2-[(2-oxo-4-phenyl-2H-chromen-7-yl)oxy]propanamide derivatives

Abstract

Disclosed are 2-[(2-oxo-4-phenyl-2H-chromen-7-yl)oxy]propanamide derivatives, where at least one of R1-R4 has an amide group. These derivatives can be used to target and eliminate cancer stem cells and can be used as therapeutic agents for the treatment of cancer, including reducing and / or preventing the likelihood of tumor recurrence and metastasis. The compounds disclosed herein demonstrate inhibition of tumorsphere formation, migration, and stemness-related signaling in cancer stem cells. These properties result from the selective inhibition of mitochondrial RNA polymerase (POLRMT) targeting mitochondrial transcription in cancer cells.

Description

[Technical field]

[0001] The present disclosure relates to 2-[(2-oxo-4-phenyl-2H-chromen-7-yl)oxy]propanamide derivatives that inhibit the proliferation of cancer stem cells (CSCs) and senescent cells. [Background technology]

[0002] The biological process of aging continues to attract significant attention in the scientific and medical research community. Physiological aging is, at least in part, associated with an increased rate of oxidative damage to cellular components, including DNA, lipids, proteins, etc. Increased oxidative damage creates an imbalance that disrupts autoregulatory processes at the cellular level. Furthermore, aging correlates with the accumulation of lipofuscin in the cytoplasm of neurons. Recent studies have also shown that aging is the result of naturally occurring DNA damage that accumulates over time, leading to abnormal DNA changes. DNA damage, both in mitochondria and nuclei, may contribute to aging indirectly by increasing apoptosis and cellular senescence, and directly by increasing cellular dysfunction. Accumulated DNA damage may lead to the loss of cellular cells and loss and mutation of gene expression in surviving cells, an effect that produces signs of aging in cells that divide less frequently. Cellular senescence occurs when aged cells stop dividing, which is thought to occur following various environmental damaging events, abnormal cell growth, autophagy, and oxidative stress, among other factors. The Senescence Associated Secretory Phenotype ("SASP") is a hallmark of senescent cells and, depending on a number of factors, leads to proteotoxic impairment of healthy cellular functions, including inflammatory or anti-inflammatory, and pro- or anti-tumor effects. The effects of SASP-associated chronic inflammation affect the immune system's normal ability to clear senescent cells, and cells that provide immune functions may be recruited to senescent cells by SASP. Biomarkers of cellular senescence have been shown to accumulate as mammals age and contribute to a variety of age-related diseases, including Alzheimer's disease, lateral sclerosis, and type 2 diabetes. For cells that divide frequently, accumulated DNA damage may also be a major cause of cancer.

[0003] Aging thus increases the likelihood of cancer development, and researchers have struggled to develop new anti-cancer and anti-aging, or senolytic, therapies. Conventional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and antimetabolites such as 5-fluorouracil) have attempted to selectively detect and eradicate fast-growing cancer cells by interfering with cellular mechanisms involved in cell growth and DNA replication. Other cancer therapies have used immunotherapy (e.g., monoclonal antibodies) that selectively bind to mutant tumor antigens on fast-growing cancer cells. Unfortunately, after these therapies, tumors often recur at the same site or at one or more different sites, indicating that not all cancer cells have been eradicated. In particular, cancer stem cells survive for various reasons, resulting in treatment failure. Recurrence may be due to insufficient doses of chemotherapy agents and / or the emergence of cancer clones resistant to therapy. Thus, new cancer treatment strategies that overcome the shortcomings of conventional therapies are needed.

[0004] Advances in mutation analysis have allowed detailed study of genetic alterations that occur during cancer development. Despite knowledge of the genomic landscape, modern oncology has had difficulty identifying key driver mutations across cancer subtypes. The harsh reality is that each patient's tumor is unique, and a single tumor may contain multiple distinct clonal cells. What is needed are new approaches that focus on commonalities between different cancer types. Targeting metabolic differences between tumor and normal cells holds promise as a new cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed increases in more than 95 mRNA transcripts related to mitochondrial biogenesis and / or mitochondrial translation. Non-Patent Document 1. Furthermore, more than 35 of the 95 upregulated mRNAs code for mitochondrial ribosomal proteins (MRPs). Similarly, proteomic analysis of human breast cancer stem cells revealed significant overexpression of multiple mitoribosomal proteins and other proteins associated with mitochondrial biogenesis.

[0005] Mitochondrial metabolism in cancer cells has been the subject of recent exploratory research in the search for both anti-cancer and senolytic therapeutic targets. Mitochondria are highly dynamic organelles that constantly divide, elongate, and interconnect to form tubular networks or fragmented granules to meet cellular requirements and adapt to the cellular microenvironment. The balance between mitochondrial fusion and fission determines mitochondrial morphology, abundance, function, and spatial distribution, thus affecting many important mitochondria-dependent biological processes, such as adenosine triphosphate (ATP) production, mitophagy, apoptosis, and calcium homeostasis. And mitochondrial dynamics can be regulated by mitochondrial metabolism, respiration, and oxidative stress.

[0006] ATP is the universal bioenergetic "currency" of all living cells and tissues, including microorganisms such as prokaryotic bacteria and eukaryotic yeast. In eukaryotes, the mitochondrial organelle functions as the cellular "powerhouse." Mitochondria generate large amounts of ATP via the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis contributes minor amounts of ATP. Conversely, mitochondrial dysfunction induces ATP depletion, leading to mitochondrially driven apoptosis (programmed cell death) and / or necrosis. Thus, the inventors proposed that ATP depletion therapy could be a viable strategy to target and eradicate even the "fittest" cancer cells.

[0007] In MCF7 breast cancer cells, under normoxic conditions, mitochondrial-driven OXPHOS contributes 80-90% of ATP production, while glycolysis contributes only the remaining 10-20%. Thus, similar to normal cells, cancer cells are highly dependent on mitochondrial ATP production. However, it remains largely unknown whether cancer cell ATP levels contribute to 3D anchorage-dependent growth and cell migration, two hallmarks of metastatic spread. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Sotgia et al., Cell Cycle, 11(23):4390‐4401 (2012) [Non-Patent Document 2] Lamb et al., Oncotarget, 5(22):11029‐11037 (2014) Summary of the Invention [Problem to be solved by the invention]

[0009] What is needed, therefore, are new anti-aging compositions and methods for treating aging at the cellular level that overcome accumulated oxidative and DNA damage, as well as the many undesirable effects of aging.

[0010] Furthermore, what is needed are therapeutic agents that target unhealthy senescent cells and the SASP, reducing the accumulation of cellular senescence and counteracting chronic aging.

[0011] Furthermore, what is needed are therapeutic agents that target a broad range of CSCs due to characteristics common to CSCs regardless of cancer type.

[0012] Furthermore, what is needed are therapeutic agents that inhibit the proliferation of CSCs, including circulating tumor cells and tumor-initiating cells, which can lead to tumor recurrence and / or metastasis.

[0013] In view of the above background, it is an object of the present disclosure to describe therapeutic agents or compounds that can be used to reduce the accumulation of cellular senescence and inhibit CSC proliferation. It is an object of the present disclosure to describe therapeutic agents for use in eradicating CSCs and senescent cells. It is an object of the present disclosure to describe therapeutic agents for use in preventing and reducing the likelihood of tumor recurrence and metastasis. It is further an object of the present disclosure to describe compositions, such as pharmaceutical compositions, and methods for the treatment and prevention of cancer, including tumor recurrence and / or metastasis. It is also an object of the present disclosure to describe compositions, such as pharmaceutical compositions, and methods for senolytic therapy. [Means for solving the problem]

[0014] 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 compounds, and methods for treating cancer. The unexpectedly advantageous advantages of the inventive approach can be demonstrated by comparing the embodiments of the therapeutic agents described herein with the formula shown below:

[0015] [ka]

[0016] A compound known as an orally active mitochondrial RNA polymerase (POLRMT) inhibitor. The compound was first described in Bonekamp, ​​NA, Peter, B., Hillen, HS et al. Small-molecule inhibitors of human mitochondrial DNA transcription. Nature 588, 712-716 (2020), which is incorporated by reference in its entirety. As a POLRMT inhibitor, the compound inhibits mitochondrial DNA expression. The IUPAC name of this embodiment is 3-piperidinecarboxylic acid, 1-[(2R)-2-[[4-(2-chloro-4-fluorophenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-1-oxopropyl]-, (3S)-, and is assigned the CAS Registry Number 2304621-06-3.

[0017] In the approach of the present invention, some embodiments of the above compounds have the general chemical structure of Formula 1, as shown below:

[0018] [ka]

[0019] or a pharma- ceutically acceptable salt thereof, wherein: R1 and R2 may be the same or different and are selected from halogen, CF2H, -CF3, -OCF2H, -OCF3, substituted or unsubstituted C5-C18 carboxyl, substituted or unsubstituted C5-C18 alkane, substituted or unsubstituted C5-C18 alkene, substituted or unsubstituted C5-C18 cyclic alkene, substituted or unsubstituted C5-C18 alkyne, substituted or unsubstituted C5-C18 ketone, substituted or unsubstituted C5-C18 aldehyde, substituted or unsubstituted C5-C18 ether, substituted or unsubstituted C5-C18 ester, substituted or unsubstituted C5-C18 amine, substituted or unsubstituted C5-C18 amide, substituted or unsubstituted C5-C18 alkylamide, monocyclic or polycyclic arene, heteroarenes, phenols, or benzoic acids; R3 and R4 may be the same or different and are selected from hydrogen, substituted or unsubstituted C2-C18-alkyl, substituted or unsubstituted C3-C8-cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C18 carboxyl, substituted or unsubstituted C2-C18 alkene, substituted or unsubstituted C2-C18 alkyne, substituted or unsubstituted C2-C18 ketone, substituted or unsubstituted C2-C18 aldehyde, substituted or unsubstituted C2-C18 ether, substituted or unsubstituted C2-C18 ester, substituted or unsubstituted C2-C18 amine, substituted or unsubstituted C2-C18 amide, substituted or unsubstituted C2-C18 alkylamide, substituted or unsubstituted phenol, or benzoic acid, or one of R3 and R4 is selected from the group:

[0020] [ka]

[0021] [ka]

[0022] [ka] or

[0023] [ka]

[0024] and the other is H or C2-C5 alkyl; or R3 and R4 together form a substituted or unsubstituted C5 or C6 heterocycle; or R3 and R4 together form the group:

[0025] [ka]

[0026] or NR3R4 is replaced by OH; provided that at least one of R1 and R2 is a substituted or unsubstituted C5-C18 amide or at least one of R3 and R4 is a group:

[0027] [ka]

[0028] [ka]

[0029] [ka] or

[0030] [ka]

[0031] and the other is hydrogen or C2-C5 alkyl.

[0032] It should be understood that the lactic acid moiety of the propanamide portion of formula 1 may be modified in some embodiments with specific side chains or methyl may be replaced with hydrogen. For example, some embodiments of compounds according to the approach of the present invention have the general chemical structure of formula 2, shown below:

[0033] [ka]

[0034] or a pharma- ceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are as described above for formula 1, and R5 and R6 may be the same or different and are selected from hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C8-cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C10 carboxyl, substituted or unsubstituted C2-C10 alkene, substituted or unsubstituted C2-C10 alkyne, substituted or unsubstituted C2-C10 ketone, substituted or unsubstituted C2-C10 aldehyde, substituted or unsubstituted C2-C10 ether, substituted or unsubstituted C2-C10 ester, substituted or unsubstituted C2-C10 amine, substituted or unsubstituted C2-C10 amide, substituted or unsubstituted C2-C10 alkylamide, substituted or unsubstituted phenol, or benzoic acid.

[0035] The following paragraphs provide various examples of the approach of the present invention. It is understood that the length of the amide chain can vary from C5 to C18. In some embodiments, for example, the therapeutic agent has the formula:

[0036] [ka]

[0037] As another example, in some embodiments, the therapeutic agent has the formula:

[0038] [ka]

[0039] As another example, some embodiments of the above therapeutic agents have the formula:

[0040] [ka]

[0041] As another example, some embodiments of the above therapeutic agents have the formula:

[0042] [ka]

[0043] By way of further example, in some embodiments, the therapeutic agent has the formula:

[0044] [ka]

[0045] As another example, some embodiments of the above therapeutic agents have the formula:

[0046] [ka]

[0047] As mentioned above, it should be understood that some embodiments of the therapeutic agent may have a C5-C18 amide. In laboratory tests, amides shorter than C5 and longer than C18 have limited therapeutic activity. In preferred embodiments, the C10-C16 amides, and in particular the C12-C15 amides, show significantly improved therapeutic activity compared to the baseline compounds identified in paragraphs

[0017] to

[0031] above.

[0048] In some embodiments, one of R3 and R4 is a group:

[0049] [ka]

[0050] [ka]

[0051] [ka] or

[0052] [ka]

[0053] and the other is H or C2-C5 alkyl. For example, in the following exemplary structure, R1 is Cl, R2 is iso-butyl carbamate, and R3 is

[0054] [ka]

[0055] and R4 is C2.

[0056] [ka]

[0057] In the above example, R2 may vary as described above. For example, in another illustrative structure, R2 may be tert-butyl carbamate.

[0058] The approach of the present invention can also be used to treat and / or prevent tumor recurrence and / or metastasis. Anti-cancer treatments often fail due to tumor recurrence or metastasis, especially 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 in situations where conventional cancer therapy has failed and / or in conjunction with or prior to anti-cancer treatment to prevent or reduce the likelihood of treatment failure due to tumor recurrence and / or metastasis. [Brief description of the drawings]

[0059] [Figure 1A] 1A-1C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [I]. [Figure 1B] 1A-1C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [I]. [Figure 1C] 1A-1C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [I]. [Figure 2A] 2A-2C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [II]. [Figure 2B] 2A-2C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [II]. [Figure 2C] 2A-2C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [II]. [Figure 3A] 3A-3C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [III]. [Figure 3B] 3A-3C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [III]. [Figure 3C] 3A-3C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [III]. [Figure 4A] 4A and 4B show the results of tumor formation and the results of the SRB assay on MCF-7 cells (2D) for an embodiment of compound [IV]. [Figure 4B] 4A and 4B show the results of tumor formation and the results of the SRB assay on MCF-7 cells (2D) for an embodiment of compound [IV]. [Figure 5A] 5A-5C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [V]. [Figure 5B] 5A-5C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [V]. [Figure 5C] 5A-5C show the results of tumor mass formation, the results of an SRB assay on MCF-7 cells, and the results of an SRB assay on BJ1-hTERT cells for an embodiment of compound [V]. [Figure 6A] 6A and 6B show the results of tumor formation and SRB assay on MCF-7 cells for an embodiment of compound [VI]. [Figure 6B]6A and 6B show the results of tumor formation and SRB assay on MCF-7 cells for an embodiment of compound [VI]. [Figure 7A] 7A and 7B show the results of tumor formation and SRB assay on MCF-7 cells for an embodiment of compound [VII]. [Figure 7B] 7A and 7B show the results of tumor formation and SRB assay on MCF-7 cells for an embodiment of compound [VII]. [Figure 8] FIG. 8 shows quantitative assessment of tumor growth using the CAM assay for various concentrations of an embodiment of the inventive approach. [Figure 9] FIG. 9 shows quantitative assessment of metastatic invasion using the CAM assay for various concentrations of an embodiment of the inventive approach. [Figure 10] FIG. 10 shows toxicity assessment using the CAM assay for various concentrations of an embodiment of the inventive approach. [Figure 11A] 11A-D show the results of mitochondrial respiration, basal respiration, maximal respiration, and ATP production for metabolic flux analysis of adherent MCF7 cells treated with compounds according to the present approach. [Figure 11B] 11A-D show the results of mitochondrial respiration, basal respiration, maximal respiration, and ATP production for metabolic flux analysis of adherent MCF7 cells treated with compounds according to the present approach. [Figure 11C] 11A-D show the results of mitochondrial respiration, basal respiration, maximal respiration, and ATP production for metabolic flux analysis of adherent MCF7 cells treated with compounds according to the present approach. [Figure 11D] 11A-D show the results of mitochondrial respiration, basal respiration, maximal respiration, and ATP production for metabolic flux analysis of adherent MCF7 cells treated with compounds according to the present approach. [Figure 12A]12A-D show the results of an analysis of glycolytic function upon treatment with compounds according to the present approach. [Figure 12B] 12A-D show the results of an analysis of glycolytic function upon treatment with compounds according to the present approach. [Figure 12C] 12A-D show the results of an analysis of glycolytic function upon treatment with compounds according to the present approach. [Figure 12D] 12A-D show the results of an analysis of glycolytic function upon treatment with compounds according to the present approach. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] In the following description, embodiments of the inventive approach are illustrated in sufficient detail to enable the inventive approach to be practiced. The inventive approach is described with reference to these specific embodiments, but it should be understood that the inventive approach may be embodied in different forms, and this description should not be construed as limiting any 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 inventive approach to those skilled in the art.

[0061] This description uses various terms that will be understood by one of ordinary skill in the art. For the avoidance of doubt, the following clarifications are provided.

[0062] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell growth. This definition includes benign and malignant cancers. Examples of cancer include multiple carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial sarcoma), neuroendocrine tumors (including but not limited to carcinoid tumors, gastrin-producing tumors and pancreatic islet cell tumors), sarcomas, schwannomas (including acoustic neuroma), medullary carcinomas, adenocarcinomas, melanomas, and leukemias or lymphomas. 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, reproductive organ cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, as well as head and neck cancer and multiple myeloma.

[0063] As used herein, the term "tumor" refers to the growth and proliferation of neoplastic cells, whether malignant or benign, including pre-cancerous and cancerous cells and tissues.

[0064] 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 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 distant locations. Metastasis is a sequential process that requires tumor cells to escape from the primary tumor, travel through the bloodstream, and arrest at a distant site. At this new site, the cells establish blood flow, grow, and potentially form life-threatening masses. Both stimulatory and inhibitory molecular pathways within the tumor cells control this behavior, and interactions between tumor cells and host cells at distant sites are also important.

[0065] The terms "treat, treated, treating" and "treatment" include the alleviation or mitigation of at least one symptom associated with or caused by the condition, disorder or disease being treated, particularly cancer. In certain embodiments, the treatment includes the alleviation or mitigation of at least one symptom associated with or caused by the cancer being treated by the compounds of the invention. In some embodiments, the treatment includes causing the death of a category of cells, such as senescent cells, SASP cells, or CSCs, which may be involved in the metastasis or recurrence of a particular cancer in the host, and this can be achieved by preventing the further proliferation of senescent cells and / or cancer cells by depriving these cells of mechanisms to generate energy, and / or by inhibiting the function of CSCs. For example, the treatment can be the alleviation 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 mitochondrial metabolism in cancer; eradicate CSCs in cancer (e.g., kill at a rate faster than the rate of proliferation); eradicate TICs in cancer; eradicate circulating tumor cells in cancer; inhibit cancer proliferation; target and inhibit CSCs; target and inhibit TICs; target and inhibit circulating tumor cells; prevent or reduce the likelihood of metastasis; prevent recurrence; sensitize cancer to chemotherapeutic agents; sensitize cancer to radiation therapy; sensitize cancer to phototherapy. As another example, the treatment can reduce accumulated senescent cells and / or reduce the rate of accumulation of senescent cells.

[0066] 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 that may be involved in recurrence or metastasis in a subject, relative to a control (i.e., in the absence of a treatment to prevent or reduce the likelihood of tumor recurrence and / or metastasis), to a level at which tumor recurrence and / or metastasis from the primary site is unlikely to occur. In effect, the treatments described herein for preventing or reducing the likelihood of tumor recurrence and / or metastasis target and inhibit or eradicate CSCs, TICs, and circulating tumor cells.

[0067] The terms "cancer stem cell" and "CSC" refer to a subpopulation of cancer cells within a tumor that have the capacity for self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. Compared to "bulk" cancer cells, CSCs have increased mitochondrial mass, enhanced mitochondrial biogenesis, and high activity of mitochondrial protein translation. As used herein, "circulating tumor cells" are cancer cells that are shed from primary tumors into the vasculature or lymphatics and transported throughout the body in the blood circulation. The CellSearch circulating tumor cell test can be used to detect circulating tumor cells.

[0068] As used herein, the phrase "pharmaceutical effective amount" refers to the amount required to be administered to a host or to a cell, tissue, or organ of a host to achieve a therapeutic result, such as regulating, modulating, or inhibiting protein kinase activity, e.g., inhibiting the activity of a protein kinase, or treating cancer. A physician or veterinarian of ordinary skill in the art can easily 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 the dosage of a compound of the present invention used in a pharmaceutical composition at a level less than the amount required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Determining a pharmaceutical effective amount is believed to be within the skill of one of ordinary skill in the art upon review of this disclosure.

[0069] As used herein, the phrase "therapeutic agent" refers to an embodiment of a compound described herein and may include pharma- ceutically acceptable salts or isotopic analogs thereof. It is to be understood that the therapeutic agent may be administered to a subject by any suitable approach known to one of ordinary skill in the art. It is also to be understood that the amount of therapeutic agent and the timing of its administration may 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 of subject-to-subject variability, any dosages described herein are intended to be initial guidelines, and the physician may titrate the dose of the therapeutic agent to achieve the treatment he or she deems appropriate for the subject. In considering the degree of treatment desired, the physician may balance a variety of factors, such as the age and weight of the subject, the presence or absence of pre-existing diseases, and the presence or absence of other diseases. Pharmaceutical formulations may be prepared for any desired route of administration, including, but not limited to, oral, intravenous, or aerosol administration, as discussed in more detail below.

[0070] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically 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 harmful to the patient. Some examples of materials that can function as pharma- ceutically 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, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) 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) buffers, 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 buffers; and (21) other non-toxic and compatible substances used in pharmaceutical preparations.

[0071] The phrase "pharmaceutically acceptable salt" refers to relatively low toxicity inorganic and organic basic addition salts of the compounds of the present approach. Pharmaceutically acceptable salts can be formed by reacting the free acid form of the compound with a base, for example, by reacting a hydroxide or carbonate of a pharmaceutically acceptable metal cation with ammonia or with 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, piperazine. Of course, it is understood that other salts may be used, and that one skilled in the art can identify suitable salt forms using methods known in the art without departing from the present approach.

[0072] As used herein, a "fatty acid" moiety is a carboxylic acid having a saturated or unsaturated aliphatic chain. Examples of fatty acids include short chain fatty acids (i.e., those having 5 or fewer carbon atoms in their chemical structure), medium chain fatty acids (i.e., those having 6-12 carbon atoms in their chemical structure), and other long chain fatty acids (i.e., those having 13-21 carbon atoms in their chemical structure). An example of a saturated fatty acid is lauric acid (CH3(CH2) 10 COOH), Palmitic acid (CH3(CH2) 14 COOH), stearic acid (CH3(CH2) 16 COOH), and myristic acid (CH3(CH2) 12Oleic acid (CH3(CH2)7CH=CH(CH2)7COOH) is an example of a naturally occurring unsaturated fatty acid. It is understood that in some embodiments, the compounds of the present approach include linear saturated fatty acids or amide groups, preferably having 5-18 carbon atoms, more preferably 10-16 carbon atoms, and even more preferably 12-15 carbon atoms. In some preferred embodiments, the linear saturated fatty acid is myristic acid, or the amide is tetradecane amide, which have 14 carbon atoms.

[0073] Recent developments have identified first-in-class inhibitors of mitochondrial RNA polymerase targeting mitochondrial transcription (POLRMT), a gene product responsible for mitochondrial gene expression and mitochondrial biogenesis in the OXPHOS system. These inhibitors were used in a 4-week study in mice. Oral treatment with these compounds showed no evidence of OXPHOS dysfunction or toxicity in mice, but demonstrated potent antitumor effects in human cancer cell xenografts.

[0074] The embodiments of the compounds disclosed herein can be used as therapeutic agents to selectively eradicate CSCs for the treatment and / or prevention of tumor recurrence and / or metastasis. 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 MCF7 cells using a tumor nodule formation assay, and cell viability using a sulforhodamine B assay (also known in the art and referred to herein as an SRB assay), which measures the amount of residual protein attached to a tissue culture dish, a surrogate marker of cell viability.

[0075] In the present approach, embodiments of therapeutic compounds have the general chemical structure of Formula 1, shown below:

[0076] [ka]

[0077] or a pharma- ceutically acceptable salt thereof, wherein: R1 and R2 may be the same or different and may be halogen, CF2H, -CF3, -OCF2H, -OCF3, substituted or unsubstituted C5-C18 carboxyl, substituted or unsubstituted C5-C18 alkane, substituted or unsubstituted C5-C18 alkene, substituted or unsubstituted C5-C18 cyclic alkene, substituted or unsubstituted C5-C18 alkyne, substituted or unsubstituted C5-C18 ketone, substituted or unsubstituted C5-C18 aldehyde, substituted or unsubstituted C5-C18 ether, substituted or unsubstituted C5-C18 ester, substituted or unsubstituted C5-C18 amine, substituted or unsubstituted C5-C18 amide, substituted or unsubstituted C5-C18 alkylamide, monocyclic or polycyclic arene, heteroarene, phenol, or benzoic acid; R3 and R4 may be the same or different and may be hydrogen, substituted or unsubstituted C2-C18-alkyl, substituted or unsubstituted C3-C8-cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C18 carboxyl, substituted or unsubstituted C2-C18 alkene, substituted or unsubstituted C2-C18 alkyne, substituted or unsubstituted C2-C18 ketone, substituted or unsubstituted C2-C18 aldehyde, substituted or unsubstituted C2-C18 ether, substituted or unsubstituted C2-C18 ester, substituted or unsubstituted C2-C18 amine, substituted or unsubstituted C2-C18 amide, substituted or unsubstituted C2-C18 alkylamide, substituted or unsubstituted phenol, or benzoic acid, or one of R3 and R4 may be a group:

[0078] [ka]

[0079] [ka]

[0080] [ka] or

[0081] [ka]

[0082] and the other is H or C2-C5 alkyl; or R3 and R4 together form a substituted or unsubstituted C5 or C6 heterocycle; or R3 and R4 together form the group:

[0083] [ka]

[0084] or NR3R4 is OH; provided that at least one of R1 and R2 is a substituted or unsubstituted C5-C18 amide or at least one of R3 and R4 is:

[0085] [ka]

[0086] and the other is H or C2-C5 alkyl.

[0087] In some preferred embodiments, at least one of R1 and R2 is a substituted or unsubstituted C5-C18 amide, or at least one of R3 and R4 is a group:

[0088] [ka]

[0089] [ka]

[0090] [ka] or

[0091] [ka]

[0092] and the other is H or C2-C5 alkyl.

[0093] Compounds having the general structure of Formula 1 can be prepared according to the following general reaction scheme, where a substituted benzoic acid may be used as the starting compound: The functional groups R1, R2, R3, and R4 may be as described above.

[0094] [ka]

[0095] It will be appreciated that other synthetic methods may be used to arrive at the compounds of the inventive approach.

[0096] The following compounds are examples of therapeutic agents according to the approach of the present invention, where R1 is a halogen and R2 is an alkylamide group. It is understood that R1 and R2 can also occupy other positions on the phenyl group without departing from the approach of the present invention.

[0097] [ka]

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] The following compounds are examples of therapeutic agents according to the approach of the present invention, which show variations in R3 and R4, and also show various enantiomers. As will be understood by one of ordinary skill in the art, R1 and R2 refer to functional groups, and the "R" and "S" symbols indicate the enantiomer of the stereocenter of a particular embodiment.

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] [ka]

[0108] [ka]

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] Compounds having the general structure of formula 1, where R1 is halogen (e.g., chloro or fluoro) and R2 is an alkylamide, can be prepared according to the following general reaction scheme, where a substituted benzoic acid may be used as the starting compound. The functional groups R1, R2, R3, and R4 may be as described above, with specific examples being described below.

[0113] [ka] Step:

[0114] It is understood that the halogens and alkylamides can occupy different positions on the phenyl other than those shown in the examples above.

[0115] As another example, some embodiments of the therapeutic agents described herein can be prepared according to the following reaction schemes.

[0116] [ka] Step:

[0117] In the embodiment shown, R1 is fluoro, R2 is alkylamido, R3 is ethyl, and R4 is (dimethylamino)propylamido. As with other exemplary embodiments described herein, it is understood that the halogen and alkylamido can occupy different positions on the phenyl other than those shown in the above example.

[0118] The compounds of the present approach exhibit significantly improved therapeutic activity compared to compound [I], which has the chemical structure shown below:

[0119] [ka]

[0120] The IC value of compound [I], 3-piperidinecarboxylic acid, 1-[(2R)-2-[[4-(2-chloro-4-fluorophenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-1-oxopropyl]-, (3S)- (CAS registration number: 2304621-06-3), against MCF-7 cells 50 is approximately 50 μM. Figures 1A-1C show the results of tumor mass formation, SRB assay results for MCF-7 cells, and SRB assay results for BJ1-hTERT cells for compound [I]. As can be seen from Figure 1A, compound [I] showed inhibition of MCF-7 cells at a concentration of 5 μM, and achieved more than 50% inhibition at a concentration of 100 μM. Figures 1B and 1C show that compound [I] showed no cytotoxicity to MCF-7 cells or BJ1-hTERT cells at all tested concentrations from 1 μM to 100 μM.

[0121] In some embodiments, the therapeutic agent can take the form of compound [II], (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid, having the chemical structure shown below:

[0122] [ka]

[0123] Compound II, (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid, can be synthesized according to the following process: In step 1, ethyl (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylate was prepared as an intermediate. To a stirred solution of (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (147 mg, 0.25 mmol) and (S)-ethylpiperidine-3-carboxylate in DMA (1 ml) at room temperature and under nitrogen atmosphere was added NMM (35.5 μl, 0.32 mmol) and EDC hydrochloride (62 mg, 0.32 mmol). The mixture was stirred for 16 h, diluted with EtOAc (40 ml), washed with 2M HCl (15 ml), water (15 ml), saturated NaHCO3 (15 ml), brine (15 ml), dried over MgSO4, filtered, and the filtrate was evaporated under reduced pressure to give the crude product. Purification on silica gel (30% EtOAc in isohexane) gave ethyl (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylate, (19.6 mg). LC-MS 709.4[M+1] + , RT6.86 minutes.

[0124] In step 2, (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid was prepared as an intermediate. To a stirred solution of ethyl (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylate (19 mg, 0.027 mmol) in THF (1.5 ml) and MeOH (0.5 ml) at room temperature, a solution of 1 M NaOH (75 μl, 0.075 mmol) was added and the mixture was stirred for 16 h. The solvent was evaporated under reduced pressure, the residue was suspended in EtOAc (30 ml) and acidified with 2M HCl (10 ml), the extract was washed with brine (10 ml), dried over MgSO4, the solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give (3S)-1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperidine-3-carboxylic acid as a solid (17.3 mg). LC-MS 681.4[M+1] + , RT6.39 minutes.

[0125] Figures 2A-2C show the results of tumor nodule formation, SRB assay results for MCF-7 cells, and SRB assay results for BJ1-hTERT cells for compound [II]. As can be seen from Figure 2A, compound [II] showed dose-dependent inhibition of MCF-7 cells at all concentrations tested, ranging from 0.1 μM to 100 μM. Compound [II] achieved more than 50% inhibition at a concentration of 10 μM and 100% inhibition at a concentration of 100 μM. With an IC50 of approximately 5 μM, compound [II] is 10 times more potent than compound [I] in inhibiting MCF-7 tumor nodule formation. Figures 2B and 2C show that compound [II] shows cytotoxicity against MCF-7 cells and BJ1-hTERT cells from 50 μM.

[0126] In some embodiments, the therapeutic agent can take the form of a compound [III], tert-butyl-N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate, having the chemical structure shown below:

[0127] [ka]

[0128] Compound [III] was prepared from 2-chloro-4-nitrobenzoic acid according to the method described in Advance Synthesis and Catalysis, 2014, vol.356, no. 11-12, p2437-2444 to give crude 2-chloro-4-nitro-benzoyl chloride as a light brown oil (11.78 g). Ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxo-propanoate was then prepared from the above crude 2-chloro-4-nitro-benzoyl chloride according to the method described in step 1 for compound [IV] to give ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxo-propanoate as a light brown solid (11.04 g). LC-MS 272.0[M+1] + , RT 5.39 min.

[0129] Then 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chromen-2-one was prepared from above ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxo-propanoate according to the method of step 2 of compound [IV] to obtain 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chromen-2-one as a purple solid (5.85g). LC-MS 318.0[M+1] + , RT5.18min.

[0130] Then 4-(4-amino-2-chloro-phenyl)-7-hydroxy-chromen-2-one was prepared from 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chromen-2-one according to the method of step 3 of compound [IV] to obtain 4-(4-amino-2-chloro-phenyl)-7-hydroxy-chromen-2-one as a dark red-brown solid (3.02 g). LC-MS 287.8[M+1] + , RT4.43 minutes.

[0131] Using the above 4-(4-amino-2-chloro-phenyl)-7-hydroxy-chromen-2-one, ethyl (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate was prepared according to the method of step 4 of compound [IV] to obtain ethyl (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate as a light brown solid (1.57g). LC-MS 388.0[M+1] + , RT 5.27 min.

[0132] Ethyl (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo-chromen-7-yl]oxypropanoate was subsequently prepared. To a stirred solution of ethyl (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate (0.26 g, 0.70 mmol) and DMAP (0.21 g, 1.70 mmol) in DCM (20 ml) at room temperature and under nitrogen atmosphere was added Boc anhydride (0.37 g, 1.70 mmol). The mixture was stirred for 16 h and the solvent was evaporated under reduced pressure to give the crude product. The residue was dissolved in EtOAc (40 ml), washed with 2M HCl (20 ml), saturated NaHCO3 (20 ml), brine (20 ml), dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. Trituration with n-pentane gave ethyl (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo-chromen-7-yl]oxypropanoate (0.54 g). LC-MS 488.0[M+1] + , RT6.60 minutes.

[0133] As the next step, (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid was prepared from ethyl (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo-chromen-7-yl]oxypropanoate according to the method of step 6 of compound [IV] to obtain (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid as a light brown solid (0.29g). LC-MS 460.0[M+1] + , RT 5.98 min.

[0134] Finally, tert-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate was prepared according to the method of step 3 of Example 6, (2R)-2-[4-[4-(tert-butoxycarbonylamino)-2-chloro-phenyl]-2-oxo- Prepared from [men-7-yl]oxypropanoic acid, tert-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate (structure shown below; "Boc" stands for tert-butyloxycarbonyl) was obtained as a light brown solid (0.15 g). LC-MS 615.2[M+1] + , RT4.38 minutes.

[0135] [ka]

[0136] Figures 3A-3C show the results of tumor mass formation, SRB assay for MCF-7 cells, and SRB assay for BJ1-hTERT cells for compound [III]. As can be seen from Figure 3A, compound [III] showed dose-dependent inhibition of MCF-7 cells at all concentrations tested, ranging from 1 μM to 10 μM. The IC50 of compound [III] was 1 μM to 2.5 μM, which is 25-fold improved compared to compound [I] at a concentration of 10 μM. Furthermore, compound [III] achieved 100% inhibition of MCF-7 cells. Figures 3B and 3C show that compound [III] exhibits cytotoxicity against MCF-7 cells and BJ1-hTERT cells from 10 μM.

[0137] In some embodiments, the therapeutic agent can take the form of compound [IV], N-[2-[7-[(1R)-2-[3-(dimethylamino)propylcarbamoyl-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]-5-fluoro-phenyl]tetradecanamide, having the chemical structure shown below:

[0138] [ka]

[0139] Compound [IV] was prepared using compound [VII], (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, as an intermediate. The synthesis of compound [VII] is described below. To a stirred solution of (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (60 mg, 0.10 mmol) in DMA (4 ml) at room temperature and under nitrogen atmosphere, NMM (23 μl, 0.21 mmol) and EDC hydrochloride (27 mg, 0.14 mmol) were added. The mixture was stirred for 16 hours and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (2.5% MeOH in DCM) gave N-[2-[7-[(1R)-2-[3-(dimethylamino)propylcarbamoyl-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]-5-fluoro-phenyl]tetradecanamide (36.8 mg, 52%). LC-MS 709.6[M+1] + , RT5.14min.

[0140] Figures 4A and 4B show the results of tumor mass formation and SRB assay for MCF-7 cells for compound [IV]. Figure 4A shows that compound [IV] shows dose-dependent inhibition of MCF-7 cells at all concentrations tested, ranging from 1 μM to 10 μM. Compound [IV] showed more than 50% inhibition at 2.5 μM and almost complete inhibition at 10 μM. Figure 4B shows that compound [IV] shows cytotoxicity against MCF-7 T cells from 5 μM.

[0141] In some embodiments, the therapeutic agent can take the form of compound [V], N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide, having the chemical structure shown below:

[0142] [ka]

[0143] Compound [V] was synthesized as follows. In step 1, tert-butyl N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-carbamate was generated as an intermediate. To a stirred solution of Boc-N-ethylglycine (1.02 g, 5.0 mmol) and NMM (0.79 ml, 7.5 mmol) in DCM (30 ml) at 0° C. and under nitrogen atmosphere, iso-butyl chloroformate (0.82 ml, 6.0 mmol) was added. The mixture was stirred for 1 hour, N,N-dimethylethylenediamine (0.83 ml, 7.5 mmol) was added, and the mixture was allowed to warm to room temperature and stirred for 60 minutes. The reaction mixture was diluted with DCM (30 ml), washed with saturated NaHCO3 (30 ml), brine (30 ml), dried over MgSO4, filtered and the filtrate was evaporated under reduced pressure to give tert-butyl N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-carbamate as a light brown oil (1.44 g). LC-MS 274.3[M+1] + , RT 0.86 min.

[0144] In step 2, N-[2-(dimethylamino)ethyl]-2-(ethylamino)acetamide hydrochloride was prepared. To a stirred solution of tert-butyl N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-carbamate (1.30 g, 4.8 mmol) in chloroform (30 ml) at room temperature under nitrogen atmosphere, 4M HCl in dioxane (23 ml, 95 mmol) was added and the mixture was stirred for 20 hours. The solvent was evaporated under reduced pressure to give N-[2-(dimethylamino)ethyl]-2-(ethylamino)acetamide hydrochloride as a light brown solid, (1.15 g). LC-MS 174.3[M+1] + , RT 0.27 min.

[0145] In step 3, N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide was prepared from the above product. To a stirred solution of (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (40 mg, 0.070 mmol) and NMM (23 μl, 0.21 mmol) in dry DCM (2 ml) at room temperature and under nitrogen atmosphere, iso-butyl chloroformate (11 μl, 0.084 mmol) was added. The mixture was stirred for 1 h, N-[2-(dimethylamino)ethyl]-2-(ethylamino)acetamide hydrochloride (27 mg, 0.11 mmol) was added, and the mixture was stirred for 17 h. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in EtOAc (20 ml), washed with water (10 ml), brine (10 ml), dried over MgSO4, filtered, and the filtrate was evaporated under reduced pressure. Purification on silica gel (5-10% MeOH in DCM; 1% 7M NH3 in MeOH) gave compound [V], N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide (14.9 mg). LC‐MS 725.6[M+1] + , RT5.35min.

[0146] Figures 5A-5C show the results of tumor mass formation, SRB assay for MCF-7 cells, and SRB assay for BJ1-hTERT cells for compound [V]. Figure 5A shows that compound [V] effectively inhibited MCF-7 cells at all concentrations tested, ranging from 0.5 μM to 10 μM. At 0.5 μM, more than 50% inhibition was observed, and at a concentration of 5 μM, compound [V] achieved 100% inhibition of MCF-7 cells. Figures 5B and 5C show that compound [V] exhibits cytotoxicity against MCF-7 cells and BJ1-hTERT cells from 5 μM.

[0147] In some embodiments, the therapeutic agent can take the form of compound [VI], (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, having the chemical structure shown below:

[0148] [ka]

[0149] Compound [VI] was synthesized according to the following approach, it being understood that alternative methods may also be used.

[0150] In step 1, methyl 2-chloro-4-(tetradecanoylamino)benzoate was produced as the initial intermediate. To a stirred suspension of methyl 4-amino-2-chlorobenzoate (1.87 g, 10.1 mmol) and NMM (1.50 ml, 13.6 mmol) in dry THF (30 ml) at room temperature and under nitrogen atmosphere, tetradecanoyl chloride (2.42 g, 9.80 mmol) in dry DCM (10 ml) was added and the mixture was stirred at room temperature for 20 h. The solvent was evaporated under reduced pressure, the residue was dissolved in EtOAc (50 ml), washed with 2M HCl (50 ml), saturated NaHCO3 (50 ml), brine (50 ml), dried over MgSO4, filtered and the filtrate was concentrated under reduced pressure to give crude methyl 2-chloro-4-(tetradecanoylamino)benzoate as a light brown solid (4.10 g). LC-MS 396.1 [M+1] + , RT6.50 minutes.

[0151] In step 2, the compound 2-chloro-4-(tetradecanoylamino)benzoic acid was prepared. To a stirred solution of methyl 2-chloro-4-(tetradecanoylamino)benzoate (3.60 g, 9.08 mmol) in THF (50 ml) and MeOH (50 ml) at room temperature, a solution of NaOH (8 g, 200 mmol) in water (50 ml) was added and the mixture was stirred for 16 h. The solvent was evaporated under reduced pressure, the residue was acidified with 6M HCl (50 ml), diluted with water (50 ml) and the product was extracted with EtOAc (100 ml). The extract was washed with brine (ClH2NaO, 50 ml), dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to give the crude product 2-chloro-4-(tetradecanoylamino)benzoic acid as a pale yellow solid (3.40 g). LC-MS 382.2[M+1] + , RT6.73 minutes.

[0152] In step 3, 2-chloro-4-(tetradecanoylamino)benzoyl chloride was prepared. To a stirred suspension of 2-chloro-4-(tetradecanoylamino)benzoic acid (2.00 g, 5.24 mmol) and DMF (4 drops) in dry DCM (100 ml) at room temperature and under nitrogen atmosphere, oxalyl chloride (0.91 ml, 10.47 mmol) was added. The mixture was stirred at room temperature for 20 h and concentrated under reduced pressure to give the crude product 2-chloro-4-(tetradecanoylamino)benzoyl chloride as a yellow solid (2.30 g). LC-MS 396.3 [M-OMe+1] + , RT7.21 minutes.

[0153] In step 4, the compound ethyl 3-[2-chloro-4-(tetradecanoylamino)phenyl]-3-oxo-propanoate was prepared from 2-chloro-4-(tetradecanoylamino)benzoyl chloride as follows: To a stirred suspension of potassium ethylmalonate (1.9 g, 11.19 mmol) in dry MeCN (30 ml) under nitrogen atmosphere at +10° C., TEA (1.65 ml, 11.74 mmol) and anhydrous magnesium chloride (1.28 g, 13.42 mmol) were added, and the mixture was stirred at room temperature for 3.5 hours. A solution of 4-fluoro-2-nitrobenzoyl chloride (1.14 g, 5.59 mmol) prepared according to Archiv der Pharmazie, 1985, vol. 318, No. 1, p. 78-84 in dry MeCN (2 ml) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a stirred mixture of saturated NH4Cl (2 ml) and EtOAc (50 ml) and the solvent was evaporated under reduced pressure. The solid residue was stirred with 4M HCl (30 ml) at room temperature for 30 min and the product was extracted with EtOAc (50 ml). The extract was washed with 4M HCl (30 ml), water (30 ml), brine (30 ml), dried over MgSO4, filtered and the filtrate was concentrated under reduced pressure to give the crude product. Purification on silica gel (15% EtOAc in iso-hexane) gave ethyl 3-[2-chloro-4-(tetradecanoylamino)phenyl]-3-oxo-propanoate as a yellow solid (2.10 g). LC-MS 452.2[M+1] + , RT7.05 minutes.

[0154] In step 5, N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide was prepared from ethyl 3-[2-chloro-4-(tetradecanoylamino)phenyl]-3-oxo-propanoate according to step 2 of the preparation of compound [VII] described below. This gave N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide as a yellow solid (0.30 g). LC-MS 498.2[M+1] +, RT6.99 minutes.

[0155] In step 6, compound ethyl (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate was prepared from N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecaneamide according to step 4 of the preparation of compound [VII] described below. This gave ethyl (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate as a yellow solid (0.25 g). LC-MS 598.3[M+1] + , RT7.83 minutes.

[0156] In step 7, (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, compound [VI], was prepared from ethyl (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate according to the method described in step 6 of the preparation of compound [VII] below. This gave a light brown solid (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, (0.15 g). LC-MS 570.4[M+1] + , RT 2.65 min.

[0157] Figures 6A and 6B show the results of tumor formation and SRB assay on MCF-7 cells for compound [VI]. Inhibitory activity was observed at all tested concentrations from 1 μM to 100 μM, with over 50% inhibition achieved at 10 μM. Significant improvement was observed increasing from 50 μM to 100 μM, and further evaluation between these concentrations is envisioned. With regard to the SRB assay, compound [VI] showed little toxicity across multiple concentrations tested, as seen in Figure 6B.

[0158] In some embodiments, the therapeutic agent can take the form of compound [VII], which has the chemical structure shown below:

[0159] [ka]

[0160] Compound [VII], (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, was synthesized according to the following approach. In step 1, ethyl 3-(4-fluoro-2-nitro-phenyl)-3-oxo-propanoate was prepared. To a stirred suspension of potassium ethylmalonate (1.9 g, 11.19 mmol) in dry MeCN (30 ml) under nitrogen atmosphere at +10°C, TEA (1.65 ml, 11.74 mmol) and anhydrous magnesium chloride (1.28 g, 13.42 mmol) were added, and the mixture was stirred at room temperature for 3.5 hours. A solution of 4-fluoro-2-nitrobenzoic acid chloride (1.14 g, 5.59 mmol) prepared according to Archiv der Pharmazie, 1985, vol. 318, No. 1, p. 78-84 in dry MeCN (2 ml) was added and the mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a stirred mixture of saturated NH4Cl (2 ml) and EtOAc (50 ml) and the solvent was evaporated under reduced pressure. The solid residue was stirred with 4M HCl (30 ml) at room temperature for 30 minutes and the product was extracted with EtOAc (50 ml). The extract was washed with 4M HCl (30 ml), water (30 ml), brine (30 ml), dried over MgSO4, filtered and the filtrate was concentrated under reduced pressure to give the crude product. Purification on silica gel (15% EtOAc in iso-hexane) gave ethyl 3-(4-fluoro-2-nitro-phenyl)-3-oxo-propanoate (1.16 g, 81%). LC-MS 256.1 [M+1]+, RT 4.08 min.

[0161] In step 2, 4-(4-fluoro-2-nitro-phenyl)-7-hydroxy-chromen-2-one was produced as an intermediate. To a stirred solution of ethyl 3-(4-fluoro-2-nitro-phenyl)-3-oxo-propanoate (0.60 g, 2.30 mmol) in methanesulfonic acid (4 ml) at room temperature under nitrogen atmosphere, resorcinol (0.31 g, 2.20 mmol) was added and the mixture was stirred at +45° C. for 1 h. The reaction mixture was cooled to room temperature and poured into a mixture of water: EtOAc (40 ml: 10 ml) and the product was extracted with EtOAc (50 ml). The extract was washed with water (40 ml), brine (40 ml), dried over MgSO4, the solid residue was removed by filtration and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (40-50% EtOAc in iso-hexane) gave 4-(4-fluoro-2-nitro-phenyl)-7-hydroxy-chromen-2-one (0.43 g, 65%). LC-MS 302.1 [M+1]+, RT 4.22 min.

[0162] In step 3, 4-(2-amino-4-fluoro-phenyl)-7-hydroxy-chromen-2-one was produced. To a stirred ice-cold solution of 4-(4-fluoro-2-nitro-phenyl)-7-hydroxy-chromen-2-one (0.21 g, 0.70 mmol) and DiPEA (0.60 ml, 3.50 mmol) in dry DCM (5 ml) under nitrogen atmosphere, a solution of trichlorosilane (0.35 ml, 3.5 mmol) in dry DCM (2 ml) was added over 10 min. The mixture was stirred at +0° C. for 1 h, quenched with saturated NaHCO3 (3 ml), and diluted with water (20 ml) and EtOAc (30 ml) to give a suspension, which was filtered through a pad of Celite. The pH was adjusted to 8-9 by adding solid NaHCO3, the organic phase was separated, washed with brine (20 ml), dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (40-50% EtOAc in iso-hexane) gave 4-(2-amino-4-fluoro-phenyl)-7-hydroxy-chromen-2-one (0.20 g, 99%). LC-MS 302.1[M+1]+, RT 4.22 min.

[0163] In step 4, ethyl (2R)-2-[4-(2-amino-4-fluoro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate was produced as an intermediate. DEAD (293 μl, 1.86 mmol) was added to a stirred ice-cold solution of 4-(2-amino-4-fluoro-phenyl)-7-hydroxy-chromen-2-one (0.33 g, 1.24 mmol), ethyl-S-lactate (212 μl, 1.86 mmol), and triphenylphosphine (0.32 ml, 1.24 mmol) in dry THF (10 ml) under nitrogen atmosphere. The mixture was stirred at +0° C. for 0.5 h, warmed to room temperature and stirred for 16 h. The solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (50% diethyl ether in iso-hexane) gave ethyl (2R)-2-[4-(2-amino-4-fluoro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate (0.43 g, 93%). LC-MS 372.3[M+1] +, RT5.12min.

[0164] In step 5, ethyl (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate was prepared. To a stirred solution of ethyl (2R)-2-[4-(2-amino-4-fluoro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate (0.26 g, 0.70 mmol) in dry pyridine (3 ml) at room temperature and under nitrogen atmosphere, tetradecanoyl chloride (380 μl, 1.40 mmol) was added in two portions. The mixture was stirred for 16 hours and the solvent was evaporated under reduced pressure to give the crude product. The residue was dissolved in iso-propanol (10 ml) and ethyl (2R)-2-[4-(2-amino-4-fluoro-phenyl)-2-oxo-chromen-7-yl]oxypropanoate (0.26 g, 0.70 mmol) in dry pyridine (3 ml) was added in two portions. The mixture was stirred for 16 hours and the solvent was evaporated under reduced pressure to give the crude product. The crude product was obtained by diluting with EtOAc (20 ml), washing with saturated NaHCO3 (20 ml), brine (20 ml), drying over MgSO4, filtering and evaporating the solvent under reduced pressure. Purification on silica gel (10-15% EtOAc in isohexane) gave ethyl (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate (0.21 g, 51%). LC-MS 580.4[M+1] + , RT7.20 minutes.

[0165] In step 6, (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid, compound [VII], was prepared. To a stirred solution of ethyl (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate (0.20 g, 0.34 mmol) in THF (4 ml) at room temperature and nitrogen atmosphere was added 1M NaOH (2 ml, 2.00 mmol). The mixture was stirred for 5 h, diluted with EtOAc (30 ml), washed with 2H HCl (10 ml), water (10 ml), brine (20 ml), dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (4% MeOH in DCM) gave (2R)-2-[4-[4-fluoro-2-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (0.15 g, 79%). LC-MS 554.5[M+1] + , RT7.00 minutes.

[0166] Figures 7A and 7B show the results of tumor formation and SRB assay on MCF-7 cells for compound [VII]. Inhibitory activity was confirmed at all tested concentrations from 1 μM to 100 μM, with over 50% inhibition achieved at 1 μM. At a concentration of 100 μM, compound [VII] showed 100% inhibition of MCF-7 cells. With regard to the SRB assay, Figure 7B shows that compound [VII] has little toxicity across multiple tested concentrations below 100 μM.

[0167] The following example illustrates the synthesis of iso-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate (shown below).

[0168] [ka]

[0169] In step 1, (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxy-N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-propanamide was prepared. To a stirred solution of tert-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate (0.125 g, 0.20 mmol) in dry DCM (3 ml) at room temperature and under nitrogen atmosphere, TFA (3 ml) was added. The mixture was stirred for 1 h and the solvent was evaporated under reduced pressure to give the crude product. The residue was dissolved in EtOAc (30 ml), saturated NaHCO3 (10 ml), brine (10 ml), dried over MgSO4, filtered and the solvent was evaporated under reduced pressure to give (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxy-N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-propanamide (0.11 g). LC-MS 515.0[M+1] + , RT 3.31 min.

[0170] In step 2, iso-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate was reacted with (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxy- Prepared from N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-propanamide, tert-butyl N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]carbamate was obtained as a light brown solid (10.1 mg). LC-MS 615.2[M+1] + , RT4.38 minutes.

[0171] The following illustrates the synthesis of N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]dodecanamide (shown below).

[0172] [ka]

[0173] To a stirred solution of (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxy-N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-propanamide (30 mg, 0.058 mmol) and pyridine (0.5 ml) in methyltetrahydrofuran (2 ml) at room temperature and under nitrogen atmosphere was added lauryl chloride (16 μl, 0.073 mmol). The mixture was stirred for 16 hours and the solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc (30 ml), water (10 ml), brine (10 ml), dried over MgSO4, filtered, the solvent was evaporated under reduced pressure, the residue was dissolved in EtOAc (20 ml), washed with water (10 ml), brine (10 ml), dried over MgSO4, filtered and the filtrate was evaporated under reduced pressure to give the crude product. Purification on silica gel (5-10% MeOH in DCM; 1-2% 7M NH3 in MeOH) gave N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]dodecanamide (27.3 mg). LC-MS 697.2[M+1] + , RT 5.47 min.

[0174] The following example illustrates the synthesis of N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]hexanamide (shown below).

[0175] [ka]

[0176] The title compound was prepared from (2R)-2-[4-(4-amino-2-chloro-phenyl)-2-oxo-chromen-7-yl]oxy-N-[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-N-ethyl-propanamide according to the method of Example 9 to give N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]hexanamide as a light brown solid (20.0 mg). LC-MS 613.1[M+1] + , RT4.93 minutes.

[0177] It should be understood that the lactic acid moiety of the propanamide portion of formula 1 may be modified in some embodiments with specific side chains or methyl may be replaced with hydrogen. For example, some embodiments of compounds according to the approach of the present invention have the general chemical structure of formula 2, shown below:

[0178] [ka]

[0179] or a pharma- ceutically acceptable salt thereof, wherein R1, R2, R3, and R4 are as described above for formula 1, R5 and R6 may be the same or different and may be hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C8-cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C10 carboxyl, substituted or unsubstituted C2-C10 alkene, substituted or unsubstituted C2-C10 alkyne, substituted or unsubstituted C2-C10 ketone, substituted or unsubstituted C2-C10 aldehyde, substituted or unsubstituted C2-C10 ether, substituted or unsubstituted C2-C10 ester, substituted or unsubstituted C2-C10 amine, substituted or unsubstituted C2-C10 amide, substituted or unsubstituted C2-C10 alkylamide, substituted or unsubstituted phenol, or benzoic acid.

[0180] Preferably, R5 and R6 are the same or different and are H or a substituent in a substituent-substituted alpha-hydroxy acid (AHH). Below are examples of structures according to the approach of the present invention, which show variations of R5, R6, including their various enantiomers and motifs. It should be understood that only a portion of the structure is shown, and that the entire structure of the given example can be understood by considering Formula 2 above.

[0181] [ka]

[0182] The synthesis of compounds according to formula 2 can be carried out generally according to the general synthetic examples shown above. Below is an example of a general scheme for the synthesis of compounds according to formula 2.

[0183] [ka]

[0184] In one exemplary embodiment, the compound may have the structure of the following compound [VIII], where R1 is a 14-carbon amide group, R2 is chloro, R3, R4 are methyl, and R5, R6 are hydrogen:

[0185] [ka]

[0186] The IUPAC name of compound [VIII] is N-[3-chloro-4-[7-[2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide. An illustrative synthesis scheme for compound [VII] (and similar embodiments of various alkyl chain lengths) is shown below.

[0187] [ka] TIFF2025513758000070.tif46170Step; Step Step 8 and 9:

[0188] It is understood that in the above scheme, one of ordinary skill in the art may identify more than one approach for carrying out one or more of the numbered steps. As an example, compound [VIII] can be synthesized from intermediate N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecaneamide using the following scheme:

[0189] [ka] Step

[0190] In step 1, compound ethyl 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxyacetate was prepared from N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecaneamide according to step 4 of the preparation of compound [VII] described above. This gave ethyl 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxyacetate as a light brown solid (0.25 g). LC-MS 584.3[M+1] + , RT6.95 minutes.

[0191] In step 2, 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxyacetic acid was prepared from ethyl 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxyacetic acid according to the method of step 6 of the preparation of compound [VII] described above. This gave a colorless solid, 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]acetic acid (0.05 g). LC-MS 556.1[M+1] + , RT6.64 minutes.

[0192] In step 3, N-[3-chloro-4-[7-[2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide [VIII] was synthesized by the following method: 2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide (VIII) was dissolved in 10 ml of dry DCM (3 ml) at room temperature under nitrogen atmosphere. To a stirred solution of N-[2-(dimethylamino)ethyl]-2-(ethylamino)acetamide trifluoroacetate (240 mg, 0.60 mmol) in dry DMF (4 ml) was added NMM (100 μl, 0.72 mmol) and EDC hydrochloride (32 mg, 0.17 mmol). The mixture was stirred for 16 h and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (5-13% MeOH in DCM; 1-2% 7M NH3 in MeOH) gave N-[3-chloro-4-[7-[2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide (50 mg, 78%). LC-MS 711.5[M+1] + , RT 5.89 min.

[0193] Another illustrative example of an embodiment is compound [IX] shown below: The IUPAC name of compound [IX] is N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide.

[0194] [ka] [IX]

[0195] Compound [IX] was synthesized as follows. In step 1, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.23 g, 1.22 mmol), 1-hydroxybenzotriazole hydrate (0.17 g, 1.22 mmol), and NMM (0.33 ml, 3.0 mmol) were added to a stirred solution of (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (see compound [VI] above) (0.35 g, 0.61 mmol) and N-ethylglycine ethyl ester trifluoroacetate (0.22 g, 1.22 mmol) in a mixture of DCM (10 ml) and DMF (5 ml) at room temperature under nitrogen atmosphere. The mixture was stirred for 16 hours and the solvent was evaporated under reduced pressure to give the crude product. Purification on silica gel (50-100% EtOAc in isohexane) gave ethyl 2-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]-ethyl-amino]acetate (0.28 g). LC-MS 683.3[M+1] + , RT6.89 minutes.

[0196] In step 2, 2-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]-ethyl-amino]acetic acid was prepared from ethyl 2-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]-ethyl-amino]acetate according to the method of step 6 of compound [IV] to obtain 2-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]-ethyl-amino]acetic acid as a light brown solid (0.16g). LC-MS 655.3[M+1] + , RT6.63 minutes.

[0197] In step 3, N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecane amide (compound [XI]) was converted to 2-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo- N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide was prepared from 1-(2-aminoethyl)pyrrolidine and N-[3-chloro-4-[7-[(1R)-2-[[2-[2-(dimethylamino)ethylamino]-2-oxo-ethyl]-ethyl-amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide was obtained as a white solid (15.9 mg). LC-MS 751.3[M+1] + , RT6.06 minutes.

[0198] Another illustrative example of an embodiment is compound [X] shown below: The IUPAC name of compound [X] is N-[3-chloro-4-[7-[(1R)-2-[ethyl-[2-[(1-methylpyrrolidin-3-yl)amino]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide.

[0199] [ka] [X]

[0200] Compound [X] was prepared from (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (compound [VI]) and 1-methylpyrrolidin-3-amine according to the method of step 1 of compound [IX] to obtain N-[3-chloro-4-[7-[(1R)-2-[ethyl-[2-[(1-methylpyrrolidin-3-yl)amino]-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide as a white solid (16.4 mg). LC-MS 737.4[M+1] + , RT 5.97 min.

[0201] Another illustrative example of an embodiment is compound [XI] shown below: The IUPAC name of compound [XI] is N-[3-chloro-4-[7-[(1R)-2-[ethyl-[2-(isopentylamino)-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide.

[0202] [ka] [XI]

[0203] Compound [XI] was prepared from (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (compound [VI]) and 3-methylbutylamine according to the method of step 1 of compound [IX] to obtain N-[3-chloro-4-[7-[(1R)-2-[ethyl-[2-(isopentylamino)-2-oxo-ethyl]amino]-1-methyl-2-oxo-ethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecaneamide as a white solid (24.9 mg). LC-MS 724.4[M+1] + , RT6.78 minutes.

[0204] The following abbreviations are used in the description of the synthetic methods and reaction schemes: acetonitrile (MeCN), ethyl acetate (EtOAc), methanol (MeOH), hydrochloric acid (HCl), magnesium sulfate (MgSO4), di-isopropylethylamine (DiPEA), dichloromethane (DCM), sodium bicarbonate NaHCO3, isopropanol (IPA), tetrahydrofuran (THF), ammonium chloride (NH4Cl), sodium hydroxide (NaOH), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC HCl), triethylamine (TEA), N-methylmorpholine (NMM), diethyl azodicarboxylate (DEAD), dimethylacetamide (DMA), dimethylformamide (DMF), ammonia (NH3), 4-dimethylaminopyridine (DMAP), trifluoroacetic acid (TFA), 1-hydroxybenzotriazole (HOBt), and ammonia (NH3).

[0205] Liquid chromatography-mass spectrometry data was prepared using a Waters Sunfire C18 30×4.6 mm column. Gradient elution: 20-100% acetonitrile / water containing 0.05% formic acid. Time: 0-10 min.

[0206] Further evaluation of both efficacy and toxicity against human breast cancer initiated from chicken embryo MDA-MB-231 cell line was performed using the chicken chorioallantoic membrane assay ("CAM assay") available from Innovation (Cambridge, Massachusetts). Compounds of the present approach showed significant effects on metastasis of MDA-MB-231 cancer cells in vivo without showing toxicity to the embryo. 6 An inoculum of MDA-MB-231 cells was added to the CAM of each egg (day E9), after which the eggs were randomly divided into groups. Tumors were detectable on day E10 and were treated for 8 days with vehicle alone (1% DMSO in PBS) or with compounds of the present approach. Eight days after drug administration, on day E18, all tumors were weighed and the lower CAM was collected to assess the number of metastatic cells analyzed by qPCR using specific primers against human Alu sequences.

[0207] FIG. 8 shows a quantitative assessment of tumor growth using the CAM assay for various concentrations of an embodiment of the inventive approach, compound [III]. These results show a dose-dependent reduction in cellular tumor growth, which indicates the pharmacological activity of the exemplary compound. Similar activity is present for other compounds disclosed herein. FIG. 9 shows a quantitative assessment of metastatic invasion using the CAM assay for various concentrations of an embodiment of the inventive approach, compound [III]. A dose-dependent reduction in metastasis is evident for the exemplary compound. Other compounds described herein have similar activity. For FIG. 8, 9, data was analyzed by t-test, P>0.05. A single * indicates P<0.05, ** indicates P<0.001, and **** indicates P>0.0001. FIG. 10 shows a toxicity assessment using the CAM assay for various concentrations of compound [III]. As can be seen, the exemplary compound has no toxicity to normal cells. Selectivity for metabolically active cancer stem cells is a common property of compounds according to the inventive approach. Toxicity analysis from the CAM assay is summarized in Table 1 below.

[0208] [Table 1]

[0209] This demonstrates that the compounds of the present approach are effective in preventing metastasis or reducing the likelihood of metastasis. It should be understood that these properties can be reproduced in embodiments of the present approach using the same CAM assay. Furthermore, little or no embryotoxicity was observed for the exemplary compounds using the CAM assay. The compounds of the present disclosure have potency as anti-metastatic agents that selectively inhibit tumor metastasis without exhibiting significant toxicity or antibiotic activity.

[0210] To examine the effects of the compounds described herein on mitochondrial respiration and aerobic glycolysis, adherent MCF7 cells were treated with exemplary compounds and the OCR and ECAR were measured. Figure 11A-D show the results of metabolic flux analysis of adherent MCF7 cells treated with 2.5 μM compound [III] for mitochondrial respiration, basal respiration, maximal respiration, and ATP production. After treatment, mitochondrial oxygen consumption rate (OCR) was measured using a Seahorse XFe96 analyzer. Data are expressed as a percentage of OCR relative to control.

[0211] Figure 12A-D shows the results of analysis of glycolytic function for treatment with 2.5 μM compound [III]. These are glycolytic function, basal glycolysis, induced glycolysis, and compensatory glycolysis, respectively. The data show that compound [III] significantly inhibited basal and maximum OCR, as well as ATP production levels, compared to vehicle-only control cells. In contrast, ECAR levels were roughly comparable between control and illustrative compounds. These results are consistent with preliminary evaluation of other compounds described herein, although evaluation of various compounds and compound concentrations is ongoing. All data was normalized to cell number. Statistical analysis was performed using one-way ANOVA.

[0212] These data directly validate the manner in which compound [III] functions specifically as an inhibitor of mitochondrial respiration and ATP production, with little or no effect on glycolysis. Furthermore, these results are consistent with the understanding that compounds of the present approach, such as compound [II], are inhibitors of mitochondrial transcription (IMT) by targeting the POLRMT enzyme.

[0213] The following paragraphs describe the materials and assays used to generate the data described herein. It will be understood that one of ordinary skill in the art can perform the same assays described herein and / or use other assays commonly known in the art to evaluate the physical, chemical, and pharmaceutical properties of the compounds as described herein.

[0214] 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. The human breast adenocarcinoma cell line MCF-7 was obtained from the American Type Culture Collection (ATCC). hTERT-BJ1 cells were obtained from Clontech, Inc. MCF-7 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.

[0215] 3D Anchorage-Dependent Growth Assay: This assay is also called the tumor formation assay. Single cell suspensions were prepared using enzymatic dissociation and manual dissociation (25g needle). Cells were then plated in tumor medium (DMEM-F12 + 1X B-27 Plus Supplement + 20ng / ml EGF + Pen / Strep) at a density of 500 cells / cm2 under non-adherent conditions in culture dishes pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.), which were called "tumor plates". Cells were grown for 5 days and kept in a humidified incubator at 37°C. After 5 days of culture, 3D tumors larger than 50 μm were counted using an eyepiece ("graticule") and the percentage of plated cells forming tumors was calculated, which was called percent tumor formation and normalized to 1 (1=100% MFE). 3D mammosphere formation efficiency (MFE) was analyzed in both low and high ATP cell subpopulations. All 3D mammosphere experiments were performed at least three times independently and in triplicate.

[0216] Statistical analysis: All analyses were performed in GraphPad Prism 6. Data are expressed as mean ± SD (or ± SEM where indicated). All experiments were performed at least three times independently, with at least four technical replicates for each experimental condition tested (unless otherwise stated, e.g., when representative data are shown). Statistical significance was determined using Student's t-test or analysis of variance (ANOVA) test. For comparisons between multiple groups, statistical significance was determined using one-way ANOVA. p<0.05 was considered significant.

[0217] The approach of the present invention includes a method to confirm cell viability. A person skilled in the art can select one or more methods to confirm cell viability suitable for a particular embodiment. We first used the sulphorhodamine (SRB) assay, which is based on the measurement of cell protein content. After 72 hours of treatment in 96-well plates, cells were fixed with 10% trichloroacetic acid (TCA) in a cold room for 1 hour and dried at room temperature overnight. Cells were then incubated with SRB for 15 minutes, washed twice with 1% acetic acid, and air-dried for at least 1 hour. Finally, the protein-binding dye was dissolved in 10 mM Tris, pH 8.8 solution and read at 540 nm using a plate reader. Using the SRB assay, we selected only compounds that depleted ATP levels without significant cytotoxicity for further analysis. "Significant cytotoxicity" was defined as less than 30% cells remaining on the plate. Of course, embodiments employing other methods of confirming cell viability may select compounds for further analysis based on other considerations, as would be known in the art.

[0218] 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 additives, such as one or more fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, as known in the art. Depending on one or more therapeutic purposes, various types of dosage unit forms can be selected. 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, as will be known in the art. To shape the pharmaceutical composition into a tablet, any known excipient may 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, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc. In addition, disintegrants such as dry starch, sodium alginate, agar powder, kelp powder, sodium bicarbonate, calcium carbonate, fatty acid ester of polyoxyethylene sorbitan, sodium lauryl sulfate, monoglyceride of stearic acid, starch, lactose, etc. Disintegration inhibitors such as white sugar, stearin, coconut butter, hydrogenated oil, etc.; absorption enhancers such as quaternary ammonium base, sodium lauryl sulfate, etc. may be used. Wetting agents such as glycerin, starch, and others known in the art may be used. For example, adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc. may be used. Lubricants such as refined talc, stearate, boric acid powder, polyethylene glycol, etc. may be used. When tablets are desired, the tablets can be further coated with common coating materials to form sugar-coated tablets, gelatin film-coated tablets, enteric-coated tablets, film-coated tablets, bilayer tablets, and multilayer tablets.Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, foams, sprays, aerosols, or oils. Such pharmaceutical compositions may contain conventional additives, including, but not limited to, preservatives, solvents to aid in drug penetration, cosolvents, emollients, propellants, viscosity modifiers (gelling agents), surfactants, and carriers.

[0219] The inventive approach can be used to prevent and / or reduce the likelihood of tumor recurrence and metastasis. Anti-cancer treatments often fail due to tumor recurrence or metastasis, especially after surgery. At least some of these causes of treatment failure are due to CSC mitochondrial activity. Embodiments of the inventive approach can be used in situations where conventional cancer therapy has failed and / or in conjunction with anti-cancer treatment to prevent or reduce the likelihood of failure due to tumor recurrence and / or metastasis.

[0220] The approach of the present invention provides a method for selectively targeting cancer cells, which may be at least one of CSCs, energetic cancer stem cells (e-CSCs), circulating tumor cells (CTCs, seed cells leading to subsequent growth of further tumors in distant organs, a mechanism responsible for the majority of cancer-related deaths), and therapy-resistant cancer cells (TRCCs, cells that have exhibited resistance to one or more of chemotherapy, radiotherapy, and other common cancer treatments).

[0221] As described in the applicant's co-pending US Provisional Patent Application Nos. 62 / 686,881 (filed June 19, 2018) and 62 / 731,561 (filed September 14, 2018), which are incorporated herein by reference in their entireties, e-CSCs represent a proliferation-associated CSC phenotype. It should be appreciated that in addition to bulk cancer cells and CSCs, the approach of the present invention can be used to target a hyperproliferative subpopulation of cells that exhibits a gradual increase in stemness markers (ALDH activity and tumorigenic activity), a large increase in mitochondrial mass, and elevated glycolytic and mitochondrial activity, which we refer to as e-CSCs.

[0222] In view of the above, it should be understood that the inventive approach can take a variety of forms depending on the embodiment. For example, an embodiment of the inventive approach can take the form of a composition, particularly a pharmaceutical composition. The therapeutic compound can be an active ingredient and can be present in a pharma- ceutical effective amount.

[0223] An embodiment of the inventive approach may also take the form of a method for preventing or reducing the likelihood of tumor recurrence and / or metastasis. In some embodiments, an effective amount of a composition comprising a compound of the inventive approach as a therapeutic agent may be administered. In some embodiments, an effective amount of a composition comprising an embodiment of a compound described herein as a therapeutic agent may be administered.

[0224] The terminology used in the description of the present invention herein is intended to describe specific embodiments only and is not intended to limit the present invention. When 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 review of the following detailed description.

[0225] Terms such as "first", "second", "third", "a)", "b)", "c)" and the like may be used herein to describe various elements of the invention, 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 invention from another. Thus, a first element discussed below can be referred to as an element aspect, and similarly, can be referred to as a "third" without departing from the teachings of the invention. Thus, the terms "first", "second", "third", "a)", "b)", "c)" and the like are not necessarily intended to impose an order or other hierarchy on the elements associated with these terms, but are used for identification purposes only. The order of operations (or steps) is not limited to the order presented in the claims.

[0226] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Furthermore, it will be understood that terms such as those defined in commonly used dictionaries shall be interpreted as having a meaning consistent with the meaning in the context of this application and the related art, and shall not be interpreted in an idealized or overly formal sense unless expressly defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only, and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety into this application. In the event of a conflict in terminology, the present specification shall control.

[0227] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the listed items associated with this term, as well as the lack of combinations when interpreted as alternatives ("or").

[0228] The terms "decrease," "lower," "lessen," and "reduce" collectively refer to the ability of a composition according to the present approach to produce and / or cause a lower physiological response (i.e., a measurable downstream effect), e.g., reduced tumor volume, compared to the response caused by a vehicle or control molecule / composition. A "decreased" or "reduced" response is typically a "statistically significant" response and may include a reduction of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or even higher (e.g., 500-fold, 1000-fold) (including all integers and decimals greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) than the response produced by a normal subject, an untreated subject, or a subject receiving a control treatment.

[0229] The phrase "treatment cycle" refers to a course of treatment, such as a dosing schedule, that is repeated on a regular or predefined basis. A treatment cycle may include multiple treatment days followed by multiple rest days. By way of example only, in a four-week treatment cycle, an agent may be administered daily for two weeks, followed by two weeks without treatment. It should be understood that a treatment cycle may depend on a number of factors, such as the individual's medical condition, age, sex, and weight, as well as the particular agent(s) and / or methodology for eliciting a desired response in the individual's body.

[0230] Unless otherwise indicated by the context, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Moreover, the invention contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For example, if a complex is described herein as comprising components A, B, and C, it is specifically contemplated that any or any combination of A, B, or C can be omitted and discarded.

[0231] The term "about" as used herein in reference to a measurable value, e.g., an amount or concentration, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of the specified amount. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​within the range.

[0232] Thus, while specific embodiments of the present invention have been described, it should be understood that the invention as defined by the appended claims is not limited to the specific details set forth in the above description, since numerous obvious variations thereof can be made without departing from the spirit or scope of the invention as hereinafter claimed.

Claims

1. Chemical structure: 【Chemistry 1】 A compound having a pharmaceutically acceptable salt thereof: R1 and R2 may be the same or different, and halogen, CF ２ H, -CF ３ , -OCF ２ H, -OCF ３ Selected from substituted or unsubstituted C5-C18 carboxyls, substituted or unsubstituted C5-C18 alkanes, substituted or unsubstituted C5-C18 alkenes, substituted or unsubstituted C5-C18 cyclic alkenes, substituted or unsubstituted C5-C18 alkynes, substituted or unsubstituted C5-C18 ketones, substituted or unsubstituted C5-C18 aldehydes, substituted or unsubstituted C5-C18 ethers, substituted or unsubstituted C5-C18 esters, substituted or unsubstituted C5-C18 amines, substituted or unsubstituted C5-C18 amides, substituted or unsubstituted C5-C18 alkylamides, monocyclic or polycyclic arenes, heteroarenes, phenols, or benzoic acid; R3 and R4 may be the same or different, and are selected from hydrogen, substituted or unsubstituted C2-C18 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C18 carboxyl, substituted or unsubstituted C2-C18 alkene, substituted or unsubstituted C2-C18 alkyne, substituted or unsubstituted C2-C18 ketone, substituted or unsubstituted C2-C18 aldehyde, substituted or unsubstituted C2-C18 ether, substituted or unsubstituted C2-C18 ester, substituted or unsubstituted C2-C18 amine, substituted or unsubstituted C2-C18 amide, substituted or unsubstituted C2-C18 alkylamide, substituted or unsubstituted phenol, or benzoic acid, or one of R3 and R4 is a group: 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 or 【Transformation 5】 The other is H or C2-C5 alkyl; or R3 and R4 form a substituted or unsubstituted C5 or C6 heterocycle; or R3 and R4 are groups: 【Transformation 6】 It forms; or NR3R4 is OH; However, at least one of R1 and R2 is a substituted or unsubstituted C5-C18 amide, or at least one of R3 and R4 is a base: 【Transformation 7】 【Transformation 8】 【Chemistry 9】 or 【Chemistry 10】 A compound in which the other is H or C2-C5 alkyl.

2. structure: 【Chemistry 11】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

3. structure: 【Chemistry 12】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

4. structure: 【Chemistry 13】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

5. structure: 【Chemistry 14】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

6. structure: 【Chemistry 15】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

7. structure: 【Chemistry 16】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

8. The compound according to claim 1, wherein one of R1 and R2 is a halogen.

9. One of R3 and R4 is based on: 【Chemistry 17】 The compound according to claim 1, wherein the other is a C2-C5 alkyl group.

10. One of R3 and R4 is based on: [Chemistry 18] The compound according to claim 1, wherein the other is a C2-C5 alkyl group.

11. R3 and R4 are based on: 【Chemistry 19】 The compound according to claim 1, which forms a compound.

12. structure: 【Chemistry 20】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

13. structure: 【Chemistry 21】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

14. structure: 【Chemistry 22】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

15. structure: 【Chemistry 23】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

16. structure: 【Chemistry 24】 The compound according to claim 1, or having a pharmaceutically acceptable salt thereof.

17. Chemical structure: 【Chemistry 25】 A compound having a pharmaceutically acceptable salt thereof: R1 and R2 may be the same or different, and halogen, CF ２ H, -CF ３ , -OCF ２ H, -OCF ３ Selected from substituted or unsubstituted C5-C18 carboxyls, substituted or unsubstituted C5-C18 alkanes, substituted or unsubstituted C5-C18 alkenes, substituted or unsubstituted C5-C18 cyclic alkenes, substituted or unsubstituted C5-C18 alkynes, substituted or unsubstituted C5-C18 ketones, substituted or unsubstituted C5-C18 aldehydes, substituted or unsubstituted C5-C18 ethers, substituted or unsubstituted C5-C18 esters, substituted or unsubstituted C5-C18 amines, substituted or unsubstituted C5-C18 amides, substituted or unsubstituted C5-C18 alkylamides, monocyclic or polycyclic arenes, heteroarenes, phenols, or benzoic acid; R5 and R6 may be the same or different, and are selected from hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C10 carboxyl, substituted or unsubstituted C2-C10 alkene, substituted or unsubstituted C2-C10 alkyne, substituted or unsubstituted C2-C10 ketone, substituted or unsubstituted C2-C10 aldehyde, substituted or unsubstituted C2-C10 ether, substituted or unsubstituted C2-C10 ester, substituted or unsubstituted C2-C10 amine, substituted or unsubstituted C2-C10 amide, substituted or unsubstituted C2-C10 alkylamide, substituted or unsubstituted phenol, or benzoic acid; R3 and R4 may be the same or different, and are selected from hydrogen, substituted or unsubstituted C2-C18 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted pyridine, substituted or unsubstituted C2-C18 carboxyl, substituted or unsubstituted C2-C18 alkene, substituted or unsubstituted C2-C18 alkyne, substituted or unsubstituted C2-C18 ketone, substituted or unsubstituted C2-C18 aldehyde, substituted or unsubstituted C2-C18 ether, substituted or unsubstituted C2-C18 ester, substituted or unsubstituted C2-C18 amine, substituted or unsubstituted C2-C18 amide, substituted or unsubstituted C2-C18 alkylamide, substituted or unsubstituted phenol, or benzoic acid, or one of R3 and R4 is a group: 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 or 【Chemistry 29】 The other is H or C2-C5 alkyl; or R3 and R4 form a substituted or unsubstituted C5 or C6 heterocycle; or R3 and R4 are groups: 【Transformation 30】 It forms; or NR3R4 is OH; However, at least one of R1 and R2 is a substituted or unsubstituted C5-C18 amide, or at least one of R3 and R4 is a base: 【Chemistry 31】 【Chemistry 32】 【Transformation 33】 or 【Transformation 34】 A compound in which the other is H or C2-C5 alkyl.

18. structure: 【Chemistry 35】 The compound according to claim 17, or having a pharmaceutically acceptable salt thereof.

19. R5 and R6 are as follows: 【Transformation 36】 A compound according to claim 17, selected from the group consisting of the following.

20. A pharmaceutical composition comprising a pharmaceutically effective amount of the compound described in any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

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