Mitochondrial-specific transcription inhibitor compounds

Abstract

Mitochondrial-specific transcription inhibitors may be used to target and eliminate cancer stem cells and may be used as therapeutic agents for cancer treatment, including reducing and / or preventing the likelihood of tumor recurrence and metastasis. The mitochondrial-specific transcription inhibitor compounds disclosed herein have demonstrated inhibition of tumor cell formation, migration, and stem cell-related signaling in cancer stem cells. These properties are attributed to selective inhibition of mitochondrial transcription targeting mitochondrial RNA polymerase (POLRMT) in cancer cells.

Description

[Technical Field]

[0001] This disclosure relates to mitochondrial-specific transcription inhibitors, or MSTI compounds, that inhibit the proliferation of cancer stem cells (CSCs) and senescent cells by selectively inhibiting mitochondrial transcription of CSCs. [Background technology]

[0002] The biological process of aging continues to attract significant attention in the scientific and medical research communities. Physiological aging is, at least in part, associated with an increased rate of oxidative damage to cellular components, including DNA, lipids, and proteins. Increased oxidative damage creates imbalances and disrupts autoregulatory processes at the cellular level. Furthermore, aging correlates with the accumulation of lipofuscin in nerve cytoplasm. Modern research also shows that aging is a result of naturally occurring DNA damage, leading to abnormal DNA changes that accumulate over time. Damage to both mitochondrial and nuclear DNA can contribute to aging indirectly through increased apoptosis and cellular senescence, and directly through increased cellular dysfunction. Accumulated DNA damage can lead to cell loss, and in viable cells, loss of gene expression and mutations, and these effects manifest as signs of aging in cells with low cell division rates. Cellular senescence occurs when aging cells stop dividing, and is thought to follow, among other things, various environmental damage events, abnormal cell proliferation, autophagy, and oxidative stress. Senescence-associated secretory phenotypes ("SASP") are characteristic of senescent cells and lead to protein toxic impairment of healthy cellular function, depending on a variety of factors, including inflammatory or anti-inflammatory, neoplastic or antitumor effects. The effects of SASP-associated chronic inflammation affect the normal ability of the immune system to eliminate senescent cells, and immune-providing cells may be induced into senescent cells by SASP. Biomarkers of cellular senescence accumulate with aging in mammals and have been found to be involved in a wide range of age-related diseases, including Alzheimer's disease, lateral sclerosis, and type 2 diabetes. Furthermore, in cells with high dividing rates, accumulated DNA damage can be a significant cause of cancer.

[0003] Therefore, aging increases the likelihood of developing cancer, and researchers have been working to develop new anti-cancer and anti-aging therapies or senescent cell removal therapies. Conventional cancer therapies (e.g., radiation, alkylating agents such as cyclophosphamide, and anti-metabolites such as 5-fluorouracil) have attempted to selectively detect and eradicate rapidly growing cancer cells by interfering with cellular mechanisms involved in cell proliferation and DNA replication. Other cancer therapies have employed immunotherapy (e.g., monoclonal antibodies) that selectively bind to mutated tumor antigens on rapidly growing cancer cells. Unfortunately, tumors often recur in the same or different locations after these therapies, indicating that not all cancer cells are eradicated. Cancer stem cells, in particular, survive for various reasons, leading to treatment failure. Recurrence may be due to insufficient chemotherapy doses and the emergence of cancer clones resistant to the therapy. Therefore, new cancer treatment strategies are needed to overcome the shortcomings of conventional therapies.

[0004] Advances in mutation analysis have enabled detailed studies of gene mutations that occur during cancer development. Despite knowledge of the genomic landscape, modern oncology has struggled to identify major driver mutations across cancer subtypes. The harsh reality seems to be that each patient's tumor is unique, and a single tumor may contain multiple different clonal cells. Therefore, what is needed is a new approach that emphasizes commonalities between different cancer types. Targeting metabolic differences between tumor cells and normal cells is a promising new cancer treatment strategy. Analysis of transcriptional profiling data from human breast cancer samples revealed upregulation of over 95 mRNA transcripts related to mitochondrial biosynthesis and / or mitochondrial translation. Sotgia et al., Cell Cycle, 11(23):4390-4401 (2012). Furthermore, more than 35 of the 95 upregulated mRNAs encoded mitochondrial ribosomal proteins (MRPs). Similarly, proteomic analysis of human breast cancer stem cells revealed significant overexpression of multiple mitrobosome proteins and other proteins involved in mitochondrial biosynthesis. Lamb et al., Oncotarget, 5(22):11029-11037 (2014).

[0005] Mitochondrial metabolism in cancer cells has been a target of recent exploratory research for both anti-cancer and senescent cell decontamination therapies. Mitochondria are highly dynamic organelles that constantly divide, elongate, and interconnect to form tubular networks or fragmented granules in order to meet cellular needs and adapt to the cellular microenvironment. The balance between mitochondrial fusion and division determines the morphology, quantity, function, and spatial distribution of mitochondria, and thus influences a variety of mitochondrial-dependent life-sustaining processes, including adenosine triphosphate (ATP) production, mitophagy, apoptosis, and calcium homeostasis. Mitochondrial dynamics are regulated by mitochondrial metabolism, respiration, and oxidative stress.

[0006] ATP is the universal bioenergy "currency" of all living cells and tissues, including microorganisms such as prokaryotic bacteria and eukaryotic yeast. In eukaryotes, mitochondrial organelles function as the cell's "powerhouse." Mitochondria produce vast amounts of ATP through the TCA cycle and oxidative phosphorylation (OXPHOS), while glycolysis contributes only a small amount. Conversely, mitochondrial dysfunction leads to ATP depletion, resulting in mitochondrial-driven apoptosis (programmed cell death) and / or necrosis. Therefore, we propose that ATP depletion therapy could be an effective strategy to target and eradicate even the "most adapted" cancer cells.

[0007] In MCF7 breast cancer cells, under normal oxygen conditions, mitochondrial-driven OXPHOS accounts for 80-90% of ATP production, while glycolysis contributes only to the remaining 10-20%. Therefore, like normal cells, cancer cells are highly dependent on mitochondrial ATP production. However, it remains largely unclear whether intracellular ATP levels in cancer cells contribute to two characteristics of metastatic diffusion: 3D scaffold-independent proliferation and cell migration.

[0008] Therefore, there is a need for novel anti-aging compositions and methods that treat aging at the cellular level and overcome accumulated oxidative and DNA damage, as well as many of the undesirable effects of aging.

[0009] Furthermore, there is a need for therapeutic agents that target unhealthy senescent cells and SASPs to reduce the accumulation of cellular senescence and counteract chronic aging.

[0010] Furthermore, there is a need for therapeutic agents that target a wide range of CSCs through common characteristics of CSCs regardless of cancer type, while minimizing or eliminating the impact on normal, healthy cells.

[0011] Furthermore, therapeutic agents are needed that inhibit the proliferation of CSCs, including circulating tumor cells and tumor-initiating cells, which can cause tumor recurrence and / or metastasis. [Prior art documents] [Non-patent literature]

[0012] [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) [Overview of the project]

[0013] In light of the above background, the purpose of this disclosure is to describe therapeutic agents or compounds that can be used to reduce the accumulation of cellular senescence and inhibit the proliferation of CSCs. The purpose of this disclosure is to describe therapeutic agents for use in eradicating CSCs and senescent cells. The purpose of this disclosure is to describe therapeutic agents for use in preventing and reducing the likelihood of tumor recurrence and metastasis. Furthermore, the purpose of this disclosure is to describe compositions and methods, such as pharmaceutical compositions, for the treatment and prevention of cancer, including tumor recurrence and / or metastasis. In addition, the purpose of this disclosure is to describe compositions and methods, such as pharmaceutical compositions, for senescent cell elimination therapeutic agents.

[0014] This specification describes mitochondrial-specific transcription inhibitors ("MSTIs"), compounds that can be used as therapeutic agents having anticancer activity, pharmaceutical compositions containing such therapeutic agents, methods for synthesizing such compounds, and methods for treating cancer. Embodiments of MSTI therapeutic agents described herein are shown below in order to demonstrate the unexpected advantages of this method:

[0015] TIFF2026525208000001.tif45170

[0016] Compounds known to be orally active mitochondrial RNA polymerase (POLRMT) inhibitors can be referred to. This compound was first described in Bonekamp, N.A., Peter, B., Hillen, H.S. et al Small-molecule inhibitors of human mitochondrial DNA transcription. Nature 588, 712-716 (2020), which is hereby incorporated by reference in its entirety as part of this specification. As a POLRMT inhibitor, this compound inhibits the expression of mitochondrial DNA. The IUPAC name of this compound is 3-piperidinecarboxylic acid, 1-[(2R)-2-[[4-(2-chloro-4-fluorophenyl)-2-oxo-2H-1-benzopyran-7-yl]oxy]-1-oxopropyl]-, (3S)-, and the CAS registration number 2304621-06-3 has been assigned.

[0017] In this method, some embodiments of the MSTI compound may have the general chemical structure of formula [I] shown below.

[0018] TIFF2026525208000002.tif76170

[0019] Wherein, · R1 is -OR 1a -, -SR 1a -, -NR 1a -, -NR 1a [[ID=2二十二]]C(O)R 1a -, -OC(O)R 1a -, -C(O)OR 1a -, -SO2R 1a , -S(O)R 1a -, -NR 1a C(O)-, -C(O)NR 1a -, -NR 1a S(O)2-, S(O)2NR 1a -, -OC(O)NR 1a -, -NR 1a C(O)OR 1a -, -NR 1a C(O)NR 1a -, -CR1a =CR 1a -, -CC-, and -CH2R 1a -Selected independently of; ·R 1a This includes H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl (where chemically acceptable, oxo, C1-C) 18 -alkyl, C2-C 18 -Alkinyl, C2-C 18 -Alkenyl, C1-C 18 -Optionally substituted with 1 to 4 groups independently selected from the haloalkyl group), OR a , NR a R b , SR a , C(O)OR a , C(O)NR a R b Halo, Cyano, Nitro, C(O)R a , and S(O)2OR a Selected independently from; ○R a In each existence, H and C1-C 18 -Selected independently from alkyl; R b In each existence, H, C1-C 18 -alkyl, C(O)C1-C 18 -alkyl and S(O)2-C1-C 18 -Selected independently of alkyl groups; R2 is independently selected from halogens, -CF2H, -CF3, -OCF2H, -OCF3, 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 allenes, heteroallenes, phenols, and benzoic acid; R3 and R4 may be the same or different, and are selected from H, 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, and benzoic acid; R5 is selected from H, -CF3, -OCF3, -OCHF2, -NO2, and -CN; L1 is -OR 2a -, -SR 2a -, -NR 2a -, -NR 2a C(O)R 2a -, -OC(O)R 2a -, -C(O)OR 2a -, -SO2R 2a ,-S(O)R 2a -, -NR 1a C(O)-, -C(O)NR 2a -, -NR 2a S(O)2-, S(O)2NR 2a -, -OC(O)NR 2a -, -NR 2a C(O)OR 2a -, -NR 1a C(O)NR 2a -, -CR 2a =CR 2a -and -CC-, -CH2R 2a -Selected independently of; ○R 2a is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl or phenyl (which may be optionally substituted with 1 to 4 groups independently selected from oxo, C1-C6-alkyl, C2-C6-alkynyl, C2-C6-alkenyl, C1-C6-haloalkyl, where chemically acceptable), OR a, NR y R b , SR y , C(O)OR y , C(O)NR y R b Halo, Cyano, Nitro, C(O)R y S(O)2OR y Selected independently from; ○R y In each presence, H and C1-C6-alkyl are independently selected; R b In each presence, the elements are independently selected from H, C1-C6-alkyl, C(O)C1-C6-alkyl, and S(O)2-C1-C6-alkyl; ·X - The anions are selected from those suitable for forming pharmaceutically acceptable salts.

[0020] It should be understood that a wide range of validated counterions are available to form pharmaceutically acceptable salts of the disclosed compounds. For example, X - The following can be selected from acetate ions, benzenesulfonate ions, benzoate ions, besylate ions, bitartrate ions, bromide ions, bamsylate, chloride ions, citrate ions, decanoate ions, edetate ions, esylate ions, fumarate ions, gluceptate ions, hexanoate ions, iodide ions, isethionate ions, lactate ions, malate ions, maleate ions, mandelate ions, mesylate ions, methylsulfate ions, mucinate ions, napsylate ions, nitrate ions, octanoate ions, oleate ions, pamoate ions, pantothenate ions, phosphate ions, propionate ions, salicylate ions, stearate ions, succinate ions, sulfate ions, tartrate ions, theoclate ions, and tosylate ions. It should be understood that the embodiments shown below as chloride salts disclose embodiments including any salt specified herein. In preferred embodiments, X - Cl - , Br - , I - , and MeSO3 -The optimal anion for a specific embodiment is currently being investigated.

[0021] This method may also be used to treat and / or prevent tumor recurrence and / or metastasis. For example, the MSTI compound of Formula I described above can be administered alone or in combination with other therapies as a pharmacopoeia to treat and / or prevent tumor recurrence and / or metastasis. Anticancer therapies are often ineffective, particularly after surgery, due to tumor recurrence or metastasis. Mitochondrial activity of CSCs is understood to be involved, at least partially, in the causes of these treatment failures. Embodiments of this method can be used to prevent or reduce the likelihood of treatment failure due to tumor recurrence and / or metastasis when conventional cancer therapies are ineffective, and / or concurrently with or prior to anticancer therapy. [Brief explanation of the drawing]

[0022] [Figure 1A] Figure 1A shows the results of mammothsphere formation at various concentrations of compound [I]. [Figure 1B] Figure 1B shows the results of SRB assays in MCF-7 cells at various concentrations of compound [I]. [Figure 1C] Figure 1C shows the results of SRB assays in BJ1-hTERT cells at various concentrations of compound [I]. [Figure 2A] Figure 2A shows the effects of various concentrations of compound [I] on MCF-7 colony formation as representative two-dimensional colony phase images. [Figure 2B] Figure 2B shows the effect of various concentrations of compound [I] on MCF-7 colony formation as colony formation rates compared to the control. [Figure 3A] Figure 3A shows the effects of various concentrations of compound [IV] on MCF-7 cells in a colony formation assay. [Figure 3B] Figure 3B shows the effects of various concentrations of the compound [IV] on hTERT-BJ1 cells in an SRB assay. [Figure 4A]Figure 4A shows the effects of various concentrations of compound [IX] on MCF-7 cells in a colony formation assay. [Figure 4B] Figure 4B shows the effects of various concentrations of compound [IX] on hTERT-BJ1 cells in an SRB assay. [Figure 5A] Figure 5A shows the effects of various concentrations of compound [X] on MCF-7 cells in a colony formation assay. [Figure 5B] Figure 5B shows the effects of various concentrations of compound [X] on hTERT-BJ1 cells in an SRB assay. [Figure 6A] Figure 6A shows the effects of various concentrations of compound [XI] on MCF-7 cells in a colony formation assay. [Figure 6B] Figure 6B shows the effects of various concentrations of compound [XI] on hTERT-BJ1 cells in an SRB assay. [Figure 7A] Figure 7A shows the effects of various concentrations of compound [XII] on MCF-7 cells in a colony formation assay. [Figure 7B] Figure 7B shows the effects of various concentrations of compound [XII] on hTERT-BJ1 cells in an SRB assay. [Modes for carrying out the invention]

[0023] The following description provides a sufficiently detailed explanation of embodiments of the Method to enable its implementation. While the Method is described with reference to these specific embodiments, it should be understood that the Method may be embodied in different forms, and this description should not be construed as limiting the appended claims to the specific embodiments shown herein. Rather, these embodiments are provided to ensure the comprehensiveness and completeness of this disclosure and to adequately convey the scope of the Method to those skilled in the art.

[0024] This specification uses various terms that should be understood by those of the ordinary level of art skilled in the art. To avoid any ambiguity, these terms are explained below.

[0025] The term "cancer" refers to a physiological condition in mammals characterized by generally uncontrolled cell proliferation. This definition includes both benign and malignant cancers. Examples of cancers include 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 islet cell carcinoma), sarcomas, schwannomas (including acoustic neuromas), medullary carcinoma, adenocarcinoma, melanoma, and leukemia or lymphocytic tumors. Specific examples of cancer include bladder cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal or gastric cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (renal cancer) or kidney cancer (renal cancer), prostate cancer, genital cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, head and neck cancer, and multiple myeloma.

[0026] As used herein, the term “tumor” refers to the proliferation and increase of new cells, including precancerous and cancerous cells and tissues, whether malignant or benign.

[0027] The term "metastasis" refers to the spread of cancer from its primary site to other parts of the body. Cancer cells escape from the primary tumor, invade the lymphatic system and blood vessels, circulate in the bloodstream, and grow in distant lesions of normal tissue in other parts of the body—that is, they "metastasize." Metastasis can be localized or distant. Metastasis is a continuous process that requires tumor cells to escape from the primary tumor, travel in the bloodstream, and settle in a distant site. In this new site, the cells can establish a blood supply and grow until they form a life-threatening mass. Both stimulative and inhibitory molecular pathways within tumor cells control this behavior, and interactions between tumor cells and host cells at the distant site are also important.

[0028] The terms “treat,” “treated,” “treating,” and “treatment” include the reduction or mitigation of at least one symptom associated with or resulting from a condition, disorder, or disease, particularly cancer, that is the subject of treatment. In certain embodiments, treatment includes reducing and / or mitigating at least one symptom associated with or resulting from the cancer of the present invention by the compounds of the present invention. In some embodiments, treatment includes inducing the death of a category of cells in the host that are associated with a particular cancer, such as senescent cells, SASP cells, or CSCs that may be involved in metastasis or recurrence, which may be achieved, for example, by preventing further proliferation of senescent cells and / or cancer cells, and / or by inhibiting CSC function, for example, by depriving such cells of energy production mechanisms. For example, treatment may be the reduction of one or more symptoms of cancer, or the complete eradication of cancer. As another example, this method can be used to inhibit mitochondrial metabolism in cancer, to eradicate cancer CSCs (e.g., kill them at a rate faster than their growth rate), to eradicate cancer TICs, to eradicate circulating tumor cells in cancer, to inhibit cancer growth, to target and inhibit CSCs, to target and inhibit TICs, to target and inhibit circulating tumor cells, to prevent or reduce the likelihood of metastasis, to prevent recurrence, to sensitize cancer to chemotherapy, to sensitize cancer to radiotherapy, and to sensitize cancer to phototherapy. As yet another example, the treatment can reduce accumulated senescent cells and / or decrease the rate of senescent cell accumulation.

[0029] With respect to tumor recurrence and / or metastasis, the terms “prevent” and “reduce the likelihood” mean reducing the presence of CSCs, TICs, and circulating tumor cells that may be involved in recurrence or metastasis in a subject to a level where tumor recurrence and / or metastasis from the primary site is unlikely to occur, compared to a control that is likely to be involved in recurrence or metastasis (i.e., no treatment is given to prevent or reduce the likelihood of tumor recurrence and / or metastasis). In practice, treatments to prevent and / or reduce the likelihood of tumor recurrence and / or metastasis as described herein inhibit or eradicate CSCs, TICs, and inhibit circulating tumor cells.

[0030] The terms “cancer stem cells” and “CSCs” refer to subpopulations of cancer cells that, when transplanted into an animal host, possess the ability to regenerate, differentiate, and create tumors within a tumor. Compared to “bulk” cancer cells, CSCs exhibit increased mitochondrial mass, enhanced mitochondrial biosynthesis, and higher activation of mitochondrial protein translation. As used herein, “circulating tumor cells” are cancer cells that have been released from a primary tumor into the blood vessels or lymphatic vessels and are transported throughout the body by blood circulation. The CellSearch circulating tumor cell test can be used to detect circulating tumor cells.

[0031] As used herein, the term "pharmaceutically effective amount" refers to the amount required to be administered to a host, or to the cells, tissues, or organs of a host, in order to achieve a therapeutic effect, such as the modulation, alteration, or inhibition of protein kinase activity, such as the inhibition of protein kinase activity, or the treatment of cancer. A physician or veterinarian with ordinary art skills can readily determine and prescribe an effective amount of a pharmaceutical composition required for a given subject using methods well known and available in the art. For example, a physician or veterinarian may start the dose of the compound of the present invention used in a pharmaceutical composition at a level lower than the amount required to achieve the desired therapeutic effect and gradually increase it until the desired effect is achieved. Determining a pharmaceutically effective amount is considered to be within the scope of those skilled in the art who have studied this disclosure.

[0032] As used herein, the term “therapeutic agent” refers to embodiments of the compounds described herein, which may include pharmaceutically acceptable salts or analogues of their isotopes. It should be understood that therapeutic agents may be administered to a subject by any suitable method, as is well known to those skilled in the art. It should also be understood that the amount and timing of administration of a therapeutic agent may vary depending on the individual subject being treated (e.g., factors such as age and weight), the mode of administration, the pharmacokinetic properties of the particular therapeutic agent, and the judgment of the prescribing physician. Therefore, due to variability among subjects, the doses described herein are intended as initial guidelines, and physicians may adjust the dose of the therapeutic agent to achieve the treatment they deem appropriate for the subject. In considering the desired degree of treatment, physicians may make a comprehensive judgment based on various factors, including the subject's age and weight, the presence of pre-existing diseases, and the presence 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 will be described in further detail below.

[0033] The term "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant. Each carrier must be "acceptable" in that it is compatible with the other components of the formulation and is harmless to the patient. Some examples of carriers that may serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth gum; (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, etc. Examples include oils; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogenic substance removal water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic suitable substances used in pharmaceutical formulations.

[0034] The term "pharmaceutically acceptable salt" refers to the relatively non-toxic inorganic and organic base addition salt compounds of this method. Pharmaceutically acceptable salts can be formed, for example, by reacting a compound in its free acid form with a pharmaceutically acceptable base, such as a hydroxide or carbonate of a metal cation, ammonia, or a pharmaceutically acceptable amine. Typical alkali salts or alkaline earth metal salts include, for example, sodium, potassium, calcium, magnesium, and aluminum salts. Examples of amines that can be used as base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine. Naturally, other salts can also be used, and those skilled in the art should understand that they can use methods known in the art to identify appropriate salt forms without departing from this method.

[0035] Suitable salts of the MSTI compounds described herein can be selected from acetates, benzenesulfonates, benzoates, besilates, hydrogen tartrates, bromides, bamsilates, chlorides, citrates, decanoates, edetates, esylates, fumarates, gluceptates, hexanoates, iodides, isethionates, lactates, malates, maleates, mandelates, mesilates, methylsulfates, mucinates, napsilates, nitrates, octanoates, oleates, pamoates, pantothenates, phosphates, propions, salicylates, stearates, succinates, sulfates, tartrates, theoclates, and tosilates. In preferred embodiments, X - Cl - , Br - , I - , and MeSO3 - Selected from.

[0036] Recent developments have identified a first-in-class inhibitor of mitochondrial transcription that targets mitochondrial RNA polymerase (POLRMT), a gene product responsible for mitochondrial gene expression and mitochondrial biogenesis of the OXPHOS system. This inhibitor was used in a 4-week study in mice. Oral administration of this compound showed no evidence of OXPHOS dysfunction or toxicity in mice, but showed a strong antitumor effect in human cancer cell xenografts.

[0037] The mitochondrial-specific transcription inhibitor (MSTI) compounds disclosed herein are a novel class of compounds with excellent selectivity and activity against cancer cells. Embodiments of the MSTI compounds disclosed herein can be used as therapeutic agents that selectively eradicate CSCs for the treatment and / or prevention of tumor recurrence and / or metastasis. The MSTI compounds disclosed herein have high anti-cancer activity compared to the aforementioned POLRMT inhibitors listed above, and data demonstrate that they 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 demonstrate anti-cancer activity by inhibition of MCF7 cells in a mammosphere formation assay and by cell viability using a sulforhodamine B assay (known in the art and also referred to herein as the SRB assay). This assay measures the amount of residual protein attached to the tissue culture dish and is an alternative marker of cell viability.

[0038] Under this approach, some embodiments of the MSTI compounds can have the general chemical structure of Formula [I] shown below.

[0039] TIFF2026525208000003.tif76170

[0040] Wherein, · R1 is -OR 1a -, -SR 1a -, -NR 1a -, -NR 1a C(O)R 1a -, -OC(O)R 1a-、 -C(O)OR 1a -、 -SO2R 1a 、 -S(O)R 1a -、 -NR 1a C(O)-、 -C(O)NR 1a -、 -NR 1a S(O)2-、 S(O)2NR 1a -、 -OC(O)NR 1a -、 -NR 1a C(O)OR 1a -、 -NR 1a C(O)NR 1a -、 -CR 1a =CR 1a -、 -CC-、 and -CH2R 1a is independently selected from; ·R 1a is optionally substituted with one to four groups independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl or phenyl (when chemically acceptable, oxo, C1-C 18 -alkyl, C2-C 18 -alkynyl, C2-C 18 -alkenyl, C1-C 18 -haloalkyl), OR a 、 NR a R b 、 SR[[ID=4⑨]] a 、 C(O)OR a ]、 C(O)NR a ]END]]R b 、 halo, cyano, nitro, C(O)R a 、 and S(O)2OR a is independently selected from; ○R a is independently selected from H and C1-C 18 -alkyl in each occurrence; R b is independently selected from H, C1-C 18 -alkyl, C(O)C1-C 18 -alkyl and S(O)2-C1-C 18 -alkyl in each occurrence;[[ID=7⑤]] R2 is independently selected from halogens, -CF2H, -CF3, -OCF2H, -OCF3, 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 allenes, heteroallenes, phenols, and benzoic acid; R3 and R4 may be the same or different, and are selected from H, 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, and benzoic acid; R5 is selected from H, -CF3, -OCF3, -OCHF2, -NO2, and -CN; L1 is -OR 2a -, -SR 2a -, -NR 2a -, -NR 2a C(O)R 2a -, -OC(O)R 2a -, -C(O)OR 2a -, -SO2R 2a ,-S(O)R 2a -, -NR 1a C(O)-, -C(O)NR 2a -, -NR 2a S(O)2-, S(O)2NR 2a -, -OC(O)NR 2a -, -NR2a C(O)OR 2a -, -NR 1a C(O)NR 2a -, -CR 2a =CR 2a -and -CC-, -CH2R 2a -Selected independently of; ○R 2a is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl or phenyl (which may be optionally substituted with 1 to 4 groups independently selected from oxo, C1-C6-alkyl, C2-C6-alkynyl, C2-C6-alkenyl, C1-C6-haloalkyl, where chemically acceptable), OR a , NR y R b , SR y , C(O)OR y , C(O)NR y R b Halo, Cyano, Nitro, C(O)R y S(O)2OR y Selected independently from; ○R y In each presence, H and C1-C6-alkyl are independently selected; R b In each presence, the elements are independently selected from H, C1-C6-alkyl, C(O)C1-C6-alkyl, and S(O)2-C1-C6-alkyl; ·X - The anions are selected from those suitable for forming pharmaceutically acceptable salts.

[0041] It should be understood that a wide range of validated counterions are available to form pharmaceutically acceptable salts of the disclosed compounds. For example, X -The following ions may be selected from acetate ions, benzenesulfonate ions, benzoate ions, besylate ions, hydrogen tartrate ions, bromide ions, bamsilate, chloride ions, citrate ions, decanoate ions, edetate ions, esylate ions, fumarate ions, gluceptate ions, hexanoate ions, iodide ions, isethionate ions, lactate ions, malate ions, maleate ions, mandelate ions, mesylate ions, methylsulfate ions, mucinate ions, napsylate ions, nitrate ions, octanoate ions, oleate ions, pamoate ions, pantothenate ions, phosphate ions, propionate ions, salicylate ions, stearate ions, succinate ions, sulfate ions, tartrate ions, theoclate ions, and tosylate ions. In a preferred embodiment, X - Cl - , Br - , I - , and MeSO3 - The optimal anion for a specific embodiment is currently being investigated.

[0042] This method can also be used to treat and / or prevent tumor recurrence and / or metastasis. For example, the MSTI compound of Formula I described above can be administered alone or in combination with other therapies as a pharmaceutical agent to treat and / or prevent tumor recurrence and / or metastasis.

[0043] One illustrative embodiment of the MSTI compound of this method, based on formula [I], is a compound having the formula shown below [I]

[0044] TIFF2026525208000004.tif31170

[0045] or another pharmaceutically acceptable salt thereof. This MSTI compound has the IUPAC name [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-6-oxo-hexyl]-triphenyl-phosphonium chloride. Naturally, it should be understood that other salts can also be used without deviation from this method.

[0046] Furthermore, this specification also describes a method for producing compounds by this method. For example, embodiments of compound [I] were prepared according to the following reaction scheme.

[0047] TIFF2026525208000005.tif38170 TIFF2026525208000006.tif142170Step; Step

[0048] The following paragraphs describe an example of the synthesis of compound [I] following the reaction scheme described above. In step 1, 2-chloro-4-nitrobenzoic acid, as shown below, is used as the starting material to synthesize 2-chloro-4-nitrobenzoyl chloride.

[0049] TIFF2026525208000007.tif26170

[0050] At room temperature under a nitrogen atmosphere, 10.3 g of 2-chloro-4-nitro-benzoic acid (50 mmol) was stirred in 200 ml of dry DCM, to which 10.6 ml of oxalyl chloride (125 mmol) and 8 drops of DMF were added. The mixture was stirred for 16 hours, and the solvent was evaporated under reduced pressure to obtain crude 2-chloro-4-nitro-benzoyl chloride as a light brown oil (11.78 g).

[0051] In Step 2, ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxopropanoate (shown below) was prepared from 2-chloro-4-nitro-benzoyl chloride.

[0052] TIFF2026525208000008.tif28170

[0053] Under a nitrogen atmosphere at +10°C, a stirring suspension of ethyl potassium malonate (17.0 g, 100 mmol) in dry MeCN (300 ml) was prepared by adding triethylamine (14.6 ml, 105 mmol) and anhydrous magnesium chloride (11.4 g, 120 mmol). The mixture was stirred at room temperature for 3 hours. A solution of crude 2-chloro-4-nitro-benzoyl chloride (11.7 g, 50 mmol) was added to dry MeCN (80 ml) over 60 minutes. The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The solvent was evaporated under reduced pressure, and the residue was diluted with aqueous NH4Cl (200 ml), pharmaceutically acceptable HCl (300 ml), and 4M HCl (120 ml). The organic phase was separated, washed with 0.5M HCl (170 ml) and brine (100 ml), dried over MgSO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product (19.8 g). Purification with silica gel (10-20% siRNA in isohexane) yielded ethyl 3-(2-chloro-4-nitrophenyl)-3-oxopropanoate as a light brown solid (12.3 g). LC-MS 272.0 [M+1] + , RT 5.56 minutes. 1 ¹H-NMR (CDCl3) d 12.50 (s,1H), 8.32-8.30 (m,1H), 8.19-8.15 (m,1H), 5.63 (s,1H), 4.30 and 4.19 (q,2H), 4.04 (s,1H), 1.35 and 1.25 (t,3H).

[0054] In step 3, 4-(2-chloro-4-nitro-phenyl)-7-hydroxy-chromen-2-one (shown below) was prepared using ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxopropanoate.

[0055] TIFF2026525208000009.tif29170

[0056] At +45°C under a nitrogen atmosphere, resorcinol (4.14 g, 41 mmol) was stirred in methane sulfonate (40 ml), to which ethyl 3-(2-chloro-4-nitro-phenyl)-3-oxopropanoate (11.6 g, 42.7 mmol) was added in small amounts over 10 minutes, and the mixture was stirred at +45°C for 1 hour. The reaction mixture was cooled to room temperature, ethanol (70 ml) was added, and this solution was poured into water (390 ml), cooled in an ice bath for 1 hour, and then warmed to room temperature. The solvent was removed by decantation, and the precipitate was washed with diethyl ether (2 × 100 m). The solid was filtered, washed with diethyl ether (2 × 100 m), DCM (2 × 50 ml), and diethyl ether (1 × 100 m), and dried under vacuum to obtain 4-(2-chloro-4-nitrophenyl)-7-hydroxychromen-2-one as a red solid (11.9 g). LC-MS 318.0 [M+1] + , RT 5.69 minutes. 1 H-NMR (DMSO-d6) d 10.80 (s,1H), 8.50 (d, 1H), 8.36 (dd, 1H), 7.89 (dd,1H), 6.87-6.83 (m, 2H), 6.73 (dd, 1H), 6.60 (s, 1H).

[0057] In step 4, 4-(4-amino-2-chlorophenyl)-7-hydroxychromen-2-one (shown below) was prepared using the product from step 3.

[0058] TIFF2026525208000010.tif27170

[0059] Under a nitrogen atmosphere, a stirred, ice-cold solution of 4-(2-chloro-4-nitrophenyl)-7-hydroxychromen-2-one (3.18 g, 10 mmol) and DiPEA (8.60 ml, 50 mmol) was added to a stirred solution of trichlorosilane (3.60 ml, 35 mmol) in a dry DCM (20 ml) over 90 minutes. The mixture was stirred at +0°C for 1 hour, quenched with water (10 ml) and MeOH (10 ml), and solid NaHCO3 (10 g) was added to adjust the pH to 8-9. The suspension was filtered through a Celite pad, the pad was washed with DCM (2 × 150 ml), and the solvent was evaporated. The residue was dissolved in 2-methyltetrahydrofuran (150 ml), washed with water (100 ml), saturated NaHCO3 (100 ml), and brine (100 ml), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to obtain 4-(4-amino-2-chlorophenyl)-7-hydroxychromen-2-one as a dark reddish-brown solid (3.02 g). LC-MS 288.0 [M+1] + , RT 4.94 minutes. 1 ¹H-NMR (DMSO-d6): d 10.60 (s,1H), 7.05 (d,1H), 6.99 (d,1H), 6.75 (s,1H), 6.72 (dd,1H), 6.63 (dd,1H), 6.05 (s,1H), 5.75 (broad s,2H).

[0060] In step 5, N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide (shown below) was prepared using 4-(4-amino-2-chlorophenyl)-7-hydroxy-chromen-2-one.

[0061] TIFF2026525208000011.tif30170

[0062] At room temperature under a nitrogen atmosphere, a stirring solution of 4-(4-amino-2-chlorophenyl)-7-hydroxy-chromen-2-one (1.0 g, 3.48 mmol) and pyridine (1.1 ml, 4.00 mmol) in 25 ml of dry 2-methyltetrahydrofuran was added to a solution of tetradecanoyl chloride (0.90 g, 3.63 mmol) in 2 ml of dry DCM. This mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours, diluted with 50 ml of 2-methyltetrahydrofuran, 70 ml of 2 M HCl, and 40 ml of brine, dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. Purified with silica gel (5-60% siRNA in isohexane), N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide was obtained as a light brown solid (1.25 g). LC-MS 498.3 [M+1] + , RT 6.47 minutes. 1 H-NMR (DMSO-d6) d 11.50 (broad s,1H), 11.20 (s, 1H), 8.00 (d, 1H) 7.60 (dd, 1H), 7.37 (d, 1H), 6.90 (d,1H), 6.79 (s, 1H) 6.74 (dd, 1H), 6.14 (s, 1H), 2.34 (t, 2H), 1.40 (m, 2H), 1.23 (m, 20H), 0.84 (m, 3H).

[0063] Next, the product from step 5 was used in step 6 to prepare (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoate ethyl as shown below.

[0064] TIFF2026525208000012.tif29170

[0065] Under a nitrogen atmosphere, ethyl-S-lactate (0.23 ml, 1.20 mmol) was added to a stirred, ice-cold solution of diisopropyl azodicarboxylic acid (0.24 ml, 1.20 mmol) and triphenylphosphine (0.31 g, 1.20 mmol) in dry 2-methyltetrahydrofuran (10 ml). This mixture was stirred for 15 minutes, and a solution of N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide (0.50 g, 1.00 mmol) was added. This mixture was stirred at +0°C for 0.5 hours, then warmed to room temperature and stirred for 64 hours. The solvent was evaporated under reduced pressure to obtain the crude product. Purification with silica gel (10-50% MTBE in isohexane) yielded ethyl (2R)-2-[4-(4-amino-2-chlorophenyl)-2-oxo-chromen-7-yl]oxypropanoate as a light brown solid (0.46 g). LC-MS 598.4 [M+1] + , RT 6.93 minutes. 1 H-NMR (DMSO-d6) d 11.25 (s, 1H), 8.01 (d, 1H) 7.60 (d, 1H), 7.39 (d, 1H), 7.00 (d,1H), 6.98 (d, 1H) 6.88 (dd, 1H), 6.27 (s, 1H), 5.70 (q, 1H), 2.34 (t, 2H), 1.60 (m, 2H), 1.54 (d, 3H), 1.23 (m, 20H), 0.85 (m, 3H).

[0066] In Step 7, 2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (shown below) was prepared from the product of Step 6.

[0067] TIFF2026525208000013.tif29170

[0068] At room temperature under a nitrogen atmosphere, 0.42 g, 1.0 mmol of ethyl (2R)-2-[4-(4-amino-2-chlorophenyl)-2-oxo-chromemen-7-yl]oxypropanoate was stirred in 5 ml of MeTHF and 5 ml of MeOH. LiOH hydrate (0.42 g, 10.0 mmol) was added to 5 ml of water. The mixture was stirred for 2 hours, diluted with 30 ml of MeTHF, washed with 20 ml of 2H HCl and brine, dried over MgSO4, filtered, and the solvent evaporated under reduced pressure to obtain (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromemen-7-yl]oxypropanoic acid as a light brown solid (0.45 g). LC-MS 570.0 [M+1] + , RT 6.70 minutes. 1 H-NMR (DMSO-d6) d 11.25 (s, 1H), 8.01 (d, 1H) 7.60 (d, 1H), 7.39 (d, 1H), 7.00 (d,1H), 6.98 (d, 1H) 6.88 (dd, 1H), 6.27 (s, 1H), 5.70 (q, 1H), 2.34 (t, 2H), 1.60 (m, 2H), 1.54 (d, 3H), 1.23 (m, 20H), 0.85 (m, 3H).

[0069] Next, step 8 included the preparation of the phosphonium moiety by the preparation of (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (shown below).

[0070] TIFF2026525208000014.tif28170

[0071] At room temperature under a nitrogen atmosphere, a stirring solution of (5-carboxypentyl)triphenylphosphonium chloride (0.46 g, 1.0 mmol) in dry DCM (20 ml) was mixed with oxalyl chloride (0.24 ml, 2.8 mmol) and dry DMF (2 drops). The mixture was stirred for 90 minutes, and the solvent was removed under reduced pressure to obtain crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (0.43 g).

[0072] Next, [6-(4-tert-butoxycarbonylpiperazin-1-yl)-6-oxo-hexyl]-triphenyl-phosphonium chloride (shown below) was prepared using the product from step 8.

[0073] TIFF2026525208000015.tif28170

[0074] At room temperature under a nitrogen atmosphere, crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (0.94 g, 2.2 mmol) was stirred in THF (30 ml) and pyridine (1 ml). Tert-butyl piperazine-1-carboxylate (0.45 g, 2.4 mmol) was added to this mixture. The mixture was stirred for 80 hours and concentrated under reduced pressure. The residue was dissolved in DCM (25 ml), washed with 1 M HCl (25 ml), saturated NaHCO3 (25 ml), and brine (25 ml), dried over MgSO4, filtered, and concentrated to obtain [6-(4-tert-butoxycarbonylpiperazin-1-yl)-6-oxo-hexyl]triphenylphosphonium chloride as a colorless solid (1.07 g). LC-MS 545.4 [M+] + , RT 5.26 minutes. 1 H-NMR (CDCl3) 7.88-7.65 (m, 15H) 3.93 (m, 2H), 3.54 (m, 2H), 3.42 (m, 2H), 3.35(m, 4H), 2.37(t, 2H), 1.78 (m, 2H), 1.68 (m, 4H), 1.46 (s, 9H). 31 H-NMR (CDCl3) d.

[0075] Using the phosphonium portion, in step 10, 6-oxo-6-piperazine-1-yl-hexyl)-triphenyl-phosphonium chloride hydrochloride (shown below) was prepared using the product from step 9.

[0076] TIFF2026525208000016.tif29170

[0077] At room temperature under a nitrogen atmosphere, a stirred solution of [6-(4-tert-butoxycarbonylpiperazin-1-yl)-6-oxo-hexyl]-triphenyl-phosphonium chloride (0.052 g, 0.09 mmol) in DCM (3 ml) was mixed with 4 M HCl (2 ml, 8 mmol) in dioxane. This mixture was stirred for 3 hours and concentrated under reduced pressure to obtain (6-oxo-6-piperazin-1-yl-hexyl)-triphenyl-phosphonium chloride hydrochloride as a light brown solid (0.052 g). LC-MS 445.3 [M+] + , RT 3.46 minutes. 1 H-NMR (CDCl3) d 9.84 (broad s, 2H) 7.90-7.76 (m, 15H), 3.67 (m, 6H), 3.01 (m, 4H), 2.30(m, 2H), 1.49 (m, 6H). 31 H-NMR (CDCl3) d.

[0078] In step 11, the products from steps 7 and 10 were used to obtain compound [I], [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-6-oxo-hexyl]-triphenyl-phosphonium chloride.

[0079] TIFF2026525208000017.tif31170

[0080] At room temperature and under a nitrogen atmosphere, a stirred solution of (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoic acid (0.065 g, 0.11 mmol) in MeTHF (5 ml), EDC (0.033 g, 0.17 mmol), HOBt (0.023 g, 0.17 mmol), and NMM (50 μl, 0.45 mmol) was mixed with a solution of (6-oxo-6-piperazine-1-yl-hexyl)-triphenyl-phosphonium chloride hydrochloride (0.052 g, 0.09 mmol) in DMF (3 ml). This mixture was stirred for 74 hours, concentrated, diluted with DCM (30 ml), washed with 1 M Na2CO3 (15 ml), 0.1 M HCl (15 ml), and brine (15 ml), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. Purified with silica gel (1-15% MeOH in DCM), [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-6-oxo-hexyl]-triphenyl-phosphonium chloride was obtained as a light brown solid (0.021 g). LC-MS 996.3 [M+] + , RT 6.45 minutes. 1 H-NMR( MeOD-d4) d 7.99 (s, 1H), 7.86 (m, 3H), 7.70-7.72 (m, 12H), 7.60 (dd, 1H), 7.30 (dd, 1H), 7.05 (d, 1H), 6.88-6.84 (m, 2H), 6.16 (s, 1H), 5.38 (m, 1H), 3.77-3.47 (m, 8H), 3.40 (m, 2H), 2.40 (m, 4H), 1.69 (m, 4H), 1.59 (m, 5H), 1.31 (m, 22H), 0.88 (t, 3H). 31 P-NMR (MeOD-d4) d 23.8 ppm.

[0081] Another illustrative embodiment is the compound [II] (shown below) having the IUPAC name [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-6-oxo-hexyl]-tris[4-trifluoromethyl)phenyl]phosphonium chloride.

[0082] TIFF2026525208000018.tif41170

[0083] In Step 1, 4-[(2S)-2-hydroxypropanoyl]piperazine-1-carboxylate tert-butyl (shown below) was prepared.

[0084] TIFF2026525208000019.tif21170

[0085] At room temperature under a nitrogen atmosphere, a stirred solution of (S)-lactic acid (2.00 g, 22.2 mmol), piperazine-1-carboxylate tert-butyl (4.11 g, 22.2 mmol), and DiPEA (4.0 ml, 22.2 mmol) in DMF (10 ml) was mixed with HATU (10.1 g, 26.2 mmol) in DMF (3 ml). This mixture was stirred for 24 hours, concentrated, diluted with 10% MeOH in DCM (220 ml), washed with 0.1 M HCl (100 ml), saturated NaHCO3 (75 ml), and brine (75 ml), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. Purification with silica gel (1-5% MeOH in MTBE) yielded 4-[(2S)-2-hydroxypropanoyl]piperazine-1-carboxylate tert-butyl as a light brown solid (1.56 g). LC-MS 261.1 [M+23] + , RT 3.94 minutes. 1 H-NMR (CDCl3) 4.45 (q, 1H), 3.76 (d, 1H), 3.72-3.30 (m, 8H), 1.46 (s, 9H), 1.32 (d, 3H).

[0086] In Step 2, 4-[(2S)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazine-1-carboxylate tert-butyl (shown below) was prepared from the product of Step 1.

[0087] TIFF2026525208000020.tif27170

[0088] Under a nitrogen atmosphere, a stirred, ice-cold solution of N-[3-chloro-4-(7-hydroxy-2-oxo-chromen-4-yl)phenyl]tetradecanamide (0.45 g, 0.90 mmol) and 4-[(2S)-2-hydroxypropanoyl]piperazine-1-carboxylate tert-butyl (0.28 g, 1.08 mmol) in dry 2-methyltetrahydrofuran (10 ml) was prepared. Diisopropyl azodicarboxylate (0.23 ml, 1.08 mmol) was added to this mixture. The mixture was stirred for 10 minutes, and triphenylphosphine (0.28 g, 1.08 mmol) was added. The mixture was stirred at +0°C for 0.5 hours, then warmed to room temperature and stirred for 16 hours. The solvent was evaporated under reduced pressure to obtain the crude product. Purification with silica gel (20-100% toluene in isohexane) yielded 4-[(2S)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazine-1-carboxylate tert-butyl as a light brown solid (0.51 g). LC-MS 738.3 [M+1] + , RT 6.99 minutes. 1 H-NMR (DMSO-d6) d 10.27(s, 1H), 7.62 (dd, 1H) 7.40 (dd, 1H), 6.97 (m, 3H) 6.80 (m, 1H), 6.25 (s, 1H), 5.45 (q, 1H), 3.51 (m, 8H), 1.99 (t, 2H), 1.60 (m, 2H), 1.46 (d, 3H), 1.40 (s, 9H), 1.20 (m, 20H), 0.83 (m, 3H).

[0089] Next, in step 3, N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide; hydrochloride was prepared.

[0090] TIFF2026525208000021.tif35170

[0091] At room temperature under a nitrogen atmosphere, a stirred solution of N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromemen-4-yl]phenyl]tetradecanamide; hydrochloride (0.20 g, 0.27 mmol) in DCM (5 ml) was added to a mixture of 4 M HCl (5 ml, 20 mmol) in dioxane. This mixture was stirred for 3 hours and concentrated under reduced pressure to obtain N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromemen-4-yl]phenyl]tetradecanamide; hydrochloride as a light brown solid (0.16 g). LC-MS 638.5 [M+1] + , RT 5.85 minutes. 1 H-NMR (DMSO-d6) d 10.44(s, 1H), 9.47 (broad s, 2H) 8.05 (dd, 1H), 7.65 (dd, 1H) 7.38 (dd, 1H), 7.01 (d, 1H) 6.99 (d, 1H), 6.85 (m, 1H), 6.25 (s, 1H), 5.49 (q, 1H), 3.96-3.05 (m, 8H), 2.36 (t, 2H), 1.59 (m, 2H), 1.46 (d, 3H), 1.23 (m, 20H), 0.83 (m, 3H).

[0092] In step 4, 5-carboxypentyl-tris[4-(trifluoromethyl)phenyl]phosphonium bromide (shown below) was prepared as a precursor for the phosphonium moiety.

[0093] TIFF2026525208000022.tif37170

[0094] A solution of tris-(4-trifluoromethyl)phenylphosphine (2.50 g, 5.4 mmol), 6-bromohexanoic acid (1.1 g, 5.4 mmol), and NaI (0.08 g, 0.84 mmol) was refluxed in dry MeCN for 64 hours. The insoluble residue was filtered off, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification with silica gel (5-15% MeOH in DCM) yielded 5-carboxypentyl-tris[4-(trifluoromethyl)phenyl]phosphonium bromide as a colorless solid (0.28 g). LC-MS 581.2 [M+] + , RT 4.86 minutes. 1 H-NMR (DMSO-d6) d 8.15 (m, 16H), 3.82 (m, 2H), 2.13 (t, 2H), 1.51 (m, 6H).

[0095] In step 5, compound [II] was prepared using the products from steps 3 and 4.

[0096] TIFF2026525208000023.tif41170

[0097] At room temperature and under a nitrogen atmosphere, NMM solution (40 μl, 0.35 mmol) was added to a stirred solution of N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide hydrochloride (0.050 g, 0.07 mmol), 5-carboxypentyl-tris[4-(trifluoromethyl)phenyl]phosphonium bromide (0.04 g, 0.06 mmol), EDC (0.017 g, 0.09 mmol), and HOBt (0.012 g, 0.09 mmol) in dry DMF (2 ml). This mixture was stirred for 16 hours, concentrated, diluted with DCM (30 ml), washed with 0.1 M HCl (15 ml) and brine (15 ml), dried over MgSO4, filtered, and the solvent was evaporated under reduced pressure to obtain the crude product. Purified with silica gel (1-10% MeOH in DCM), [6-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-6-oxo-hexyl]-tris[(4-trifluoromethyl)phenyl]phosphonium chloride was obtained as a light brown solid (0.0442 g). LC-MS 1200.5 [M+] + , RT 6.24 minutes. 1 H-NMR( MeOD-d4) d 8.09 (m, 16H), 7.98 (s, 1H), 7.60 (m, 1H), 7.30 (m, 1H), 7.05 (dd, 1H), 6.86 (m, H), 6.17(s, 1H), 5.39 (m, 1H), 3.65-3.54 (m, 10H), 2.40 (m, 4H), 1.74-1.62 m,6H), 1.58 (d, 3H), 1.34 (m, 20H), 0.87 (t, 3H). 31 P-NMR (MeOD-d4) d 25.2 ppm. 19 F-NMR (MeOD-d4) d 65.1 ppm.

[0098] Another illustrative embodiment of this method is compound [III] (shown below), which has the IUPAC name [5-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-5-oxo-pentyl]-triphenyl-phosphonium chloride. Compound [II] was prepared according to the following method.

[0099] TIFF2026525208000024.tif28170

[0100] Compound [III] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide; hydrochloride and (4-carboxybuta-1-yl)triphenylphosphonium bromide according to the method of step 5 of Compound 2 in Example, to obtain [5-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazine-1-yl]-5-oxo-pentyl]-triphenylphosphonium; chloride as a light brown solid (0.0351 g). LC-MS 968.3 [M+] + , RT 6.44 minutes. 1 H-NMR( MeOD-d4) d 7.99 (s, 1H), 7.91-7.72 (m, 16H), 7.61 (dd, 1H), 7.33 (dd, 1H), 7.07 (d, 1H), 6.88-6.85 (m, 2H), 6.19 (s, 1H), 5.39 (m, 1H), 3.85-3.36 (m, 10H), 2.69 (t, 2H), 2.42 (t, 2H), 1.95 (m, 2H), 1.72 (m, 2H), 1.60 (d, 3H), 1.37 (m, 20H), 0.88 (t, 3H). 31 P-NMR (MeOD-d4) d 23.7 ppm.

[0101] As another example of this method, compound [IV] (shown below), which has the IUPAC name [4-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-4-oxo-butyl]-triphenyl-phosphonium chloride, was prepared as follows.

[0102] TIFF2026525208000025.tif28170

[0103] Compound [IV] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide; hydrochloride and (3-carboxypropa-1-yl)triphenylphosphonium bromide according to the method of step 5 of Compound 2 in Example, to obtain [4-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazine-1-yl]-4-oxo-butyl]-triphenylphosphonium; chloride as a light brown solid (0.024 g). LC-MS 982.5 [M+] + , RT 6.39 minutes. 1 H-NMR( MeOD-d4) d 7.99 (s, 1H), 7.91-7.72 (m, 16H), 7.60 (d, 1H), 7.32 (dd, 1H), 7.07 (d, 1H), 6.88 (s, 1H), 6.19 (d, 2H), 5.38 (m, 1H), 3.83-3.35 (m, 10H), 2.47 (m, 2H), 2.41 (t, 2H), 1.84 (m, 2H), 1.72 (m, 2H), 1.60 (d, 3H), 1.37 (m, 21H), 0.88 (t, 3H). 31 P-NMR (MeOD-d4) d 23.7 ppm.

[0104] Figures 3A and 3B show the effects of compound [iV] at various concentrations on MCF-7 cells in a colony formation assay and hTERT-BJ1 cells in an SRB assay, respectively. As can be seen from Figure 3A, compound [IV] showed anticancer activity at 0.5 μM and eradicated MCF-7 cells at a concentration of 2.5 μM. In contrast, Figure 3B shows that compound [IV] has no activity against hTERT-BJ1 cells, a non-cancerous normal cell line useful for demonstrating the non-toxicity of pharmaceutical compounds.

[0105] As another example of this method, compound [V] (shown below), which has the IUPAC name [3-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazin-1-yl]-3-oxo-propyl]-triphenyl-phosphonium chloride, was prepared as follows.

[0106] TIFF2026525208000026.tif28170

[0107] Compound [V] was prepared from N-[3-chloro-4-[7-[(1S)-1-methyl-2-oxo-2-piperazine-1-ylethoxy]-2-oxo-chromen-4-yl]phenyl]tetradecanamide; hydrochloride and (2-carboxyeta-1-yl)triphenylphosphonium bromide according to the method of step 5 of compound [II] in examples, to obtain [3-[4-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]piperazine-1-yl]-3-oxo-propyl]-triphenylphosphonium chloride as a light brown solid (0.0243 g). LC-MS 954.5 [M+] + , RT 6.39 minutes. 1H-NMR(MeOD-d4) d 7.99 (s, 1H), 7.93-7.70 (m, 15H), 7.61 (dd, 1H), 7.33 (dd, 1H), 7.07 (d, 1H), 6.86 (m, 2H), 6.20 (s, 1H), 5.35 (m, 1H), 3.78-3.35 (m, 10H), 2.93 (t, 2H), 2.42 (t, 2H), 1.72 (m, 2H), 1.57 (d, 3H), 1.37 (m, 20H), 0.89 (t, 3H). 31 P-NMR (MeOD-d4) d 24.8 ppm.

[0108] As another example of this method, compound [VI] (shown below), which has the IUPAC name [6-[[1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]-4-piperidyl]amino]-6-oxo-hexyl]-triphenyl-phosphonium chloride, was prepared as follows.

[0109] TIFF2026525208000027.tif27170

[0110] Compound [VI] was prepared as follows: In step 1, the intermediate phosphonium compound [6-[(1-tert-butoxycarbonyl-4-piperidyl)amino]-6-oxo-hexyl]-triphenyl-phosphonium chloride (shown below) was prepared.

[0111] TIFF2026525208000028.tif28170

[0112] At room temperature under a nitrogen atmosphere, crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (0.94 g, 2.2 mmol) was stirred in THF (30 ml) and pyridine (1 ml). Tert-butyl-4-amino-piperidine-1-carboxylate (0.48 g, 2.4 mmol) was added to this mixture. The mixture was stirred for 80 hours and concentrated under reduced pressure. The residue was dissolved in DCM (25 ml), washed with 1 M HCl (25 ml), saturated NaHCO3 (25 ml), and brine (25 ml), dried over MgSO4, filtered, and concentrated to obtain [6-[(1-tert-butoxycarbonyl-4-piperidyl)amino]-6-oxo-hexyl]-triphenylphosphonium chloride as a light brown solid (0.84 g). LC-MS 559.5 [M+] + , RT 5.24 minutes. 1 H-NMR (CDCl3) d 8.26 (d, 1H), 7.86-7.68 (m, 15H) 4.01 (m, 2H), 3.84 (m, 1H), 3.74 (m, 2H), 2.85 (m, 2H), 2.34(t, 2H), 1.85 (m, 4H), 1.65 (m, 6H), 1.43 (s, 9H). 31 ¹H-NMR (CDCl3) d 24.8 ppm.

[0113] In step 2, the aforementioned product was converted to [6-oxo-6-(4-piperidylamino)hexyl]-triphenyl-phosphonium chloride (shown below).

[0114] TIFF2026525208000029.tif28170

[0115] At room temperature under a nitrogen atmosphere, a stirred solution of [6-[(1-tert-butoxycarbonyl-4-piperidyl)amino]-6-oxo-hexyl]-triphenyl-phosphonium chloride (0.24 g, 0.09 mmol) in DCM (10 ml) was mixed with 4 M HCl (5 ml, 20 mmol) in dioxane. This mixture was stirred for 90 minutes and concentrated under reduced pressure to obtain [6-oxo-6-(4-piperidylamino)hexyl]-triphenyl-phosphonium chloride hydrochloride as a light brown solid (0.28 g). LC-MS 459.3 [M+] + , RT 3.58 minutes. 1 H-NMR (CDCl3) d 9.31 (broad s, 2H), 8.15 (d, 1H), 7.90-7.75 (m, 15H), 3.78 (m, 1H), 3.60 (m, 2H), 3.20 (m, 2H), 2.90 (m, 2H), 2.05 (t, 2H), 1.84 (m, 2H), 1.65 (m, 2H), 1.48 (m, 6H). 31 ¹H-NMR (CDCl3) d 24.0 ppm.

[0116] In Step 3, compound [VI] was prepared using the product from Step 2 and (2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromemen-7-yl]oxypropanoic acid according to the method of Step 11 of Compound 1 in Example, and [6-[[1-[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromemen-7-yl]oxypropanoyl]-4-piperidyl]amino]-6-oxo-hexyl]-triphenyl-phosphonium chloride was obtained as a light brown solid (0.0267 g). LC-MS 1010.4 [M+] + , RT 6.42 minutes. 1H-NMR( MeOD-d4) d 8.02 (m, 1H), 7.88-7.74 (m, 16H), 7.61 (m, 1H), 7.32 (d, 1H), 7.08 (d, 1H), 6.85 (m, 2H), 6.18 (d, 1H), 5.38 (m, 1H), 4.39 (m, 1H), 4.15 (m, 1H), 3.90 (m, 1H), 3.40 (m, 4H), 2.86 (m, 1H), 2.42 (t, 2H), 2.16 (m, 2H), 1.92 (m, 1H), 1.64 (m, 12H), 1.28 (m, 22H), 0.88 (t, 3H). 31 ¹H-NMR (CDCl3) d 23.8 ppm.

[0117] TIFF2026525208000030.tif35170

[0118] As another example of this method, compound [VII] (shown below), which has the IUPAC name [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromen-7-yl]oxypropanoyl]amino]-1-piperidyl]-6-oxo-hexyl]-triphenyl-phosphonium chloride, was prepared as follows.

[0119] TIFF2026525208000031.tif45170

[0120] In Step 1, the phosphonium intermediate compound [6-[4-(tert-butoxycarbonylamino)-1-piperidyl]-6-oxo-hexyl]-triphenyl-phosphonium chloride (shown below) was prepared.

[0121] TIFF2026525208000032.tif36170

[0122] At room temperature under a nitrogen atmosphere, crude (6-chloro-6-oxo-hexyl)triphenylphosphonium chloride (1.30 g, 3.0 mmol) was stirred in THF (35 ml) and pyridine (1 ml). N-(4-piperidyl)carbamate tert-butyl (0.86 g, 4.3 mmol) was added to this mixture. The mixture was stirred for 3 hours and concentrated under reduced pressure. Purified with silica gel (3-12% MeOH in DCM), [6-[4-(tert-butoxycarbonylamino)-1-piperidyl]-6-oxo-hexyl]-triphenylphosphonium chloride was obtained as a colorless solid (1.30 g). LC-MS 559 [M+] + , RT 5.26 minutes. 1 H-NMR (CDCl3) d 7.86-7.65 (m, 15H) 4.66 (m, 1H), 4.12 (d, 1H), 3.84 (m, 3H), 3.63 (m, 1H), 3.05 (t, 1H), 2.66 (t, 1H), 2.33 (m, 2H), 2.02-1.53 ​​(m, 10H), 1.43 (s, 9H). 31 ¹H-NMR (CDCl3) d 24.3 ppm.

[0123] In Step 2, the product from Step 1 was converted to [6-(4-amino-1-piperidyl)-6-oxo-hexyl]-triphenyl-phosphonium chloride.

[0124] TIFF2026525208000033.tif30170

[0125] At room temperature under a nitrogen atmosphere, a stirred solution of [6-[4-(tert-butoxycarbonylamino)-1-piperidyl]-6-oxo-hexyl]-triphenyl-phosphonium chloride (0.25 g, 0.42 mmol) in DCM (5 ml) was added to 4 M HCl (5 ml, 20 mmol) in dioxane. This mixture was stirred for 90 minutes and concentrated under reduced pressure to obtain [6-(4-amino-1-piperidyl)-6-oxo-hexyl]-triphenyl-phosphonium chloride as a colorless solid (0.29 g). LC-MS 459.3 [M+] +, RT 3.61 minutes. 1 H-NMR (DMSO-d6) d 8.43 (b broad s, 2H), 7.82-7.76 (m, 15H), 4.35 (d, 1H), 3.86 (d, 1H), 3.60 (m, 3H), 3.20 (m, 1H), 3.01 (m, 1H), 2.58 (m, 1H), 2.26 (m, 2H), 1.92 (m, 2H), 1.65 (m, 2H), 1.48 (m, 8H). 31 ¹H-NMR (CDCl3) d 24.1 ppm.

[0126] In Step 3, using the product from Step 2, compound [VII], [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromemen-7-yl]oxypropanoyl]amino]-1-piperidyl]-6-oxo-hexyl]-triphenyl-phosphonium chloride was prepared according to the method of Step 11 for compound [I], and [6-[4-[[(2R)-2-[4-[2-chloro-4-(tetradecanoylamino)phenyl]-2-oxo-chromemen-7-yl]oxypropanoyl]amino]-1-piperidyl]-6-oxo-hexyl]-triphenyl-phosphonium chloride was obtained as a light brown solid (0.0375 g). LC-MS 1010.5 [M+] + , RT 6.41 minutes. 1 H-NMR( MeOD-d4) d 8.00 (d, 1H), 7.92-7.71 (m, 16H), 7.61 (dd, 1H), 7.32 (dd, 1H), 7.06 (d, 1H), 6.91 (m, 2H), 6.17 (d, 1H), 4.37 (m, 1H), 3.91 (m, 2H), 3.42 (m, 2H), 3.13 (q, 1H), 2.75 (t, 1H), 2.42 (t, 2H), 2.38 (m, 2H), 1.57 (m, 14H), 1.28 (m, 22H), 0.89 (t, 3H). 31 P-NMR (MeOD-d4) d 23.8 ppm.

[0127] These synthesis examples can be used by those skilled in the art to prepare compounds of formula [I] with obvious modifications. It should be understood that other synthetic methods are also available to obtain compounds using this method. The following structures are examples of further compounds produced by this method.

[0128] TIFF2026525208000034.tif95170 TIFF2026525208000035.tif94170 TIFF2026525208000036.tif90170 TIFF2026525208000037.tif195170 TIFF2026525208000038.tif105170 TIFF2026525208000039.tif115170 TIFF2026525208000040.tif128170 TIFF2026525208000041.tif119170 TIFF2026525208000042.tif89170 TIFF2026525208000043.tif106170 TIFF2026525208000044.tif97170 TIFF2026525208000045.tif106170

[0129] The data obtained for the embodiments of this method indicate that these compounds exhibit potent anticancer activity in both inhibiting tumor growth and preventing metastasis, while being non-toxic to normal cells. The following paragraphs summarize the data for compound [I]. Preliminary results for other embodiments are consistent with those for compound [I], and it should be noted that analyses of compounds [I] to [VII], etc., are currently underway.

[0130] Figures 1A-1C show the results of mammothsphere formation in MCF-7 cells, SRB assay results, and SRB assay results in BJ1-hTERT cells for various concentrations of compound [I]. Asterisks indicate P values. ** P<0.01, *** P<0.001, **** This indicates P<0.0001. As can be seen from Figure 1A, compound [I] inhibited the MCF-7 sphere at all concentrations, and nearly complete inhibition at 2.5 μM. Compound [I] achieved inhibition of over 50% at concentrations of 0.5–1.0 μM, demonstrating the remarkable efficacy of compounds in this method.

[0131] Figures 1B and 1C show the results of SRB assays on (B) MCF-7 cells and (C) hTERT-BJ1 cells. These data indicate that compound [I] showed cytotoxicity starting at less than 1.0 μM in MCF-7 cells, but showed almost no cytotoxicity in hTERT-BJ1 cells up to around 10 μM.

[0132] Figures 2A and 2B show the effects of various concentrations of compound [I] on MCF-7 colony formation in a Petri dish (A) and the effect expressed as colony formation compared to the control group (B). As can be seen from the representative two-dimensional colony phase image in Figure 2A, treatment with compound [I] significantly reduced MCF-7 colony formation. MCF-7 colony formation showed complete inhibition starting at compound [I] 1.0 μM. The significant inhibitory activity on MCF-7 colony formation is shown in Figure 2B, where compound [I] is IC2. 50 The graph clearly shows that the concentration is less than 0.5 μM, and complete inhibition is observed at 1.0 μM. Preliminary results suggest that other compounds tested using this method exhibit equivalent or superior efficacy and selectivity against cancer stem cells.

[0133] Figures 4A and 4B show the effects of compound [IX] at various concentrations on MCF-7 cells in a colony formation assay and hTERT-BJ1 cells in an SRB assay, respectively. Compound [IX] showed anticancer activity starting at concentrations below 0.5 μM, but remained nontoxic to hTERT-BJ1 cells even at high concentrations of 10 μM.

[0134] TIFF2026525208000046.tif38170

[0135] Figures 5A and 5B show the effects of compound [X] at various concentrations on MCF-7 cells in a colony formation assay and hTERT-BJ1 cells in an SRB assay, respectively. Compound [X] showed anticancer activity starting at low concentrations such as 0.25 μM, but remained nontoxic to hTERT-BJ1 cells even at high concentrations such as 10 μM.

[0136] TIFF2026525208000047.tif49170

[0137] Figures 6A and 6B show the effects of various concentrations of compound [XI] on MCF-7 cells in a colony formation assay and hTERT-BJ1 cells in an SRB assay, respectively. Compound [XI] showed anticancer activity at 0.5 μM and complete inhibition at 2.5 μM, but was nontoxic to hTERT-BJ1 cells at all test concentrations.

[0138] TIFF2026525208000048.tif38170

[0139] Figures 7A and 7B show the effects of compound [XII] at various concentrations on MCF-7 cells in a colony formation assay and hTERT-BJ1 cells in an SRB assay, respectively. Compound [XII] showed anticancer activity at 0.25 μM and complete inhibition at 0.5 μM, but did not show toxicity to hTERT-BJ1 cells up to concentrations above 1 μM.

[0140] TIFF2026525208000049.tif38170

[0141] In the description of the synthesis method and reaction scheme, the following abbreviations are used: acetonitrile (MeCN), ethyl acetate (RINKAN), methanol (MeOH), hydrochloric acid (HCl), magnesium sulfate (MgSO4), diisopropylethylamine (DiPEA), tert-butyloxycarbonyl (Boc), dichloromethane (DCM), sodium bicarbonate (NaHCO3), isopropanol (IPA), tetrahydrofuran (THFF), 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), and 4-dimethylaminopyridine (DMAP), trifluoroacetic acid (TFA), 1-hydroxybenzotriazole (HOBt), and ammonia (NH3).

[0142] Liquid chromatography-mass spectrometry data were prepared using a Waters Sunfire C1830 × 4.6 mm column. Gradient elution agent: 20-100% acetonitrile / water (containing 0.05% formic acid). Time: 0-10 minutes.

[0143] NMR spectra were recorded using a Bruker 500MHz Avance III HD spectrometer equipped with a broadband prodigy cryoProbe (1H, 13C, 15N, 19F, 31P) running TopSpin version 3.6, patch level 4, in a Windows 10 environment.

[0144] The following paragraphs describe the materials and assays used to generate the data described herein. It should be understood that those with the ordinary level of the art can perform assays similar to those described herein, and / or other assays commonly known in the art are available to evaluate the physical, chemical, and pharmaceutical properties of the compounds described herein.

[0145] Reagents and Model Cell Lines: Please note that other cell lines can be used without deviating from this method. The human mammary cancer cell line MCF-7 was from the American Type Culture Collection (ATCC). The hTERT-BJ1 cells were 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 culture medium was changed 2-3 times per week.

[0146] 3D Scaffold-Independent Growth Assay: This assay is also called the mammothsphere formation assay. Single-cell suspensions were prepared by enzymatic dissociation and manual dissociation (25g needle). Next, cells were seeded at a density of 500 cells / cm2 in mammothsphere medium (DMEM-F12 + 1X B-27 Plus Supplement + 20ng / ml EGF + Pen / Strep) under non-adhesion conditions in culture dishes pre-coated with (2-hydroxyethyl methacrylate) (poly-HEMA, Sigma Aldrich Inc.), called "mammothsphere plates". The cells were grown for 5 days and maintained at 37°C in a humidified incubator. After 5 days of culture, 3D mammothspheres larger than 50 μm were counted using an eyepiece ("scale plate"), and the percentage of seeded cells that formed spheres was calculated. This is called the mammothsphere formation rate (MFE) and was normalized to 1 (1 = 100% MFE). The rate of 3D mammothsphere formation (MFE) was analyzed in both low- and high-ATP cell subpopulations. All 3D mammothsphere experiments were performed independently at least three times with three replicates.

[0147] Statistical Analysis: All analyses were performed using GraphPad Prism 6. Data were expressed as mean ± standard deviation (SD) (or ± SEM if specified). All experiments were conducted independently at least three times, with more than three technical replicates for each experimental condition (unless otherwise specified, such as when showing representative data). Statistical significance was determined using Student's t-test or analysis of variance (ANOVA). For comparisons between multiple groups, one-way ANOVA was used to determine statistical significance. p < 0.05 was considered statistically significant.

[0148] This method includes a method for confirming cell viability. Those skilled in the art can select one or more methods for confirming cell viability suitable for a particular embodiment. The inventors initially used a sulforhodamine (SRB) assay based on the measurement of intracellular protein content. After treating cells in a 96-well plate for 72 hours, the cells were fixed in 10% trichloroacetic acid (TCA) for 1 hour in a cold room and dried overnight at room temperature. Subsequently, the cells were incubated with SRB for 15 minutes, washed twice with 1% acetic acid, and air-dried for at least 1 hour. Finally, the protein-bound dye was dissolved in 10 mM Tris pH 8.8 buffer and measured at 540 nm using a plate reader. Using the SRB assay, the inventors selected only compounds that depleted ATP levels without significant cytotoxicity for further analysis. Significant cytotoxicity was defined as less than 30% of cells remaining on the plate. Naturally, in embodiments employing other cell viability confirmation methods, compounds for further analysis can be selected based on other considerations known to those skilled in the art.

[0149] Therapeutic agents can be used in the form of pharmaceutical compositions that can be prepared using one or more known methods. For example, pharmaceutical compositions can be prepared using one or more fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, or other diluents or excipients well known to those skilled in the art. Depending on the therapeutic purpose, various types of dosage unit forms can be selected. Examples of forms of pharmaceutical compositions, but not limited to, include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injectable preparations (solutions and suspensions), topical creams, and other forms well known to those skilled in the art. For the purpose of forming pharmaceutical compositions in tablet form, any known additives can be used, such as carriers such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, cyclodextrin, crystalline cellulose, and silicic acid; and binders such as water, ethanol, propanol, monosaccharide syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, and polyvinylpyrrolidone. Furthermore, disintegrants such as dried starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, starch, and lactose can also be used. Disintegration inhibitors such as white sugar, stearin, coconut butter, and hydrogenated oil; absorption enhancers such as quaternary ammonium bases and sodium lauryl sulfate can also be used. Wetting agents such as glycerin, starch, and others well known to those skilled in the art can also be used. Adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid can also be used. Lubricants such as refined talc, stearate, boric acid powder, and polyethylene glycol can also be used. If tablets are desired, they can be further coated with conventional coating materials to produce sugar-coated tablets, gelatin film-coated tablets, enteric-coated tablets, film-coated tablets, bilayer tablets, and multilayer tablets. Pharmaceutical compositions suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, foams, sprays, aerosols, or oils.Such pharmaceutical compositions may include, but are not limited to, conventional additives such as preservatives, drug penetration aids, cosolvents, emollients, propellants, viscosity modifiers (gelling agents), surfactants, and carriers.

[0150] This method can be used to prevent and / or reduce the likelihood of tumor recurrence and / or metastasis. Anticancer therapies often fail because tumors recur or metastasize, particularly after surgery. Mitochondrial activity in CSCs is at least partially involved in the causes of these treatment failures. Embodiments of this method can be used in combination with anticancer therapies in situations where conventional cancer therapies are ineffective, and / or to prevent or reduce the likelihood of failure due to tumor recurrence and / or metastasis.

[0151] This method provides a way to selectively target cancer cells. Target cancer cells may be at least one of the following: CSCs, energetic cancer stem cells (e-CSCs), circulating tumor cells (CTCs, seed cells that cause further tumor growth in subsequent distant organs, a mechanism responsible for the majority of cancer-related deaths), and treatment-resistant cancer cells (TRCCs: cells that have acquired resistance to one or more chemotherapy, radiotherapy, and other common cancer treatments).

[0152] As described in the applicant's concurrently pending U.S. Provisional Patent Application No. 62 / 686,881 filed June 19, 2018, and No. 62 / 731,561 filed September 14, 2018 (both of which are incorporated herein by reference), e-CSC represents a CSC phenotype associated with proliferation. In addition to bulk cancer cells and CSCs, it should be understood that this method can be used to target a hyperproliferative cell subpopulation that exhibits a gradual increase in stem cell markers (ALDH activity and mammothsphere formation activity), a significantly increased mitochondrial volume, and increased glycolytic and mitochondrial activity, which the inventors refer to as e-CSCs.

[0153] From the above, it should be understood that this method can take various forms depending on the embodiment. For example, embodiments of this method can take the form of a composition, particularly a pharmaceutical composition. The therapeutic compound may be an active ingredient and may be present in a pharmaceutically effective amount.

[0154] Embodiments of this method may also take the form of a method for preventing or reducing the possibility of at least one of tumor recurrence and metastasis. In some embodiments, an effective amount of a composition containing the compound of this method may be administered as a therapeutic agent. In some embodiments, an effective amount of a composition containing embodiments of the compound described herein may be administered as a therapeutic agent.

[0155] The terms used in this description of the invention are for the purpose of describing specific embodiments and are not intended to limit the invention. Where used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context clearly indicates otherwise. The invention includes numerous options, modifications, and equivalents, which will become apparent by considering the following detailed description.

[0156] Terms such as “First,” “Second,” “Third,” “a),” “b),” and “c)” may be used herein to describe various elements of the present invention, but it will be understood that these terms should not limit the scope of the claims. These terms are used solely to distinguish the elements of the present invention from one another. Accordingly, the first element described below may also be called an elemental aspect without departing from the teachings of the present invention, and so the third element. Accordingly, terms such as “First,” “Second,” “Third,” “a),” “b),” and “c)” are used solely for identification purposes and are not necessarily intended to indicate an order or other hierarchy of the related elements. The order of operations (or steps) is not limited to the order shown in the claims.

[0157] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Furthermore, terms as defined in commonly used dictionaries should be interpreted in a way consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. The terms used in describing the invention herein are intended solely to describe specific embodiments and are not intended to limit the invention. All publications, patent applications, patents and other references cited herein are incorporated by reference as part of this specification. In the event of any conflict in terminology, this specification shall prevail.

[0158] Furthermore, as used herein, “and / or” encompasses all possible combinations of one or more of the enumerated items, as well as the absence of any combination (“or”) where it is interpreted as a choice.

[0159] The terms “reduce,” “decrease,” “mitigate,” and “reduce” generally refer to a composition produced by this method resulting in and / or causing a smaller physiological response (i.e., a measurable downstream effect) compared to the response caused by either the vehicle or the control molecule / composition, e.g., a reduction in tumor volume. A “reduced” or “reduce” response is typically a “statistically significant” response and may include reductions of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, or more (e.g., 500, 1000) (including any integer greater than 1 and integers and decimals greater than or equal to 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the response produced by a normal subject, an untreated subject, or a control-treated subject.

[0160] The term "treatment cycle" refers to a treatment process, such as a scheduled administration of medication repeated at regular or predetermined intervals. A treatment cycle may include several days of treatment followed by several days of rest. For example, in a four-week treatment cycle, a drug may be administered daily for two weeks, followed by a two-week rest period. It should be understood that a treatment cycle may depend on several factors, such as the individual's disease state, age, sex, and weight, as well as the specific drugs and / or methodologies used to elicit the desired response in the individual.

[0161] Unless the context indicates otherwise, the various features of the invention described herein are particularly intended to be usable in any combination. Furthermore, the present invention intends that in some embodiments of the invention, any of the features or combinations of features shown herein may be excluded or omitted. For example, if the present invention states that a complex comprises components A, B, and C, it is particularly intended that any one of A, B, or C, or any combination thereof, may be omitted or abandoned.

[0162] For example, when used herein to refer to measurable values ​​such as quantity or concentration, the term “about” means to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of a particular quantity. The ranges indicated herein for measurable values ​​may include any other range and / or individual values ​​within it.

[0163] 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 above, as many obvious modifications are possible without departing from its spirit or scope as claimed below.

Claims

1. Chemical structure: [In the formula, R １ａ is -OR １ａ -, -SR １ａ -, -NR １ａ -, -NR １ａ C(O)R １ａ -, -OC(O)R １ａ -, -C(O)OR １ａ -, -SO ２ R １ａ , -S(O)R １ａ -, -NR １ａ C(O)-, -C(O)NR １ａ -, -NR １ａ S(O) ２ -, S(O) ２ NR １ａ -, -OC(O)NR １ａ -, -NR​​​​​​​​​​​​​​​​ R １ａ This includes H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl, or phenyl (where chemically acceptable, oxo, C) １ -C １８ - Alkyl, C ２ -C １８ - Alkinyl, C ２ -C １８ - Alkenil, C １ -C １８ -Optionally substituted with 1 to 4 groups independently selected from haloalkyl groups), OR ａ , NR ａ R ｂ , SR ａ , C(O)OR ａ C(O)NR ａ R ｂ Halo, Cyano, Nitro, C(O)R ａ , and S(O) ２ OR ａ Selected independently from; R ａ In each existence, H and C １ -C １８ - Independently selected from alkyl groups; R ｂ In each existence, H, C １ -C １８ - Alkyl, C(O)C １ -C １８ - Alkyl and S(O) ２ -C １ -C １８ - Selected independently from alkyl; R ２ is halogen, -CF ２ H, -CF ３ , -OCF ２ H, -OCF ３ Independently 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 allenes, heteroallenes, phenols, and benzoic acid; R ３ and R ４ These may be the same or different, and are selected from H, 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, and benzoic acid; R ５ H, -CF ３ , -OCF ３ , -OCHF ２ , -NO ２ Selected from , and -CN; L １ is -OR ２ａ -, -SR ２ａ -, -NR ２ａ -, -NR ２ａ C(O)R ２ａ -, -OC(O)R ２ａ -, -C(O)OR ２ａ -, -SO ２ R ２ａ , -S(O)R ２ａ -, -NR １ａ C(O)-, -C(O)NR ２ａ -, -NR ２ａ S(O) ２ -, S(O) ２ NR ２ａ -, -OC(O)NR ２ａ -, -NR ２ａ C(O)OR ２ａ -, -NR １ａ C(O)NR ２ａ -, -CR ２ａ =CR ２ａ - and -CC-, -CH ２ R ２ａ is independently selected from; R ２ａ is independently selected from 1 to 4 groups optionally substituted with groups independently selected from H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, heteroaryl or phenyl (when chemically acceptable, oxo, C １ -C ６ -alkyl, C ２ -C ６ -alkynyl, C ２ -C ６ -alkenyl, C １ -C ６ -haloalkyl), OR ａ , NR ｙ R ｂ , SR ｙ , C(O)OR ｙ , C(O)NR ｙ R ｂ , halo, cyano, nitro, C(O)R ｙ , S(O) ２ OR ｙ and is independently selected from; R ｙ In each existence, H and C １ -C ６ - Independently selected from alkyl groups; R ｂ In each existence, H, C １ -C ６ - Alkyl, C(O)C １ -C ６ - Alkyl and S(O) ２ -C １ -C ６ - Selected independently from alkyl; X － [These are selected from anions suitable for forming pharmaceutically acceptable salts.] A compound having the following properties.

2. X － The compound according to claim 1, wherein the compound is selected from the group consisting of acetate ion, benzenesulfonate ion, benzoate ion, besylate ion, hydrogen tartrate ion, bromide ion, bamsilate, chloride ion, citrate ion, decanoate ion, edetate ion, esylate ion, fumarate ion, gluceptate ion, hexanoate ion, iodide ion, isethionate ion, lactate ion, malate ion, maleate ion, mandelate ion, mesylate ion, methyl sulfate ion, mucinate ion, napsylate ion, nitrate ion, octanoate ion, oleate ion, pamoate ion, pantothenate ion, phosphate ion, propionate ion, salicylate ion, stearate ion, succinate ion, sulfate ion, tartrate ion, theoclate ion, and tosylate ion.

3. X － Cl － , Br － , I － , and MeSO ３ － A compound according to any one of claims 1 to 2, selected from the group consisting of the following.

4. R ５ CF ３ The compound according to any one of claims 1 to 3.

5. and The compound according to claim 1, having a structure selected from the group consisting of the following.

6. The compound according to claim 1, having a structure selected from the group consisting of the following.

7. and The compound according to claim 1, having a structure selected from the group consisting of the following.

8. R ５ CF in each existence ３ The compound according to claim 1.

9. R ５ The compound according to claim 1, wherein is H in each presence.

10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, wherein the carrier is selected from the group consisting of sugar, lactose, glucose, sucrose, starch, corn starch, potato starch, cellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, tragacanth gum, malt, gelatin, talc, cocoa butter, glycol, propylene glycol, polyol, glycerin, sorbitol, mannitol, polyethylene glycol, ester, ethyl oleate, ethyl laurate, agar, buffer, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogenic substance removal water, isotonic physiological saline, Ringer's solution, ethyl alcohol, and phosphate buffer.

12. The pharmaceutical composition according to claim 10, wherein the excipient is selected from the group consisting of lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, cyclodextrin, crystalline cellulose, and silicic acid.

13. A method for treating or preventing tumor recurrence and / or metastasis, comprising administering to a patient at risk of tumor recurrence and / or metastasis a pharmaceutically effective amount of a pharmaceutical composition comprising a compound according to any one of claims 1 to 9.

14. The method according to claim 13, wherein the administration is performed at at least one time point among before cancer treatment, concurrently with cancer treatment, and after cancer treatment.

15. A method for inhibiting the proliferation of cancer stem cells in a patient, comprising administering to the patient a pharmaceutically effective amount of a pharmaceutical composition containing a compound according to any one of claims 1 to 9.

16. The method according to claim 15, wherein the administration is performed at at least one time point among before cancer treatment, concurrently with cancer treatment, and after cancer treatment.

17. A method for treating cancer, comprising administering to a person having cancer a pharmaceutically effective amount of a pharmaceutical composition containing a compound described in any one of claims 1 to 9.

18. The method according to claim 17, wherein the administration is performed at at least one time point among before cancer treatment, concurrently with cancer treatment, and after cancer treatment.

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