Macrolide anti-aging compounds

Abstract

Disclosed herein are macrolide compounds having anti-aging activity. These compounds selectively target senescent cells and may be used to reduce or eliminate senescent cells and / or delay the onset of aging in a subject. These compounds also inhibit the proliferation of cancer stem cells and can be used to treat cancer, and in particular to inhibit the proliferation of cancer stem cells responsible for metastasis and tumor recurrence. Formulae (I) and (II): # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] N / A Technical Field

[0003] The present invention relates to macrolide compounds that selectively eradicate senescent cells and inhibit the proliferation of cancer stem cells (CSCs). Background Art

[0004] The biological process of aging continues to receive great attention from the scientific and medical research community. Physiological aging is at least partially related to an increase in the rate of oxidative damage to cellular components (including DNA, lipids, proteins, etc.). The increase in oxidative damage creates an imbalance that disrupts the self-regulatory process at the cellular level. Further, aging is associated with the accumulation of lipofuscin in the cytoplasm of neurons. Modern research also shows that aging is the result of naturally occurring DNA damage, which causes abnormal DNA changes to accumulate over time. Damage to both mitochondrial and nuclear DNA can indirectly cause aging by increasing apoptosis and cell senescence and directly by increasing cell dysfunction. Accumulated DNA damage can lead to cell loss, and in surviving cells, loss of gene expression and mutations can have an impact in cells that do not divide frequently, thereby producing signs of aging. When aging cells stop cell division, the result of cell senescence is believed to occur after various environmental damage events, abnormal cell growth, autophagy and oxidative stress, among other factors. The Senescence Associated Secretory Phenotype (SASP) is a characteristic of senescent cells and leads to proteotoxic damage to healthy cell functions, including inflammatory or anti-inflammatory and tumor or anti-tumor effects, depending on a range of factors. The effects of chronic inflammation associated with the SASP can affect the immune system's normal ability to remove senescent cells, and cells that provide immune function can be recruited into senescent cells by the SASP. Biomarkers of cellular senescence have been found to accumulate with the age of mammals and lead to a range of age-related diseases, including Alzheimer's disease, lateral sclerosis, and type 2 diabetes. And for cells that divide frequently, accumulated DNA damage can be an important cause of cancer.

[0005] Therefore, aging increases the likelihood of developing cancer, and researchers have worked hard to develop new anti-cancer and anti-aging or anti-senescence treatments (senolytic treatment). Traditional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) have attempted to selectively detect and eliminate rapidly growing cancer cells by interfering with the cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies have used immunotherapy that selectively binds mutated tumor antigens to rapidly growing cancer cells (e.g., monoclonal antibodies). Unfortunately, tumors often recur after these treatments, at the same or different one or more sites, indicating that not all cancer cells are eliminated. In particular, cancer stem cells survive and cause treatment failure for a variety of reasons. Recurrence can be due to insufficient chemotherapy doses and / or the emergence of cancer clones that are resistant to treatment. Therefore, new cancer treatment strategies are needed to overcome the shortcomings of traditional therapies.

[0006] Advances in mutation analysis have allowed for in-depth studies of genetic mutations that occur during cancer progression. Despite an understanding of the genomic landscape, modern oncology has difficulty identifying major driver mutations between cancer subtypes. The harsh reality seems to be that each patient's tumor is unique and that a single tumor can contain multiple different clonal cells. Therefore, new approaches that emphasize the commonalities between different cancer types are needed. Targeting metabolic differences between tumors and normal cells holds promise as a new cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples showed elevated transcription of more than 95 mRNAs associated with mitochondrial biogenesis and / or mitochondrial translation. Sotgia et al. Cell Cycle, 11(23):4390-4401(2012). In addition, of the 95 upregulated mRNAs, more than 35 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs). Proteomic analysis of human breast cancer stem cells also showed significant overexpression of several mitotic ribosomal proteins and other proteins associated with mitochondrial biogenesis. Lamb et al. Oncotarget, 5(22): 11029-11037 (2014).

[0007] Relative to the search for anticancer therapeutic targets and anti-aging therapeutic targets, cancer cell mitochondrial metabolism is the target of recent exploratory research. Mitochondria are extremely dynamic organelles that constantly divide, elongate and connect to each other to form tubular networks or fragmented particles in order to meet the needs of the cell and adapt to the cellular microenvironment. The balance of mitochondrial fusion and fission stipulates the morphology, abundance, function and spatial distribution of mitochondria, and therefore affects a large number of important mitochondrial-dependent biological processes, such as adenosine triphosphate (ATP) production, mitosis, apoptosis and calcium homeostasis. In turn, the dynamics of mitochondria can be regulated by mitochondrial metabolism, respiration and oxidative stress.

[0008] ATP is the universal bioenergetic "currency" of all living cells and tissues, including microorganisms such as prokaryotic bacteria and eukaryotic yeast. In eukaryotes, mitochondrial organelles serve as the "powerhouse" of the cell. Mitochondria produce large amounts of ATP via the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis contributes a small amount of ATP. Conversely, mitochondrial dysfunction induces ATP depletion, which leads to mitochondrial-driven apoptosis (programmed cell death) and / or necrosis. Therefore, we propose that ATP depletion therapy may be a viable strategy for targeting and eliminating cancer cells, even the "fittest" ones.

[0009] In MCF7 breast cancer cells, mitochondria-driven OXPHOS contributes 80%-90% of ATP production under normoxic conditions, while glycolysis contributes only the remaining 10%-20%. Thus, like normal cells, cancer cells are highly dependent on mitochondrial ATP production. However, whether ATP levels in cancer cells contribute to 3D anchorage-independent growth and cell migration, two hallmarks of metastatic spread, remains largely unknown.

[0010] Therefore, there is a need for new anti-aging compositions and methods that treat aging at the cellular level, overcoming accumulated oxidative and DNA damage and the many adverse effects of aging.

[0011] Further, there is a need for therapeutic agents that target unhealthy senescent cells and the SASP, reduce the accumulation of cellular senescence, and counteract chronic aging.

[0012] Further, therapeutic agents that target a wide range of CSCs through their common characteristics regardless of cancer type are needed.

[0013] Further, there is a need for therapeutic agents that inhibit the proliferation of CSCs, including circulating tumor cells and tumor-initiating cells that have the potential to lead to tumor recurrence and / or metastasis. Summary of the invention

[0014] In view of the foregoing background, the purpose of the present disclosure is to describe therapeutic agents or compounds that can be used to reduce the accumulation of cellular senescence and inhibit CSC proliferation. The purpose of the present disclosure is to describe therapeutic agents for eliminating CSC and senescent cells. The purpose of the present disclosure is to describe therapeutic agents for preventing tumor recurrence and metastasis and reducing the possibility of tumor recurrence and metastasis. Another purpose of the present disclosure is to describe compositions (such as pharmaceutical compositions) and methods for treating and preventing cancers including tumor recurrence and / or metastasis. Another purpose of the present disclosure is to describe compositions (such as pharmaceutical compositions) and methods for anti-aging therapeutic agents.

[0015] Described herein are compounds that are useful as therapeutic agents having anti-cancer activity, pharmaceutical compositions comprising such therapeutic agents, methods for synthesizing such compounds, and methods for treating cancer.

[0016] The method can also be used to treat and / or prevent tumor recurrence and / or metastasis. Anticancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. It is understood that CSC mitochondrial activity is at least partly responsible for the failure of treatment. Embodiments of the method can be used in situations where conventional cancer treatments have failed and / or in combination with or before anticancer treatments, thereby preventing treatment failure due to tumor recurrence and / or metastasis, or reducing the likelihood of treatment failure due to tumor recurrence and / or metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the SRB assay on both control MRC5 cells and MRC5 cells treated with BrdU were compared for different concentrations of Azithromycin and Compound [I] of the present method (also known as Azm-Gal).

[0018] Figure 2 Shown are xCELigence data comparing the effects of compound [I] of the present method on control MRC5 cells and MRC5 cells treated with BrdU.

[0019] Figure 3 A and Figure 3 B shows graphs of MRC-5 fibroblasts treated with compound [I] at a concentration of 50 μM without and with BrdU pretreatment, respectively.

[0020] Figure 4AThe results of mammosphere formation assay for MCF-7 cells treated with compound [I] are shown, and Figure 4B Shown are the results of a mammary cell sphere formation assay on MCF-7 cells treated with azithromycin.

[0021] Figure 5 The results of the SRB assay for compound [III] on control MRC5 cells and MRC5 cells treated with BrdU were compared.

[0022] Figure 6 The results of the SRB assay for azithromycin and compound [IV] on control MRC5 cells and MRC5 cells treated with BrdU were compared.

[0023] Figure 7 The results of the SRB assay for Compound [V] and Compound [VI] on control MRC5 cells and MRC5 cells treated with BrdU were compared. DETAILED DESCRIPTION

[0024] The following description fully describes the embodiments of the method in detail to achieve the practice of the method. Although the method is described with reference to these specific embodiments, it should be understood that the method can be implemented in different forms, and this specification should not be interpreted as limiting any of the appended claims of the specific embodiments set forth herein. On the contrary, these embodiments are provided so that the disclosure will be comprehensive and complete, and will fully convey the scope of the method to those skilled in the art.

[0025] This specification uses various terms that should be understood by those of ordinary skill in the art. For the avoidance of doubt, the following clarification is made.

[0026] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. This definition includes both benign and malignant tumors. Examples of cancer include cancer types, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (carcinoid, gastrin-producing including but not limited to tumors and islet cell carcinomas), sarcomas, schwannomas (including acoustic neuromas), medullary carcinomas, adenocarcinomas, melanomas, and leukemias or lymphocytic tumors. Specific examples of cancer include bladder cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung cancer including squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer or gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal cancer (kidney cancer) or renal cancer (kidney cancer), prostate cancer, genital cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, as well as head and neck cancer and multiple myeloma.

[0027] As used herein, the term "tumor" refers to the growth and proliferation of neoplastic cells, whether malignant or benign, including precancerous and cancerous cells and tissues.

[0028] The term "metastasis" refers to the spread of cancer from the primary site to other parts of the body. Cancer cells can escape from the primary tumor, penetrate lymphatic and blood vessels, circulate through the blood, and grow or "metastasize" in distant lesions of normal tissues in other parts of the body. Metastasis can be local or distant. Metastasis is a continuous process that requires tumor cells to escape from the primary tumor, travel through the blood, and stop at a distant site. At this new site, the cells can establish a blood supply and grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cells control this behavior, and the interaction between the tumor cells and the host cells at the distant site is also important.

[0029] The terms "treat", "treated", "treating" and "treatment" include the diminishment or alleviation of at least one symptom associated with or caused by the condition, disorder or disease being treated, particularly cancer. In certain embodiments, treatment includes alleviation and / or alleviation of at least one symptom associated with or caused by the cancer being treated by the compounds of the present invention. In some embodiments, treatment includes causing the death of a class of cells, such as senescent cells, SASP cells or CSCs that may be involved in the metastasis or recurrence of a particular cancer in a host, and may be achieved by preventing further proliferation of senescent cells and / or cancer cells, and / or inhibiting CSC function by, for example, depriving such cells of mechanisms for energy production. For example, treatment can be the alleviation of one or more symptoms of a cancer, or the complete elimination of a cancer. As another example, the present method can be used to inhibit mitochondrial metabolism in cancer, eliminate (e.g., kill at a rate greater than the proliferation rate) CSCs in cancer, eliminate TICs in cancer, eliminate circulating tumor cells in cancer, inhibit proliferation of cancer, target and inhibit CSCs, target and inhibit TICs, target and inhibit circulating tumor cells, prevent metastasis or reduce the likelihood of metastasis, prevent recurrence, sensitize cancer to chemotherapy, sensitize cancer to radiotherapy, sensitize cancer to phototherapy. As another example, the treatment can reduce the accumulation of senescent cells, and / or reduce the rate at which senescent cells accumulate.

[0030] In the context of tumor recurrence and / or metastasis, the terms "prevention" and "reducing (reducing) likelihood" refer to reducing the presence of CSCs, TICs, and circulating tumor cells that may be involved in recurrence or metastasis in a subject to a level that is less likely to recur and / or metastasize from the primary site relative to a control group (i.e., no treatment to prevent tumor recurrence and / or metastasis, or to reduce the likelihood of tumor recurrence and / or metastasis). In practice, the treatments described herein to prevent tumor recurrence and / or metastasis, and / or reduce the likelihood of tumor recurrence and / or metastasis target and inhibit or eliminate CSCs, TICs, and inhibit circulating tumor cells.

[0031] The terms "cancer stem cells" and "CSC" refer to a subpopulation of cancer cells within a tumor that have the ability to self-renew, differentiate, and become tumorigenic when transplanted into an animal host. CSCs have increased mitochondrial mass, enhanced mitochondrial biogenesis, and increased activity of mitochondrial protein translation compared to "bulky" cancer cells. As used herein, "circulating tumor cells" are cancer cells that shed from a primary tumor into the vasculature or lymphatics and are carried throughout the body in the blood circulation. The CellSearch Circulating Tumor Cell Test can be used to detect circulating tumor cells.

[0032] As used herein, the expression "pharmaceutically effective amount" means the amount required to administer to a host or a cell, tissue or organ of the host to obtain a therapeutic result (such as, regulating, adjusting or inhibiting protein kinase activity, for example, protein kinase activity inhibition, or treatment of cancer). A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required for a given subject using methods known and available in the art. For example, a physician or veterinarian can start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. After studying this disclosure, the determination of the pharmaceutically effective amount is considered to be within the authority of a person with ordinary skills in the art.

[0033] As used herein, the expression "therapeutic agent" refers to the embodiment of the compound described herein, which may include a pharmaceutically acceptable salt or an isotopic analog thereof. It should be understood that, as known to the general level of the art, the therapeutic agent may be administered to the subject by any appropriate method. It should also be understood that the amount of the therapeutic agent and its administration time may depend on the individual subject being treated (e.g., age and weight, and other factors), the mode of administration, the pharmacokinetic properties of the specific therapeutic agent, and the judgment of the doctor who prescribes the prescription. Therefore, due to the variability between subjects, any dosage described herein is intended to be used as an initial guide, and the doctor can titrate the dosage of the therapeutic agent to achieve the treatment that the doctor considers suitable for the subject. When considering the degree of treatment required, the doctor can balance various factors (such as, the age and weight of the subject, the presence of pre-existing diseases and other diseases). Pharmaceutical preparations can be prepared for any desired route of administration, including but not limited to, oral, intravenous or aerosol administration, as discussed in more detail below.

[0034] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and Soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0035] The expression "pharmaceutically acceptable salt" is a relatively nontoxic inorganic and organic base addition salt of the compound of the present method. Pharmaceutically acceptable salts can be formed by, for example, reacting the compound with a base (such as, a pharmaceutically acceptable metal cation hydroxide or carbonate) in the form of a free acid, with ammonia or a pharmaceutically acceptable amine. For example, representative alkali salts or alkaline earth salts include sodium salts, potassium salts, calcium salts, magnesium salts and aluminum salts. Examples of amines that can be used as base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine. Of course, it should be understood that other salts can also be used, and those of ordinary skill in the art can use methods known in the art to identify suitable salt forms without departing from the present method.

[0036] Senescence is a distinct feature of normal biological aging. Senescence involves a potentially irreversible cell cycle arrest via the induction of CDK-inhibitors (such as p16-INK4A, p19-ARF, p21-WAF, and p27-KIP1) and the onset of SASP (senescence-associated secretory phenotype) and the induction of key lysosomal enzymes (β-galactosidase) and lipofuscin (established aging pigments). Interestingly, SASP leads to the secretion of a large number of inflammatory cytokines (such as IL-1-β and IL-6), which allow senescent cells to "spread" the senescence phenotype from one cell type to another cell type via chronic inflammation, systemically infecting the whole body. This chronic inflammation can also promote the onset of cancer and promote the recurrence and metastasis of tumors.

[0037] Described herein are compounds that have potency and high selectivity against senescent cells. The compounds of the present method can be used as anti-aging agents, such as therapeutic agents for eliminating senescent cells. In addition, the compounds of the present method show significantly lower antibacterial activity compared to the reference compound. Therefore, the compounds of the present method do not cause potential antibacterial resistance.

[0038] Some embodiments of the present method take the form of a compound having the structure of compound [I] shown below, wherein 'Ac' represents an acetyl group.

[0039]

[0040] Compound [I] is also known as AZM-Gal and its IUPAC name is [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclodecane] [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-meth [oxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate). Galactose is joined via the hydroxide group of the desosamine ring.

[0041] As shown in the structure below, compound [II] (also called deacetylated AZM-Gal) has comparable activity to compound [I].

[0042]

[0043] The compounds disclosed herein have shown anti-aging activity via clinical assays. For example, in one assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine) (also known as BrdU) was used to induce senescence of a cell population. BrdU is an analog of the nucleoside thymidine and is commonly used to identify proliferating cells. BrdU induces controlled DNA damage and efficiently drives cell senescence. In the BrdU assay, normal fibroblasts were cultured for a long time (8 days) in the presence of 100 μM BrdU to induce controlled DNA damage and senescence. In an exemplary embodiment, the inventors used two independent normal, non-immortalized human fibroblast cell lines, MRC-5 lung cells, in a BrdU-based assay. Anti-aging activity was assessed using a sulforhodamine B assay (also known as an SRB assay in the art). This assay measures the amount of residual protein attached to a tissue culture dish and is a surrogate marker for cell viability.

[0044] Figure 1 Results of the SRB assay were compared for different concentrations of azithromycin (100 μM and 50 μM) and compound [I] (100 μM and 50 μM) on control MRC-5 cells and MRC-5 cells treated with BrdU. For these data, MRC-5 cells were pretreated with BrdU for 8 days (to induce senescence) and then exposed to azithromycin or compound [I] ( Figure 1 The drug was then added to the fibroblasts (labeled "Azi-Gal") for 5 days. Afterwards, normal MRC-5 cells were used as a control group and the SRB assay was performed to determine the effect of the drug on cell viability. As can be seen, 100 μM of azithromycin had no effect on the viability of normal MRC-5 lung fibroblasts, but selectively killed senescent MRC-5 fibroblasts. However, 50 μM of azithromycin had no effect on either control group cells or senescent cells. On the contrary, compound [I] showed special selectivity for senescent MRC-5 fibroblasts at 50 μM, and was effective for both control group cells and senescent cells at 100 μM.

[0045] Senescent cells undergo the so-called senescence-associated secretory phenotype (SASP), which involves a dramatic increase in protein synthesis and secretion. The xCELLigence assay system was used to assess whether protein measurement assays underestimate the anti-aging activity of test compounds. The xCELLigence assay system is protein-independent and uses electrical impedance to continuously measure cell proliferation in real time. Therefore, the real-time xCELigence assay system complements the more static SRB assay and provides a more direct visualization of the potential anti-aging effects of compounds during drug screening.

[0046] The anti-senescence activity of compound [I] was determined using the xCELLigence assay. The assay was performed using MRC-5 fibroblasts. Figure 2 xCELLigence data comparing the effects of compound [I] of the present method on control MRC-5 cells and MRC-5 cells treated with BrdU are shown. The data are expressed as the final cell index, which is the mean ± standard error of 50 μM compound [I] for control cells, control cells treated with compound [I], control cells treated with BrdU, and fibroblasts treated with BrdU. Senescent MRC-5 cells were pretreated with BrdU for 8 days to induce senescence and then exposed to compound [I] for an additional 5 days. Compared with the control group, compound [I] treatment had no effect on the survival rate of normal fibroblasts, but killed more than 90% of BrdU-treated fibroblasts. These data confirm that compound [I] has excellent anti-aging activity.

[0047] Figure 3 A and Figure 3 B shows graphs of MRC-5 fibroblasts treated with compound [I] at a concentration of 50 μM without and with BrdU pretreatment, respectively. These graphs show that compound [I] has little effect on normal MRC-5 cells, but induces cell death in senescent MRC-5 cells. Figure 3 B and Figure 3 The scale bar in the upper right corner of C represents 20 μm.

[0048] These results show that the compounds disclosed herein have anti-aging activity and can be used as anti-aging agents in pharmaceutical compositions. For example, the compounds can be used to eliminate senescent cells in a subject. Some embodiments of the present method can take the form of a method for delaying the onset of age-related diseases in a subject. Age-related diseases can be at least one of atherosclerosis, arthritis, cancer, cardiovascular disease, cataracts, dementia, diabetes, hair loss, hypertension, inflammatory diseases, kidney disease, muscle atrophy, nervous system diseases, osteoarthritis, osteoporosis, lung disease, intervertebral disc degeneration and alopecia. For example, age-related diseases can be nervous system diseases such as mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease and macular degeneration. In such embodiments, an anti-aging agent as described herein can be administered to the subject in a therapeutic amount. In some embodiments, the anti-aging agent can be administered together with another therapeutic agent, as described herein. It can be administered at the time of onset, i.e., when or shortly after the diagnosis of an age-related disease. Alternatively, the anti-aging agent can be routinely administered after diagnosis, and the frequency and dosage can be determined using techniques known in the art. In some embodiments, anti-aging agents may be administered prior to the onset of a disease, particularly where an age-related disease is expected or likely to occur in a subject (eg, due to genetic or other biological markers).

[0049] The compounds disclosed herein can also be used as therapeutic agents to selectively eliminate CSCs for the treatment and / or prevention of tumor recurrence and / or metastasis. The data show that the compounds disclosed herein have anticancer activity and are suitable for use as therapeutic agents for anticancer treatment (including treatment and / or prevention of tumor recurrence and metastasis). The data described herein demonstrates its anticancer activity by inhibiting MCF-7 cells via a mammary cell sphere formation assay. This assay measures the amount of residual protein adhered to a tissue culture dish and is a surrogate marker for cell viability.

[0050] Figure 4A The results of the mammary cell sphere formation assay for MCF-7 cells treated with compound [I] are shown, and Figure 4B The results of mammary cell sphere formation assay on MCF-7 cells treated with azithromycin are shown. The data show that the IC of compound [I] 50 The IC50 of azithromycin was 118 μM, and it inhibited MCF-7 cells by only about 30% even at a concentration of 200 μM compared to the control group. These results indicate that compound [I] has superior potency in inhibiting MCF-7 proliferation.

[0051] However, modification of known anti-aging compounds with galactose moieties does not always result in improved anti-aging activity. Compound [III], also known as AZM-Succinate or AZM-Succ, was synthesized as described herein. It can be seen that in compound [III], galactose is attached to the hydroxide group of desaccharamine via the succinate group. However, as described below, the anti-aging activity of compound [III] is significantly lower than that of azithromycin and compound [I].

[0052]

[0053] Figure 5 The results of SRB assays for compound [III] on control MRC5 cells and MRC5 cells treated with BrdU are shown. The addition of the galactose moiety reduced the potency of the parent compound azithromycin by about half. This suggests that simply conjugating the galactose moiety does not improve the anti-aging activity of the base compound.

[0054] Furthermore, modification with a galactose moiety does not always improve the selectivity of the parent compound for senescent cells. Compound [IV] (shown below and referred to as Des-AZM-Gal) is illustrative. It can be seen that the galactose is attached via the amine of the desaccharide amine ring of the macrolide structure.

[0055]

[0056] Figure 6 The results of the SRB assay for azithromycin and compound [IV] on control MRC5 cells and MRC5 cells treated with BrdU were compared. It can be seen that compound [IV] shows no little selectivity for senescent cells over normal, non-senescent cells.

[0057] Conjugation to galactose causes some compounds to not only lose their anti-aging and antimicrobial activities, but also target and kill normal, non-senescent cells. Compound [V] (shown below, and also referred to as Roxy-Gal) is a conjugate of roxithromycin and galactose.

[0058]

[0059] Compound [VI] (shown below and referred to as Erythro-Gal) is a conjugate of erythromycin and galactose. Compounds [V] and [VI] were conjugated using the same general method as compound [I] via the hydroxide group of the desaccharide ring of the macrolide structure. However, the resulting activities were very different and illustrate the unique properties of compounds [I] and [II]:

[0060]

[0061] Figure 7 The results of the SRB assay for compound [V] and compound [VI] on control MRC5 cells and MRC5 cells treated with BrdU were compared. Compared with roxithromycin and erythromycin, these compounds showed a preference for normal, non-senescent control cells and had little effect on senescent cells. These data are in sharp contrast to the activity shown by compounds [I] and [II].

[0062] The compounds of the present method (compound [I] and compound [II]) have lower antibacterial activity than existing macrolide antibiotics (such as azithromycin and erythromycin). This is advantageous because the use of the compounds of the present method as therapeutic agents will have less impact on the development of antimicrobial resistance. For example, embodiments of the present method are screened for antimicrobial activity using an in vitro broth microdilution assay. In this assay, the minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits the visible growth of microorganisms in vitro. The assay conditions described in clinical and laboratory standard studies are used to prepare inocula, growth media, and endpoint readings. The test substance is dissolved in 100% DMSO, completely suspended by vortexing, and diluted in the same medium using a 2-fold serial titration for a total of 11 assay concentrations. In the wells of a 96-well plate, 4 μL of each dilution is added to 196 μL of broth medium inoculated with a microbial suspension (final bacterial count: 2-8×10(5) colony forming units / mL). The final volume per well was 200 μL, and the final DMSO concentration was 2%. The test concentrations were 0.1 to 100 μM. After incubation, the test plates were visually inspected and the wells were scored for growth or complete growth inhibition to determine the minimum inhibitory concentration. Each test substance was evaluated in duplicate. The excipient control group and the active reference agent were used as blank and positive controls. The results are shown in Table 1. It is worth noting that MRSA stands for methicillin-resistant Staphylococcus aureus, and VRE stands for vancomycin-resistant enterococci.

[0063]

[0064] Table 1 Antimicrobial efficacy results.

[0065] Table 1 shows that the antimicrobial potency (expressed as MIC) of the embodiments tested was higher than that of the azithromycin control. The MICs for compound [I]AZM-Gal were quite high for almost all species tested. This is exemplary for embodiments of the present method - the compounds disclosed herein have lower antimicrobial activity compared to macrolide antibiotics.

[0066] The following paragraphs provide exemplary synthesis schemes for embodiments of the present method. The synthetic products were confirmed using liquid chromatography and mass spectroscopy (LC-MS). The LC column was Waters Sunfire C18 30x4.6 mm, using a 20-100% acetonitrile / water gradient eluent containing 0.05% formic acid. Time: 0-10 min. The following abbreviations are used in the synthetic examples: acetonitrile (MeCN), methanol (MeOH), dichloromethane (DCM), dichloroethane (DCE), isopropanol (IPA), sodium borohydride (NaBH4), sodium carbonate Na2CO3, ammonium chloride (NH4Cl), 4-dimethylaminopyridine (DMAP), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl), triethylamine (TEA).

[0067] Example 1 - Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0068]

[0069] At room temperature, a solution of 4-hydroxy-3-nitrobenzaldehyde (3.10 g, 7.55 mol) in acetonitrile (35 ml) was added to a stirred suspension of Ag2O (8.0 g, 34.52 mmol) and tetra-O-acetyl-aD-glucopyranosyl bromide (1.26 g, 7.55 mmol) in MeCN (50 ml). The mixture was stirred at room temperature for 4 hours, the solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give a crude product. Purification on silica gel (2-4% methanol in DCM) gave [(2R, 3R, 4S, 5R, 6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl] acetic acid methyl ester (3.61 g). LC-MS 515.2 [M+H2O] + , RT 4.25min.

[0070] Example 2 - Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0071]

[0072] Methyl [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]acetate (3.00 g, 6.03 mmol) was added to a mixed ice-cold solution of dry DCM (30 ml) and dry IPA (8.5 ml), NaBH4 (0.50 g, 13.22 mmol) was added under nitrogen atmosphere, and the mixture was stirred for 3.5 hours. Saturated NH4Cl solution (70 ml) was added to the stirred mixture, stirring was continued for 5 minutes, the product was extracted twice with DCM, the combined extracts were washed with brine, dried over Na2CO3, the solid residue was removed by filtration and evaporated under reduced pressure to give [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester (2.60 g). LC-MS 517.2 [M+H2O] + , RT 3.91min.

[0073] Example 3 - Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0074]

[0075] Under nitrogen atmosphere, excess phosgene (1.60 ml, 3.00 mmol) was added to an ice-cold suspension of [(2R, 3R, 4S, 5R, 6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetate (0.25 g, 0.50 mmol) and KCO (0.44 g, 3.00 mmol) stirred in dry MeCN (5 ml), and the mixture was stirred at +5° C. for 2 hours. The solid residue was removed by filtration through Celite, and the solvent was evaporated under reduced pressure to give the crude product [(2R, 3R, 4S, 5R, 6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetate.

[0076] Example 4 - Intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0077]

[0078] The title compound was prepared according to Ghosh Ajit, K. et al. A daunorubicin b-galactoside prodrug for use in conjunction with gene-directed enzyme prodrug therapy Tetrahedron Lett. 2000, 41, 4871-4874. LC-MS 515.2 [M+H2O] + , RT 4.16min.

[0079] Example 5 - Intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0080]

[0081] The title compound was prepared according to Ghosh Ajit, K. et al. A daunorubicin b-galactoside prodrug for use in conjunction with gene-directed enzyme prodrug therapy Tetrahedron Lett. 2000, 41, 4871-4874. LC-MS 517.2 [M+H2O] + , RT 3.81min.

[0082] Example 6 - Intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy)tetrahydropyran-2-yl]acetic acid methyl ester.

[0083]

[0084] The crude title compound was prepared according to the procedure described above in Example 3 for the intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester.

[0085] Example 7 - Compound [I], [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S ,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester. It can be seen that the galactose moiety is attached to compound [I] via the hydroxide on the desosamine ring.

[0086]

[0087] Under nitrogen atmosphere, (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyl-tetrahydropyran-2-yl]oxy-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptahydro A solution of methyl-1-oxa-6-azacyclopentadecan-15-one (0.23 g, 0.3 mmol) in dry MeCN (4 ml) was added to a stirred ice-cold suspension of crude [(2R, 3S, 4S, 5R, 6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester (0.28 g, 0.50 mmol) and K2CO3 (0.14 g, 1.00 mmol) in dry MeCN (6 ml). The reaction mixture was stirred at room temperature for 16 hours. The solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give the crude product. Silica gel (2-10% MeOH in DCM) to give [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R ,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester (0.090g). LC-MS 638.1[M / 2+1] + , 1274.7[M] + , RT 4.20min.

[0088] Example 8-(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-11-[(2S,3R,4S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one.

[0089]

[0090] The title compound was prepared according to Novel tandem reaction for the synthesis of N'-Substituted 2-Imino-1,3-oxazolidines from Vicinal (sec-ortert-) Amino Alcohol of Desosamine by Vujasinovic, Ines et al. Eur. J. Org. Chem. 2011, 2507-2518. LC-MS 735.3 [M+H] + , RT 0.97min.

[0091] Example 9 - Compound [IV], whose IUPAC name is [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[[(2S,3R,4S,6R)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5 S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-3-hydroxy-6-methyl-tetrahydropyran-4-yl]-methyl-carbamoyl]oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester.

[0092]

[0093] The title compound (0.064 g) was prepared according to the method of Example 7 using (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-11-[(2S,3R,4 S,6R)-3-hydroxy-6-methyl-4-(methylamino)tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester. LC-MS1260.5[M] + , RT 5.06min.

[0094] Example 11-4-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxy-4-oxo-butyric acid.

[0095]

[0096] DMAP (0.024 g, 0.2 mmol) and succinic anhydride (0.20 g, 2.00 mmol) were added to a stirred solution of (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyl-tetrahydropyran-2-yl]oxy-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-1-oxa-6-azacyclopentadecan-15-one (0.795 g, 1.00 mmol) in anhydrous DCM (3 ml). The reaction mixture was stirred at room temperature under nitrogen atmosphere for 20 hours. The solvent was evaporated under reduced pressure to give 4-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxy-4-oxo-butyric acid as a white solid (0.84 g). LC-MS 425.5 [M / 2+1] + ,849.6[M] + ,RT 2.40min.

[0097] Example 12-O 4-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy -3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]O1-[[3-nitro-4-[(2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-phenyl]methyl]succinate.

[0098]

[0099] At room temperature and nitrogen atmosphere, EDC HCl (0.10 g, 0.52 mmol), TEA (50 μl, 0.35 mmol) were added to 4-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadecan-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxy-4-oxo-butanoic acid (0.30 g, 0.35 mmol). ol) and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester (0.175g, 0.35mmol) in dry DCM (8ml). The reaction mixture was stirred for 72 hours under nitrogen atmosphere at room temperature. The solvent was evaporated under reduced pressure to give a crude product. Silica gel (5-10% IPA in DCM) purification gave O4-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran -2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]O1-[[3-nitro-4-[(2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-phenyl]methyl]succinate. (0.239g). LC-MS 660.3[M / 2+1] + , 1330.9[M] + , RT 3.33min.

[0100] Example 13 - Compound [VI], (2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-14-ethyl-7,12,13-trihydroxy-4-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,7,9,11,13-hexamethyl-2,10-dioxo-oxacyclotetradec-6-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetate.

[0101]

[0102] The title compound (0.107 g) was prepared from (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-6-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyl-tetrahydropyran-2-yl]oxy-14-ethyl-7,12,13-trihydroxy-4-[(2S,4R,5S ,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,7,9,11,13-hexamethyl-oxacyclotetradecane-2,10-dione and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester. LC-MS1259.8[M] + , RT 4.06min.

[0103] Example 14 - Compound [V], [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(3R,4S,5S,6R,7R,9R,10E,11S,12R,14R)-14-ethyl-7,12,13,13-tetrahydroxy-4-[(2S, 4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-10-(2-methoxyethoxymethoxyimino)-3,5,7,9,11-pentamethyl-2-oxo-oxacyclotetradec-6-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester.

[0104]

[0105] The title compound (0.123 g) was prepared from (3R,4S,5S,6R,7R,9R,10E,11S,12R,14R)-6-[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyl-tetrahydropyran-2-yl]oxy-14-ethyl-7,12,13,13-methoxyethoxymethoxyimino-4-[(2S,4R,5S,6S)-5-hydroxy] ... -4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-10-(2-methoxyethoxymethoxyimino)-3,5,7,9,11-pentamethyl-oxatetradecane-2-one and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester, using IPA instead of MeOH as the chromatographic eluent. LC-MS682.3[M / 2+1] + , 1363.0[M] + , RT 4.32min.

[0106] Example 15-[(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R ,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester.

[0107]

[0108] The title compound (0.046 g) was prepared from [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester and (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-11-[(2S,3R,4S,6R)-4-(dimethyl)-1-[[(2R,3R,4S,6R)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]acetate] Prepared by using IPA instead of MeOH as the chromatographic eluent. LC-MS 638.3[M / 2+1] + ,1274.9[M] + ,RT 3.30min.

[0109] Example 16-[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl][3-nitro-4-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl]carbonate.

[0110]

[0111] Under nitrogen atmosphere, 0.5 M sodium methoxide solution (0.4 ml, 0.20 mmol) was added to a stirred [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5- Hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]acetic acid methyl ester (0.050 g, 0.04 mmol) was stirred in ice-cold suspension in dry THF (1 ml), and the mixture was stirred at 0°C for 2.5 hours. The reaction mixture was treated with DOWEX 50WX2 (100 mg) was treated at 0°C for 0.5 h, the solid resin was removed by filtration, washed with DCM (10 ml) and the solvent was evaporated under reduced pressure to give [(2S,3R,4S,6R)-4-(dimethylamino)-2-[[(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-13-[(2S,4R,5S,6S)-5-hydroxy -4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl]oxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-1-oxa-6-azacyclopentadeca-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl] [3-nitro-4-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-phenyl] carbonate methyl ester (0.050 g). LC-MS 554.0 [M / 2+1] + , RT 3.52min.

[0112] The therapeutic agent can be used in the form of a pharmaceutical composition, which can be prepared using one or more known methods. For example, the pharmaceutical composition can be prepared by using a diluent or excipient, such as, for example, one or more fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, etc., which are well known in the art. Various types of administration unit forms can be selected according to the therapeutic purpose. Examples of the form of the pharmaceutical composition include, but are not limited to, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injection preparations (solutions and suspensions), topical creams, and other forms known in the art. In order to form a pharmaceutical composition in the form of a tablet, any known excipient can be used, for example: carriers, such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, cyclodextrin, crystalline cellulose, silicic acid, etc.; binders, such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc. In addition, disintegrants such as dry starch, sodium alginate, agar powder, kelp powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, starch, lactose, etc. can also be used. Disintegration inhibitors such as white sugar, stearic acid, coconut oil, hydrogenated oil can be used; absorption promoters such as quaternary ammonium bases, sodium lauryl sulfate, etc. can be used. Wetting agents such as glycerol, starch and other wetting agents known in the art can be used. Adsorbents such as, for example, starch, lactose, kaolin, bentonite, colloidal silicic acid, etc. can be used. Lubricants such as pure talc, stearate, boric acid powder, polyethylene glycol, etc. can be used. If tablets are needed, they can be further coated with commonly used coating materials to make the tablets into sugar-coated tablets, gelatin film-coated tablets, enteric-coated tablets, film-coated tablets, double-layer tablets and multi-layer tablets. The pharmaceutical composition suitable for topical application can be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, foam, spray, aerosol or oil. Such pharmaceutical composition can include conventional additives, including but not limited to preservatives, solvents that assist drug penetration, cosolvents, emollients, propellants, viscosity modifiers (gelling agents), surfactants and carriers.

[0113] The present method can be used to prevent tumor recurrence, metastasis and / or reduce the possibility of tumor recurrence and metastasis. Anticancer treatments often fail due to tumor recurrence or metastasis (especially after surgery). The activity of CSC mitochondria is at least partly responsible for the failure of these treatments. Embodiments of the present method can be used in the case of conventional cancer treatment failure and / or in combination with anticancer treatment to prevent failure due to tumor recurrence and / or metastasis or reduce the possibility of failure due to tumor recurrence and / or metastasis.

[0114] The present method provides a method for selectively targeting cancer cells. The targeted cancer cells can be at least one of CSC, energetic cancer stem cells (e-CSC), circulating tumor cells (CTC, seed cells that lead to subsequent growth of more tumors in distant organs, a mechanism that leads to a large part of cancer-related deaths) and therapy-resistant cancer cells (TRCC, cells that have become resistant to one or more chemotherapy, radiotherapy and other common cancer treatments).

[0115] As described in Applicant's co-pending U.S. Provisional Patent Application Nos. 62 / 686,881, filed on June 19, 2018, and 62 / 731,561, filed on September 14, 2018, which are incorporated herein by reference in their entirety, e-CSCs represent a CSC phenotype associated with proliferation. In addition to bulk cancer cells and CSCs, it should be understood that the present method can be used to target a highly proliferative subpopulation of cells that the inventors call e-CSCs, which show a gradual increase in stemness markers (ALDH activity and mammary cell sphere formation activity), a high increase in mitochondrial mass, and an increase in glycolysis and mitochondrial activity.

[0116] In view of the above, it should be understood that, depending on the embodiment, the present method can take a variety of forms. For example, the embodiment of the present method can take the form of a composition, and in particular the form of a pharmaceutical composition. The compound of the present method can be the active ingredient in the composition, and can be present in a pharmaceutically effective amount (pharmaceutically effective amount, pharmaceutically-effective amount) together with one or more pharmaceutically acceptable excipients. The embodiment of the present method can also take the form of a method for preventing at least one of tumor recurrence and metastasis or reducing the possibility of at least one of tumor recurrence and metastasis. In some embodiments, a pharmaceutically effective amount of the composition can be administered, the composition having the compound described in the present method as a therapeutic agent and one or more pharmaceutically acceptable excipients. In some embodiments, an effective amount of the composition can be administered, the composition having the embodiment of the compound described herein as its therapeutic agent.

[0117] The following paragraphs describe the materials and tests used to generate the data described herein. It should be understood that a person skilled in the art with an ordinary level of skill in the art can perform the same tests as described herein, and / or can utilize other tests known in the art to evaluate the physical, chemical and pharmaceutical properties of the compounds described herein.

[0118] Reagents and Model Cell Lines: Obviously, other cell lines can be used without departing from the present method. MRC-5 ( CCL-171) human lung fibroblasts, BJ ( CRL2522) human skin fibroblasts and MCF-7 human breast cancer cells were purchased from ATCC (American Type Culture Collection). hTERT-BJ1 cells were from MCF-7 of Clontech, and hTERT-BJ-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum, GlutaMAX and 1% penicillin-streptomycin, and incubated at 37°C in a humidified 5% CO2 incubator. The culture medium was changed 2-3 times / week. Gibco brand cell culture medium (MEM) was purchased from Life Technologies. Bromodeoxyuridine, azithromycin, roxithromycin and erythromycin were purchased from Sigma-Aldrich.

[0119] BrdU test: cells were seeded into 24-well plates. The next day, half of the plates were treated with 100 μM BrdU, while control wells were treated with vehicle (DMSO) only and incubated at 37°C in a humidified atmosphere of 5% CO2 for 8 days. After 8 days of BrdU treatment, cells were treated with different test compounds or drugs (e.g., azithromycin, roxithromycin, telithromycin, erythromycin, etc.) for another 3-5 days. BrdU or vehicle treatment was also continued during drug treatment.

[0120] Sulphorhodamine B assay: After incubation of the plate cells, cell viability was measured using the Sulphorhodamine B (SRB) assay. This assay is based on the measurement of cellular protein content. Cells were fixed with 10% trichloroacetic acid (TCA) at 4°C for 1 hour and dried overnight at room temperature. The plates were then incubated with SRB for 30 minutes, washed twice with 1% acetic acid, and air-dried for at least 1 hour. Finally, the protein-bound dye was dissolved in a solution of 10 mM Tris, pH 8.8, and read at 540 nm using a plate reader.

[0121] xCELLigence Assay System: xCELLigence RTCA System (ACEA Biosciences). Briefly, mRC-5 lung fibroblasts (vehicle alone and / or treated with 100 μM BrdU) were seeded in each well and the efficacy of azithromycin was evaluated using RTCA (real-time cell analysis) via measurement of cell-induced electrical impedance. This method allows quantification of the onset and kinetics of cellular responses. The experiment was repeated several times independently, using four samples in each case.

[0122] Autophagy and cell cycle analysis: Autophagy was performed according to the manufacturer's instructions (using Muse TM Autophagy LC3-Antibody Kit, Merck Millipore) and Cell Cycle ( Cell cycle kit, Merck Millipore) experiment.

[0123] β-Gal staining: β-galactosidase staining was performed on BrdU-treated MRC-5 cells using the Senescence β-galactosidase staining kit (#9860, Cell Signating Technology Co., Ltd.) according to the manufacturer's protocol.

[0124] 3D anchorage independent growth test: This test is also called a mammary cell sphere formation test. Single cell suspensions were prepared using enzymes and manual dissociation (25-g needle). Then, under non-adherent conditions, cells were seeded at a density of 500 / cm2 in mammary cell sphere culture medium (DMEM-F12+1X B-27Plus supplement+20ng / ml EGF+penicillin / streptomycin) in a culture dish called a "mammary cell sphere plate" pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.). The cells were grown for 5 days and maintained in a humidified 37°C incubator. After 5 days of culture, 3D-mammary cell spheres of >50 μm were counted using an eyepiece ("grid"), and the percentage of cells that formed mammary cell spheres was calculated, and was called the percentage of mammary cell sphere formation, and normalized to 1 (1=100% MFE). 3D mammosphere formation efficiency (MFE) was analyzed in low-ATP and high-ATP subpopulations of cells. All 3D mammosphere experiments were performed three times, at least three times independently.

[0125] Statistical analysis: All analyses were performed using GraphPad Prism 6. Data are presented as mean ± standard deviation (or ± SEM, where indicated). All experiments were performed at least 3 times independently, with >3 technical replicates per experimental condition (unless otherwise indicated, e.g., when representative data are shown). Statistically significant differences were determined using Student's t-test or analysis of variance (ANOVA) tests. For comparisons between multiple groups, one-way ANOVA was used to determine statistical significance. p < 0.05 was considered significant.

[0126] The terms used in the description of the present invention herein are only used to describe specific embodiments and are not intended to limit the present invention. As used in the description of the present invention and the description of the appended claims, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" are also intended to include plural forms. The present invention includes many replacements, modifications and equivalents, as will become apparent from consideration of the following detailed description.

[0127] It should be understood that although the terms "first", "second", "third", "a)", "b)" and "c)" etc. can be used to describe various elements of the present invention herein, and the claims should not be limited by these terms. These terms are only used to distinguish one element of the present invention from another element. Therefore, the first element discussed below can be referred to as element aspect, and similarly, the third element can be referred to as element aspect without departing from the teachings of the present invention. Therefore, the terms "first", "second", "third", "a)", "b)" and "c)" etc. are not intended to convey a sequence or other hierarchy to the relevant elements, but are only used for identification purposes. The order of operations (or steps) is not limited to the order proposed in the claims.

[0128] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. It will also be understood that terms, such as those defined in common dictionaries, should be interpreted as having the same meaning as that in the context of the present application and the related art, and unless clearly defined herein, should not be interpreted in an idealized or overly formal sense. The terms used in the present description herein are only used to describe specific embodiments and are not intended to limit the present invention. All publications, patent applications, patents and other references mentioned herein are all incorporated herein by reference. In the event of a conflict in terms, this specification is controlling.

[0129] Furthermore, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when construed in the alternative ("or").

[0130] The terms "decrease," "lower," "lessen," and "reduce" generally refer to the ability of a composition according to the present method to produce and / or elicit a lesser physiological response (i.e., a measurable downstream effect) than that elicited by a vehicle or control molecule / composition, e.g., a reduction in tumor volume. A "decrease" or "reduced" response is generally a "statistically significant" response, and can include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more fold (e.g., 500, 1000 fold) decrease (including all integers and decimals between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response produced by a normal, untreated or control treated subject.

[0131] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein can be excluded or omitted. For illustration, if the specification states that a composite includes components A, B, and C, it is specifically intended that any one or combination of A, B, or C can be omitted and disclaimed.

[0132] As used herein, the term "about" refers to a measurable value, such as, for example, an amount or concentration, etc., and is intended to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0133] Having thus described certain embodiments of the present invention, it should be understood that the invention defined by the appended claims is not limited to the specific details set forth in the foregoing description, since many obvious variations thereof are possible without departing from the spirit or scope thereof, as hereinafter required.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has the chemical structure of one of compound [I] and compound [II]:

2. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein the compound has the chemical structure of one of compound [I] and compound [II].

3. A method for treating or preventing tumor recurrence and / or metastasis, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 2 to a patient at risk of tumor recurrence and / or metastasis.

4. The method according to claim 3, wherein: The administering is performed at least one of before cancer treatment, concomitantly with cancer treatment, and after cancer treatment.

5. A method for inhibiting the proliferation of cancer stem cells in a patient, the method comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 2 to the patient.

6. The method according to claim 5, wherein: The administering is performed at least one of before cancer treatment, concomitantly with cancer treatment, and after cancer treatment.

7. A method for treating cancer, the method comprising: A pharmaceutically effective amount of the pharmaceutical composition according to claim 2 is administered to a human suffering from cancer.

8. The method according to claim 7, wherein: The administering is performed at least one of before cancer treatment, concomitantly with cancer treatment, and after cancer treatment.

9. A method for eliminating senescent cells, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 2 to a patient.

10. A method for minimizing the accumulation of senescent cells in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition according to claim 2.

11. A method for delaying the onset of aging in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition according to claim 2.

12. A method for treating the effects of aging in a subject, the method comprising administering to the subject a pharmaceutically effective amount of the pharmaceutical composition of claim 2.

13. An anti-aging treatment method, comprising administering a pharmaceutically effective amount of the pharmaceutical composition according to claim 2 to a subject.

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