Macrolide senescent cell-removing compound

Abstract

Disclosed herein are macrolide compounds having senescent cell elimination activity. These compounds selectively target senescent cells and can be used to reduce or eradicate senescent cells in a subject's body and / or delay the onset of aging. These compounds also inhibit the proliferation of cancer stem cells and can be used to treat cancer, particularly to inhibit the proliferation of cancer stem cells that cause metastasis and tumor recurrence.

Description

Technical Field

[0001] Related Applications None

[0002] The present disclosure relates to a macrolide compound that selectively eradicates senescent cells and inhibits the proliferation of cancer stem cells (CSCs).

Background Art

[0003] The biological process of aging has continued to attract significant attention in the scientific and medical research communities. Physiological aging is at least partially associated with an increased rate of oxidative damage to cellular components, including DNA, lipids, proteins, etc. The increased oxidative damage creates an imbalance that disrupts the self-regulatory processes at the cellular level. Furthermore, aging correlates with the accumulation of lipofuscin in the cytoplasm of neurons. Recent research has also shown that aging is the result of spontaneous DNA damage that leads to abnormal DNA changes accumulating over time. DNA damage in both mitochondria and the nucleus can contribute to aging, indirectly through increased apoptosis and cellular senescence, and directly through increased cellular dysfunction. The accumulated DNA damage can lead to the loss of cells, the loss and mutation of gene expression in surviving cells, i.e., the effects that generate signs of aging in cells that do not divide as frequently. Cellular senescence occurs when aging cells stop dividing, which is thought to occur, among other things, following various environmental damage events, abnormal cell growth, autophagy, and oxidative stress. The Senescence Associated Secretory Phenotype (SASP) is a characteristic of senescent cells and can lead to proteotoxic impairment of the functions of healthy cells, including inflammatory or anti-inflammatory, and tumor or anti-tumor effects, depending on a number of factors. The effects of SASP-related chronic inflammation affect the normal ability of the immune system to remove senescent cells, and cells that provide immune function can be attracted to senescent cells by the SASP. Biomarkers of cellular senescence are known to accumulate as mammals age and contribute to various age-related diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, and type 2 diabetes. Also, with regard to cells that divide frequently, accumulated DNA damage can be a major cause of cancer.

[0004] Aging increases the likelihood of cancer development in this way, and researchers have struggled to develop new anti-cancer therapies, anti-aging therapies, or senolytic therapies. Conventional cancer therapies (e.g., radiation therapy, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) attempt to selectively detect and eradicate fast-growing cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies use immunotherapies (e.g., monoclonal antibodies) that selectively bind to mutant tumor antigens on fast-growing cancer cells. Unfortunately, tumors often recur at the same site or at one or more different sites after these therapies, indicating that not all cancer cells have been eradicated. In particular, cancer stem cells survive for various reasons, leading to treatment failure. Recurrence may be due to insufficient doses of chemotherapy agents and / or the emergence of cancer clones resistant to the therapy. Therefore, a new cancer treatment strategy that overcomes the drawbacks of conventional therapies is needed.

[0005] Advances in mutation analysis have made it possible to study in detail the genetic mutations that occur during cancer development. Despite knowledge of the genomic landscape, in modern oncology, it has been difficult to identify the major driver mutations across multiple cancer subtypes. The harsh reality is that each patient's tumor is unique and a single tumor may contain multiple different clonal cells. What is needed, therefore, is a new approach that focuses on the commonalities between different cancer types. Targeting the metabolic differences between tumor cells and normal cells is expected as a new cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed an increase in over 95 mRNA transcripts related to mitochondrial biogenesis and / or mitochondrial translation. Non-Patent Document 1. Furthermore, more than 35 of the 95 upregulated mRNAs encode mitochondrial ribosomal proteins (MRPs). Similarly, proteomic analysis of human breast cancer stem cells revealed significant overexpression of multiple mitochondrial ribosomal proteins and other proteins related to mitochondrial biogenesis. Non-Patent Document 2.

[0006] The mitochondrial metabolism of cancer cells has been the subject of recent exploratory research regarding both the search for anti-cancer treatment targets and treatment targets for the removal of senescent cells. Mitochondria are extremely dynamic organelles that constantly divide, elongate, and interconnect to form a tubular network or fragmented granules in order to meet the requirements of the cell and adapt to the cellular microenvironment. The balance between mitochondrial fusion and fission affects a number of important mitochondrial-dependent biological processes such as adenosine triphosphate (ATP) production, mitophagy, apoptosis, and calcium homeostasis, as it determines the morphology, abundance, function, and spatial distribution of mitochondria. And the dynamics of mitochondria can be regulated by mitochondrial metabolism, respiration, and oxidative stress.

[0007] ATP is the universal biological energy "currency" of all living cells and tissues, including microorganisms such as prokaryotic bacteria and eukaryotic yeast. In eukaryotes, the mitochondrial organelle functions as the "powerhouse" of the cell. Mitochondria generate 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, leading to apoptosis (programmed cell death) and / or necrosis driven by mitochondria. Therefore, the inventors have proposed that ATP depletion therapy could be a feasible strategy for targeting and eradicating even the "fittest" cancer cells.

[0008] In MCF-7 breast cancer cells, under normoxic conditions, OXPHOS driven by mitochondria contributes 80-90% of ATP production, while glycolysis contributes only 10-20% of the remainder. Thus, like normal cells, cancer cells strongly depend on mitochondrial ATP production. However, little is still known about whether the ATP level in cancer cells contributes to 3D matrix-dependent growth and cell migration, which are two characteristics of the spread of metastasis.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] What is needed, therefore, are new anti-aging compositions and methods for treating aging at the cellular level that overcome the accumulated oxidative and DNA damage, as well as many of the undesirable effects of aging.

[0011] Furthermore, what is needed is a therapeutic agent that targets unhealthy senescent cells and SASP, reduces the accumulation of cellular senescence, and counteracts chronic aging.

[0012] Furthermore, what is needed is a therapeutic agent that targets a wide range of CSCs based on characteristics common to CSCs regardless of cancer type.

[0013] Furthermore, what is needed is a therapeutic agent that inhibits the proliferation of CSCs, including circulating tumor cells and tumor-initiating cells, which may cause tumor recurrence and / or metastasis.

[0014] In view of the above background, an object of the present disclosure is to describe a therapeutic agent or compound that can be used for reducing the accumulation of cellular senescence and inhibiting CSC proliferation. An object of the present disclosure is to describe a therapeutic agent for use in eradicating CSCs and senescent cells. An object of the present disclosure is to describe a therapeutic agent for use in preventing and reducing the likelihood of tumor recurrence and metastasis. Furthermore, an object of the present disclosure is to describe compositions and methods, such as pharmaceutical compositions, for the treatment and prevention of cancer, including tumor recurrence and / or metastasis. Another object of the present disclosure is to describe compositions and methods, such as pharmaceutical compositions, for an agent for senescent cell removal therapy.

Means for Solving the Problems

[0015] Described herein are compounds that can be used as therapeutic agents having anti-cancer activity, pharmaceutical compositions containing the therapeutic agents, methods for synthesizing the above compounds, and methods for treating cancer.

[0016] The approach of the present invention can also be used for the treatment and / or prevention of tumor recurrence and / or metastasis. Anticancer treatment often fails, especially because tumors recur or metastasize after surgery. At least some of these causes of treatment failure are understood to be due to CSC mitochondrial activity. Embodiments of the approach of the present invention can be used to prevent or reduce the likelihood of treatment failure due to tumor recurrence and / or metastasis in situations where conventional cancer therapies have failed and / or concurrently with or prior to anticancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1

Figure 2

Figures 3A-3B

Figure 4A

Figure 4B

Figure 5

Figure 6

Figure 7

[0018] In the following description, embodiments of the approach of the present invention are illustrated in sufficient detail to enable the practice of the approach of the present invention. The approach of the present invention will be described with reference to these specific embodiments, but it should be understood that the approach of the present invention can be implemented in different forms and this description should not be construed as limiting any of the appended claims to the specific embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the approach of the present invention to those skilled in the art.

[0019] This description uses various terms that should be understood by those of ordinary skill in the art. The following clarifications are made to avoid ambiguity.

[0020] The term "cancer" refers to a mammalian physiological state characterized by uncontrolled cell growth. This definition includes both benign and malignant cancers. Examples of cancer include multiple cancer types, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrin-producing tumors, and pancreatic islet cell tumors, among others), sarcomas, schwannomas (including acoustic neuromas), medullary carcinoma, adenocarcinoma, melanoma, and leukemia or lymphoma. Specific examples of cancer include bladder cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung cancer including squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer or stomach 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, kidney cancer or renal cancer, prostate cancer, genital cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancer and multiple myeloma.

[0021] As used herein, the term "tumor" refers to the growth and proliferation of neoplastic cells, including precancerous and cancerous cells and tissues, whether malignant or benign.

[0022] The term "metastasis" refers to the spread of cancer from its primary site to other parts of the body. Cancer cells can escape from the primary tumor, invade the lymphatic and blood vessels, circulate through the bloodstream, and grow or "metastasize" in distant lesions of normal tissue elsewhere in the body. Metastasis can be local or at a distant location. Metastasis is a continuous process that requires the tumor cells to escape from the primary tumor, move through the bloodstream, and stop at a distant site. At this new site, the cells can establish a blood supply, grow, and form life-threatening masses. This behavior is regulated by both stimulatory and inhibitory molecular pathways within the tumor cells, and the interaction between the tumor cells and host cells at the distant site is also important.

[0023] The terms "treat", "treated", and "treating" and "treatment" include the reduction or alleviation of at least one symptom associated with or caused by a condition, disorder, or disease to be treated, particularly cancer. In certain embodiments, the treatment includes the reduction or alleviation of at least one symptom associated with or caused by the cancer to be treated by a compound of the invention. In some embodiments, the treatment includes causing the death of a certain category 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 this can be achieved by depriving these cells of the mechanism by which they generate energy, thereby preventing the further proliferation of senescent cells and / or cancer cells, and / or inhibiting the function of CSCs. For example, the treatment can be the reduction of one or more symptoms of cancer or the complete eradication of cancer. As another example, the approach of the present invention can be used to: inhibit the mitochondrial metabolism of cancer; eradicate CSCs of cancer (e.g., kill at a rate faster than the growth rate); eradicate TICs of cancer; eradicate circulating tumor cells of cancer; inhibit the growth of cancer; target and inhibit CSCs; target and inhibit TICs; target and inhibit circulating tumor cells; prevent or reduce the likelihood of metastasis; prevent recurrence; increase the sensitivity of cancer to chemotherapeutic agents; increase the sensitivity of cancer to radiation therapy; increase the sensitivity of cancer to phototherapy. As another example, the treatment can reduce the accumulated senescent cells and / or reduce the rate of accumulation of senescent cells.

[0024] In the context of tumor recurrence and / or metastasis, the terms "prevent" and "reduce the likelihood of" refer to reducing the abundance of CSCs, TICs, and circulating tumor cells in a subject's body that may be involved in recurrence or metastasis to a level at which there is no likelihood of tumor recurrence and / or metastasis from the primary site occurring, compared to a control (i.e., a state in which there is no treatment to prevent or reduce the likelihood of tumor recurrence and / or metastasis). In practice, the treatments described herein to prevent or reduce the likelihood of tumor recurrence and / or metastasis target and inhibit or eradicate CSCs, TICs, and circulating tumor cells.

[0025] The terms "cancer stem cell" and "CSC" refer to a subpopulation of cancer cells within a tumor that have the ability to self-renew, differentiate, and form tumors when transplanted into an animal host. Compared to "bulk" cancer cells, CSCs have an increased mitochondrial mass, enhanced mitochondrial biogenesis, and higher activity of mitochondrial protein translation. As used herein, "circulating tumor cell" refers to a cancer cell that has shed from a primary tumor into the vasculature or lymphatics and is carried throughout the body in the bloodstream. Circulating tumor cells can be detected using the CellSearch circulating tumor cell assay.

[0026] As used herein, the phrase "pharmaceutically effective amount" refers to the amount that needs to be administered to a host, or to the cells, tissues, or organs of a host, to achieve a therapeutic result, such as the modulation, regulation, or inhibition of protein kinase activity, e.g., the inhibition of the activity of a protein kinase, or the treatment of cancer. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of a pharmaceutical composition required for a given subject using methods known and available in the art. For example, a physician or veterinarian can start with a level of the dosage of the compounds of the invention used in the pharmaceutical composition that is less than the amount required to achieve the desired therapeutic effect and incrementally increase the dosage until the desired effect is achieved. The determination of a pharmaceutically effective amount is considered to be within the scope of skill of those of ordinary skill in the art having considered the present disclosure.

[0027] As used herein, the phrase "therapeutic agent" refers to embodiments of the compounds described herein and may include pharmaceutically acceptable salts or isotopically-labeled analogs thereof. It should be understood that a therapeutic agent can be administered to a subject by any suitable approach known to those of ordinary skill in the art. Also, it should be understood that the amount of a therapeutic agent, and the timing of its administration, can depend on the individual subject being treated (e.g., age and weight among other factors), the method of administration, the pharmacokinetic characteristics of the particular therapeutic agent, and the judgment of the prescribing physician. Thus, because there is variability from subject to subject, the dosages described herein are all intended to be initial guidelines, and a physician can titrate the dosage of a therapeutic agent to achieve a treatment that the physician deems suitable for the subject. In considering the desired degree of treatment, a physician can balance a variety of factors such as the age and weight of the subject, the presence or absence of existing diseases, and the presence or absence of other diseases. Pharmaceutical formulations can be prepared to suit any desired route of administration, including but not limited to oral, intravenous, or aerosol administration, as will be considered in more detail below.

[0028] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating agent. 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 function as pharmaceutically acceptable carriers are: (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 carboxymethyl cellulose, 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 glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solution; and (21) other non-toxic and compatible substances used in pharmaceutical formulations.

[0029] The phrase "pharmaceutically acceptable salt" refers to relatively less toxic inorganic and organic basic addition salts of the compounds of the approach of the present invention. Pharmaceutically acceptable salts can be formed, for example, by reacting a compound in free acid form with a base such that a hydroxide or carbonate of a pharmaceutically acceptable metal cation is reacted with ammonia or a pharmaceutically acceptable amine. Representative alkali or alkaline earth salts include, for example, sodium, potassium, calcium, magnesium, and ammonium salts. Examples of amines that can be used for basic addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine. It should of course be understood that other salts may be used and that those skilled in the art can identify a suitable salt form using methods known in the art without departing from the approach of the present invention.

[0030] Senescence is a distinct feature of normal aging over time. Senescence involves a potentially irreversible cell cycle arrest due to the induction of CDK inhibitors such as p16 - INK4A, p19 - ARF, p21 - WAF, and p27 - KIP1, as well as the development of SASP (senescence - associated secretory phenotype), and the induction of important lysosomal enzymes (β - galactosidase) and lipofuscin, an established aging pigment. Interestingly, SASP results in the secretion of diverse inflammatory cytokines such as IL - 1 - β and IL - 6, whereby senescent cells can "contagiously" spread the senescent phenotype systemically from one cell type to another via chronic inflammation. Such chronic inflammation can also potentially promote the development of cancer and drive tumor recurrence and metastasis.

[0031] Described herein are compounds having efficacy against senescent cells and high selectivity. The compounds of the approach of the present invention can be used as senescent cell-removing agents, for example, as therapeutic agents for eradicating senescent cells. Further, the compounds of the approach of the present invention exhibit significantly lower antibiotic activity when compared to reference compounds. Thus, the compounds of the approach of the present invention do not contribute to potential antibiotic resistance.

[0032] Some embodiments of the approach of the present invention take the form of compounds having the structure of compound [I] shown below, where "Ac" represents an acetyl group.

[0033]

Chemical Structure

[0034] The IUPAC name of compound [I], also called AZM-Gal, 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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate. Galactose is linked via the hydroxide of the desosamine ring.

[0035] Compound [II], also called deacetylated AZM-Gal, having the structure shown below, has activity comparable to that of compound [I].

[0036]

Chemical Structure

[0037] The compounds disclosed in this specification exhibited senescent cell elimination activity in clinical assays. For example, in one assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine), also known as BrdU, was used to induce senescence in a cell population. BrdU is an analog of thymidine, a nucleoside commonly used to identify proliferating cells. BrdU induces controlled DNA damage and efficiently senesces cells. In the BrdU assay, normal fibroblasts were subjected to long-term culture (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 the BrdU-based assay. The senescent cell elimination activity was evaluated using the sulforhodamine B assay, also known in the art as the SRB assay. This assay measures the amount of protein remaining attached to the tissue culture dish and is a surrogate marker of cell viability.

[0038] Figure 1 compares the results of SRB assays regarding various concentrations of azithromycin (100 μM and 50 μM) and compound [I] of the approach of the present invention (100 μM and 50 μM) in both 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 further exposed to azithromycin or compound [I] (labeled as "Azi-Gal" in Figure 1) for 5 more days. Subsequently, an SRB assay was performed, using normal MRC-5 cells as a control to determine the effect of the above drugs on cell viability. As can be seen from the figure, 100 μM of azithromycin did not affect the viability of normal MRC-5 lung fibroblasts, but selectively killed senescent MRC-5 fibroblasts. However, at 50 μM, azithromycin did not affect either the control cells or the senescent cells. In contrast, compound [I] showed significant selectivity for senescent MRC-5 fibroblasts at 50 μM and was effective against both control cells and senescent cells at 100 μM.

[0039] Senescent cells exhibit a so-called senescence-associated secretory phenotype (SASP), which is accompanied by a dramatic increase in protein synthesis and secretion. Using the xCELLigence assay system, it was evaluated whether the protein measurement assay underestimated the senescent cell elimination activity of the test compounds. Instead of relying on proteins, the xCELLigence assay system uses electrical impedance to continuously measure cell proliferation in real time. Therefore, the real-time xCELLigence assay system complements the more static SRB assay and provides a more direct visualization of the potential senescent cell elimination effect of compounds during drug screening.

[0040] Using the xCELLigence assay, the senescent cell elimination activity of compound [I] was confirmed. MRC-5 fibroblasts were used in the above assay. Figure 2 shows the xCELLigence data comparing the effects of compound [I] of the approach of the present invention on control MRC-5 cells and MRC-5 cells treated with BrdU. The above data are represented as the final cell index, i.e., mean ± standard error of the mean, for each of control cells, control cells treated with compound [I], BrdU-treated control cells, and BrdU-treated fibroblasts exposed to 50 μM of compound [I]. Senescent MRC-5 cells were pretreated with BrdU for 8 days to induce senescence and then further exposed to compound [I] for 5 days. Compared with the control, treatment with compound [I] did not affect the viability of normal fibroblasts, but killed more than 90% of the BrdU-treated fibroblasts. These data confirm that compound [I] has prominent senescent cell elimination activity.

[0041] Figures 3A and 3B are images of MRC-5 fibroblasts without and with BrdU pretreatment, respectively, treated with compound [I] at a concentration of 50 μM. These images show that although compound [I] had little effect on normal MRC-5 cells, it induced cell death in senescent MRC-5 cells. The scale bars in the upper right of Figures 3B and 3C represent 20 μm.

[0042] These results indicate that the compounds disclosed herein have senescent cell elimination activity and can be used as senescent cell eliminators in pharmaceutical compositions. For example, the above compounds can be used to eradicate senescent cells in the body of a subject. Some approaches of the present invention may take the form of methods 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, cataract, dementia, diabetes, hair loss, hypertension, inflammatory diseases, kidney disease, muscular atrophy, neurological diseases, osteoarthritis, osteoporosis, lung diseases, intervertebral disc degeneration, and alopecia. For example, age-related diseases can be neurological diseases such as mild cognitive impairment, motor neuron dysfunction, Alzheimer's disease, Parkinson's disease, and macular degeneration. In such embodiments, a therapeutically effective amount of the senescent cell eliminator described herein may be administered to a subject. In some embodiments, the above senescent cell eliminator may be administered together with another therapeutic agent described herein. The above senescent cell eliminator may be administered at the onset of an age-related disease, i.e., at the time of diagnosis or immediately after diagnosis. Alternatively, the above senescent cell eliminator may be administered periodically after diagnosis, and the frequency and dosage can be determined using techniques known in the art. In some embodiments, the above senescent cell eliminator can be administered before the onset, especially when an age-related disease is expected to occur or is likely to occur in the body of a subject (e.g., by a genetic marker or other biological marker).

[0043] By using the compounds disclosed herein as therapeutic agents, CSCs can also be selectively eradicated for the treatment and / or prevention of tumor recurrence and / or metastasis. The data show that the compounds disclosed herein have anti-cancer activity and are suitable for use as therapeutic agents for anti-cancer treatment, including the treatment and / or prevention of tumor recurrence and metastasis. The data described herein show anti-cancer activity by inhibition of MCF-7 cells as demonstrated by a tumor spheroid formation assay. This assay measures the amount of residual protein, a surrogate marker of cell viability, attached to a tissue culture dish.

[0044] Figure 4A shows the results of a tumoroid formation assay for MCF-7 cells treated with compound [I], and Figure 4B shows the results of a tumoroid formation assay for MCF-7 cells treated with azithromycin. The above data 50 indicates that the IC

[0045] of compound [I] was 60 μM and reached nearly complete inhibition at a concentration of 100 μM. On the other hand, the IC50 of azithromycin was 118 μM, and even at a concentration of 200 μM, it only inhibited about 30% of MCF-7 cells compared to the control. These results demonstrate the excellent efficacy of compound [I] in inhibiting MCF-7 proliferation.

[0046]

Chemical Structure

[0047] Figure 5 compares the results of an SRB assay for compound [III] in both control MRC5 cells and MRC5 cells treated with BrdU. The addition of the galactose moiety decreased the potency of the parent compound, azithromycin, by approximately half. This demonstrates that simply conjugating the galactose moiety does not improve the senescent cell elimination activity of the base compound.

[0048] Furthermore, modification by the galactose moiety does not always improve the selectivity of the parent compound for senescent cells. Compound [IV], also called Des-AZM-Gal, shown below, is a demonstration example thereof. As can be seen from below, galactose is linked via the amine of the desosamine ring of the macrolide structure.

[0049] [Chemical formula]

[0050] Figure 6 compares the results of the SRB assay for azithromycin and compound [IV] in both control MRC5 cells and MRC5 cells treated with BrdU. As can be seen from the figure, compound [IV] showed little selectivity for senescent cells over normal non-senescent cells.

[0051] Conjugation with galactose causes some compounds not only to lose their senescent cell elimination and antibiotic activities, but also to target and kill normal non-senescent cells. Compound [V], also called Roxy-Gal, shown below, is a conjugate of roxithromycin and galactose.

[0052] [Chemical formula]

[0053] Compound [VI], also called Erythro-Gal, shown below, is a conjugate of erythromycin and galactose. Compounds [V] and [VI] are conjugated via the hydroxide of the desosamine ring of the macrolide structure using the same general approach as compound [I]. However, the resulting activities are quite different, showing the unique properties of compounds [I] and [I].

[0054] [Chemical formula]

[0055] Figure 7 compares the results of the SRB assay for compounds [V] and [VI] in both control MRC5 cells and MRC5 cells treated with BrdU. Compared with roxithromycin and erythromycin, these compounds showed preference for normal non-senescent control cells and had little effect on senescent cells. These data are in stark contrast to the activity demonstrated by compound [I].

[0056] Compounds of the approach of the present invention, Compound [I] and Compound [I], have lower antibiotic activity than existing macrolide antibiotics such as azithromycin and erythromycin. Using the compounds of the approach of the present invention as therapeutic agents is advantageous because it has little effect on the development of antibiotic resistance. For example, embodiments of the approach of the present invention were screened for antibiotic activity using an in vitro broth microdilution assay. In this assay, the minimum inhibitory concentration (MIC) is defined as the lowest concentration of an agent that completely inhibits the visible growth of microorganisms in vitro. Assay conditions described by the Clinical and Laboratory Standards Institute were used for inoculum preparation, growth medium, and endpoint reading. The test substance was dissolved in 100% DMSO, completely suspended by vortexing, and diluted by serial two-fold titration with the same vehicle to obtain a total of 11 test concentrations. A 4 μL aliquot of each dilution was added to 196 μL of broth medium seeded with the above biological suspension in the wells of a 96-well plate (bacterial count: 2 - 8×10(5) colony forming units / final mL). The final volume was 200 μL in each well and the final DMSO concentration was 2 percent. The test concentrations were 0.1 - 100 μM. After incubation, the test plates were visually inspected and the wells were scored for growth or complete growth inhibition to define the minimum inhibitory concentration. Each test substance was evaluated repeatedly. The vehicle control and active reference agent were used as blank control and positive control, respectively. The results are shown in Table 1. Note that MRSA represents methicillin-resistant staphylococcus aureus and VRE represents vancomycin resistant Enterococcus.

[0057]

Table 1

[0058] Table 1 shows that the antibacterial efficacy (expressed as MIC) of the tested embodiments was higher than that of azithromycin as a control. The MIC of compound [I] AZM-Gal is quite high for almost all species tested. This demonstrates an embodiment of the approach of the present invention, and the antibiotic activity of the compounds disclosed herein is lower compared to macrolide antibiotics.

Example

[0059] The following paragraphs provide exemplary synthetic schemes related to embodiments of the approach of the present invention. The synthetic products were confirmed using liquid chromatography and mass spectroscopy (LC-MS). The LC column was a Waters Sunfire C18 30×4.6 mm, and 20 - 100% acetonitrile / water containing 0.05% formic acid was used as the gradient eluent. The time was 0 - 10 minutes. In the examples of synthesis, the following abbreviations are used: acetonitrile (MeCN), methanol (MeOH), dichloromethane (DCM), isopropanol (IPA), sodium borohydride (NaBH4), sodium carbonate Na2CO 3、 ammonium chloride (NH4Cl), 4-dimethylaminopyridine (DMAP), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC HCl), triethylamine (TEA).

[0060] Example 1 Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]methyl acetate

[0061]

Chemical formula

[0062] A solution of 4-hydroxy-3-nitrobenzaldehyde (3.10 g, 7.55 mmol) in acetonitrile (35 ml) was added to a stirred suspension of Ag2O (8.0 g, 34.52 mmol) and tetra-O-acetyl-α-D-glucopyranosyl bromide (1.26 g, 7.55 mmol) in MeCN (50 ml) at room temperature. The mixture was stirred at room temperature for 4 h, the solid residue was removed by filtration, and the solvent was evaporated under reduced pressure to give the crude product. Purification by silica gel (2 - 4% MeOH in DCM) gave [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]methyl acetate (3.61 g). LC-MS 515.2 [M+H2O] + , RT 4.25 min.

[0063] Example 2 Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0064]

Chemical Structure

[0065] A stirred ice-cold solution of [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]methyl acetate (3.00 g, 6.03 mmol) in a mixture of dry DCM (30 ml) and dry IPA (8.5 ml) under a nitrogen atmosphere was treated with NaBH4 (0.50 g, 13.22 mmol), and the mixture was stirred for 3.5 h. A solution of saturated NH4Cl (70 ml) was added to the stirred mixture, stirring was continued for 5 min, 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 the solvent was evaporated under reduced pressure to give [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate (2.60 g). LC-MS 517.2 [M+H2O] + , RT 3.91 min.

[0066] Example 3 Intermediate [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0067] [Chemical formula]

[0068] A stirred ice-cold suspension of [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate (0.25 g, 0.50 mmol) and K2CO3 (0.44 g, 3.00 mmol) in dry MeCN (5 ml) under a nitrogen atmosphere was treated with an excess of phosgene (1.60 ml, 3.00 mmol), and the mixture was stirred at +5 °C for 2 h. 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]methyl acetate.

[0069] Example 4 Intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-(4-formyl-2-nitro-phenoxy)tetrahydropyran-2-yl]methyl acetate

[0070]

Chemical formula

[0071] The title compound was prepared according to Ghosh Ajit, K., at 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 4.16 min.

[0072] Example 5 Intermediate [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(hydroxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0073] [Chemistry]

[0074] The labeled compound was prepared according to Ghosh Ajit, K., at 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 3.81 minutes.

[0075] Example 6 Intermediate [(2R,3S,4S,5R,6S)‐3,4,5‐triacetoxy‐6‐[4‐(chlorocarbonyloxymethyl)‐2‐nitro‐phenoxy]tetrahydropyran‐2‐yl]methyl acetate

[0076] [Chemistry]

[0077] The crude labeled compound was prepared from the intermediate [(2R,3S,4S,5R,6S)‐3,4,5‐triacetoxy‐6‐[4‐(hydroxymethyl)‐2‐nitro‐phenoxy]tetrahydropyran‐2‐yl]methyl acetate according to the method of Example 3 above.

[0078] 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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate. As can be seen from the following, the galactose moiety is attached to Compound [I] via the hydroxide of the desosamine ring.

[0079]

Chem.

[0080] A 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-azacyclopentadecane-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]methyl acetate (0.28 g, 0.50 mmol) and K2CO3 (0.14 g, 1.00 mmol) in dry MeCN (6 ml) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. The solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give the crude product. Purification by silica gel (2 - 10% MeOH in DCM) gave [(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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyloxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate (0.090 g). LC-MS 638.1 [M / 2+1] + , 1274.7 [M] + , RT 4.20 min.

[0081] (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-azacyclopentadecane-15-one

[0082] [Chemical formula]

[0083] The title compound was prepared according to Vujasinovic, Ines et al. Novel tandem Reaction for the Synthesis of N’-Substituted 2-Imino-1,3-oxazolidines from Vicinal (sec-or tert-)Amino Alcohol of Desosamine. Eur. J. Org.Chem. 2011, 2507-2518. LC-MS 735.3[M+H] + , RT 0.97 min.

[0084] Example 9 The IUPAC name of compound [IV] 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,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-azacyclopentadec-11-yl]oxy]-3-hydroxy-6-methyl-tetrahydropyran-4-yl]-methyl-carbamoyl]oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate.

[0085]

Chemical formula

[0086] The labeled compound (0.064 g) was prepared from (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-azacyclopentadecane-15-one, and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate according to the method of Example 7 above. LC-MS 1260.5 [M] + , RT 5.06 minutes.

[0087] 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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxy-4-oxo-butanoic acid

[0088] [Chemical formula]

[0089] 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 - azacyclopentadecane - 15 - one (0.795 g, 1.00 mmol) in dry DCM (3 ml) were added DMAP (0.024 g, 0.2 mmol) and succinic anhydride (0.20 g, 2.00 mmol). The reaction mixture was stirred at room temperature for 20 h under a nitrogen atmosphere. 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 - azacyclopentadec - 11 - yl]oxy] - 6 - methyl - tetrahydropyran - 3 - yl]oxy - 4 - oxo - butanoic acid as a white solid (0.84 g). LC - MS 425.5[M / 2+1] + , 849.6[M] + , RT 2.40 min.

[0090] Example 12 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-azacyclopentadec-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] butanedioate

[0091] [Chemical formula]

[0092] 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-Azacyclopentadec-11-Yl]Oxy]-6-Methyl-Tetrahydropyran-3-Yl]Oxy-4-Oxo-Butanoic acid (0.30 g, 0.35 mmol) and [(2R,3S,4S,5R,6S)-3,4,5-Triacetoxy-6-[4-(Hydroxymethyl)-2-Nitro-Phenoxy]Tetrahydropyran-2-Yl]Methyl Acetate (0.175 g, 0.35 mmol) in dry DCM (8 ml) at room temperature under a nitrogen atmosphere were stirred, and EDC HCl (0.10 g, 0.52 mmol) and TEA (50 μl, 0.35 mmol) were added. The reaction mixture was stirred at room temperature for 72 hours under a nitrogen atmosphere. The solvent was evaporated under reduced pressure to obtain the crude product. Purification by silica gel (5 - 10% IPA in DCM) 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-Azacyclopentadec-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]Butanedioate (0.239 g). LC-MS 660.3 [M / 2+1] + , 1330.9 [M] + , RT 3.33 min.

[0093] 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]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0094]

Chem.

[0095] 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]methyl acetate according to the method of Example 7. LC-MS 1259.8 [M] + , RT 4.06 min.

[0096] 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]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0097]

Chemical formula

[0098] The title compound (0.123 g) was prepared according to the method of Example 7, except that IPA was used instead of MeOH as the eluent for chromatography 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-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-oxacyclotetradecan-2-one and [(2R,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate. LC-MS 682.3 [M / 2+1] + , 1363.0 [M] + , RT 4.32 minutes.

[0099] 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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate

[0100]

Chem.

[0101] The title compound (0.046 g) was prepared according to the method of Example 7, except that IPA was used instead of MeOH as the eluent for chromatography from [(2R,3R,4S,5R,6S)-3,4,5-triacetoxy-6-[4-(chlorocarbonyloxymethyl)-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate and (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. LC-MS 638.3 [M / 2+1] + , 1274.9 [M] + , RT 3.30 min.

[0102] 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 - azacyclopentadec - 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]methyl carbonate

[0103] [Chemical formula]

[0104] In a stirred ice-cold suspension of [(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-azacyclopentadec-11-yl]oxy]-6-methyl-tetrahydropyran-3-yl]oxycarbonyl-oxymethyl]-2-nitro-phenoxy]tetrahydropyran-2-yl]methyl acetate (0.050 g, 0.04 mmol) in dry THF (1 ml) under a nitrogen atmosphere, 0.5 M sodium methoxide solution (0.4 ml, 0.20 mmol) was added and the mixture was stirred at 0 °C for 2.5 hours. The reaction mixture was treated with DOWEX 50WX2 (100 mg) at 0 °C for 0.5 hour, 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-azacyclopentadec-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]methyl carbonate (0.050 g). LC-MS 554.0 [M / 2+1] + , RT 3.52 min.

[0105] The therapeutic agent can be used in the form of a pharmaceutical composition that can be prepared using one or more known methods. For example, the pharmaceutical composition can be prepared using diluents or excipients such as one or more fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, etc., as known in the art. Depending on one or more therapeutic purposes, various types of dosage unit forms can be selected. Examples of forms of the pharmaceutical composition include, but are not limited to, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injectable preparations (solutions and suspensions), topical creams, and other forms, as would be known in the art. To shape the pharmaceutical composition into the form of a tablet, any known excipient can be used, such as carriers like lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, cyclodextrin, crystalline cellulose, silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc. Further, disintegrants such as dried starch, sodium alginate, agar powder, kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyoxyethylene sorbitan, sodium lauryl sulfate, monoglycerides of stearic acid, starch, lactose, etc. can be used. Disintegration inhibitors such as sucrose, stearin, coconut butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium base, sodium lauryl sulfate, etc. can be used. Wetting agents such as glycerin, starch, and others known in the art can be used. Adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc. can be used. Lubricants such as purified talc, stearate, boric acid powder, polyethylene glycol, etc. can be used. If tablets are desired, the tablets can be further coated with a common coating material to make the tablets into sugar-coated tablets, gelatin film-coated tablets, tablets coated with enteric coating, film-coated tablets, double-layer tablets, and multi-layer tablets.A pharmaceutical composition adapted for topical administration can be formulated as an ointment, cream, suspension, lotion, powder, solution, paste, gel, foam, spray, aerosol, or oil. Such pharmaceutical compositions may contain conventional additives, including, but not limited to, preservatives, solvents that assist drug penetration, co-solvents, emollients, propellants, viscosity modifiers (gelling agents), surfactants, and carriers.

[0106] The approach of the present invention can be used for the prevention of tumor recurrence, metastasis, and / or reduction of the likelihood thereof. Anticancer treatments often fail, especially after surgery, because tumors recur or metastasize. At least some of these causes of treatment failure are due to CSC mitochondrial activity. Embodiments of the approach of the present invention can be used to prevent or reduce the likelihood of failure due to tumor recurrence and / or metastasis in situations where conventional cancer therapies have failed and / or concurrently with anticancer treatment.

[0107] The approach of the present invention provides a method for selectively targeting cancer cells. The target cancer cells may be at least one of cancer stem cells (CSCs), energetic cancer stem cells (e-CSCs), circulating tumor cells (CTCs, seed cells that lead to subsequent growth of further tumors in distant organs, the mechanism responsible for most cancer-related deaths), and therapy-resistant cancer cells (TRCCs, cells that exhibit resistance to one or more of chemotherapy, radiotherapy, and other common cancer treatments).

[0108] As described in U.S. Provisional Patent Application No. 62 / 686,881, filed Jun. 19, 2018, and U.S. Provisional Patent Application No. 62 / 731,561, filed Sep. 14, 2018, both of which are hereby incorporated by reference in their entirety, e-CSCs represent a CSC phenotype associated with proliferation. In addition to bulk cancer cells and CSCs, using the approach of the present invention, the inventors have identified a hyperproliferative cell subpopulation, termed e-CSCs, that exhibits a progressive increase in stemness markers (ALDH activity and tumorsphere-forming activity), a substantial increase in mitochondrial mass, and elevated glycolytic and mitochondrial activities.

[0109] In view of the above, it should be understood that the approach of the present invention can take various forms depending on the embodiment. For example, an embodiment of the approach of the present invention can take the form of a composition, particularly a pharmaceutical composition. A compound of the approach of the present invention may be an active ingredient in the composition and may be present in a pharmaceutically effective amount together with one or more pharmaceutically acceptable excipients. An embodiment of the approach of the present invention can also take the form of a method for preventing or reducing the likelihood of at least one of tumor recurrence and metastasis. In some embodiments, a pharmaceutically effective amount of a composition comprising a compound of the approach of the present invention as a therapeutic agent and one or more pharmaceutically acceptable excipients can be administered. In some embodiments, an effective amount of a composition comprising an embodiment of the compounds described herein as a therapeutic agent can be administered.

[0110] In the following paragraphs, the materials and assays used to generate the data described herein are explained. It should be understood that one of ordinary skill in the art can perform the same assays as those described herein and / or utilize other assays generally known in the art to evaluate the physical, chemical, and pharmaceutical properties of the compounds as described herein.

[0111] Reagents and model cell lines: It will be apparent that other cell lines can be used without departing from the approach of the present invention. MRC-5 (ATCC® CCL-171) human lung fibroblasts, BJ (ATCC® CRL2522) human skin fibroblasts, and MCF-7 human breast cancer cells were purchased from ATCC (American Type Culture Collection). hTERT-BJ1 cells were obtained from Clontech, Inc. MCF-7 cells and hTERT-BJ1 cells were grown 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 medium was changed 2-3 times a week. Gibco brand cell culture medium (MEM) was purchased from Life Technologies. Bromodeoxyuridine, azithromycin, roxithromycin, and erythromycin were purchased from Sigma-Aldrich.

[0112] BrdU assay: Cells were plated in 24-well plates. The next day, half of the plates were treated with 100 μM BrdU and the control wells were treated with vehicle only (DMSO), and incubated at 37°C for 8 days in a humidified atmosphere of 5% CO2. Eight days after BrdU treatment, the cells were treated with various test compounds or drugs (e.g., azithromycin, roxithromycin, telithromycin, erythromycin, etc.) for an additional 3-5 days. BrdU or vehicle treatment was continued during the drug treatment as well.

[0113] Sulforhodamine B assay: After incubation of the plates, cell viability was measured by the sulforhodamine 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. Subsequently, the plates were 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 10 mM Tris, pH 8.8 solution and read at 540 nm using a plate reader.

[0114] xCELLigence assay system: xCELLigence RTCA System (ACEA Biosciences Inc.). Briefly, MRC-5 lung fibroblasts (vehicle only and / or treated with 100 μM BrdU) were seeded in each well and used to evaluate the efficacy of azithromycin by RTCA (real-time cell analysis) based on the measurement of electrical impedance induced by the cells. This approach enables quantification of the onset and kinetics of cell responses. The experiments were repeated independently multiple times and four samples were used for each condition.

[0115] Analysis of autophagy and cell cycle: Autophagy (using the Muse™ Autophagy LC3 Antibody-Based Kit (Merck Millipore)) experiments and cell cycle (Muse® Cell Cycle Kit (Merck Millipore)) experiments were performed according to the manufacturer's instructions.

[0116] β-Gal staining: β-Galactosidase staining of BrdU-treated MRC-5 cells was performed using the Senescence β-Galactosidase Staining Kit (#9860, Cell Signalling Technology Inc.) according to the manufacturer's protocol.

[0117] 3D Scaffold-Dependent Growth Assay: This assay is also called the tumoroid formation assay. A single-cell suspension was prepared using enzymatic dissociation and manual dissociation (25g needle). Subsequently, the cells were plated at a density of 500 cells / cm2 in tumoroid medium (DMEM-F12 + 1X B-27 Plus Supplement + 20 ng / ml EGF + Pen / Strep) under non-adherent conditions in a culture dish pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.), which was called the "tumoroid plate". The cells were grown for 5 days and maintained in a humidified incubator at 37°C. After 5 days of culture, 3D tumoroids larger than 50 μm were counted using an eyepiece ("graticule"), and the percentage of plated cells forming tumoroids was calculated, which was called percent tumoroid formation and normalized to 1 (1 = 100% MFE). The 3D tumoroid formation efficiency (mammosphere formation efficiency: MFE) was analyzed in both the low-ATP cell subpopulation and the high-ATP cell subpopulation. All experiments on 3D tumoroids were performed in triplicate at least 3 times independently.

[0118] Statistical Analysis: All analyses were performed using GraphPad Prism 6. Data were presented as mean ± SD (or ± SEM if specified). All experiments were performed at least 3 times independently, and for each experimental condition tested, more than 4 technical replicates were performed (except when otherwise stated, as in the case of representative data shown). Statistical significance was determined using the Student's t-test or analysis of variance (ANOVA) test. For comparisons between multiple groups, one-way ANOVA was used to determine statistical significance. p < 0.05 was considered significant.

[0119] The terminology used in this specification to describe the present invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used in the description of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The present invention includes numerous alternatives, modifications, and equivalents, as will become apparent upon consideration of the following detailed description.

[0120] In order to describe various elements of the present invention, the terms "first," "second," "third," "a)," "b)," "c)," etc. may be used herein, but it will be understood that the claims are not limited by these terms. These terms are used only to distinguish one element of the present invention from another. Thus, the first element discussed below could be referred to as an element aspect, and similarly, could be referred to as "third" without departing from the teachings of the present invention. Thus, the terms "first," "second," "third," "a)," "b)," "c)," etc. are not necessarily intended to impart an order or other hierarchy to the elements associated with these terms and are used for purposes of identification only. The order of operations (or steps) is not limited to the order presented in the claims.

[0121] Unless otherwise defined, all terms (including technical and scientific terms) used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, terms such as those defined in commonly used dictionaries shall be interpreted as having a meaning that coincides with the meaning in the context of the present application and related technologies, and it will be understood that they should not be interpreted in an idealized or overly formal sense unless clearly defined herein. The terminology used herein in the description of the present invention is for the purpose of describing particular embodiments only and is not intended to limit the present invention. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict in terminology, the present specification shall prevail.

[0122] Also, as used herein, "and / or" shall be interpreted to mean any possible combination of one or more of the listed items associated with this term, as well as the absence of a combination when interpreted as an alternative ("or"), and shall include these.

[0123] The terms "decrease", "lower", "lessen", and "reduce" as a whole refer to the ability to produce and / or cause a lower physiological response (i.e., a measurable downstream effect), such as a reduced tumor volume, of a composition according to the approach of the present invention compared to the response caused by a vehicle or a control molecule / composition. A "decreased" or "reduced" response is typically a "statistically significant" response, which is 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, or even higher multiples (e.g., 500 times, 1000 times) (including all integers and decimals greater than 1 between these, such as 1.5, 1.6, 1.7, 1.8, etc.) lower than the response produced by a normal subject, an untreated subject, or a subject receiving a control treatment.

[0124] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the invention is also contemplated to be able to exclude or omit any feature or combination of features described herein in some embodiments of the invention. For example, herein, when a complex is described as including components A, B, and C, it is specifically intended that any one of A, B, or C or a combination thereof can be omitted and discarded.

[0125] When used herein in reference to a measurable value such as an amount or a concentration, the term "about" is intended to encompass a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the stated amount. The ranges provided herein with respect to measurable values may include any other range and / or individual value within that range.

[0126] Thus, while specific embodiments of the present invention have been described, it is to be understood that the invention as defined by the appended claims is not limited by the specific details set forth in the above description. This is because numerous obvious variations are possible without departing from the spirit or scope of the invention as claimed below.

Claims

1. Compounds [I] and [II]: 【Chemical Formula 1】 , 【Chemical Formula 2】 A compound having the chemical structure of one of them, or a pharmaceutically acceptable salt thereof.

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

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

4. The method according to claim 3, wherein the administering step is carried out at least one of before cancer treatment, simultaneously 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 to the patient a pharmaceutically effective amount of the pharmaceutical composition according to claim 2.

6. The method according to claim 5, wherein the administering step is carried out at least one of before cancer treatment, simultaneously with cancer treatment, and after cancer treatment.

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

8. The method according to claim 7, wherein the administering step is carried out at least one of before cancer treatment, simultaneously with cancer treatment, and after cancer treatment.

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

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 according to claim 2.

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

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