness, and rippling (depth changes).

[0281] Administration of the anti-aging agents described herein can extend survival compared to expected survival if the subject had not received treatment. Subjects in need of treatment include subjects already suffering from the disease or abnormality, as well as subjects susceptible to or at risk of developing the disease or abnormality, and subjects in which prophylactic treatment of the disease, disorder or abnormality is required. Subjects may have a genetic predisposition to develop the disease or abnormality, who would benefit from the removal of senescent cells, or may not be young, in which case receiving an anti-aging agent will provide a clinical benefit to delay the development of the disease (including age-related diseases or abnormalities) or reduce its severity.

[0282] In other embodiments, methods for treating diseases or abnormalities associated with aging are provided, further comprising identifying subjects who will benefit from treatment with anti-aging agents 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). Biological samples can be obtained from subjects, such as blood samples, serum or plasma samples, biopsy samples, body fluids (e.g., lung lavage fluid, ascites, mucosal washes, synovial fluid, vitreous fluid, spinal fluid), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation from a 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); markers associated with aging such as β-galactosidase (SA-β-gal), p16INK4a, p21, PAI-1, or any one or more SASP factors (e.g., IL-6, MMP3) associated with aging. Senescent cells and non-senescent cells of biological samples can also be used for in vitro cell assays, in which cells are exposed to any of the anti-senescent agents described herein to determine the ability of the anti-senescent 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 the subject before, during, and after treatment with an anti-senescent agent. In certain embodiments, the presence of senescent cells can be detected (e.g., by determining the level of senescent cell marker expression of mRNA), and the course of treatment and / or non-treatment interval can be adjusted accordingly.

[0283] Lung diseases and disorders

[0284] In some embodiments, provided is a method for treating or preventing a disease or disorder (i.e., reducing the likelihood of the disease or disorder occurring) by killing senescent cells associated with the disease or disorder (i.e., identified senescent cells) in a subject suffering from a disease or disorder associated with aging through administration of an anti-aging agent as described herein, wherein the disease or disorder associated with aging is a pulmonary disease or disorder. Pulmonary diseases and disorders associated with aging include, for example, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, bronchiectasis, and emphysema.

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

[0286] The processes that cause lung damage include oxidative stress, such as that produced by high concentrations of free radicals in tobacco smoke; the release of cytokines due to inflammatory responses to irritants in the airways; and damage to proteases by tobacco smoke and free radicals, allowing them 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 alpha-1-antitrypsin production 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, the production of extracellular matrix proteins increases, and transdifferentiation into contractile myofibroblasts contributes to wound contraction. The temporary matrix plugs the damaged epithelium and provides a scaffold for epithelial cell migration (involving epithelial-mesenchymal transition (EMT)). Blood loss associated with epithelial damage induces platelet activation, the production of growth factors and acute inflammatory responses. Normally, the epithelial barrier heals and the inflammatory response subsides. However, in fibrotic diseases, fibroblastic reactions persist, leading to unresolved wound healing. The formation of fibroblastic foci is a feature of the disease, reflecting the location of ongoing fibrogenesis. As the name suggests, the cause of IPF is unknown. The involvement of cellular senescence in IPF is shown by the observation that the incidence of the disease increases with age and that lung tissue of IPF patients is enriched 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 suggested by reports that SASP components of senescent cells (such as IL-6, IL-8, and IL-1β) promote fibroblast to myofibroblast differentiation and epithelial-mesenchymal transition, leading to extensive remodeling of the extracellular matrix of the alveolar and interstitial spaces (see, e.g., Minagawa et al., supra).

[0288] Subjects at risk for 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 medications (e.g., amiodarone, bleomycin, busulfan, methotrexate, and nitrofurantoin); subjects who have radiation therapy to the chest; and subjects whose family members have pulmonary fibrosis.

[0289] 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 because of excess mucus in the lungs; a chronic cough that produces sputum that may be clear, white, yellow, or green; bluish lips or nail beds (cyanosis); frequent respiratory infections; lack of energy; unexpected weight loss (observed in the later stages of the disease). Subjects with COPD may also experience exacerbations, during which symptoms worsen and last for several days or longer. Symptoms of pulmonary fibrosis are known in the art and include shortness of breath, especially during exercise; frequent dry coughs; rapid shallow breathing; gradual, unexpected weight loss; tiredness; painful joints and muscles; and clubbing (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 routinely practiced in the art. The effects of one or more anti-aging agents administered to subjects with lung disease or at risk of developing lung disease can be monitored using methods commonly used for diagnosis. Typically, one or more of the following examinations or tests can be performed: physical examination, patient's medical history, patient's family history, chest X-ray, lung function test (such as spirometry), blood test (e.g., arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CT scan, and exercise test.

[0291] Other lung diseases or disorders that can be treated by using anti-aging agents include, for example, emphysema, asthma, bronchiectasis, and cystic fibrosis (see, for example, Fischer et al., Am J Physiol Lung Cell Mol Physiol. 304 (6) (2013) L394-400). These diseases can also be aggravated 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 lead to inflammation. Emphysema is sometimes considered a subgroup of COPD.

[0292] Bronchiectasis is caused by damage to the airways, which causes them to widen and become flaccid and scarred. Bronchiectasis is often caused by medical conditions that damage the airway walls or inhibit the airways from clearing mucus. Examples of such conditions include cystic fibrosis and primary ciliary dyskinesia (PCD). When only a portion of the lungs is affected, the abnormality may be caused by obstruction rather than the medical condition.

[0293] The method for treating or preventing lung diseases or abnormalities associated with aging as described herein (i.e., reducing the possibility or occurrence of lung diseases or abnormalities associated with aging) can also be used to treat subjects who are aging and have loss of lung function (or degeneration) (i.e., lung function declines or is impaired compared to young subjects) and / or lung tissue degeneration. The respiratory system undergoes various anatomical, physiological and immune changes with age. These structural changes include chest wall and thoracic vertebral deformities, which can damage the compliance of the entire respiratory system, resulting in increased respiratory force. The respiratory system undergoes structural, physiological and immune changes with age. Compared with young people, the increased ratio of neutrophils and the reduced percentage of macrophages are found in the bronchoalveolar lavage fluid (BAL) of the elderly. Continuous low-grade inflammation in the lower respiratory tract can lead to proteolysis and oxidant-mediated damage to the lung matrix, resulting in the loss of alveolar units and impaired gas exchange across the alveolar membrane (occurring with age). Continuous lower respiratory tract inflammation can make the elderly more susceptible to toxic environmental exposures and accelerate the decline of 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 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 lead to tissue and organ damage during chronic inflammation (see, e.g., Demedts et al., Respir. Res. 7 (2006) 53-63). By administering anti-aging agents to aging subjects, including asymptomatic middle-aged humans, the decline in lung function can be slowed or inhibited by killing senescent cells and removing them from the airways.

[0294] The effectiveness of anti-aging agents can be easily determined by technicians in the medical and clinical fields. One or any combination of diagnostic methods (including physical examination, evaluation and monitoring of clinical symptoms, and the execution of analytical tests and methods described herein) can be used to monitor the health status of the subject. Using techniques known in the art, such as comparing the symptoms of patients with lung disease or risk of lung disease who have been treated with no such treatment or with placebo, the therapeutic effect of anti-aging agents or pharmaceutical compositions including the agents can be analyzed. In addition, methods and techniques for evaluating the mechanical function of the lungs, such as techniques for measuring lung capacity, elasticity, and airway hypersensitivity, can be performed. In order to determine lung function and monitor lung function throughout treatment, any of a number 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, 25% to 75% of forced expiratory flow and maximum spontaneous ventilation (MVV), peak expiratory flow (PEF), slow vital capacity (SVC). Total lung capacity includes total vital capacity (TLC), vital capacity (VC), residual volume (RV), and functional residual capacity (FRC). Gas exchange across the alveolar capillary membrane can be measured using the diffusion capacity of carbon monoxide (DLCO). Peripheral capillary blood oxygen saturation (SpO2) can also be measured; normal oxygen levels are usually between 95% and 100%. SpO2 levels below 90% indicate that the subject has hypoxemia. Values ​​below 80% are considered critical values, and intervention is required to maintain brain and heart function and avoid cardiac or respiratory arrest.

[0295] Metastatic disease

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

[0297] Diseases or abnormalities associated with senescent cells include metastatic lesions, and subjects with cancer may benefit from the administration of anti-aging agents for inhibiting metastatic lesions as described herein. When administered to subjects with cancer according to the methods described herein, such anti-aging agents can inhibit tumor proliferation. When cancer cells (i.e., tumor cells) spread beyond the anatomical site of origin and initial colonization to other areas of the subject's body, metastatic lesions of cancer occur. Tumor proliferation can be determined by tumor size, which can be measured in various ways familiar to those skilled in the art, such as, for example, by PET scans, MRI, CAT scans, biopsies. The effect of therapeutic agents on tumor proliferation can also be assessed 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 characterized by cells that are generally characterized by abnormal cell proliferation. The term cancer is generally used to describe malignant tumors or disease states caused by tumors. Alternatively, abnormal growth may be referred to as a tumor in the art. The term tumor, such as when it relates to tissue, generally refers to any abnormal tissue growth characterized at least in part by excessive and abnormal cell proliferation. Tumors may be metastatic and can spread beyond the anatomical site of their origin and initial colonization to other areas of the subject's body. Cancer may include solid tumors or may include "liquid" tumors (e.g., leukemias and other blood cancers).

[0299] Cells are induced to become senescent by cancer therapies such as radiation and certain chemotherapy 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 small-sized tumors 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 the occurrence of metastatic lesions), inhibiting or delaying metastatic lesions in subjects with cancer by administering 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 or 14 days. In yet 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 yet other embodiments, the treatment course is one day. In yet other embodiments, the anti-aging agent is administered on two or more days within a treatment window of no more than 7 or 14 days.

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

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

[0303] Chemotherapy may be referred to as chemotherapy, chemotherapeutic agents or chemotherapeutic drugs. Many chemotherapeutic agents are compounds known as small organic molecules. Chemotherapy is a term also used to describe a combination of chemotherapeutic drugs administered to treat a particular cancer. As will be appreciated by those skilled in the art, chemotherapy may also refer to a combination of two or more chemotherapeutic molecules administered in concert, and it may be referred to as combined 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] Cancer that can be transferred can be solid tumor, or can be liquid tumor (for example blood cancer, for example leukemia).Cancer as liquid tumor is classified as those occurring in blood, bone marrow and lymph node in the art, and generally comprises leukemia (myeloid and lymphatic), lymphoma (for example Hodgkin lymphoma) and melanoma (comprising multiple myeloma).Leukemia comprises for example acute lymphocytic 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 more frequently in humans include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer (including squamous cell skin cancer), kidney cancer, head and neck cancer, laryngeal cancer, squamous cancer that forms on the moist mucosal lining of the nose, mouth, throat, etc., bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In certain specific embodiments, the 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 a melanoma cell, a prostate cancer cell, a testicular cancer cell, a breast cancer cell, a brain cancer cell, a pancreatic cancer cell, a colon cancer cell, a thyroid cancer cell, a stomach cancer cell, a lung cancer cell, an ovarian cancer cell, a Kaposi's sarcoma cell, a skin cancer cell, a kidney cancer cell, a head or neck cancer cell, a laryngeal cancer cell, a squamous cell cancer cell, a bladder cancer cell, an osteosarcoma cell, a cervical cancer cell, an endometrial cancer cell, an esophageal cancer cell, a liver cancer cell, or a kidney cancer cell.

[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 (tumor) include the following: adrenocortical carcinoma, childhood adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytoma, childhood brain stem glioma, childhood atypical teratoid / rhabdoid tumor of the central nervous system, childhood embryonal tumor of the central nervous system, 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, unknown primary cancer, childhood unknown primary cancer, childhood cardiac (heart) tumor, cervical cancer, childhood cervical cancer, childhood chordoma, chronic bone marrow Hyperplastic dysplasia, Colon cancer, Colorectal cancer, Colorectal cancer in children, Extrahepatic bile duct cancer, Ductal carcinoma in situ (DCIS), Endometrial cancer, Esophageal cancer, Esophageal cancer in children, Olfactory neuroblastoma in children, Eye cancer, Malignant fibrous histiocytoma of bone, Gallbladder cancer, Gastric (Stomach) cancer, Gastric (Stomach) cancer in children, Gastrointestinal stromal tumor (GIST), Gastrointestinal stromal tumor (GIST) in children, Extracranial germ cell tumor in children, Extragonadal germ cell tumor, Gestational trophoblastic tumor, Glioma, Head and neck cancer, Head and neck cancer in children, Hepatocellular (liver) cancer, Hypopharyngeal cancer, Kidney cancer, Renal cell renal carcinoma, Wilms tumor, Kidney tumor in children, Langerhans cell histiocytosis, Laryngeal cancer, Laryngeal cancer in children, Leukemia, Acute lymphoblastic leukemia ALL, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma (CNS), melanoma, childhood melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, metastatic squamous neck carcinoma with occult primary, middle-aged and elderly patients with NUT gene involvement Tract cancer, oral cancer, multiple endocrine neoplasia syndrome in children, mycosis fungoides, myelodysplastic syndrome, myelodysplastic tumor, myeloproliferative tumor, multiple myeloma, nasal cancer, nasopharyngeal cancer, nasopharyngeal cancer in children, neuroblastoma, oral cancer, oral cancer in children, oropharyngeal cancer, ovarian cancer, ovarian cancer in children, epithelial ovarian cancer, low malignant potential tumor ovarian cancer, pancreatic cancer, pancreatic cancer in children, pancreatic neuroendocrine tumor (islet cell tumor), childhood papilloma, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma in children, prostate cancer, rectal cancer, renal pelvic transitional cell carcinoma, retinoblastoma, salivary gland cancer,Childhood salivary gland cancer, Ewing sarcoma family of tumors, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, childhood skin cancer, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, laryngeal cancer, thymoma and thymic cancer, childhood thymoma and thymic cancer, thyroid cancer, childhood thyroid cancer, ureteral transitional cell carcinoma, urethral cancer, endometrial cancer, vaginal cancer, vulvar cancer, and Waldenstrom's macroglobulinemia.

[0306] Side effects of chemotherapy and radiation therapy

[0307] In other embodiments, the abnormality or condition associated with senescent cells is a chemotherapy side effect or a radiation therapy side effect. Examples of chemotherapeutic agents that induce senescence of non-cancer cells include anthracyclines (such as doxorubicin, daunorubicin); Taxol (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 chemotherapy side effects or radiation therapy side effects (i.e., reduce their likelihood or occurrence). The removal or destruction of senescent cells can improve the acute toxicity of chemotherapy or radiation therapy, including acute toxicity including energy imbalance. Acute toxic side effects include, but are not limited to, gastrointestinal toxicity (e.g., nausea, vomiting, constipation, anorexia, diarrhea), peripheral neuropathy, fatigue, discomfort, low physical activity, blood toxicity (e.g., anemia), liver toxicity, 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 effects caused by chemotherapy or radiation therapy. For example, toxic side effects caused by radiation therapy or chemotherapy can be improved by the methods described herein. Therefore, 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)) or reducing the severity of its toxic side effects (i.e., harmful side effects) of chemotherapy or radiation therapy or both 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 chemotherapy or radiotherapy side effects or reducing the severity of chemotherapy or radiotherapy side effects can be accomplished by the same course of treatment 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 agents are administered during non-therapy or non-therapy time intervals or after the chemotherapy or radiotherapy treatment regimen has been completed.

[0308] In more specific embodiments, acute toxicity is acute toxicity including energy imbalance, and may include one or more of weight loss, endocrine changes (e.g., hormone imbalance, hormone signaling changes), and changes in body composition. In certain embodiments, acute toxicity including energy imbalance involves a decrease or reduction in the subject's physical activity capacity, as indicated by a decrease or reduction in energy expenditure than would be observed in a subject not receiving the drug therapy. As a non-limiting example, such acute toxic effects including energy imbalance include low physical activity. In other embodiments, energy imbalance includes fatigue or discomfort.

[0309] In some embodiments, the side effect of chemotherapy treated or prevented (i.e., the likelihood of occurrence is reduced) by the anti-aging agents described herein is cardiotoxicity. Subjects with cancer being treated with anthracyclines (such as doxorubicin, daunorubicin) can be treated with one or more anti-aging agents described herein that reduce, improve or reduce the cardiotoxicity of anthracyclines. 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 dose that a subject can receive for survival is limited. Administration of one or more anti-aging agents can reduce cardiotoxicity, so that additional amounts of anthracyclines can be administered to the subject, resulting in an improved prognosis associated with the cancer disease. In some embodiments, cardiotoxicity is caused by the administration of anthracyclines such as doxorubicin. Doxorubicin is an anthracycline topoisomerase inhibitor that is approved for the treatment of patients with ovarian cancer after failure of platinum therapy; Kaposi's sarcoma after failure of primary systemic chemotherapy or intolerance to therapy; or multiple myeloma in patients who have not previously received bortezomib or have previously received at least one therapy. If the patient's overall survival dose exceeds 550 mg / m 2 If the patient also receives mediastinal radiation or other cardiotoxic drugs, cardiotoxicity may occur even at lower doses.

[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 usually caused by multiple exposures to chemotherapy or radiotherapy or administration of chemotherapy or radiotherapy over a long period of time. Certain toxic effects occur for a long time after treatment (also referred to as late toxic effects) and are caused by damage to organs or systems by therapy. Organ dysfunction (e.g., neurological, pulmonary, cardiovascular and endocrine dysfunction) has been observed in patients receiving cancer treatment in 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, especially normal cells that have been induced to age by chemotherapy or radiotherapy, the likelihood of chronic side effects can be reduced, or the severity of chronic side effects can be reduced or reduced, or the onset of chronic side effects can be delayed. Chronic and / or late toxic side effects that occur in subjects receiving chemotherapy or radiation therapy include, by way of non-limiting examples, cardiomyopathy, congestive heart disease, inflammation, early menopause, osteoporosis, infertility, impaired cognitive function, peripheral neuropathy, secondary cancers, cataracts and other vision problems, hearing loss, chronic fatigue, reduced lung capacity, and lung disease.

[0311] In addition, by killing or removing senescent cells in subjects with cancer through the administration of anti-aging agents, 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, when an anti-aging agent is administered to a subject treated with the corresponding chemotherapy or radiotherapy, the development of chemotherapy or radiotherapy resistance can be inhibited.

[0312] Neurological diseases and disorders

[0313] Diseases or disorders associated with aging that can be treated by administering the anti-aging agents described herein include neurological diseases or disorders. Such diseases and disorders associated with aging include Parkinson's disease, Alzheimer's disease (and other dementias), motor neuron dysfunction (MND), mild cognitive impairment (MCI), Huntington's disease, and ocular diseases and disorders, such as age-related macular degeneration. Other ocular 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 brain disorder characterized by slow movements (bradykinesia), tremors, rigidity and loss of balance in the later stages. Many of these symptoms are due to the loss of certain nerves in the brain, which leads to a lack 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, the deep loss of dopamine in the striatum and / or the presence of intracytoplasmic inclusions (Lewy bodies), which are mainly composed of α-synuclein and ubiquitin. Parkinson's disease also has motor function defects, such as tremor, rigidity, bradykinesia and / or postural instability. Subjects at risk of developing Parkinson's disease include subjects with a family history of Parkinson's disease and subjects exposed to pesticides (e.g., rotenone or paraquat), herbicides (e.g., Agent Orange) or heavy metals. Senescence of dopamine-producing neurons is thought to contribute to the cell death observed in PD through the generation of reactive oxygen species (see, e.g., Cohen et al., J. Neural Transm. Suppl. 19 (1983) 89-103); therefore, the methods and anti-senescence agents described herein may 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 initiating or completing voluntary movements, jerks, rigid movements, muscle atrophy, tremors (tremors), and heart rate changes, but normal reflexes, bradykinesia, and postural instability. It is increasingly recognized that people diagnosed with Parkinson's disease may have cognitive impairment, including mild cognitive impairment, in addition to their physical symptoms.

[0316] Alzheimer's disease (AD) is a neurodegenerative disease that manifests as a slow progressive mental degeneration, accompanied by memory failure, dysphagia and confusion, leading to deep dementia. Age is the single largest risk factor for the development of AD, which is the main cause of dementia in the elderly (see, e.g., Hebert, et al., Arch. Neural. 60 (2003) 1119-1122). Early clinical symptoms show significant similarities with mild cognitive impairment (see below). As the disease progresses, impaired judgment, confusion, behavioral changes, dysphagia, 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 areas 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 mainly located in the neocortex, hippocampus and nucleus basalis of Meinert. 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. There are subjects with the risk of developing Alzheimer's disease, including subjects of advanced age, subjects with a family history of Alzheimer's disease, subjects with genetic risk genes (such as ApoE4) or deterministic gene mutations (such as APP, PS1 or PS2), and subjects with head trauma or heart / vascular disease (such as, hypertension, heart disease, stroke, diabetes, high cholesterol, etc.) history.

[0318] Many behavioral and histopathological assays are known in the art for assessing Alzheimer's disease phenotypes, for characterizing therapeutic agents, and for assessing treatment. Histological analysis is usually 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 sliced ​​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 are also described. BSB ((trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)phenylvinylbenzene) and PET tracers 11 C-labeled Pittsburgh compound-B (PIB) binds to Aβ plaques (see, for example, Skovronsky et al., Proc. Natl. Acad. Sci. USA 97 (2000) 7609-7614; Klunk et al., Ann. Neurol. 55 (2004) 306-319). 19The amyloidophilic Congo red-type compound FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)phenylvinylbenzene) of F allows visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., Nature Neurosci. 8 (2005) 527-533). Radiolabeled, putrescine-modified amyloid β peptides label amyloid deposits in a mouse model of Alzheimer's disease (see, e.g., Wengenack et al., Nat. Biotechnol. 18 (2000) 868-872).

[0319] Glial fibrillary acid protein (GFAP) increased by astrocytes is a marker of astrocyte activation and gliosis during neurodegeneration. AP plaques are associated with GFAP-positive activated astrocytes and can be visualized by GFAP staining (see, e.g., Nagele et al., Neurobiol. Aging 25 (2004) 663-674; Mandybur et al., Neurology 40 (1990) 635-639; Liang et al., J. Biol. Chem. 285 (2010) 27737-27744). Neurofibrillary tangles can be identified by immunohistochemistry using thioflavin-S fluorescence microscopy and Gallyas silver staining (see, e.g., Gotz et al., J. Biol. Chem. 276 (2001) 529-534; U.S. Patent No. 6,664,443). Axonal staining using electron microscopy and axonal transport studies can be used to observe neuronal degeneration (see, eg, Ishihara et al., Neuron 24 (1999) 751-762).

[0320] The subject suffering from Alzheimer's disease can be identified using the standard diagnostic method of Alzheimer's disease known in the art.Usually, the diagnosis of Alzheimer's disease is based on the patient's symptoms (e.g., progressive decline in memory function, gradual withdrawal and frustration to normal activities, apathy, excitement or irritability, aggressiveness, anxiety, sleep distress, irritability, abnormal motor behavior, disinhibition, social withdrawal, loss of appetite, hallucinations, dementia), medical history, neuropsychological tests, neurology and / or physical examination.Cerebral spinal fluid can also be tested for various proteins associated with Alzheimer's pathology, including taurine, amyloid beta peptide and AD7C-NTP.Gene testing can also be used for early-onset familial Alzheimer's disease (eFAD), a kind of autosomal dominant genetic disease.Clinical gene testing can be used for individuals with AD symptoms or high-risk family members of patients with early-onset diseases.In the U.S., the mutation of PS2 and APP can be tested in a laboratory approved by the clinical laboratory improvement amendments or the federal government.The commercial test for PS1 mutation is also available (Elan Pharmaceuticals).

[0321] Zhang et al. have reported that in the brains of patients with AD and in AD mouse models, oligodendrocyte progenitor cells (OPCs) expressing Olig2 and NG2 associated with Aβ plaques, but not astrocytes, microglia, or oligodendrocytes, exhibit an aging-like phenotype characterized by upregulation of p21 / CDKN1A, p16 / INK4 / CDKN2A proteins and aging-associated β-galactosidase activity (see Nature Neurosci. 22 (2019) 719-728). Molecular interrogation of the Aβ plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregated Aβ triggers cellular senescence. Treatment of AD mice with an anti-aging cocktail including dasatinib and quercetin selectively removed senescent cells from the plaque environment, reduced neuroinflammation, reduced Aβ load, and improved cognitive deficits. These findings suggest a role for Aβ-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit for anti-aging treatments.

[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 easily determined by those skilled in the medical and clinical fields. One or any combination of diagnostic methods (including physical examination, evaluation and monitoring of clinical symptoms, and execution of analytical tests and methods described herein) can be used to monitor the health status of the subject. The effect 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 the symptoms of patients who have not received such treatment or who have received a placebo.

[0323] Mild cognitive impairment (MCI) is a brain function syndrome, which involves the onset and evolution of cognitive impairment, and its degree exceeds the expectation based on individual age and education, but is not enough to significantly interfere with individual daily activities. MCI is an aspect of cognitive aging, which is considered to be a transitional state between normal aging and the dementia it may be converted into (see, Pepeu, Dialogues in Clinical Neuroscience 6 (2004) 369-377). The MCI that mainly affects memory is called "amnestic MCI". The person suffering from amnestic MCI may begin to forget important information that he or she used to be easy to recall, such as recent events. Amnestic MCI is often regarded as the prodromal stage of Alzheimer's disease. The MCI that affects the thinking ability other than memory is called "non-amnestic MCI". This type of MCI affects thinking skills, such as the ability to make correct decisions, the ability to judge the time or step sequence required to complete complex tasks, or visual perception ability. The individual suffering from non-amnestic MCI is considered to be more likely to change into other types of dementia (for example, dementia with Lewy bodies).

[0324] There is a growing awareness in the medical field that people diagnosed with Parkinson's disease may have MCI in addition to their physical symptoms. Recent studies suggest that 20-30% of people with Parkinson's disease have MCI, and their MCI tends to be of the non-amnestic type. Parkinson's disease patients with MCI sometimes develop full dementia (Parkinson's disease with dementia).

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

[0326] Motor neuron dysfunction (MND) is a group of progressive neurological abnormalities that destroy motor neurons, i.e. cells that control basic voluntary muscle activity 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-Weiland disease) and Kennedy disease), post-polio syndrome and hereditary spastic paraplegia. In adults, the most common MND is amyotrophic lateral sclerosis (ALS), which affects upper and lower motor neurons. It can affect the arms, legs or facial muscles. Primary lateral sclerosis is a disease of upper motor neurons, while progressive muscular atrophy only affects lower motor neurons in the spinal cord. In progressive bulbar palsy, the motor neurons at the bottom of the brainstem are most affected, resulting in slurred speech and difficulty chewing and swallowing. Mild abnormal signs are almost always present in the arms and legs. Patients with MND exhibit a Parkinson's phenotype (e.g., with tremor, rigidity, bradykinesia and / or postural instability). Methods for detecting, monitoring or quantifying movement and / or other defects 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 deficits and histopathological deficits associated with MND are known in the art, including histopathological, biochemical and electrophysiological studies and 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 abnormal neurofilament accumulation in degenerating motor neurons. In addition, reactive astrocytes and microglia are often detected in diseased tissues. Patients with MND show one or more motor deficits, including muscle weakness and emaciation, uncontrollable twitching, spasms, slow and laborious movements, and overactive tendon reflexes.

[0328] Eye diseases and abnormalities

[0329] In certain embodiments, the disease or abnormality associated with aging is an eye disease, abnormality or condition, such as presbyopia, macular degeneration or cataract. In certain other embodiments, the disease or abnormality associated with aging is glaucoma. Macular degeneration is a neurodegenerative disease that causes the loss of photoreceptor cells in the central part of the retina (called the macula). Macular degeneration is generally divided into two types: dry type and wet type. Dry form is more common than wet form, and about 90% of patients with age-related macular degeneration (ARMD or AMD) are diagnosed as dry form. The wet form of this disease usually causes more serious vision loss. Although the exact cause of age-related macular degeneration is not clear, the number of aging retinal pigment epithelial (RPE) cells increases with age. Age and certain genetic and environmental factors are risk factors for developing ARMD (see, e.g., Lyengar et al., Am. J. Hum. Genet. 74 (2004) 20-39; Kenealy et al., Mol. Vis. 10 (2004) 57-61; Gorin et al., Mol. Vis. 5 (1999) 29). Environmental susceptibility factors include 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 lead to a cellular senescence profile.

[0330] Dry ARMD is associated with atrophy of the RPE layer, and atrophy of the RPE layer causes 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 pigment in the macula. Aging seems to suppress the duplication and migration of RPE simultaneously, resulting in the permanent depletion of RPE in the macula of dry AMD patients (see, for example, 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, producing blind spots in central vision. Different forms of macular degeneration may also occur in young patients. The cause of disease unrelated to age may be associated with heredity, diabetes, nutritional deficiencies, head injuries, infection or other factors.

[0331] Decreased vision noted by the patient or ophthalmologist during a routine eye exam may be the first indicator of macular degeneration. The formation of sub-Bruch's membrane exudates or "warts" in the macula is often the first physical sign that macular degeneration may be developing. Symptoms include perceived distortion of straight lines, and in some cases, the center of vision appears more distorted than the rest of the scene; the appearance of a dark, fuzzy area or "blank" in the center of vision; and / or altered or diminished color perception. Diagnosis and monitoring of subjects with macular degeneration can be accomplished by a person skilled in the art of ophthalmology based on art-accepted procedures for regular eye examinations and the subject's reporting of symptoms.

[0332] Presbyopia is an age-related condition in which the ability of the eye to focus on nearby objects gradually decreases as the speed and amplitude of normal eye accommodation decrease with age. Loss of lens elasticity and loss of ciliary muscle contractility have been considered to be 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 indicate that the mechanical strength of the posterior lens capsule decreases significantly with age (see, e.g., Krag et al., Invest. Ophthalmol. Vis. Sci. 44 (2003) 691-696; Krag et al., Invest. Ophthalmol. Vis. Sci. 38 (1997) 357-363).

[0333] The laminar 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, supra, and references cited therein). The major structural component of the lens capsule is basement membrane type IV collagen, which is organized into a three-dimensional molecular network (see, e.g., Cummings et al., Connect. Tissue Res. 55 (2014) 8-12; Veis et al., Coll. Relat. Res. 1 (1981) 269-286). Type IV collagen consists of six homologous α chains (α1-6), which are combined into heterotrimeric type IV collagen protomers, each of which includes a specific chain combination of α112, α345 or α556 (see, e.g., Khoshnoodi et al., Microsc. Res. Tech. 71 (2008) 357-370). The protomer shares structural similarity with the triplet peptide sequence of Gly-XY in the triple helical collagen domain (Timpl et al., Eur. J. Biochem. 95 (1979) 255-263), terminating in a globular C-terminal region known as the non-collagen 1 (NC1) domain. The N-terminus consists of a helical domain known as 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 basement membrane beneath the epithelium, 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 following years after cataract surgery (see, e.g., Awasthi et al., Arch. Ophthalmol. 127 (2009) 555-562). PCO is caused by 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). 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 down or prevent (delay, inhibit, delay) the destruction of the type IV collagen network. Removing senescent cells and thereby removing 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 post-operative treatment of cataracts to reduce the likelihood of PCO.

[0336] Although direct evidence that cellular senescence is associated with cataract development has not been obtained from human studies, BubR1 hypomorphic mice develop bilateral posterior subcapsular cataracts early in life, suggesting that aging may play a role (see, e.g., Baker et al., Nat. Cell Biol. 10 (2008) 825-836). Cataracts are clouding of the lens of the eye that causes blurred vision and can lead to blindness if not treated. Surgery is effective and routinely performed to remove cataracts. Administration of one or more 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 ocular examination using methods routinely performed by those skilled in the art of ophthalmology.

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

[0338] In certain embodiments, the disease or abnormality associated with aging is glaucoma. Glaucoma is a broad term used to describe a group of diseases that cause visual field loss, 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 subjects with glaucoma do not become blind, their vision is often severely impaired. Normally, clear fluid flows in and out of the front of the eye (called the anterior chamber). In individuals with open-angle / wide-angle glaucoma, this fluid drains too slowly, causing increased pressure within the eye. If not treated, this high pressure subsequently damages the optic nerve and can lead to complete 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. When the cell network required for fluid outflow was subjected to SA-β-Gal staining, a four-fold increase in aging was observed in glaucoma patients (see, e.g., Liton et al., Exp. Gerontol. 40 (2005) 745-748).

[0339] To monitor the effect of therapy on inhibiting 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). Other methods include gonioscopy (examining the trabecular meshwork and the angle at which fluid flows out of the eye); imaging techniques such as scanning laser tomography (e.g., HRT3), laser polarization measurement (e.g., GDX), and ocular coherence tomography); ophthalmoscopy; and pachymetry measurement to determine central corneal thickness.

[0340] Metabolic diseases or disorders

[0341] Diseases or disorders associated with aging that can be treated by administering anti-aging agents 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 levels of blood sugar caused by defects in insulin production, insulin action, or both. Most (90% to 95%) adult diabetes diagnosis cases are type 2 diabetes, which is characterized by the gradual loss of insulin secretion by the pancreas. In the U.S., diabetes is the main cause of new cases of renal failure, non-traumatic lower limb amputation, and adult blindness. Diabetes is the main cause of heart disease and stroke, and is the seventh leading cause of death in the U.S. (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")). Anti-aging agents as described herein can be used to treat type 2 diabetes, particularly type 2 diabetes associated with age, diet, and obesity.

[0343] Senescent cells are involved in metabolic diseases, such as obesity and type 2 diabetes, and have been considered to be a response to injury or metabolic dysfunction (see, e.g., Tchkonia et al., Aging Cell 9 (2010) 667-684). Adipose tissue from obese mice showed induction of senescence markers SA-β-Gal, p53, and p21 (see, e.g., Tchkonia et al., above; Minamino et al., Nat. Med. 15 (2009) 1082-1087). Concomitant upregulation of proinflammatory cytokines (such as tumor necrosis factor α and Ccl2 / MCP1) was observed in the same adipose tissue (see, e.g., Minamino et al., above). The induction of senescent cells in obesity is potentially clinically significant, as proinflammatory SASP components are also thought to contribute to type 2 diabetes (see, e.g., Tchkonia et al., above). Similar upregulation patterns of senescence markers and SASP components are associated with diabetes, both in mice and in humans (see, e.g., Minamino et al., above). Therefore, the methods described herein comprising administering an anti-aging agent 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 senescent preadipocytes with an anti-aging agent thereby killing senescent preadipocytes can provide clinical and health benefits to a person suffering from any of diabetes, obesity or metabolic syndrome.

[0344] The experimenter suffering from type 2 diabetes can be identified using standard diagnostic methods known in the art for type 2 diabetes. In general, 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 healing of ulcers or frequent infection and / or darkening of the skin), medical history and / or patient's physical examination. The experimenter with development type 2 diabetes risk comprises an experimenter with a family history of type 2 diabetes and an experimenter with other risk factors, such as overweight, fat distribution, inactivity, race, age, prediabetes and / or gestational diabetes.

[0345] The effectiveness of anti-aging agents can be easily determined by technicians in the medical and clinical fields. One or any combination of diagnostic methods (including physical examination, evaluation and monitoring of clinical symptoms, and the execution of analytical tests and methods, such as those described herein) can be used to monitor the health status of the subject. For example, it is possible to monitor the subjects who receive one or more anti-aging agents described herein for the treatment or prevention of diabetes by measuring glucose and insulin tolerance, energy expenditure, body composition, adipose tissue, skeletal muscle and liver inflammation and / or fat toxicity (muscle and liver lipids by in vivo imaging and muscle, liver, bone marrow and pancreatic beta cell lipid accumulation and inflammation by histology). Other features or phenotypes of type 2 diabetes are known and can be measured 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 mass is significantly greater than the ideal value in terms of their height and stature. Body mass index (BMI) is a measurement tool for determining overweight weight, and is calculated by the height and weight of the subject. When a person has a BMI of 25-29, the person is considered to be overweight; when a person has a BMI of 30-39, the person is considered to be obese; and when a person has a BMI of>40, the person is considered to be severely obese. Therefore, 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 that is not captured by BMI is referred to in the art as "abdominal obesity", which is related to the extra fat found in the middle of the subject, which is an important factor in health, even unrelated to BMI. The simplest and most commonly used measure of abdominal obesity is waist circumference. Generally speaking, abdominal obesity in women is defined as a waist circumference of 35 inches or more, and abdominal obesity in men is defined as a waist circumference of 40 inches or more. 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 diabetic ulcers (i.e., diabetic wounds). 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 show elevated presence of cellular senescence at the site of chronic wounds (see, e.g., Stanley et al., J. Vas. Surg. 33 (2001) 1206-1211). Chronic inflammation is also observed at the site of chronic wounds, such as diabetic ulcers (see, e.g., Goren et al., Am. J. Pathol. 168 (2006) 65-77), suggesting that the pro-inflammatory cytokine phenotype of senescent cells plays a role in pathology.

[0348] Experimenters who suffer from type 2 diabetes or have a risk of developing type 2 diabetes may suffer from metabolic syndrome. Human metabolic syndrome is usually associated with obesity, and is characterized by one or more of cardiovascular disease, hepatic steatosis, hyperlipidemia, diabetes and insulin resistance. Experimenters who suffer from metabolic syndrome may have a series of metabolic abnormalities or abnormalities, which for example can include one or more of hypertension, type 2 diabetes, hyperlipidemia, dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulin resistance, hepatic steatosis (steatohepatitis), hypertension, atherosclerosis and other metabolic abnormalities.

[0349] Abnormal renal function

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

[0351] Dermatological diseases or abnormalities

[0352] Diseases or abnormalities associated with aging that can be treated by administering the anti-aging agents described herein include dermatological diseases or abnormalities. Such diseases and abnormalities associated with senescent cells include psoriasis and eczema, which are also inflammatory diseases and are discussed in more detail above. Other dermatological diseases and abnormalities associated with aging include wrinkles (wrinkles caused by aging); itching (associated with diabetes and aging); dysesthesia (a side effect of chemotherapy 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 rashes (commonly observed in elderly patients and associated with side effects of certain drugs). Other dermatological diseases and abnormalities associated with aging include eosinophilic dermatosis (associated with certain types of hematological cancers); reactive neutrophilic dermatosis (associated with underlying diseases such as inflammatory bowel syndrome); pemphigus (an autoimmune disease in which autoantibodies against desmoglein are formed); pemphigoid and other immune bullous dermatoses (autoimmune blisters of the skin); fibrohistiocytic proliferation of the skin, which is associated with aging; and cutaneous lymphomas that are more common in the elderly population. Another dermatological disease that can be treated according to the methods described herein includes cutaneous lupus, which is a symptom of lupus erythematosus. Late-onset lupus may be associated with a decline in the function of T cells and B cells (i.e., reduction) and cytokines associated with aging (immunosenescence).

[0353] Inflammatory and autoimmune diseases and disorders

[0354] In certain embodiments, the disease or disorder associated with aging is an inflammatory disease or disorder, such as, by way of non-limiting example, osteoarthritis, which can be treated or prevented (i.e., reduced likelihood of occurrence) according to the methods described herein including administration of an anti-aging agent. Other inflammatory or autoimmune diseases or disorders that can be treated by administration of 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 disease, COPD and idiopathic pulmonary fibrosis.

[0355] Osteoarthritis is a degenerative joint disease characterized by cartilage fibrillation, bone sclerosis, and thickening of the synovium and joint capsule at sites of high mechanical stress. Fibrillation is a localized surface disintegration involving splitting of the surface layer of cartilage. Early splits are tangential to the cartilage surface, following 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 destruction ensues. Predisposing risk factors for developing osteoarthritis include increasing age, obesity, previous joint injuries, overuse of joints, thigh muscle weakness, and heredity. Symptoms of osteoarthritis include joint pain or stiffness, especially in the hips, knees, and lower back after inactivity or overuse; stiffness after rest that disappears after exercise; and pain that is more severe after activity or toward the end of the day. Osteoarthritis may also affect the neck, little toe joints, base of the thumb, ankles, and big toes. Chronic inflammation is considered the main age-related factor leading to osteoarthritis. In combination with age, joint overuse and obesity appear to promote osteoarthritis.

[0356] By selectively killing senescent cells, anti-aging agents prevent (i.e., reduce the likelihood of occurrence), reduce or inhibit the loss or erosion of proteoglycan layers in joints, reduce inflammation in affected joints, and promote (i.e., stimulate, enhance, induce) the production of collagen (e.g., type 2 collagen). The removal of senescent cells results in a reduction in the amount (i.e., level) of inflammatory cytokines such as IL-6 produced in joints, and inflammation is alleviated. Provided herein is a method for treating osteoarthritis, selectively killing senescent cells in osteoarthritic joints of subjects, and / or inducing the production of collagen (such as type 2 collagen) in subject joints by administering at least one anti-aging agent (which can be combined with at least one pharmaceutically acceptable excipient to form a pharmaceutical composition) to a subject. Anti-aging agents can also be used to reduce (inhibit, reduce) the production of metalloproteinase 13 (MMP-13) that degrades collagen in joints, and to restore the proteoglycan layer or inhibit the loss and / or degradation of the proteoglycan layer. Therefore, treatment with anti-aging agents can also prevent bone erosion (i.e., reduce the likelihood of bone erosion occurring), 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, anti-aging agents are directly administered to osteoarthritic joints (e.g., by intra-articular, topical, transdermal, intradermal or subcutaneous delivery). Treatment with anti-aging agents can also restore, improve or inhibit the deterioration of joint strength. In addition, methods including administration of anti-aging agents can reduce joint pain and are therefore useful for pain control of osteoarthritic joints.

[0357] The effectiveness of one or more anti-aging agents for treating or preventing osteoarthritis in a subject and 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 (such as determining tenderness, swelling or redness of the affected joint), evaluation and monitoring of clinical symptoms (such as pain, stiffness, range of motion), and execution of analytical tests and methods described herein and practiced in the art (e.g., determining the level of inflammatory cytokines or chemokines; X-ray images to determine the loss of cartilage, as shown by the narrowing of the space between bones in the joint; magnetic resonance imaging (MRI), which provides detailed images of bones and soft tissues (including cartilage)), can be used to monitor the health of a subject. The therapeutic effect of one or more anti-aging agents can be analyzed by comparing the symptoms of patients who have an inflammatory disease or disorder (such as osteoarthritis) or are at risk for an inflammatory disease or disorder (who have received treatment) with the symptoms of patients who have not received such treatment or have received a placebo treatment.

[0358] In certain embodiments, anti-aging agents can be used to treat and / or prevent rheumatoid arthritis (RA) (i.e., reduce or reduce the likelihood of rheumatoid arthritis occurring). Abnormal regulation of innate and adaptive immune responses is a characteristic of rheumatoid arthritis (RA), which is an autoimmune disease whose incidence increases with age. Rheumatoid arthritis is a chronic inflammatory abnormality that usually 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, causing painful swelling, which can lead to bone erosion and joint deformity. RA can also sometimes 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 usually begins to develop after the age of 40. This abnormality 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 aging-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 usually occurs after osteoporosis weakens the spine to the point where it breaks and compresses. Several types of kyphosis target 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 aging. The ability of anti-aging agents to treat kyphosis can be determined in preclinical animal models used in the art. For example, TTD mice develop kyphosis (see, e.g., de Boer et al., Science 296 (2002) 1276-1279); other mice that can be used include Bub1 H / H Mice, which are also known to develop kyphosis (see, e.g., Baker et al., Nature 479 (2011) 232-236). Kyphosis development is measured visually over time. 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 decrease in bone mass and bone density, which may lead to an increased risk of fractures, which can be treated or prevented by administering the anti-aging agents described herein. Bone mineral density (BMD) decreases, bone microarchitecture deteriorates, and the amount and type of protein in the bone changes. Osteoporosis is usually diagnosed and monitored by bone mineral density testing. Postmenopausal women or women with reduced estrogen are at greatest risk. Although both men and women over 75 are at risk, women are twice as likely to develop osteoporosis as men. By detecting the presence of one or more markers associated with senescent cells, such as by SA-β-Gal staining, the level of senescent cells reduced by treatment with anti-aging agents can be determined.

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

[0362] Other inflammatory or autoimmune diseases that can be treated or prevented (i.e., reduced likelihood of occurrence) by the use of 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 induced by radiation in some cases). Certain organ fibrosis or fibrotic conditions (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 abnormality associated with senescent cells is an inflammatory abnormality 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 excess and rapid growth of the epidermal layer of the skin. The diagnosis of psoriasis is usually 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, skin and systemic overexpression of various pro-inflammatory cytokines (such as IL-6, a key component of SASP) is observed. Eczema is an inflammation of the skin characterized by redness, swelling of the skin, itching and dryness, crusting, flaking, blistering, cracking, exudation or bleeding. The effectiveness of anti-aging agents for treating psoriasis and eczema and the monitoring of subjects receiving such anti-aging agents can be easily determined by a technician in the medical or clinical field. One or any combination of diagnostic methods comprising physical examination (such as skin appearance), monitoring 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., determining the levels of pro-inflammatory cytokines).

[0364] Other immune abnormalities or conditions that can be treated or prevented (i.e., reduced likelihood of occurrence) with the anti-aging agents described herein include conditions caused by the host's immune response to an organ transplant (e.g., a 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 disorders

[0366] In other embodiments, the disease or disorder associated with aging treated by the methods described herein is a cardiovascular disease. The cardiovascular disease can be any one or more of angina, arrhythmia, atherosclerosis, cardiomyopathy, congestive heart failure, coronary artery disease (CAD), carotid artery disease, endocarditis, heart attack (coronary thrombosis, myocardial infarction [MI]), high blood pressure / hypertension, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, diastolic dysfunction, hypercholesterolemia / hyperlipidemia, mitral valve prolapse, peripheral vascular disease (e.g., peripheral arterial disease (PAD)), cardiac stress resistance, and stroke.

[0367] In certain embodiments, methods for treating cardiovascular diseases associated with or caused by atherosclerosis (i.e., hardening of the arteries) are provided. Cardiovascular diseases can be any one or more of atherosclerosis (e.g., coronary artery disease (CAD) and carotid artery disease); angina, congestive heart failure, and peripheral vascular disease (e.g., peripheral artery disease (PAD)). Methods for treating cardiovascular diseases associated with or caused by atherosclerosis can reduce the likelihood of high blood pressure / hypertension, angina, stroke, and heart attack (i.e., coronary thrombosis, myocardial infarction (MI)). In certain examples, methods for stabilizing atherosclerotic plaques in a subject's blood vessels (e.g., arteries) are provided, thereby reducing the likelihood 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) the lipid content of atherosclerotic plaques in a subject's blood vessels (e.g., arteries) and / or increasing the thickness of the fibrous cap (i.e., causing an increase in the fibrous cap, enhancing or promoting the thickening of the fibrous cap).

[0368] Atherosclerosis is characterized by patchy intimal plaques (atheromas) that invade the lumen of medium and large arteries; plaques contain lipids, inflammatory cells, smooth muscle cells, and connective tissue. Atherosclerosis can affect large and medium-sized arteries, including coronary arteries, carotid arteries and cerebral arteries, the aorta and its branches, and major arteries of the limbs. In some embodiments, a method for inhibiting the formation of atherosclerotic plaques (or reducing, reducing the formation of atherosclerotic plaques, causing a reduction in the formation of atherosclerotic plaques) by administering an anti-aging agent is provided. In other embodiments, a method for reducing (lowering, reducing) the amount (i.e., level) of plaques is provided. The reduction in the amount of plaques in a blood vessel (e.g., an artery) can be determined, for example, by a reduction in the surface area of ​​the plaques, or by a reduction in the extent or degree (e.g., percentage) of the occlusion of a blood vessel (e.g., an artery), which can be determined by angiography or other visualization methods used in the cardiovascular field. Also provided herein is a method 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 disease can be identified using standard diagnostic methods known in the field of cardiovascular disease. Typically, the diagnosis of atherosclerosis and other cardiovascular diseases is based on symptoms (e.g., chest pain or pressure (angina), numbness or weakness in arms or legs, difficulty speaking or slurred speech, facial muscle drooping, leg pain, hypertension, renal failure and / or erectile dysfunction), medical history and / or physical examination of the patient. Diagnosis can be confirmed by angiography, ultrasonography or other imaging tests. Subjects with risk of developing cardiovascular disease include subjects with any one or more susceptibility factors (such as a family history of cardiovascular disease) and subjects 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 as a disease / abnormality associated with senescent cells is atherosclerosis.

[0370] The effectiveness of one or more anti-aging agents for treating or preventing cardiovascular disease (e.g., atherosclerosis) (i.e., reducing or reducing 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, evaluation and monitoring of clinical symptoms, and the performance of analytical tests and methods described herein and practiced in the art (e.g., angiography, electrocardiogram, stress testing, non-stress testing) can be used to monitor the health status of the subject. The therapeutic effect of an anti-aging agent or a pharmaceutical composition comprising the anti-aging agent can be analyzed using techniques known in the art, such as comparing the symptoms of patients with cardiovascular disease or at risk of cardiovascular disease who have received treatment with the symptoms of patients who have not received such treatment or who have received a placebo.

[0371] Combination therapy

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

[0373] For example, the anti-aging agent can be administered in combination with a chemotherapeutic agent or after the administration of 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 senescent tumor cells. Examples of chemotherapeutic drugs that can be used for tumor treatment and anti-aging compound combinations 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 above-mentioned 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] Also provided herein is a pharmaceutical composition 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 composition may be a sterile aqueous or non-aqueous solution, suspension, or emulsion (e.g., a microemulsion). The excipients described herein are exemplary and by no means limiting. An effective amount or therapeutically effective amount refers to the amount of one or more anti-aging agents administered to a subject as a single dose or as part of a series of doses that 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 abnormality as described herein, each anti-aging agent can be formulated into a separate pharmaceutical composition. A pharmaceutical preparation comprising each separate pharmaceutical composition can be prepared (for convenience, it can 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 can be administered at the same time (i.e., simultaneously) and by the same route of administration, or can be administered at different times by the same or different routes of administration. Alternatively, two or more anti-aging agents can be formulated into a single pharmaceutical composition together.

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

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

[0380] By using the techniques conventionally practiced 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., tablet, capsule), a semi-solid (e.g., gel), a liquid, or a gas (aerosol). In certain other specific embodiments, the anti-aging agent (or a pharmaceutical composition comprising the anti-aging agent) is administered in the form of a pill infusion. In certain embodiments, when the anti-aging agent is delivered by infusion, the anti-aging agent is delivered to an organ or tissue comprising senescent cells to be killed through a blood vessel according to the techniques conventionally performed by technicians in the medical field.

[0381] Pharmaceutically acceptable excipients are well known in the medical field and are described, for example, in Rowe et al., Handbook of Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties, and Safety, 5 th Ed., 2006 and Remington: The Science and Practice of Pharmacy (Gennaro, 21 st Ed.Mack Pub.Co., Easton, Pa. (2005)). Exemplary pharmaceutically acceptable excipients include sterile saline and phosphate buffered saline at physiological pH. Preservatives, stabilizers, dyes, buffers, etc. can be provided in the pharmaceutical composition. In addition, antioxidants and suspending agents can 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 composition described herein can be formulated as a lyophilizate. The composition described herein can be lyophilized or otherwise formulated into a lyophilized product, and one or more suitable excipient solutions are used to solubilize and / or dilute the medicament of the composition when it is administered. In other embodiments, the medicament can be encapsulated in liposomes using techniques known and practiced in the art. The pharmaceutical composition can be formulated for any suitable mode of administration described herein and in the art.

[0382] The pharmaceutical composition can be delivered to a subject in need thereof by any of several approaches known to those skilled in the art. As a non-limiting example, the composition can be delivered orally, intravenously, intraperitoneally, by infusion (e.g., pill infusion), subcutaneously, enterally, rectally, intranasally, by inhalation, oral, sublingual, intramuscularly, transdermally, intradermally, topically, intraocularly, vaginally, rectally, or by intracranial injection or any combination thereof. In certain embodiments, as described above, the dose is administered by intravenous, intraperitoneal, direct access to the target tissue or organ, or subcutaneous routes. In certain embodiments, the delivery method comprises a drug-coated or infiltrated stent in which the drug is an anti-aging agent. Preparations 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 directly administered to a target tissue or organ including senescent cells that contribute to the manifestation of a disease or abnormality. In a specific embodiment of treating osteoarthritis, the at least one anti-aging agent is directly administered to an osteoarthritic joint (i.e., intra-articularly) of a subject in need thereof. In other specific embodiments, the anti-aging agent may be administered to a joint by a topical, transdermal, intradermal, or subcutaneous route. In certain other embodiments, provided herein is a method 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 lung diseases or abnormalities 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, the anti-aging agent (or a pharmaceutical composition comprising an anti-aging agent) can be delivered directly to the eye by injection (e.g., intraocular or intravitreal) or by applying a cream, ointment, gel, or eye drops to the conjunctiva under the eyelid. In more specific embodiments, the anti-aging agent or a pharmaceutical composition comprising an anti-aging agent can be formulated as a timed release (also known as sustained release, controlled release) composition or can be administered as a bolus infusion.

[0384] The pharmaceutical composition (e.g., for oral administration or for injection, infusion, subcutaneous delivery, intramuscular delivery, intraperitoneal delivery or other methods) can be in liquid form. The liquid pharmaceutical composition can include, for example, one or more of the following: a sterile diluent, such as water, a saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a fixed oil that can be used as a solvent or suspension medium, polyethylene glycol, glycerol, propylene glycol or other solvent; an antibacterial agent; an antioxidant; a chelating agent; a buffer and an agent for adjusting tension such as sodium chloride or glucose. Parenteral compositions can be enclosed in an ampoule, a disposable syringe or a multi-dose vial made of glass or plastic. It is preferred to use physiological saline, and the injectable pharmaceutical composition is preferably sterile. In other embodiments, in order to treat eye conditions or diseases, the liquid pharmaceutical composition can be applied to the eye in the form of eye drops. The liquid pharmaceutical composition can 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 appropriate additives to prepare tablets, powders, granules or capsules, and if necessary, can be used in combination with diluents, buffers, wetting agents, preservatives, colorants and flavorings. 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 for oral delivery, for example, in a liquid, solid or semi-solid formulation and / or with an enteric coating.

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

[0387] In certain embodiments, the pharmaceutical composition comprising the anti-aging agent is formulated for transdermal, intradermal or topical administration. The composition can be applied in the form of powder / talcum powder or other solids, liquids, sprays, aerosols, ointments, foams, creams, gels, pastes using a syringe, bandage, transdermal patch, insert or syringe-shaped 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 the area to be treated or in the area to be treated (intradermal or subcutaneous). 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 parahydroxybenzoate, ethyl parahydroxybenzoate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, thimerosal and combinations thereof.

[0388] The pharmaceutical composition including the anti-aging agent can be formulated into an emulsion for topical application. The emulsion contains a liquid that is distributed in the body 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 aqueous phase can contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers and other excipients. The oil phase can contain excipients approved by other oily drugs. Suitable surfactants include but are not limited to anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants. The composition for topical application can also include at least one suitable suspending agent, antioxidant, chelating agent, emollient or wetting agent.

[0389] Ointments and creams can be prepared, for example, with aqueous or oily bases, with the addition of suitable thickeners and / or gelling agents. Lotions can be prepared with aqueous or oily bases, and will usually also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or coloring agents. Liquid sprays can be delivered from pressurized packaging, for example, by closures of special shapes. Oil-in-water emulsions can also be used in compositions, patches, bandages and products. These systems are semisolid emulsions, microemulsions or foam emulsion systems.

[0390] Controlled release or sustained release transdermal or topical preparations can be realized by adding the available timed release additives (such as polymer structure, matrix) in this area.For example, compositions can be applied by using hot melt extrusion products (such as bioadhesive hot melt extrusion films).Described preparation can include crosslinked polycarboxylic acid polymer preparation.Crosslinking agent can exist with the amount that enough adhesion is provided, to allow system to keep being attached to target epithelium or endothelial cell surface and continue the long enough time, to allow the required release of compound.

[0391] The insert, transdermal patch, bandage or article may include a mixture of polymers or coatings that provide for release of the active agent at a constant rate 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 may be mixed with a water-insoluble polymer to increase the durability of the insert and prolong the release of the active ingredient.

[0392] Polymer formulations can also be used to provide controlled release or sustained release. Bioadhesive polymers described in the art can be used. For example, sustained release gels and compounds can be incorporated into polymer matrices (such as hydrophobic polymer matrices). Examples of polymer matrices include microparticles. Microparticles can be microspheres, and cores can be materials different from polymer shells. Alternatively, polymers can be cast into thin plates or films, powders or gels (such as hydrogels) produced by grinding or other standard techniques. Polymers can also be in the form of a part for a coating or bandage, stent, catheter, vascular graft or other device, for the convenience of delivery of anti-aging agents. Matrix can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.

[0393] Kits are provided with unit doses of one or more of the agents described herein, typically oral or injectable doses. Such kits may include a container containing the unit dose, an informational package insert describing the use of the drug in treating diseases associated with senescent cells and the attendant benefits, and optionally an implement or device for delivering the composition.

[0394] All references and patent documents are hereby incorporated in their entirety for all purposes.

[0395] Example

[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] 2-(2-Methyl-1H-indol-3-yl)ethyl-1-amine (28) (400 mg, 2.2 mmol) was dissolved in a mixture of THF: dichloroethane: methanol (5:5:0.5). To this solution was added (E)-3-(4-formylphenyl)acrylic acid (27) (367 mg, 2.09 mmol, 0.95 equiv), triacetoxyborohydride (2320 mg, 11 mmol, 5.0 equiv) and 3 drops of acetic acid. The mixture was allowed to stir overnight and the volatiles were removed under vacuum. LC / MS showed the presence of the desired product. Water was added and a solid precipitated from the solution. After the pH was adjusted to 7 with dilute NaHCO3, the aqueous solution was washed with ethyl acetate. The white solid was filtered and washed with water, ether and hexanes 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 (1400mg, 6.5mmol, 1.0 equivalent) and 2-chloropyrimidine-5-carboxylic acid methyl ester (1180mg, 6.8mmol, 1.05 equivalent) in dioxane (28.0mL, 0.23M) were treated with cesium carbonate (5.27g, 16.2mmol, 2.5 equivalents) and Pd (dba) 2 acetone (440mg, 0.48mmol, 0.075 equivalents) was added. The solution was purged with nitrogen (3x). Xanthene (558mg, 0.96mmol, 0.15 equivalents) was then added in one portion. Within a few minutes, the suspension changed from dark red to yellow-green. It was then heated at 70°C for 30min, when LC / MS analysis showed the presence of the desired product. The mixture was cooled to room temperature and filtered through a diatomaceous earth pad and washed with dichloromethane (20mL, 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 2-[4-(tert-butoxycarbonylamino-methyl)-piperidin-1-yl]-pyrimidine-carboxylic acid methyl ester as an off-white solid. (1650 mg). LC / MS: RT=3.23 min; m / z=351.6 [M+H] + .

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

[0404] 1-Methyl-1H-indole-3-carboxaldehyde (600mg, 3.77mmol, 0.95 equivalent) was added in one portion to 2-(4-aminomethyl-piperidin-1-yl)-pyrimidine-5-carboxylic acid methyl ester (1080mg, 3.77mmol, 1.0 equivalent) and triethylamine (1.5mL, 10.5mmol, 2.5 equivalent) in THF:DCE (1:1) 5% methanol 18mL. Sodium triacetoxyborohydride (6300mg, 30.6mmol, 8.0 equivalent) was added with 4 drops of acetic acid. NMP (1.8mL) was added, and the mixture was stirred at room temperature for 2 days. LC / MS analysis showed the formation of the desired product. Water was added, the pH was adjusted to 7 with sodium bicarbonate, and the white solid was filtered and washed with water and ethyl acetate. The product was dried under high vacuum to give methyl 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylate (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] Crude 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid methyl ester (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 the reaction was complete. The solvent was concentrated to remove excess alcohol, and the mixture was acidified to pH 5 and washed with water and then hexanes. The gray solid was dried under high vacuum to give pure 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxylic acid (220 mg). LC / MS: RT = 2.41 min; m / z = 380.6 [M+H] + .

[0406] 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 eq) and sodium bicarbonate (165 mg, 1.9 mmol, 3.8 eq) were suspended in dioxane:water (3:1) (1.4 mL). Fmoc chloride (136 mg, 0.5 mmol, 1.0 eq) was added in portions until the solution was clear. LC / MS analysis showed the desired product. The pH of the solution was adjusted to 2, and ethyl acetate was added. The mixture was extracted with water (3x) and washed with brine. The combined organic layers were dried over sodium sulfate and concentrated to dryness to give a white foam which was ground with dichloromethane methanol-hexane (1:5, 3x). Thorough drying gave 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) as a white foam. 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 equiv) was dissolved in dichloromethane (80 mL). To this solution was added 2-hydroxyisoindoline-1,3-dione (2600 mg, 16.05 mmol, 1.0 equiv). Tetrabutylammonium hydrogen sulfate (1090 mg, 3.21 mmol, 0.2 equiv) in 1 M sodium carbonate (32.0 mL, 32 mmol, 2.0 equiv) was slowly added at ice bath temperature. The mixture was stirred at room temperature overnight. TLC analysis showed the appearance of new spots at a lower rf compared to the starting material with PMA staining. 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:hexanes. The product fractions were collected, combined and concentrated to give pure (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((1,3-dioxoisoindolin-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white foam. (2000 mg, 55%). LC / MS: RT=2.96 min; m / z=494.4 [M+H] + .

[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 equiv) was dissolved in methanol (35 mL, 0.1 M). Hydrazine hydrate (0.20 mL, 4.25 mmol, 1.05 equiv) was added slowly. After five minutes, LC / MS analysis showed the desired product. Dichloromethane (125 mL) was added and the solution was washed with saturated NaHCO3 (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:hexanes. The product fractions were collected, combined and concentrated to give the title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) as a white foam. (1170 mg, 79% yield). LC / MS: RT = 1.98 min; m / z = 364.3 [M+H] + .

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

[0412]

[0413] (3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (5000mg, 17mmol) was dissolved in THF (100.0mL). DAST (18.0mL, 137mmol, 8.0 equivalents) was added at -30°C and cooled with a dry ice-methanol bath. The reaction mixture was allowed to warm to room temperature and stirred at this temperature for 1h. Another portion of DAST (4mL) was added at -30°C and allowed to warm to room temperature again and stirred for 1h. LC / MS analysis showed that the reaction was complete. Methanol was added at -20°C and the solvent was evaporated. NaHCO3 was added and the solution was extracted with dichloromethane (3x). The combined organic layers were washed with water. Dilute HCl was added to decompose the residual DAST, and the solution was extracted with ethyl acetate (2x). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated to give the crude product (4000mg) as an oil. The brown oil was subjected to normal phase purification, eluting with hexane-ethyl acetate (0 to 40%). The product fractions were collected, combined and concentrated to give (3S,4R,5R,6S)-2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (30) as a white foam solid. (2000 mg). LC / MS: RT=2.45 min; m / z=310 [M+H2O] + .

[0414] (3S, 4R, 5R, 6S) -2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (30) (1800 mg, 6.16 mmol, 1.0 equiv) was dissolved in acetonitrile (30 mL). N-hydroxyphthalimide (1100 mg, 6.78 mmol, 1.1 equiv) was added followed by TEA (1.15 mL, 6.78 mmol, 1.05 equiv). BF3: Et2O (1.1 mL, 6.78 mmol, 1.05 equiv) was added dropwise and the reaction product was stirred for 1 h, at which time LC / MS analysis showed the presence of the desired product. The mixture was poured into 10% NaHCO3 / 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 that the reaction was complete 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 NaHCO3 (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 equiv) and 1-hydroxybenzotriazole (HOBt) (91 mg, 0.59 mmol, 1.33 equiv) 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 equiv) in N,N-dimethylformamide (DMF) (1.4 mL) and stirred at room temperature for 30 min. Then, (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(aminooxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (24) (244 mg, 0.67 mmol, 1.5 eq.) and DIPEA (0.078 mL, 0.449 mmol, 1.4 eq.) were added to the mixture at ice bath temperature. The mixture was stirred at room temperature overnight. LC / MS analysis showed the desired product. The mixture was quenched with cold saturated NH4Cl solution. The white precipitate thus formed was filtered and washed with water (2x). The white solid was redissolved in ethyl acetate and washed with water, NaHCO3 and brine. The combined organic layers were dried over sodium sulfate and concentrated to give pure (2S,3R,4S,5S,6R)-2-(((E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (116) as a light 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 equiv) was dissolved in methanol (3.0 mL) and 25% sodium methoxide in methanol (0.1 mL, 0.49 mmol, 3.0 equiv) was added slowly at ice bath temperature. After 20 min, LC / MS showed the desired product. The reaction was quenched by the addition of 10% acetic acid. Water was removed and replaced with methanol. Insoluble salts were removed by filtration twice and the crude product after removal of solvent 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-d4): δ7.59-7.45 (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=8.0Hz,1H),3.85(m,4H),3.78-3.67(m,2H),3.65-3.60(m,1H),3.57(d d, J=9.6, 3.4Hz, 1H), 2.97 (dd, J=7.9, 5.8Hz, 2H), 2.90 (t, J=7.3Hz, 2H), 2.36 (s, 3H).

[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 equiv) was dissolved in dichloromethane (5 mL). Piperidine (10% in DCM) (7.0 mL, 8.2 mmol, 10.0 equiv) was added and the mixture was stirred at room temperature for 3 h. LC / MS analysis showed the reaction was complete. EtOAc (200 mL) was added and the solution was washed with NaHCO3 (2x). The organic layer was washed with brine, dried over sodium sulfate, and concentrated to dryness to give the crude product. The title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(((E)-3-(4-(((2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (113) was isolated as its white hydrochloride salt (550 mg, 80% yield) after HPLC purification using a buffer containing HCl. LC / MS: RT = 4.38 min; m / z = 680.7 [M+H] + . 1 HNMR (500MHz, methanol-d4): δ7.61-7.48(m,3H),7.40(dt,J=7.8Hz,1.0Hz,1H),7.33(d,J=7.9Hz,2H),7 .25(d,J=8.0Hz,1H),7.02(t,J=7.5Hz,1H),6.95(t,J=7.4Hz,1H),6.48(d,J=16.1Hz,1H),5.43(d ,J=3.3Hz,1H),5.32-5.19(m,2H),5.02(d,J=8.0Hz,1H),4.27-4.13(m,3H),3.89(s,2H),3.11(t, J=5.7Hz,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 eq.) and HOBt (344 mg, 2.2 mmol, 1.4 eq.) were added to (29) (900 mg, 1.6 mmol, 1.0 eq.) in DMF (8.0 mL). The mixture was stirred for 10 min. Compound (32) (500 mg, 1.63 mmol, 1.05 eq.) was added, and DIEA (418 μL, 1.5 eq.) was then added at ice bath temperature. The reaction product was stirred at room temperature overnight. LC / MS analysis showed the desired product. Saturated ammonium chloride solution (20 mL) was added, and a white solid was precipitated. The solid was filtered, washed with water (2x), and dried to obtain 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%) (0.085 mL, 2.6 equiv) in methanol (1.0 mL) were mixed at ice bath temperature. The mixture was stirred at room temperature for 2 h. 1N HCl (1.0 equiv) was added, 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 (500MHz, methanol-d4): δ7.69(d,J=8.5Hz,3H),7.53(d,J=8.2Hz,2H),7.43(d,J=7.8Hz, 1H),7.28(d,J=8.0Hz,1H)7.08-6.97(m,2H),6.59(d,J=15.8Hz,1H),4.57(d,J=7.9Hz ,1H),4.28(s,2H),3.74(q,J=6.4Hz,1H),3.68-3.61(m,2H),3.57(dd,J=9.7,3.3Hz,1 H), 3.30-3.22 (m, 2H), 3.14 (dd, J = 9.3, 6.6Hz, 2H), 2.42 (s, 3H), 1.33 (d, J = 6.4Hz, 3H).

[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 compound (117) (640 mg, 0.75 mmol, 1.0 equiv) in DCM:DMF (1:1) (10 mL) was added 50% triethylamine / DCM (10.0 mL). The mixture was stirred at room temperature overnight. LC / MS analysis 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. LC / MS now 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 salt as a white solid. LC / MS: RT=1.89 min; m / z=622.4 [M+H] + . 1H NMR (500MHz, methanol-d4): δ7.73-7.61(m,3H),7.54(d,J=8.1Hz,2H),7.44(d,J=7.8Hz,1H),7.28(d,J =8.0Hz,1H),7.06(t,J=7.5Hz,1H),7.00(t,J=7.4Hz,1H),6.55(d,J=15.9Hz,1H),5.48-5.31(m, 3H),5.16(dd,J=11.3,3.9Hz,1H),4.63(s,1H),4.28(s,2H),3.26(dd,J=9.5,6.5Hz,2H),3.14(d d,J=9.3,6.5Hz,2H),2.42(s,3H),2.18(s,3H),2.16(s,3H),2.01(s,3H),1.20(d,J=6.5Hz,3H).

[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 ice bath temperature. The solution was stirred at room temperature overnight. 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 give (2S,3R,4S,5S,6R)-2-((2-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (111) (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 equivalent) was slowly added at ice bath temperature. After stirring at room temperature for 2 h, the Fmoc protecting group was not completely removed. An additional 0.2 equivalent of sodium methoxide was added at ice bath temperature, and the reaction was completed in 20 min. HCl / dioxane (1 N, 1.2 equivalent) was added to adjust the pH to 5-6. The solvent was concentrated to dryness, and the residue was subjected to HPLC purification to give the title compound 2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (112) (80 mg). LC / MS: RT=1.33 min; m / z=557.3 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ8.72(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=7.8Hz,1H),4.45,(s 2H),3.88-3.81(m,5H),3.77(dd,J=11.3,4.6Hz,1H),3.71(dd,J=9.6,8.0Hz,1H),3.64(m,1H ),3.57(m,1H),3.05-2.96(m,4H),2.10(m,1H),2.06(s,2H),1.92-1.85(m,2H),1.26(m,2H).

[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 that the reaction was complete. 1N HCl / dioxane (1.0 equiv) was added. The solution was concentrated to dryness and the crude residue was subjected to HPLC purification to give 60 mg of the title compound (2R, 3S, 4S, 5R, 6S)-2-(acetoxymethyl)-6-((2-(4-((((1-methyl-1H-indol-3-yl)methyl)amino)methyl)piperidin-1-yl)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (114). LC / MS: RT=1.89min; m / z=725.3[M+H] + . 1 H NMR (500MHz, methanol-d4): δ8.64(s,2H),7.69(m,1H),7.40(m,1H),7.31(s,1H),7.23(m,1H),7. 13(m,1H),5.42(d,J=3.5Hz,1H),5.34(m,1H),5.21(m,1H),5.07(d,J=8.3Hz,1H),4.81(d,J =13.3Hz,2H),4.26-4.13(m,5H),3.82(s,3H),2.94(t,J=12.8Hz,2H),2.77(d,J=6.8Hz,2H ), 2.13 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.97 (s, 3H), 1.85 (d, J = 11.6Hz, 2H), 1.19 (m, 2H).

[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 equivalents) were added to a solution of compound (110) (96 mg, 0.31 mmol, 1.4 equivalents) in DMF (0.5 mL). The solution was stirred at room temperature for 15 min, and a portion of compound (32) (96 mg, 0.37 mmol, 1.4 equivalents) was added. DIEA (1.5 equivalents, 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 out and washed with water. Ethyl acetate was added to the solution, and washed with salt water (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 foam 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] Compound (115) (70 mg, 0.079 mmol, 1.0 eq.) and sodium methoxide (25%) (2.0 eq.) were dissolved in methanol (0.8 mL) at ice bath temperature. The mixture was stirred at room temperature for 2 h. 1N HCl (1.0 eq.) 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] + . 1H NMR (500MHz, methanol-d4): δ8.71 (s, 2H), 7.74 (d, J = 8.0Hz, 1H), 7.51-7.43 (m, 2H), 7 .30(m,1H),7.21(m,1H),4.93(d,J=3.9Hz,1H),4.59(d,J=7.9Hz,1H),4.45(s,2 H),3.87(s,3H),3.77-3.71(m,1H),3.68-3.61(m,2H),3.57(m,1H),3.05-2.95( m, 4H), 2.09 (m, 1H), 1.88 (d, J = 12.4Hz, 2H), 1.33 (d, J = 6.5Hz, 3H), 1.26 (m, 2H).

[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 tripropropionate (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 continued for another 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 give (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 eq) was added to a solution of ethylenediamine (1.78 mL, 24.7 mmol, 1.2 eq) in THF (200 mL). A solid was 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 eq) was added in one portion. The reaction mixture was stirred at room temperature overnight, at which point LC / MS analysis showed mainly unreacted starting material. The reaction product was reprocessed and subjected to the same reaction conditions again, stirring overnight at room temperature. A second batch was repeated on the same scale. LC / MS analysis showed that the reaction was complete. Water was added and the two layers were separated. Dilute HCl (2%) and ethyl acetate were added and the organic layer was washed twice with water and then with brine. The solution was dried over sodium sulfate and the solvent was removed to give (2R,3S,4R,5R,6S)-2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl triprolate as a white sticky 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 before, two additional DAST (3.0 mL, 3.0 mL each) were added, and the reaction product was stirred until the starting material was completely consumed (about 8 h in total). The solution was cooled to -30 ° C, methanol (15 mL) was slowly added, and the mixture was allowed to warm to room temperature. Cold NaHCO3 solution was added, and the mixture was diluted with dichloromethane. The organic layer was separated and washed with 1N HCl (2x), then washed with brine. The solution was dried over sodium sulfate, and the solvent was removed to give a yellow oil (4.1 g). TLC analysis showed that the product was impure. The crude product was subjected to normal phase purification, with a gradient elution of 100% hexane to 15% ethyl acetate in hexane for 60 min. The product fractions were collected, and the solvent was removed to give pure (2S, 3S, 4R, 5R, 6S) -2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (2.4 g) as a colorless oil. 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 (1500mg, 4.49mmol, 1.0 equiv) was dissolved in acetonitrile (15mL). N-hydroxyphthalimide (915mg, 5.61mmol, 1.25 equiv) was added, followed by triethylamine (0.81mL, 5.61mmol, 1.25 equiv). The solution turned dark red. BF3-etherate (1.03mL, 8.08mmol, 1.8 equiv) was added, and the solution became clear. Stir at room temperature for 1h. LC / MS showed the desired product. A cold solution of NaHCO3 was added, and extracted twice with ethyl acetate. The solution turned dark red. The combined organic layers were washed twice with NaHCO3 until no color was detected. The organic solvent was dried over sodium sulfate. The solvent was concentrated to give a yellow oil (2000 mg). The crude mixture was subjected to normal phase purification using a 25 g silica gel column, eluting with a gradient of 100% hexanes to 25% ethyl acetate in hexanes over 40 minutes. 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 1 Comparison of the H NMR spectrum with the triacetoxy analogue characterized in the literature (31) identified peak #1 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 triprolate. It was isolated as a white solid (500 mg).

[0453] (2S,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (500 mg, 1.04 mmol, 1.0 equiv) was dissolved in methanol (10 mL). Hydrazine hydrate (65%, 0.090 mL, 1.04 mmol, 1.0 equiv) was added dropwise at ice bath temperature. The mixture was stirred for 30 minutes and the solution turned 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 NaHCO3. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was concentrated to give (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triprolate (280 mg) as a white solid. LC / MS: RT = 2.34 min; m / z = 348.4 [M+H] + .

[0454] (E)-3-(4-(((((9H-fluoren-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylic acid (29) (281 mg, 0.503 mmol, 1.0 equiv) was dissolved in DMF (1.5 mL). EDC (124 mg, 0.653 mmol, 1.3 equiv) and 1-hydroxybenzotriazole (100 mg, 0.65 mmol, 1.3 equiv) were added and the mixture was stirred for 10 min. (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl tripropionate (226 mg, 0.503 mmol, 1.0 equiv) was added followed by DIPEA (1.4 equiv). After stirring at room temperature for 2 h, LC / MS showed the desired product. Saturated NH4Cl solution was added, followed by ethyl acetate, and the organic phase was extracted twice with NaHCO3. The combined organic layer was washed with saline, and the solvent was concentrated to obtain a pale yellow foam. (490 mg). The product was purified using normal phase purification on a 40 g silica gel column, with a gradient elution of 100% hexane to 50% ethyl acetate in hexane for 45 minutes. The product fractions were collected, and the solvent was concentrated to obtain (2S, 3S, 4R, 5R, 6S)-2-(((E)-3-(4-(((((9H-fluorenes-9-yl)methoxy)carbonyl)(2-(2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)acrylamido)oxy)-6-methyltetrahydro-2H-pyrans-3,4,5-triyl tripropionate as a white foamy solid. (200 mg). LC / MS: RT=7.48min; m / z=886.7[M+H] + .

[0455] (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) was dissolved in 50% NEt3 in DCM / DMF. The solution was stirred at room temperature for 3 days to remove the Fmoc protecting group. LC / MS showed the reaction was complete. The solvent was concentrated, saturated NH4Cl solution was added, and the mixture was extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate. The crude mixture was triturated twice with hexanes. The final product was concentrated to give a white foamy solid. (138 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 triprolate (121) was isolated as its hydrochloride salt as a white solid. LC / MS: RT=4.98 min; m / z=664.7 [M+H] + . 1 HNMR (500MHz, methanol-d4): δ7.70-7.68 (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=7.6Hz,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-methylpropionate) (122)

[0457]

[0458] L-fucose (3 g, 18.4 mmol, 1.0 equivalent) is dissolved in pyridine (13 mL) and chloroform (19.5 mL). Under a nitrogen atmosphere, isobutyryl chloride (13.14 mL, 123.8 mmol, 6.7 equivalent) is slowly added by a syringe at ice bath temperature. The reaction mixture is allowed to warm to room temperature and then stirred for 48 h. The solution is concentrated to dryness under high vacuum and then quenched with 2M HCl (15 mL). Ethyl acetate (200 mL) is added and the solution is washed twice with water. The organic layer is washed with 2M HCl (20 mL) again, then with saturated sodium bicarbonate solution (30 mL) and then water (30 mL). The organic layer is dried with sodium sulfate solution. The solvent was removed to give (2S,3S,4R,5R,6S)-6-methyltetrahydro-2H-pyran-2,3,4,5-tetrayltetra(2-methylpropanoate) (9.8 g) as a light 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-methylpropionate) (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 reprocessed and re-experienced the same reaction conditions, this time stirring for a total of five days, at which time water was added. The layers were separated, and dilute HCl (2%) was added, followed by ethyl acetate (200 mL). After shaking, the layers were separated, and the organic layer was washed twice with water. The organic layer was further washed with brine and dried over sodium sulfate. The solvent was removed to give (2R, 3S, 4R, 5R, 6S) -2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropionate) as a yellow oil. The crude product was dry loaded onto an 80 g silica gel column and purified using a gradient of 100% hexanes to 30% ethyl acetate in hexanes for 1 hour. The product fractions were collected and concentrated to give (2R, 3S, 4R, 5R, 6S) -2-hydroxy-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris(2-methylpropionate) as a colorless oil. (2.89 g). LC / MS: RT = 3.19 min; m / z = 392.5 [M+18].

[0460] To a solution of (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 3 portions of 1% ethyl acetate (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) at 0°C over 30 min.

[0461] DAST (3.69 mL, 27.5 mmol, 4.3 equiv) was added dropwise. The mixture was stirred for another 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 slowly added. The mixture was stirred for another 15 min, at which time 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 washed with saturated sodium bicarbonate solution, and finally washed with brine. After drying with sodium sulfate, the solvent was removed to obtain a yellow oil (2.5 g). Using a 25 g silica gel column, the crude product was subjected to normal phase purification, eluting with a gradient of 100% hexane to 25% ethyl acetate in hexane. The product fractions were collected and the solvent was removed to obtain (2RS, 3S, 4R, 5R, 6S) -2-fluoro-6-methyltetrahydro-2H-pyran-3,4,5-triyl tris (2-methylpropionate) (2.0 g) as white bubbles. LC / MS: RT=3.64,3.78min; 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-methylpropionate) (2000 mg, 5.3 mmol, 1.0 equiv) and N-hydroxyphthalimide (1100 mg, 6.89 mmol, 1.3 equiv) in acetonitrile (4.0 mL) was added triethylamine (0.98 mL, 6.8 mmol, 1.3 equiv). BF3.Et2O (2.1 mL, 15.9 mmol, 3.0 equiv) was slowly added via a syringe. The solution turned from red to a clear light yellow. After 30 min, LC / MS analysis showed that the reaction was complete to give two isomeric products. Dichloromethane was added and the solution was poured into a 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 give 2500 mg of crude (2RS,3S,4R,5R,6S)-2-((1,3-dioxoisoindolin-2-yl)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyltris(2-methylpropionate). 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 1 Comparison of the H NMR spectrum with the triacetoxy analogue characterized in the literature (31) identified peak #1 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-methylpropionate). 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-methylpropionate). 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-methylpropionate) (900 mg, 2.3 mmol, 1.0 eq) was dissolved in methanol (20 mL). Hydrazine hydrate (65%, 2.3 mL, 2.3 mmol, 1.0 eq) was added dropwise at ice bath temperature and the reaction mixture was stirred for 30 min. The solution turned from clear to turbid. LC / MS analysis showed that the reaction was complete. Dichloromethane (25 mL) was added and the separated organic layer was washed twice with cold saturated NaHCO3 solution. The organic layer was washed with brine and dried over sodium sulfate. The solvent was removed to give (2S,3S,4R,5R,6S)-2-(aminooxy)-6-methyltetrahydro-2H-pyran-3,4,5-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 analyzes the formation of display required product.Add ethyl acetate, and the mixture is washed twice with saturated NH4Cl solution.Ethyl acetate layer is washed twice with aqueous NaHCO3, and finally washed with salt water.Remove solvent, to obtain light yellow foam (590mg).Use 10g silica gel column, use normal phase purification method to purify product, with 100% hexane to the gradient elution 45min of 30% ethyl acetate in hexane.Collect product fractions, and remove solvent, to obtain (2S, 3S, 4R, 5R, 6S)-2-(((E)-3-(4-(((((9H-fluorenes-9-yl) methoxy) carbonyl)(2-(2-methyl isophthalic acid H-indol-3-yl) ethyl) amino) methyl) phenyl) acrylamido) oxygen base)-6-methyltetrahydrochysene-2H-pyrans-3,4,5-triyl tris(2-methyl propionate) (250mg) as white foam. LC / MS: RT=8.13min; 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 stirred at room temperature overnight to remove the Fmoc protecting group. LC / MS showed the desired product. The mixture was triturated with hexanes, ethyl acetate was added, and the mixture was extracted twice with saturated NH4Cl. The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was concentrated to give a yellow foam. (300 mg). The residue was triturated with hexanes / 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-triyltris(2-methylpropanoate) (122) was isolated as its hydrochloride salt as a white solid. LC / MS: RT=5.67 min; m / z=706.9 [M+H] + . 1 H NMR (500 MHz, methanol-d4): δ7.72-7.61 (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=3.4,1.4Hz,1H),5.33(d,J=3.9Hz,1H),5.24(d,J=10.3Hz,1H),4.70(br.s,1H),4.28(s,2H),3.26(m,2H) ,3.17-3.10(m,2H),2.73(m,1H),2.67(s,2H),2.47(m,1H),2.42(s,3H),1.31-1.15(m,15H),1.12(d,J=7.0Hz,6H).

[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 2M 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 provide 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) followed by HOBt (321 mg, 2.1 mmol) and the resulting mixture was stirred at room temperature for 5 min. Compound (24) (700 mg, 1.92 mmol) was added in one portion. The reaction mixture was cooled in an ice / water bath and Et3N (0.3 mL, 2.1 mmol) was added. The reaction mixture was gradually warmed to room temperature and stirred for 12 h. Saturated aqueous NH4Cl (6 mL) was added and the resulting precipitate was filtered and washed with water (10 mL). The residue was dissolved in EtOAc (20 mL), washed with brine, dried over anhydrous Na2SO4, filtered and concentrated to give the crude product as a white foamy solid (1 g, 80% yield, 90% purity). 350 mg of this crude material was purified on reverse phase HPLC to give 146 mg of the title compound (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl(methyl)amino)pyrimidine-5-carboxamido)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (123). LC / MS: RT=1.92 min; m / z=839.4 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ9.08(d,J=2.6Hz,1H),8.79(s,2H),8.51(dd,J=8.8,2.6Hz,1H), 7.56(s,1H),7.04(d,J=8.7Hz,1H),5.45(d,J=3.3Hz,1H),5.33(s,2H),5.30(dd,J=10.4 ,8.1Hz,1H),5.24(dd,J=10.4,3.4Hz,1H),5.06(d,J=8.1Hz,1H),4.29-4.16(m,7H),4.0 6(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 solution of compound (123) (650 mg, 0.77 mmol) in MeOH (7.7 mL) was added dropwise with 25% NaOMe solution in MeOH (0.21 mL, 1 mmol). 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 give the title compound 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl(methyl)amino)-N-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (124) (267 mg) as a white powder. LC / MS: RT=1.34 min; m / z=671.6 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ9.08(dd,J=2.7,0.7Hz,1H),8.85(s,2H),8.51(dd,J=8.9,2.6Hz,1H ),7.58(d,J=1.0Hz,1H),7.05(dd,J=8.9,0.7Hz,1H),5.36-5.34(m,2H),4.63(d,J=7.9Hz,1 H),4.27(m,4H),4.07(s,3H),3.94-3.82(m,6H),3.78(dd,J=11.4,4.6Hz,1H),3.71(dd,J=9 .7,7.9Hz,1H),3.65(ddd,J=7.6,4.6,1.1Hz,1H),3.58(dd,J=9.6,3.4Hz,1H),3.38(s,3H).

[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] To a solution of 2-(N-((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-N-methylamino)pyrimidine-5-carboxylic acid (700 mg, 1.4 mmol) in DMF (4.6 mL) was added EDC.HCl (377 mg, 1.9 mmol) in one portion at room temperature followed by 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 eq) were added at 0°C. The resulting mixture was left at room temperature and stirred for 2 h. Cold saturated aqueous NH4Cl 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) and washed with brine, dried over anhydrous Na2SO4, 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 on reverse phase HPLC to give 153 mg of the title compound (2S,3S,4R,5R,6S)-2-((2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl(methyl)amino)pyrimidine-5-carboxamido)oxy)-6-methyltetrahydro-2H-pyran-3,4,5-triyl triacetate (125). LC / MS: RT=1.96 min; m / z=781.5 [M+H] + . 1H NMR (500MHz, methanol-d4): δ9.08(dd,J=2.6,0.8Hz,1H),8.80(s,2H),8.52(dd,J=8.8,2. 6Hz,1H),7.51(d,J=0.9Hz,1H),7.00(d,J=8.8Hz,1H),5.46-5.36(m,3H),5.32(s,2H ),5.17(dd,J=11.1,4.0Hz,1H),4.70(d,J=6.7Hz,1H),4.20(m,4H),4.05(s,3H),3.9 0(m,4H),3.37(s,3H),2.19(s,3H),2.17(s,3H),2.01(s,3H),1.19(d,J=6.5Hz,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 solution of compound (125) (750 mg, 0.96 mmol) in MeOH (9.6 mL) was added dropwise with a 25% NaOMe solution in MeOH (0.22 mL, 1.06 mmol). 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 give the title compound 2-(((2-(6-methoxypyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl(methyl)amino)-N-(((2S,3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)pyrimidine-5-carboxamide (126) (359 mg) as a white powder. LC / MS: RT=1.40 min; m / z=655.4 [M+H] + . 1H NMR (500MHz, methanol-d4): δ9.08(dd,J=2.7,0.7Hz,1H),8.81(s,2H),8.51(dd,J =8.8,2.6Hz,1H),7.57(d,J=0.9Hz,1H),7.05(dd,J=8.8,0.7Hz,1H),5.34(m ,2H),5.15(d,J=3.7Hz,1H),4.42(m,1H),4.27(m,4H),4.07(s,3H),3.95-3 .84(m,6H),3.76(dd,J=3.0,1.3Hz,1H),3.38(s,3H),1.26(d,J=6.5Hz,3H).

[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] β-D-galactosyl conjugates (46) and (47) were synthesized by adapting the synthetic protocol of Wadzinski et al (Nature Chem. Biol., 10 (2018) 644-652) for rapid O-phenolic glycosylation in aqueous medium. Starting from commercially available (2,4-dihydroxy-5-isopropylphenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone AT13387 (36), treatment with α-D-galactopyranosyl fluoride in the presence of Ca(OH)2 afforded a mixture of regioisomers of β-D-galactosides that were 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 to a plastic bottle cooled in an ice / water bath by syringe. 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 NaHCO3 aqueous solution (150 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated in vacuo, and dried under high vacuum to obtain a crude residue.

[0483] Anhydrous MeOH (130mL) is added to the above-mentioned residue, and the mixture of gained is stirred for 15min.Then the reaction mixture is cooled in ice / water for 10min, NaOMe (139mg, 2.57mmol) is added, and after 10min, ice / water bath is removed, and the reaction mixture is gradually warmed to room temperature in 3h.The reaction mixture is cooled again in an ice / water bath, and silica gel (11g) is added.The suspension of gained is concentrated into thick paste.The solution of 7:3EtOAc:MeOH (50mL) is added, fully stirred for 5min and filtered.The residue is washed with 7:3EtOAc:MeOH (60mL), and the filtrate combined is concentrated to dryness using a rotary evaporator.The residue is dried overnight under high vacuum to obtain the desired product α-D-pyranosyl fluoride (4.2g, 91%) as a foamy solid, which is used for 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 clear conversion to the desired compounds (46) and (47). The reaction mixture was quenched by the addition of aqueous 1M HCl to adjust the pH to 8, and the resulting mixture was concentrated in vacuo to remove water. A DMSO solution of the residue was directly purified by reverse phase HPLC, and two regioisomeric products were isolated. The regiochemical characteristics of the products were inferred by correlating the retention times on reverse phase LC / MS with the calculated LogP values ​​(the less lipophilic isomer was calculated to be assigned to the earlier eluting peak on HPLC).

[0485] The earlier eluting peak yielded 34 mg of (2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (46) as a white solid (cLogP=1.8). LC / MS: RT=1.13 min; m / z=572.5 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ7.41-7.35(br s,2H),7.33(s,1H),7.30(s,2H),5.01-4.90(m,6H),4.10-4.00(m,2H),3.83-3. 63(m,9H),3.29(m,4H),2.88(s,3H),1.25(d,J=6.9Hz,3H)1.24(d,J=6.9Hz,3H).

[0486] The subsequent eluting peak yielded 22 mg of (4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)(5-((4-methylpiperazin-1-yl)methyl)isoindolin-2-yl)methanone (47) as a white solid (cLogP=2.0). LC / MS: RT=1.20 min; m / z=572.5 [M+H] + . 1 HNMR (500MHz, methanol-d4): δ7.42-7.35(m,1H),7.35-7.27(m,1H),7.25-7.18(m,1H),7.12(d,J=1.7Hz,1H),6.78(s,1H),4.98-4.78(m,6H),4.71(m ,1H),3.92-3.86(m,1H),3.85-3.62(m,6H),3.55(m,1H),3.30-3.22(m, 7H), 2.88(d,J=8.8Hz,3H), 1.24(d,J=6.8Hz,3H), 1.22(d,J=6.8Hz,3H).

[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] 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-phenolic glycosylation in aqueous media. Starting from the commercially available resorcinol compound NVP-AUY922 (37), treatment with α-D-galactopyranosyl fluoride in the presence of Ca(OH)2 provided a mixture of regioisomers of β-D-galactosides, which were 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 showed clear conversion to the desired compounds (48) and (49). The reaction mixture was quenched by the addition of aqueous 1 M HCl to adjust to pH 8 and the resulting mixture was directly purified by reverse phase HPLC to give two regioisomeric products. The regiochemical identity of the products was inferred by correlating retention times on reverse phase LC / MS with calculated LogP values ​​(the less lipophilic isomer was calculated to be assigned to the earlier eluting peak on HPLC).

[0491] The earlier eluting peak yielded 7 mg of N-ethyl-5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (48) as a white solid (cLogP=0.88). LC / MS: RT=1.29 min; m / z=628.4 [M+H] + . 1 HNMR (500MHz, methanol-d4): δ7.44(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,4 H), 3.71 (m, 1H), 3.59 (m, 1H), 3.39-3.15 (m, 11H), 1.23 (t, J = 7.3Hz, 3H), 1.06 (d, J = 6.9Hz, 3H), 1.05 (d, J = 6.9Hz, 3H).

[0492] The subsequent eluting peak yielded 22 mg of N-ethyl-5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (49) as a white solid (cLogP=1.02). LC / MS: RT=1.43 min; m / z=628.4 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ7.45(s,4H),7.05(s,1H),6.72(s,1H),4.71(d,J=7.7Hz,1H),4.35(q,J=7.3Hz,2H),4.06(br.s,1H),3.86(m,1H ),3.77-3.70(m,3H),3.60(m,1H),3.48-3.20(m,10H),3.16(m,1H),1.23(t,J=7.3Hz,3H),1.10(d,J=6.9Hz,3H),1.09(d,J=6.9Hz,3H).

[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 CsCO (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 (3×50 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was dried overnight under high vacuum.

[0496] MeOH (5 mL) and NaOMe (25% solution in MeOH, 25 μL, 0.11 mmol) were added to the above residue, and the resulting mixture was stirred at room temperature for 1 h. LC / MS showed significant conversion to the desired compounds (127) and (128). The reaction mixture was quenched by addition of 4M HCl in dioxane solution to adjust to pH 8. The reaction mixture was then concentrated in vacuo and purified by reverse phase HPLC to give two regioisomer products. The regiochemical characteristics of the product were inferred by correlating the retention time on reverse phase LC / MS with the calculated LogP value (the calculated less lipophilic isomer was assigned to the earlier elution peak on HPLC).

[0497] The earlier eluting peak yielded 25 mg of 5-(2-hydroxy-5-isopropyl-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (127) as a white solid (cLogP=1.96). LC / MS: RT=1.30 min; m / z=527.2 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ7.53-7.46(m,2H),7.29(d,J=3.2Hz,1H),7.08(dd,J= 8.6,2.1Hz,1H),6.71(s,1H),6.67(s,1H),6.49(dd,J=3.1,0.8Hz,1H),4.80( d,J=7.8Hz,1H),3.90(d,J=2.9Hz,1H),3.86(s,3H),3.82-3.71(m,3H),3.67( m, 1H), 3.55 (dd, J = 9.7, 3.4Hz, 1H), 3.14 (m, 1H), 0.73 (dd, J = 6.9, 1.7Hz, 6H). The subsequent eluting peak yielded 35 mg of 5-(4-hydroxy-5-isopropyl-2-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (128) as a white solid (cLogP=2.09). LC / MS: RT=1.54 min; m / z=527.2 [M+H] + . 1 H NMR (500MHz, methanol-d4): δ7.55-7.49(m,2H),7.30(d,J=3.1Hz,1H),7.09(dd,J=8.6 ,2.0Hz,1H),6.59(s,1H),6.51(d,J=3.0Hz,1H),6.29(s,1H),5.27(d,J=9.1Hz, 1H),4.40(m,1H),3.98(d,J=3.3Hz,1H),3.87(s,3H),3.85-3.75(m,3H),3.72(d d,J=9.6,3.3Hz,1H),2.91(m,1H),0.67(d,J=6.9Hz,3H),0.65(d,J=6.9Hz,3H).

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

[0499] Normal human fibroblasts (IMR90) and patient-derived β-galactosidase (GLB1) or α-fucosidase (FUCA1) deficient cells were used to determine changes in cell viability after exposure to compounds. Cells were cultured with DMEM and 10% heat-inactivated FBS under a controlled atmosphere of 5% carbon dioxide and 5% oxygen. Induced by cell contact inhibition and confirmed by the absence of senescence-associated β-galactosidase (SA-β-Gal) staining and the ability to re-enter the cell cycle. Senescence was induced by treatment with doxorubicin and confirmed by SA-β-Gal positive staining and 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 test compounds. To generate dose-response curves, data were fit to a four-parameter Hill function and IC 50 The anti-senescence index (SI) of each compound was determined by comparing the IC 50 Divide by IC for senescent cells 50 The anti-aging index data are shown in Table 1, where the compounds have SI<1(-), SI>1(+), SI>5(++) and SI>10(+++).

[0500]

[0501]

[0502] Example 24: Anti-aging 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 7 days of incubation in culture medium, senescence was confirmed by SA-β-Gal staining (Itahana et al., Methods Mol. Biol. 371 (2007) 21-31) and lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation (see Yu et al., J. Immunol. Methods 350 (2009) 29-35).

[0504] Proliferating or senescent MEFs 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 assessed by the ratio of PI-positive to PI-negative nuclei. Figure 1A The results shown in show that the active drug (102) significantly reduced cell viability, whereas the prodrug (101) was completely nontoxic to proliferating cells at concentrations up to 4 mM. In contrast, compounds (101) and (102) were equally toxic to senescent MEFs, as shown in Figure 1B As shown, this suggests that the prodrug (101) is efficiently converted to cytotoxic FUR (102) by hydrolases in the senescent cell population.

[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 to two groups of mice (N=3 per group) by single-dose intraperitoneal injection. After six days of treatment, blood cell counts ( Figure 2A ), count bone marrow cells from femur ( Figure 2B ), and spleen weight was determined ( Figure 2C ) to compare the toxicity of the two drugs. Prodrug (101) showed negligible toxicity to platelets, neutrophils, lymphocytes, and myeloid cells, while FUR (102) induced a significant reduction in all cell populations. Prodrug (101) also showed minimal effect on spleen weight compared to FUR (102).

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

[0508] Two groups of C57BL / 6 mice (N=5 / group) were injected intraperitoneally with doxorubicin (25 mg / kg) to induce hepatocyte senescence. After 4 and 6 days, each group was injected with PBS or FURGal (140 mg / kg) (101). A third group of 5 animals served as controls. After another 4 days, the livers were removed and sectioned (30 μm) (6 fields / animal were evaluated) before SA-β-Gal staining ( Figure 3A and 3B ).like Figure 3C As shown in Figure 5, quantitative analysis revealed a numerical trend of a decrease in senescent hepatocytes after FURGal treatment. The average body weight of mice on the day of analysis was Figure 3D Shown in.

[0509] Example 27: Effects 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 hepatocytes of aged C57BL / 6 mice after in vivo administration

[0510] Two groups of C57BL / 6 mice (N=9 / group) were intraperitoneally injected with doxorubicin (20 mg / kg) to induce hepatocyte senescence. After 4 and 6 days, each group was intraperitoneally injected with PBS or compound (113) (also known as RBO-013) (2x20 mg / kg). A third group of animals was used as a control. After an additional 2 days, the livers were removed and sectioned (30 μm) before SA-β-Gal staining as described above ( Figure 4A and 4B ).like Figure 4C As shown, quantification revealed a decrease in senescent hepatocytes after treatment with (113) (as visualized by SA-β-Gal staining). INK4a This anti-aging effect was also confirmed by the mRNA levels of IL-6 (note that expression of Cdkn2a was only detected in animals receiving doxorubicin). Expression levels are reported relative to Actb as a reference gene. Figure 4D and 4E Significant decreases in Cdkn2a and IL-6 gene expression were shown, respectively.

[0511] Example 28: Anti-aging effects 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 of C57BL / 6 mice after in vivo administration

[0512] Four groups of C57BL / 6 mice (N=9 / group) were injected intravenously 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 another 3 days, the lungs were removed and the left lobe was sectioned before SA-β-Gal staining as described above ( Figure 5A and 5B ).like 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 PRC to detect Cdkn2a (p16 INK4a The expression levels are reported relative to Actb as a reference gene. Figure 5D A dose-dependent decrease in Cdkn2a gene expression in the lung is shown.

Claims

1. Compounds having the following structures: or a pharmaceutically acceptable salt.

2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable excipient.

3. Compounds having the following structures: or a pharmaceutically acceptable salt.

4. A pharmaceutical composition comprising the compound of claim 3 and a pharmaceutically acceptable excipient.

5. Compounds having the following structures: or a pharmaceutically acceptable salt.

6. A pharmaceutical composition comprising the compound of claim 5 and a pharmaceutically acceptable excipient.

7. Compounds having the following structure: or a pharmaceutically acceptable salt.

8. A pharmaceutical composition comprising the compound of claim 7 and a pharmaceutically acceptable excipient.

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